The repellent potential of the celery seed oil was evaluated against adult *Ae. aegypti* using human-bait technique. Five human volunteers, non-allergic to mosquito bites, were invited from different institutes and a consent letter regarding the experiment was taken from each of them. The letters were deposited in the institute for reference, if needed. Starved and 3–10 days old females of *Ae. aegypti* were released in groups of 25 into separate laboratory cages (45 cm × 45 cm × 40 cm) for the investigations. During repellency bioassays, the arms of the human volunteers were thoroughly cleaned, washed with neutral soap without any fragrance, thoroughly rinsed with distilled water, and allowed to dry for 10 min before the application of extracts. A square of 5 cm × 5 cm size was marked on each forearm of human volunteers using a permanent marker. One forearm of all the volunteers was used for repellency bioassay and approximately 0.1 mL of the essential seed oil of *A. graveolens* was applied to the marked area. The other forearm of each volunteer was considered control and the marked area was applied with ethanol. The rest of the area of each forearm was covered by a paper sleeve thus leaving only the marked area open. Both the control and treated arms were introduced simultaneously into the cage. Any attempt of inserting the stylets by a female mosquito was considered a bite. The numbers of bites occurred in 3 min were scored every 15 min, for 3 h; from 11:00 to 14:00 hours.

Protection time was recorded as the time elapsed between the application of essential oil and the time of a confirmed bite. The tests, with none of the adult *Ae. aegypti* landing on the control arm or attempting to bite, were discarded. These tests were carried out again with a fresh batch of adults to ensure that failure to bite was due to repellence potential of oil and not because of the mosquitoes being pre-disposed to get a blood meal. Three replicates of each experiment were carried out. Each replicate was conducted in separate cages and with different volunteers to negate the effect of skin variability on repellency, if any.

The percent protection from the mosquito bite provided by the oil was calculated by using the following formula:

$$\% \text{Protein} = \frac{\text{Number of bits on the control arm} - }{\text{Number of bits on the treated arm}}$$

$$\text{Number of bits on the control arm} \times 100$$

### **CONTACT IRRITANCY ASSAY**

The contact irritancy assay was performed on 3-day-old nonblood females of *Ae. aegypti.* The oil-impregnated papers were prepared with Whatman filter paper no. 1, which were cut out in circles of 8 cm diameter. The papers were impregnated with 10 and 1% of celery seed essential oil and then allowed to shade-dry. The completely dried paper was placed on a glass plate, and a perspex funnel with a hole on the top was kept inverted over the impregnated paper. Single female adult was released in the funnel and per-conditioned for 3 min. Thereafter, the time at which first flight was taken was recorded. The experiment was continued for 15 min and the total number of flights undertaken by each female adult was scored. Three replicates were carried out for each treatment. Parallel control tests were performed with papers impregnated with acetone. The relative irritability caused by the seed oil was calculated with respect to control by the following formula:

#### **PHYTOCHEMICAL ANALYSIS**

The essential oil extracted from *A. graveolens* seeds was analyzed for the presence of phytochemical components using standard procedures as illustrated by Harborne (19). The qualitative biochemical assays were performed to identify the presence of secondary metabolites; alkaloids, carbohydrates, flavonoids, phenolic compounds, phlobatannins, saponins, tannins, and terpenoids.

## **RESULTS**

The potential of essential oil extracted from the seeds of celery plant, *A. graveolens* was evaluated as larvicidal and repellent agent against dengue vector,*Ae. aegypti*. The investigations validated the significant potential of essential oil as the probable agent for the control and management of rising *Ae. aegypti* population.

#### **LARVICIDAL BIOASSAY**

The 24 h exposure of early fourth instars of *Ae. aegypti* to the essential seed oil of *A. graveolens* resulted in quite low LC50 and LC90 values of 16.10 and 29.08 ppm, respectively (**Table 1**). The toxicity potential of the oil increased by 1.2-fold (LC50 = 13.22 ppm) on exposure of the larvae to the oil for another 24 h. The bioassay did not cause the formation of pupa or larval–pupal intermediates resulting in complete mortality of the larvae. The control treatments did not cause any mortality till 48 h (**Table 1**).

#### **BEHAVIORAL STUDIES IN OIL-TREATED AE. AEGYPTI LARVAE**

The larvae were scrutinized carefully during the treatment period for any behavioral modifications. The observations revealed that *A. graveolens* oil did not cause immediate or quick mortality. Initial exposure of the larvae to the essential oil did not affect the larvae and all larvae were found moving normally and exhibited a typical appearance. The restlessness in the larval behavior was noticed after 10 min of treatment. The principal lethal effects of the seed oil were observed after approximately 20–25 min of treatment in the form of incapability of rising to the water surface, body tremors, and convulsions. The symptoms of paralysis were clearly visible in 20% of the larvae after an hour leading to death of these larvae. Continued exposure of the larvae to oil caused mortality in approximately 50% larvae after 5–6 h, and the death of most of the larvae was observed within 10 h.

#### **ADULT REPELLENCY BIOASSAY**

Investigations conducted on the repellency potential of celery seed oil against adults *Ae. aegypti* revealed it as a promising and notable repellent. The oil resulted in 100% protection against bites by female *Ae. aegypti* in the first 150 min as compared to the ethanol that did not cause any repellency against mosquito bites (**Table 2**). When the experiment was continued for next 15 min, only one bite was recorded as compared to the nine bites scored on the control arm. It clearly reveals the reduced protection from 100 to 88.8% in the 165th-min. The percent protection to adult *Aedes* further decreased to 77% with two bites recorded after 180 min of exposure to the oil (**Table 2**). It is significant to note that direct application of the *A. graveolens* oil did not induce any dermal irritation during the experiment as well as afterward.

**Table 2 | Percent repellency and percent protection to the bites of Aedes aegypti after application of the essential oil of Apium graveolens on the arms of human volunteers**.

#### **CONTACT IRRITANCY ASSAY**

A significant elicit response was observed in the adults of *Ae. aegypti* when subjected to contact irritancy assays. The exposure to 10% oil led to complete knockdown of adults when released for acclimatization in the funnel for 3 min. Conversely, exposure to 1% seed oil caused first flight of the female adult after a mean time of 4 s (**Table 3**). The average total of 63.66 take-offs were observed after 15 min of exposure to 1% oil as compared to only 2.36 flights when exposed to ethanol-impregnated paper resulting in the relative irritability of 26.97.

#### **PHYTOCHEMICAL ANALYSIS**

The qualitative biochemical analysis for the secondary metabolites present in the essential oil of *A. graveolens* seeds revealed the presence of terpenoids, lactones, and flavonoids as the main constituents. Other tested components were not detected in the essential seed oil of *A. graveolens* (**Table 4**).

### **DISCUSSION**

Mosquito-borne diseases are increasing each year in tropical and sub-tropical countries. Since many decades, chemical insecticides have been used to combat the mosquito menace. However, the continued and frequent use of these insecticides has caused adverse effects, including toxicity to non-target organisms and humans, environment pollution, and increased development of resistance in the mosquito population. Botanicals have now become favorite agents among researchers as suitable alternatives to the toxic chemical insecticides. A few reports are available regarding the larvicidal and repellent potency of essential oils, volatiles extracted from different plants, against *Ae. aegypti* (20–22).

Keeping these in view, present studies were conducted to assess the probable role of celery seed oil as larvicidal, repellent, and irritancy agent for the control and management of *Ae. aegypti* population. Our investigations on early fourth instars of *Ae. aegypti* **Table 3 | Response of 3-day-old adult females of Aedes aegypti in the contact irritancy assays to celery seed essential oil-impregnated papers**.

aMean ± SEM.

**Table 4 | Phytochemical screening of the essential oil of the seeds of Apium graveolens.**

showed that 24 h of exposure to the oil resulted in an LC50 and LC90 value of 16.10 and 29.08 ppm, respectively (*p* > 0.05). It was also revealed that when the larvae were exposed to the oil for 48 h, the toxicity potential of the oil rose by 1.2-fold. However, keeping in view the large population of *Ae. aegypti* and enormous heterogeneity in their population*;* the chi-square distribution and insignificant *p* values obtained in our investigations suggest bioassays with more random selection of larvae and increased number of replicates to confirm the larvicidal potential of celery seed oil. Similar larvicidal activity of the ethanol-extracted *A. graveolens* was reported by Choochote et al. (23) against a Thailand strain of *Ae. aegypti,* the fourth instars exhibiting LD50 and LD95 values of 81.0 and 176.8 mg/L, respectively. The significant larvicidal activity of the volatile oils of *A. graveolens* has also been reported by Pitasawat et al. (24) against the two mosquito species, *Ae. aegypti* and *Anopheles stephensi* after an exposure to 24 h.

Several researchers have evaluated the larvicidal potential of various other essential oils against mosquitoes. The excellent larvicidal efficiency of the essential oil extracted from *Mentha piperita* revealing an LC50 and LC90 value of 111.9 and 295.18 ppm, respectively after 24 h of exposure has been reported against dengue vector (8). They also showed that the toxicity of the peppermint oil increased by 11.8% on exposure to the oil for 48 h. Similarly, Warikoo et al. (25) observed essential oil isolated from *Pinus longifolia* as the efficient larvicidal agent against *Ae. aegypti*. On exposure to commercially available pine oil, the early fourth instar larvae showed an LC50 value of 0.330 mg L−1 and an LC90 value of 1.118 mg L−1 . Recently, Liu et al. (10) established the larvicidal potential of essential oil derived from the roots of *Toddalia asiatica* and the constituents isolated from the oil against *Ae. albopictus.* The essential oil extracted from the leaves of *Feronia limonia* showed remarkable larvicidal activity against *An. stephensi* with LC50 value of 15.03 ppm after 24 h, while against *Ae. aegypti* and *Cx*. *quinquefasciatus*, the LC50 values reported were 11.59 and 22.49 ppm, respectively (26). Lee (27) evaluated the larvicidal activity of essential oils derived from 11 aromatic medicinal plants against early fourth instars of *Ae. aegypti* and reported 100% mortality on exposure to all oils at 100 ppm. Cheng et al. (28) investigated the larvicidal potential of the essential oils from the leaves of eight provenances of indigenous cinnamon (*Cinnamomum osmophloeum* Kaneh.) and reported the excellent inhibitory effect of the essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type against the IV instars of *Ae. aegypti*. Choochote et al. (23) have observed significant larvicidal activity of the volatile oils of *Curcuma aromatica* against the fourth instars of *Ae. aegypti* with an LC50 value of 36.30 ppm. Pushpanathan et al. (29) reported the LC50 values of 50.78 ppm when the essential oil extracted from *Zingiber officinalis* was tested against *Cx. quinquefasciatus*. Tiwary et al. (30) tested the essential oil of *Zanthoxylum armatum* against three species of mosquitoes and reported *Cx. quinquefasciatus* to be the most susceptible against oil with an LC50 value of 49 ppm followed by *Ae. aegypti* and *An. stephensi* with LC50 values in the range of 54–58 ppm.

Present study demonstrated the delayed toxicity of *A. graveolens* oil against early fourth instars of *Ae. aegypti* instead of quick larval mortality. Similarly, Choochote et al. (23) suggested the delayed larval killing effect of the ethanol-extracted celery seed oil *A. graveolens* against *Ae. aegypti*. The larvae treated with celery seed essential oil exhibited excitation and aggressive vertical and horizontal movements. These symptoms suggest the probable impact of oil on the neuro-muscular co-ordination in chemical synapses. These findings are in conformity with few earlier studies (17, 23, 31).

The exposure of the adults *Ae. aegypti* to celery seed oil established the promising and remarkable repellency potential. The oil provided 100% protection to human volunteers in the first 150 min followed by only one to two bites in the next 30 min of exposure. Likewise, the crude seed extract of celery has been reported to exhibit repellent activity against *Ae. aegypti* with ED50 and ED95 values of 2.03 and 28.12 mg/cm2 , respectively, providing the biting protection time of 3 h on application at a concentration of 25 g% (23). The mosquito repellent potential of celery, *A. graveolens*, has also been compared with commercial repellents by Tuetun et al. (32). Kumar et al. (8) reported the repellent properties of essential oil extracted from *M. piperita* against adults *Ae. aegypti* with 100% protection till 150 min after which only one to two bites were recorded during the next 30 min, as compared to eight to nine bites on the control arm. The skin repellent tests performed at 1.0, 2.0, 3.0, and 4.0 mg/cm2 with essential oil extracted from *Z. officinalis* gave 100% protection against *Cx. quinquefasciatus* up to 120 min (29). The repellent activities of the essential oil of *Cinnamomum zeylanicum*, *Z. officinale*, and *Rosmarinus officinalis* have been also reported by Prajapati et al. (33) against *An. stephensi, Ae. aegypti,* and *Cx. quinquefasciatus*.

In the present investigations, the contact irritancy assays with the celery seed oil resulted in a significant elicit response in the adults of *Ae. aegypti*. Exposure to 1% oil led to average number of 63.66 take-offs in 15 min with first flight after a mean time of 4 s. The behavioral modifications in the mosquitoes through chemical actions in contact irritancy may diminish population that may ultimately reduce disease transmission (34). Exposure of the female adults of *Ae. aegypti* to the crude leaf extracts of *Parthenium hysterophorus* showed a similar significant repellency behavior (9). Nour et al. (35) suggested the utilization of 0.1% essential oils of *Ocimum basilicum* as promising natural repellents against *Anopheles* mosquito.

Studies have shown that secondary metabolites; steroids, alkaloids, terpenoids, saponins, phenolics, essential oil, etc., of plants play a major role in the mosquito control being associated with a wide range of bioefficacy. The phytochemical analysis of the essential oil of *A. graveolens* seeds showed the presence of terpenoids, lactones, and flavonoids as the main constituents. Fazal and Singla (36) have reported d-limonene (80%) as the prime constituent in celery seed oils. They have also reported selinene, various sesquiterpene alcohols, b-elemne, linalool, *N*-butylphthalide, sedanenolide, and sedanonic anhydride as the other secondary metabolites in celery seed oil. However, the larvicidal and repellency potential of the essential celery seed oil reported in the present study need to be further investigated for identification of the compound responsible. It is further suggested that the efficacy observed may be because of the individual or the synergistic effects of various compounds present in them, identified or unidentified.

The potential role of *A. graveolens* seed essential oil as larvicide and repellent has been investigated against *Ae. aegypti*. Nevertheless, this is an explorative evaluation and the studies have suggested the potential bioefficacy of celery seed oil. However, keeping in view the heterogeneous and large population of *Ae. aegypti*,further complex investigations with more random selection of larvae and increased number of replicates are needed to ascertain the efficacy of celery seed oil against *Ae. aegypti.* Moreover, the identification of bioactive components present in the oil and understanding their mode of action is essential for its use as mosquito control agent. Field trials are recommended before the use of *A. graveolens* seed oil as an anti-mosquito natural, environment-friendly product in the mosquito-management program.

## **ACKNOWLEDGMENTS**

The authors are highly grateful to University Grants Commission for providing financial assistance to carry out the present investigations. Thanks are extended to Dr. Savithri Singh, Principal, Acharya Narendra Dev College for providing infrastructure and research facilities.

#### **REFERENCES**

*mitis*. *Trans Royal Soc Trop Med Hyg* (2004) **98**:451–5. doi:10.1016/j.trstmh. 2003.10.008

for mosquito control. *Fitoterapia* (2007) **78**:205–10. doi:10.1016/j.fitote.2007. 01.003

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 June 2014; accepted: 02 September 2014; published online: 18 September 2014.*

*Citation: Kumar S, Mishra M, Wahab N and Warikoo R (2014) Larvicidal, repellent, and irritant potential of the seed-derived essential oil of Apium graveolens against dengue vector, Aedes aegypti L. (Diptera: Culicidae). Front. Public Health 2:147. doi: 10.3389/fpubh.2014.00147*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Kumar, Mishra, Wahab and Warikoo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## New records of mosquitoes (Diptera: Culicidae) from Bolívar State in South Eastern Venezuela, with 27 new species for the state and 5 of them new in the country

## **Jesús Berti \*, Hernán Guzmán,Yarys Estrada and Rodrigo Ramírez**

Laboratory of Entomology, Center for Endemic Diseases Studies, Maracay, Venezuela

#### **Edited by:**

Juan-Carlos Navarro, Universidad Central de Venezuela, Venezuela

#### **Reviewed by:**

Thomas James Zavortink, University of California Davis, USA Gustavo Carlos Rossi, Centro de Estudios Parasitológicos y de Vectores, Argentina

#### **\*Correspondence:**

Jesús Berti, Laboratory of Entomology, Center for Endemic Diseases Studies, Las Delicias, Av. Bermudez Sur, Maracay, Aragua, Venezuela e-mail<EMAIL_ADDRESS>
**INTRODUCTION**

The occupation of Amazonia in Brazil and Venezuela, has formed part of the integration processes that both governments are implementing in their common border, which makes evident that both governments are aware of the importance that the Amazonia has in our present world (1). Diseases such as malaria, dengue, Chikungunya, Yellow Fever, Mayaro virus, West Nile virus, and several emerging and reemerging arboviruses, which are responsible for millions of cases of sickness and death among people living in the tropical regions, continue to be of great concern to the World Health Organization authorities in our present world (2). Dengue is an important arbovirus that affects humans; it is transmitted by *Aedes aegypti* (Linnaeus) and *Aedes albopictus* Skuse. *A. aegypti* and *A. albopictus* species have been involved in Dengue transmission in the Manaus rural areas, Brazilian Amazon (3)*. A. albopictus* a secondary dengue vector in Asia, has spread to America and Europe largely, due to the international trade of used tires (a typical larval habitat), timber, and other goods such as"lucky bamboo" (a decorative house plant that is marketed worldwide). This species has a wide geographical distribution; it is particularly resistant, and can survive in both rural and urban environments*.* Mosquito's eggs are highly resistant and can remain viable throughout dry season, and can survive in cold temperature regions of Europe (3)*. A. albopictus* and *A. terrens* (Walker) species are two potential Yellow Fever vectors in jungle. These species utilize a wide variety of natural larval microhabitats, such as tree-holes, bamboo internodes, and artificial containers and may be found in the same natural environments as *Haemagogus* species (1). *Haemagogus* species have been involved in Sylvain yellow fever transmission in Venezuela (4). Human malaria, is one of the most serious parasitic diseases in tropical ecosystems, it is caused by parasites of the genus *Plasmodium* (Apicomplexa: Plasmodidae) and transmitted

This is the first part of a series of studies related to mosquito ecological and biogeographic aspects. A total of 69 mosquito species (Diptera: Culicidae) was collected in 16 localities sampled in the Gran Sabana Municipality, Canaima National Park, and Venezuela. Twentyseven mosquito species are recorded for the first time from Bolívar State, Venezuela. Five of them species are reported for the first time in Venezuela: Anopheles malefactor Dyar and Knab (1907); Chagasia bonneae Root (1927); Chagasia ablusa Harbach (2009); Culex anduzei Lane (1944), and Uranotaenia leucoptera Theobald (1907). Their medical importance is commented, and ecological and epidemiological aspects are discussed. A checklist of the mosquito species reported in the Gran Sabana County is given.

**Keywords: mosquitoes, vectors, inventory, emerging diseases, Bolívar State, Gran Sabana,Venezuela**

among human hosts by bites of the infected *Anopheles* female (1). Five parasite species cause malaria in humans. *Plasmodium falciparum* and *P. vivax* are the two most common. *P. falciparum* is the most dangerous, with the highest rates of mortality (1).

In 2012, Venezuela reported the highest recorded incidence of malaria in its history with 51,264 cases. In Bolívar State, incidence cases increased to 44,180 (86.2% of the country); with three counties (Sifontes, Gran Sabana, and Cedeño) in "epidemic" and two counties (Piar and Sucre) in "alarm" status (5). *Anopheles darlingi* Root has been considered as human malaria's principal vector in South America. In Amazonas and Bolívar states, it is responsible for 90% of malaria cases reported in Venezuela (1, 6).

Mosquito borne diseases such as malaria, dengue, Venezuelan Equine Encephalitis (VEE), West Nile virus and others equine encephalitis, Mayaro, or Chikungunya are zoonoses with increasing incidence in the current decade in tropical and temperate countries. These diseases emerge as a consequence of changes made to terrains that often increase the natural and artificial mosquito larval habitats. Many of these mosquito species are of public health importance. Mosquito's population increases result in a risk increased of tropical diseases transmission (1). There are many factors that can accelerate the emergence of zoonoses, such as environmental changes, habitat modifications, variations of human and animal demography, deterioration of strategies of vector control, or changes in pathogen genetics (1). Efforts to control such species that transmit emerging diseases have primarily been concentrated on the use of synthetic insecticides. Unfortunately, this has resulted in the appearance of physiological mosquito resistance, toxicity problems to human, environmental contamination, ecological imbalance, and economic burden (1). Such problems have created the need to look for alternative, environmentally friendly control mechanisms, based on those found in nature.

These include the essential oils from plants, some of which have been used by people for medicinal purposes; also biological control (e.g., *Bacillus sphaericus* against *Anopheles*) and biochemical control with synthetics juvenile hormones (7, 8).

It is well known that the rate of species lost worldwide surpasses that by which taxonomic knowledge is increased (9). Thus, it is necessary to intensify studies focusing on diversity and from areas with an acceptable level of conservation (e.g., Canaima National Park, Venezuela). The Gran Sabana Region (Canaima National Park) is an undulating plain grass-dominated upland savanna covering close to 18,000 km2 , with altitudes ranging from 750 to 1,450 m (10). Most of the Gran Sabana uplands have a humid submontane climate, with average annual temperatures ranging between 18 and 24°C, average annual rainfall between 2,000 and 3,000 mm, and a short dry season occurring from December to March. This area is drained by tributaries of the Orinoco River (Venezuelan part of the igneous metamorphic Guyana Shield), most of them black-water Rivers, with very acidic and low mineral waters (10). The ecological studies in the Gran Sabana are relatively scarce, especially for short and long-term evaluations on possible changes induced by human activities (e.g., climatic change). Additionally, ecological studies on richness and distribution at regional levels are few. It is well known that Culicidae larvae are dependent on habitat characteristics and that they are sensitive to biotic factors, as predators and also to several abiotic factors (pH, temperature, dissolved oxygen, salinity, and conductivity).

The present article, refers to the finding of 69 species and 17 genera of mosquito (Diptera: Culicidae), collected in the Gran Sabana county, Bolívar State, Venezuela. Twenty-seven mosquito species are recorded for the first time from Bolívar State, and five species are reported for the first time in the country.

## **MATERIALS AND METHODS**

#### **STUDY AREA**

The study area is located in the southern of Gran Sabana Municipality, Canaima National Park, a natural protected area in southeastern Venezuela, Amazonian Region that borders Brazil and Guyana (**Figures 1**–**3**). This study is based on material collected in indigenous territory (Pemón ethnic group) of Gran Sabana,

Canaima National Park, located in Bolívar State, and roughly occupies the same area as the Gran Sabana region (11). The park was established on 12 June 1962. It is the second largest park in the country, after Parima-Tapirapecó in Amazonas State, and sixth biggest national park in the world (1). About 65% of the park is occupied by plateaus of rock called tepuis, which are a kind of plateau of 1.7–1.8 billion years old. The oldest Roraima sandstone is estimated to be 1.6 billion years old. These ancient mountains of the Guiana Shield (**Figure 1**) in the Amazonian Region (12) constitute a unique biological environment and are of great geological interest, which makes them one of the oldest formations in the world (11).

**FIGURE 2 | Relative situations of the Bolivar State in Venezuela**.

**FIGURE 3 | (A)** Relative situation of the municipalities in the Bolivar State; and **(B)** situations of the study area and Santa Elena of Uairén.

The park is home to indigenous Pemón Indians (Pemón ethnic group), part of the Carib linguistic group (11). Pemón Indians have an intimate relationship with the tepuis, and believe they are the home of the "Mawari" spirits. Most transport within the park is done by light plane, from airstrips built by various Capuchin missions, or by foot and canoe. Pemón indigenous have developed some basic and luxurious camps, which are mainly visited by tourists from around the world and Venezuelan tourists. In 1994, Canaima National Park was named a World Heritage Site by UNESCO, as a natural reserve that has abrupt relief special and unique around the world,"The Tepuis," The park includes the entire watershed of the right bank of the Caroní River, and two of the highest waterfalls in the world, the Angel Falls and the Kukenán Falls and plenty of waterfalls of lower altitude (11).

Field surveys of mosquito adults (human bait) and mosquito larvae sampling, were carried out in the Gran Sabana County. The annual mean temperature is 22°C (18–24°C); the total annual rainfall is 1,500–5,700 mm, with annual average rainfall between 2,000 and 3,000 mm, with altitudes ranging from 750 to 1,450 m and annual mean of 205 days with rain per year, and a very short dry season occurring from December to March (10). The estimated population is 39,000, mainly concentrated in the capital, Santa Elena of Uairén (**Figures 2** and **3**).

Sixteen localities and four rivers were sampled for mosquitoes in the Gran Sabana County near the common border Brazil– Venezuela, between August 2011 and November 2013. These sites were selected as representative areas of the Gran Sabana, Venezuelan Canaima National Park. All localities are at altitudes >600 m. These altitudes are variables and can range from 600 to 1,450 m. These localities and rivers are: Santa Elena de Uairén, Waramasén, Manak-Krú, Maurak, Colinas de la Laguna,Altamira, San Antonio, Kinok-Pon-Parú, La Primavera, Chiricayén, Chiririka, Uaiparú, Betania, Kamaiwa, El Paraíso, El Paují, Chiririka, Uairén, Uaiparú, and Kukenán rivers.

## **COLLECTION AND IDENTIFICATION OF MOSQUITOES**

A sampling program of mosquito larvae was carried out between August 2011 and November 2013 by visiting the study area periodically (dry and rainy season every year). Mosquito samples were taken with a standard dipper. The natural aquatic habitats (not-Phytotelmata) were *a priori* classified into four categories: lagoons, streams, rivers, and freshwater herbaceous swamps. At each breeding site, 30 dips for mosquito larvae samples were made. Most of the mosquito collections were made in tropical humid forests and edges of streams, rivers, and herbaceous swamps (morichales) of savanna areas in Gran Sabana County. Additionally, mosquito larvae were collected in artificial and natural containers. In Phytotelmata, larvae were collected by extracting water with a plastic pipettes from tree-holes, cut bamboo internodes, leaf axils of bromeliads, foliar axils of Araceae, fallen leaves of Musaceae, fallen palm spates, especially the "Moriches" palms (*Mauritia flexuosa*), and from floral bracts of Heliconiaceae. Immature mosquitoes were collected from 40 samples (water-holding structures) per plant species per locality. Collected immature specimens (IV instars larvae) from half the samples (20 samples) were preserved in ethanol (90%) for identification purposes. Specimens (larvae and pupae) from the rest of samples (50%) were pooled, transported, and reared in the field laboratory to obtain the associated specimens. The species presents, were recognized on the basis of correlated anatomical features in associated life stages. Sampling to adult collections on human landing catches (**Figures 4A,B**) were carried out in the same localities and rivers. The taxonomic determination of the specimens was based on direct observation of morphological characters, through a stereoscopic microscope (adults) and transmitted light microscope (larvae), using several taxonomic keys and descriptions and re-descriptions of species. The abbreviations employed for mosquito genera and subgenera are those proposed by Reinert (13).

All diagnostic and differential characters were confirmed by using several taxonomic keys and using descriptions and redescriptions of species (4, 14–28, 29). All immature and adults specimens are deposited at the Collection of Center for Endemic Disease studies, located in Las Delicias, Maracay, Venezuela.

## **RESULTS**

A list of mosquito taxa of the Gran Sabana Municipality is presented. A total of 69 mosquito species (Diptera: Culicidae) and 17 mosquito genera from the indigenous territory (Pemón ethnic group) are reported (**Table 1**). A total of 19 species of anophelines were collected; 17 of them belonging to genus *Anopheles*

**FIGURE 4 | (A)** Adult collections on human landing catch during the day. **(B)** Adult collections on human landing catch in streams edges during the nights.

## **Table 1 | Inventory of Culicidae from Gran Sabana Municipality, Venezuela**.

#### **Culicidae: 1.  Chagasia Chagasia bonneae Root (1927)b Chagasia ablusa Harbach (2009)b

#### **Culicidae: 2. Subfamily Culicinae**

2.1. Culex (VEE vectors and West Nile virus vectors) Culex (Phenacomyia) corniger Theobald (1903) Culex (Culex) quinquefasciatus Say (1823) Culex (Culex) brevispinosus Bonne-Wester and Bonne (1920) Culex (Culex) coronator Dyar and Knab (1906) Culex (Culex) nigripalpus Theobald (1901) Culex (Culex) pinarocampa Dyar and Knab (1908)a Culex (Melanoconion) dunni Dyar (1918) (VEE vector)a Culex (Melanoconion) educator Dyar and Knab (1906) Culex (Melanoconion) spissipes Theobald (1903) (VEE vector) Culex (Melanoconion) mistura Komp and Rozeboom (1951) Culex (Carrollia) urichii Coquillet (1906) Culex (Carrollia) anduzei Lane (1944)b Culex (Lutzia) bigoti Bellardi (1862) (predators)a

2.2. Aedes (Dengue, Mayaro, Chikungunya, VEE, and Yellow Fever vectors) Aedes (Stegomyia) aegypti Linnaeus (1762) (Dengue, Chikungunya, Mayaro, and Yellow Fever vector)

Aedes (Finlaya) terrens Walker (1856) (potential Yellow Fever vector) Aedes (Ochlerotatus) scapularis Rondani (1848) (EEV vector) Aedes (Ochlerotatus) serratus Theobald (1901) (EEV vector) Aedes (Ochlerotatus) fulvus Wiedemann (1828) (EEV vector)

Aedes (Ochlerotatus) angustivittatus Dyar and Knab (1907) (EEV vector)a

2.3. Psorophora (VEE vectors)

Psorophora (Janthinosoma) cyanescens Coquillet (1902) Psorophora (Janthinosoma) albipes Theobald (1907) Psorophora (Janthinosoma) ferox Von Humboldt (1819) Psorophora (Psorophora) ciliata Fabricius (1794)a Psorophora (Psorophora) lineata Von Humboldt (1819) Psorophora (Grabhamia) cingulata Fabricius (1805)

(Continued)

### 2.4. Mansonia (VEE vectors)

Mansonia (Mansonia) pseudotitillans Theobald (1901)a Mansonia (Mansonia) titillans Walker (1848)a

2.5. Coquilletidia (VEE vectors and West Nile virus vectors) Coquilletidia (Rhynchotaenia) juxtamansonia Chagas (1907)a Coquilletidia (Rhynchotaenia) venezuelensis Theobald (1912)a Coquilletidia (Rhynchotaenia) nigricans Coquillet (1904)a

2.6. Haemagogus (Mayaro and Sylvain Yellow Fever vectors) Haemagogus anastasionis Dyar (1921)a Haemagogus janthinomys Dyar (1921)a Haemagogus celeste Dyar and Núñez-Tovar (1927)

2.7. Uranotaenia (Avían Malaria vectors)

Uranotaenia (Uranotaenia) typhlosomata Dyar and Knab (1907)a Uranotaenia (Uranotaenia) calosomata Dyar and Knab (1907)a Uranotaenia (Uranotaenia) geometrica Theobald (1901) Uranotaenia (Uranotaenia) pulcherrima Arribalzaga (1891) Uranotaenia (Uranotaenia) nataliae Arribalzaga (1891)a Uranotaenia (Uranotaenia) leucoptera Theobald (1907)b Uranotaenia (Uranotaenia) lowii Theobald (1901)a

2.8. Aedeomyia (Avían Malaria vectors) Aedeomyia (Aedeomyia) squamipennis Arribalzaga (1878)

#### **Culicidae: Subfamily Culicinae: 3.Tribe: Sabethini**

3.1. Sabethes (Mayaro and Sylvain Yellow Fever vectors) Sabethes purpureus Theobald (1907)

3. 2. Limatus (potential arbovirus vectors) Limatus asulleptus Theobald (1903)a Limatus durhami Theobald (1901)

3.3. Wyeomyia (potential arbovirus vectors) Wyeomyia (Wyeomyia) celaenocephala Dyar and Knab (1906)a

3.4. Runchomyia (facultative predators) Runchomyia (Ctenogoeldia) frontosa Theobald (1903)

3.5. Johnbelkinia (facultative predators, arbovirus vectors) Johnbelkinia ulopus Dyar and Knab (1906)a

3.6. Trichoprosopon (potential arbovirus vectors) Trichoprosopon digitatum Rondani (1848)

#### **Culicidae:Toxorhynchitinae (agents biological control: predators)**

4.1. Toxorhynchites (predators) Toxorhynchites (Lynchiella) theobaldi Dyar and Knab (1906) Toxorhynchites (Lynchiella) haemorroidalis Fabricius (1787)a

aNew for the State, bNew for the country.

Meigen and 2 species belonging to genus *Chagasia* Cruz. Additionally, 50 species of culicines were collected, belonging to 14 genera of *Culicinae* and one genus (*Toxorhynchites*) of *Toxorhynchitinae* (**Table 1**).

Special attention should be placed in the new species records for Bolívar State and for Venezuela. A total of 27 mosquito species are recorded for the first time in Bolívar State. Five of them are new for the country (**Tables 1** and **2**) and they namely: *Anopheles (Anopheles) malefactor* Dyar and Knab (1907); *Chagasia bonneae* **Table 2 | (A) GPS collection coordinates in localities with new records; (B) GPS collection coordinates in localities with new records**.

(Continued)

were identified, according to keys proposed by Wilkerson (26)

and Harbach and Howard (20). Larval specimens of the three species were collected in Waramasén (**Figure 5**) and reared to obtain associated specimens; larval habitats of the three species are streams edges, especially sites with algae and partial shade (**Figure 5**). The same larval habitats for *A. malefactor* and *Anopheles punctimacula* Dyar and Knab (1906) were found by Wilkerson (26). The geographical distribution of *A. malefactor* was restricted to Panamá and northwestern Colombia (26). With this new record (*A. malefactor*) their geographical distribution in Central and South America now includes Panamá, Colombia, and Venezuela. Literature records indicate that *A. punctimacula* was found naturally infected with malaria parasites in Panamá and Colombia (26). However, *A. malefactor* is not a potential malaria vector in South America (26).

The species *Anopheles (Kertezsia) cruzii* Dyar and Knab (1908) and *Anopheles (Stethomyia*) *nimbus* Theobald (1902) are reported for the first time in Bolívar State (**Table 1**). Larvae of *A. nimbus* were collected in river edges of Chiririka River (**Figure 6**) especially in habitats with algae and partial shade. This species does not transmit diseases of medical importance to man. Adults were not collected. Larvae were reared to obtain the associated specimens. *A. nimbus* was identified, according to key proposed by Navarro (22). Females of *A. cruzii* are very aggressive crepuscular biters and they are potential malaria vectors (31); their larvae live in water of leaf axils of bromeliads (31). Adult females were captured on human landing catches, in the forest in El Paraíso and El Paují, between 16:30 and 18:45 h. Immature specimens were not collected. Diagnostic and differential characters of *A. cruzii* were confirmed in adults specimens, using descriptions of Wilkerson and Peyton in basis to 11 females collected in Iguape, Brazil (28). *Anopheles darlingi* Root (Major malaria vector in the southern Bolívar State) is reported for the first time in the Gran Sabana Municipality; also reports for the first time in South America, the two highest mosquito records for *A. darlingi.* These altitude records are: Colinas de la Laguna, with 870 MSL and Santa Elena, with 893 MSL. Larvae of *A. darlingi* were only collected in shaded pools in the forest. When the water level dropped during the dry season, pools formed in or near the river bed (Uaiparú and Uairén rivers). These localities with *A. darlingi* larval habitats are at altitudes >600 m. These altitudes can range from 600 to 1,200 m. The lower altitude record for *A. darlingi* in the Gran Sabana was found in Uaiparú River, with 628 m, and the highest altitude record was found in Santa Elena, near of the Uairén River (**Figures 6** and **7**) with 893 m. The same larval habitats of *A. darlingi* were found in Suriname by Rozendaal (32).

In this article, we also report for the first time in Venezuela, the presence of *Culex (Carrollia) anduzei*. With this new record, their geographical distribution in South America now includes Brazil and Venezuela. Immature specimens were identified, according to keys proposed by Valencia (25).*C. anduzei* females do not transmit diseases of medical importance to man (25). Larvae were collected in plastic and metallic artificial containers in Chiririka (**Figure 6**), El Paraíso, El Paují, and Santa Elena (**Figure 7**). Adult specimens of *C. anduzei* were not captured. Additionally, we report for the

**FIGURE 5 | Map of Waramasén, showing sites where larvae of Culex bigoti, Chagasia bonneae, Chagasia ablusa, Uranotaenia leucoptera, and Anopheles malefactor were collected for the first time; and**

**showing sites where adults of Johnbelkinia ulopus, Limatus asulleptus,Wyeomyia celaenocephala, and Culex dunni were captured for the first time**.

first time the presence of *Culex* (*Lutzia*) *bigoti* Bellardi in Bolívar State. This species does not transmit diseases of medical importance to man. Larval specimens were collected in Waramasén and were found in artificial containers (tires, and plastic and metallic containers). It is well known that *Cx. bigoti* larvae are predators in several artificial containers (tires, tin cans, old paint cans, plastic and metallic containers). In Paraná State, Brazil, larval specimens of *C. bigoti* were found in tires, cans, and cut bamboo (33). In Venezuela, this species was collected in artificial containers (tires and plastic containers) but, was not collected in epiphytic and terrestrial bromeliads (12, 34). *Toxorhynchites* (*Lynchiella*) *theobaldi* Dyar and Knab (1906) and *Toxorhynchites* (*Lynchiella*) *haemorroidalis* Fabricius (1787) were collected in tires and tree-holes; they are a potential predators used for mosquito control. A longterm investigation in Florida (USA), demonstrated a reduction in tree-holes, mosquitoes, attributable to predation by*Toxorhynchites rutilus* (35).

Additionally, *Culex dunni* Dyar (1918); *Aedes* (*Ochlerotatus*) *angustivittatus* Dyar and Knab (1907); and *Psorophora* (*Psorophora*)*ciliata* Fabricius (1794) are reported for the first time in Bolívar State. These three species are potential vectors of the VEE (36–38). In addition, we reports three new species of the genus *Coquilletidia* and two new species of *Mansonia* for Bolívar State; they are namely: *Mansonia titillans* Walker (1848), *Mansonia pseudotitillans* Theobald (1901), *Coquilletidia juxtamansonia* Chagas (1907), *Coquilletidia venezuelensis* Theobald (1912), and *Coquilletidia nigricans* Coquillet (1904). The two species of the genus *Mansonia* are potential vectors of the epizootic cycle of the VEE (36) and *Coquilletidia venezuelensis* is a potential vector of enzootic cycle of the West Nile virus in Venezuela (39).

The genus *Haemagogus* Williston includes mosquitoes with diurnal activity and immature habitats on Phytotelmata (treeholes and cut bamboo internodes). Adult females bite in forests during the day (40). *Haemagogus* species have been involved in yellow fever transmission, a virus circulating in forest areas in Latin America among arboreal primates and marsupials by means of mosquito bite (40). The genus comprises 28 species; 9 of them are present in Venezuela (4). The presence of *Haemagogus anastasionis* Dyar (1921), *Haemagogus janthinomys* Dyar (1921), and *Haemagogus celeste* Núñez-Tovar (1927) was detected in forest areas of Chiririka and Santa Elena (**Figures 6** and **7**). Adult specimens were identified, according to photographical key proposed by Liria and Navarro (4). These three species were captured on human landing catches in the forest, between 16:30 and 18:45 h, near of the Uairén and Chiririka rivers edges (**Figures 6** and **7**). Immature specimens were not collected. *H. anastasionis* and *H. janthinomys* species are two potential vectors of jungle yellow fever and jungle cycle of Mayaro virus in Venezuela (4); and both are new species records for the Gran Sabana and the Bolívar State. With these two new records, the genus *Haemagogus* in Bolívar State now includes six species: *H. celeste* Dyar & Núñez-Tovar, *H. equinus* Theobald, *H. leucocelaenus* (Dyar & Shannon),

*H. albomaculatus* Theobald, *H. anastasionis* Dyar, and *H. janthinomys* Dyar. The species *Sabethes purpureus* Theobald (1907) was collected on human landing catches near of the Uairén River edge (**Figure 7**). This species is also a potential vector of sylvatic yellow fever and jungle cycle of Mayaro virus. The Mayaro alpha virus produces non-specific, sub-lethal disease symptoms, often confused with dengue, but with symptoms of arthralgias (arthrosis) that can cause incapacitating disability (40). The Mayaro virus shows a great plasticity in vertebrate host infection, whereas high specificity in the mosquitoes of Culicidae family, vectors in the jungle cycle. Risk factors of infection are associated with forest areas of northern South America and the rainy season (40). The enzootic cycle is similar to the jungle cycle of yellow fever, which involves *Haemagogus* mosquitoes and monkeys as reservoirs (40). However, the involvement of others secondary vectors (e.g., *Sabethes*) and others hosts may be important in spread of the virus. Humans may have high levels of viremia and efficient experimental transmission has been demonstrated in *Aedes aegypti*, *Aedes albopictus*, and *Aedes scapularis*, suggesting a significant risk to public health in urban, rural, and domestic locations close to enzootic foci of Mayaro virus (40). In addition, we collected adults of *A. aegypti* and *A. scapularis* Rondani (1848) in urban and periurban areas of Santa Elena, Waramasén, Chiririka and Maurak, representing a significant risk to the inhabitant's populations of theses localities. *A. albopictus* specimens were not collected in the Gran Sabana.

*Aedes aegypti* and *A. albopictus* species have been involved in dengue transmission in the Manaus rural areas, Brazilian Amazon (3). The presence of *A. albopictus* in the Brazilian Amazon represents a potential risk of transmission of Chikungunya and Mayaro virus in urban, rural, domestic, and wild environments (1). The occurrence of its larvae and pupae, breeding in the same containers with other domestic species, associated with several sources of blood meal available in urban, rural, and wild environments, reveal its gradual establishment in the indoor of households and its potential involvement in the transmission of zoonotic pathogens to humans. The ability that their eggs may remain viable in nature for long periods of diapauses and the demonstrated transovarial transmission occurrence of several arboviruses has raised the need to expand the strategies directed toward combating*A. albopictus* in Vector Control Programs in South American (1). In Venezuela, *A. albopictus* was detected for the first time in 2009 (41), suggesting a significant risk to public health in urban, rural, domestic, and wild environments (1). Dormancy of the egg stage (and drought resistance) is considered to be a reproductive strategy for the long-term survival of multivoltine mosquitoes that develop in temporary habitats, such as tree-holes and other natural water containers that are subject to water fluctuations (42). Egg diapause involves a long stable arrest of hatching, even when environmental conditions are favorable for hatching (42).

Additionally, in this article we also report for the first time three new records of *Sabethini* Tribe mosquitoes for the Bolívar State (**Table 1**); and they are: *Wyeomyia celaenocephala* Dyar and Knab (1906), *Limatus asulleptus* Theobald (1903), and *Johnbelkinia ulopus* Dyar and Knab (1906). Adult females were collected on human landing catch during the day in Waramasén and Chiririka (**Figures 5** and **6**). These three species are potential arboviruses vectors in Venezuela (43) and *J. ulopus* larvae are facultative predators (43).

Species of the genus *Runchomyia* (**Table 1**) does not transmit diseases of medical importance to man; their larvae live in leaf axils of epiphytic and terrestrial bromeliads and in floral bracts (19). In 1986, the species *Runchomyia frontosa* Theobald was reported in carnivorous bromeliads from the Gran Sabana, Venezuela. The immature mosquitoes were collected living in the fluids held by the carnivorous bromeliad *Brocchinia reducta* Baker (44). The larvae of *Runchomyia frontosa* is a facultative predator, filter feeding, or consuming large prey that it captures with its enlarged maxillae in carnivorous bromeliads (44). Adult females of *Runchomyia frontosa* were collected on human landing catch during the day in El Paraíso and Waramasén (**Table 1**). This species does not transmit diseases of medical importance to man (19).

In addition we report, five new records of the genus *Uranotaenia* in Bolívar State, they are: *Uranotaenia typhlosomata* Dyar and Knab (1907); *Uranotaenia calosomata* Dyar and Knab (1907); *Uranotaenia nataliae* Arribalzaga (1891); *Uranotaenia leucoptera* Theobald (1907); and *Uranotaenia lowii* Theobald (1901). Species of genus *Uranotaenia* were found mainly in ground-water habitats, including springs, stream margins, herbaceous swamps, and temporary pools with vegetation (19). Some species have been found in tree-holes, plant axils, and artificial containers. Females of some species are known to feed on frogs, birds, and mammals, but are normally not attracted to humans (19). They are avian malaria vectors in Venezuela. In the Llanos of Venezuela, was found a high endemicity of avian malaria (45, 46). Immature specimens were collected in lagoons and herbaceous swamps (Morichales) in Santa Elena de Uairén, Waramasén, Maurak, Colinas de la Laguna, Altamira, San Antonio, Kinok-Pon-Parú, La Primavera, Chiricayén, Manak-Krú, Maurak, and Uaiparú. *Uranotaenia leucoptera* was collected only in tree-holes in Waramasén and Manak-Krú. This species is recorded for the first time from Venezuela. This study also extends the geographical distributions of *Uranotaenia leucoptera* in South America to Venezuela.

These findings shows the importance of further studies related to mosquito's geographical distribution, ecological aspects, arbovirus detection, epidemiological surveillance, and possible epidemiological link with emerging and reemerging arboviruses in the common border of Brazil and Venezuela. The entomological surveillance has an important role among the preventive measures against emerging diseases transmitted by insects, particularly by mosquitoes.

### **ACKNOWLEDGMENTS**

The authors appreciate the valuable collaboration of the following people and institutions: Dr. Jonathan Liria. Universidad de Carabobo. Facultad de Ciencias y Tecnología. Valencia, Venezuela. Dr. Yasmín Rubio-Palis, Dr. Darjaniva Molina, and Dr. María Naranjo. Servicio Autónomo Instituto de Altos Estudios Dr. Arnoldo Gabaldon (IAE). Maracay, Venezuela. This work was financed by Dirección de Investigación. Servicio Autónomo Instituto de Altos Estudios Dr. Arnoldo Gabaldon. Av. Bermúdez, Maracay, Venezuela.

#### **REFERENCES**

1. Berti JA. *Mosquitos (Diptera: Culicidae) de la Gran Sabana, Venezuela*. España: Editorial Académica Española (2012).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 July 2014; accepted: 18 November 2014; published online: 13 March 2015. Citation: Berti J, Guzmán H, Estrada Y and Ramírez R (2015) New records of mosquitoes (Diptera: Culicidae) from Bolívar State in South Eastern Venezuela, with 27 new species for the state and 5 of them new in the country. Front. Public Health 2:268. doi: 10.3389/fpubh.2014.00268*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2015 Berti, Guzmán, Estrada and Ramírez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Australian public is still vulnerable to emerging virulent strains of West Nile virus

#### **Natalie A. Prow 1,2, Elise K. Hewlett 1,2, Helen M. Faddy 3 , Flaminia Coiacetto4 ,WenqiWang4 ,Tarnya Cox 5,6 , Roy A. Hall 1,2 and Helle Bielefeldt-Ohmann2,4\***

1 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia

2 Australian Infectious Diseases Research Centre, The University of Queensland, St Lucia, QLD, Australia

3 Research and Development, Australian Red Cross Blood Service, Kelvin Grove, QLD, Australia

4 School of Veterinary Science, The University of Queensland, Gatton, QLD, Australia

5 Vertebrate Pest Research Unit, NSW Department of Primary Industries, Orange, NSW, Australia

6 Invasive Animals Cooperative Research Centre, University of Canberra, Bruce, ACT, Australia

#### **Edited by:**

Rubén Bueno-Marí, University of Valencia, Spain

#### **Reviewed by:**

James W. Hardin, University of South Carolina, USA Kalliopi-Stavroula S. Chatzigeorgiou, National and Kapodistrian University of Athens, Greece

#### **\*Correspondence:**

Helle Bielefeldt-Ohmann, School of Veterinary Science, The University of Queensland, Gatton Campus, QLD 4343, Australia e-mail: h.bielefeldtohmann1@ uq.edu.au

The mosquito-borneWest Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans and horses with particularly virulent strains causing recent outbreaks in Eastern Europe, the Middle East, and North America. In Australia, a strain ofWNV, Kunjin (WNVKUN), is endemic in the north and infection with this virus is generally asymptomatic. However, in early 2011, following extensive flooding, an unprecedented outbreak ofWNVKUN encephalitis in horses occurred in South-Eastern Australia, resulting in more than 1,000 cases and a mortality of 10–15%. Despite widespread evidence of equine infections, there was only a single mild human case reported during this outbreak. To understand why clinical disease was seen in horses without similar observations in the human population, a serosurvey was conducted using blood donor samples from areas where equine cases were reported to assess level of flavivirus exposure. The seroprevalence to WNVKUN in humans was low before the outbreak (0.7%), and no significant increase was demonstrated after the outbreak period (0.6%). Due to unusual epidemiological features during this outbreak, a serosurvey was also conducted in rabbits, a potential reservoir host. Out of 675 animals, sampled across Australia between April 2011 and November 2012, 86 (12.7%) were seropositive for WNVKUN, with the highest prevalence during February of 2012 (28/145; 19.3%). As this is the first serological survey for WNVKUN in Australian feral rabbits, it remains to be determined whether wild rabbits are able to develop a high enough viremia to actively participate inWNV transmission in Australia. However, they may constitute a sentinel species for arbovirus activity, and this is the focus of on-going studies. Collectively, this study provides little evidence of human exposure toWNVKUN during the 2011 outbreak and indicates that the Australian population remains susceptible to the emergence of virulent strains of WNV.

**Keywords:West Nile virus, equine encephalitis, seroprevalence, humans, rabbits**

## **INTRODUCTION**

Flaviviruses are a group of medically important arboviruses causing large disease outbreaks around the world with approximately 50 million cases per year. Mosquito-borne flaviviruses in the Japanese encephalitis virus (JEV) serogroup, including JEV, West Nile virus (WNV), and Murray Valley encephalitis virus (MVEV), cause severe, potentially fatal neurological disease in humans, horses, and some avian species. WNV has traditionally been associated with outbreaks of viral encephalitis in Europe and Africa (1). In 1999, WNV appeared for the first time in the USA, associated with an outbreak of a fatal or debilitating disease in humans and equines, and extremely high levels of morbidity and mortality in several species of native birds in New York (2, 3). Since its introduction into the USA, WNV has caused more than 16,196 human cases of neuroinvasive disease and more than 1549 deaths in the USA alone and spread to most parts of North, Central, and South America via mosquito-bird transmission cycles (4).

The Kunjin strain of WNV (WNVKUN) is a closely related virus from Australia. Although WNVKUN was initially considered a separate species in the flavivirus genus, studies by our laboratory and collaborators revealed that it shared a high degree of antigenic and genetic homology to WNV strains, justifying re-classification of the virus as a subtype of WNV (5–7). Until recently, the relatively benign WNVKUN, had only been associated with a few cases of non-fatal encephalitis in humans and a small number of equine cases since it was first isolated in 1960 (8). However, in early 2011 an unprecedented outbreak of equine encephalitis occurred in South-EastAustralia, causing mortality of 10–15% of horses infected (9). Upon isolation, a new equine virulent strain of WNVKUN was confirmed, being the first strain to cause a major outbreak in Australia (9). Symptoms of equine infection with this strain included ataxia, muscle paralysis and tremors, changes in temperament, incoordination, and general weakness, which are consistent with clinical signs of the equine WNV encephalitis caused by virulent North American (WNVNY99) and European strains (10, 11). Notably, this new WNV strain, named WNVNSW2011, arose in regions of Southern Australia where WNVKUNV had not been observed previously, including coastal and inland cities of New SouthWales (NSW) (9).

Abundant rainfall in the latter half of 2010 and extending into the first quarter of 2011 led to extensive flooding of inland areas of NSW. This unforeseen event provided ideal climatic conditions for breeding of freshwater mosquito populations, which recorded a sixfold increase in number compared to the previous season (12). This prolific population growth of the primary vector for WNVKUN is presumed to have spurred the major outbreak in 2011 (13, 14). Nevertheless, only a small number of human infections were recorded during the time period of the equine encephalitis epidemic. Perhaps even more curiously, a large number of the equine cases occurred east of the Great Dividing Range in the much dryer coastal regions where no flooding was experienced during the same period and mosquito populations were low (14). This prompted us to consider epidemiological factors other than a bird–mosquito–human transmission chain. One animal species that occurs in abundance in the main affected regions is the rabbit (*Oryctolagus cuniculus*). Feral rabbits have previously been shown to sustain a brief viremia sufficient to transmit to mosquitoes when experimentally infected with MVEV (15). Similarly, Eastern cottontail rabbits (*Sylvilagus floridanus*) develop a viremia sufficient for mosquito transmission when experimentally infected with WNVNY99 (16), and preliminary work in our group had shown that domestic rabbits (*O. cuniculus*) can become infected with WNVNSW2011 (17).

We therefore carried out a serological survey on humans from east-coast regions of NSW with high incidences of equine encephalitis cases, using plasma samples obtained from the Australian Red Cross Blood Service (Blood Service), to assess human exposure during the outbreak and evaluate the on-going risk of virulent strains of WNVKUN. In addition, we tested feral rabbits sampled by the NSW Department of Primary Industries (NSW DPI) from two areas in NSW and one area in all other states during and up to 1.5 years after the equine epidemic. Collectively, our data suggest that an overwhelming proportion of the Australian human population remains susceptible to the emergence of virulent strains of WNV, and that feral rabbits may represent a possible reservoir, at least in some areas of South-Eastern Australia.

### **MATERIALS AND METHODS**

#### **HUMAN PLASMA SAMPLES**

Plasma samples were acquired from healthy Australian blood donors after routine infectious disease testing was complete. Samples were selected from donors residing in eastern NSW coinciding with locations of WNVNSW2011 equine infection [**Figure 1**; see also Figure 3 in Ref. (14)], with samples collected from November 2009 to November 2010 (*n* = 148) forming the pre-2011 sample group and those from September 2013 (*n* = 168) forming the post-2011 sample group. Samples in the pre-2011 group were collected into EDTA plasma preparation tubes [Becton, Dickson and Company (BD) Biosciences, San Diego, USA], while samples collected in the post-2011 group were collected into EDTA tubes (BD Vaccutainer® Whole Blood Collection tube). All samples were centrifuged and plasma aliquots archived at −30°C. Donor demographic data were obtained for each sample (age, gender, suburb, and postcode). This study was carried out under approval from the Blood Service Human Research Ethics Committee.

### **RABBIT SERUM SAMPLES**

Serum samples derived from rabbits captured or shot as part of surveys conducted by NSW DPI in all states of Australia were made available for testing. Collections were performed in two central areas of NSW (Euchareena and Oaky Creek in the central tablelands), and one area in each of the other states and the Northern Territory (near Hattah-Kulkyne National Park in Victoria; near Coorong in South Australia; south-east Queensland; South-West Western Australia; **Figure 2**) during seven time periods between April 2011 (Autumn) and November 2012 (Spring). A total of 675 samples were available for testing. The age of the rabbits was determined by lens dry weight measurements as described previously (18).

#### **EPITOPE BLOCKING ELISA**

Virus-specific antibodies to WNVKUN or MVEV were detected in human plasma and rabbit sera using a blocking ELISA (bELISA) (19) as detailed in Prow et al. (20). Seropositivity is defined as inhibition of the binding of virus-specific monoclonal antibodies (mAb) by more than 30%. All samples were initially screened for flavivirus antibodies using the anti-flaviviral E protein mAb 4G2 as the competing antibody (20). All flavivirus positive samples were then tested for WNVKUN (both human and rabbit samples) and MVEV (human samples only) using the same bELISA protocols with the mAbs 3.1112G and 10C6, respectively. Horse sera previously tested to be positive and negative for the virus of interest (20) were employed as controls for the bELISA on human plasma,while serum from naïve SPF-bred New Zealand White rabbits and rabbits experimentally infected with WNVNSW2011 (kindly provided by W. Suen, University of Queensland) were used as negative and positive controls for the bELISA involving rabbit sera. Controls consisting of no sera and no antigen (coating buffer only) were also included in each bELISA.

#### **VIRUS NEUTRALIZATION ASSAY**

Samples positive in the bELISA were tested for neutralizing activity against WNVKUN (rabbit and human samples) and MVEV (human samples only) in a microtiter serum neutralization assays, using MVEV (strain 1–51) or WNVKUN (strain MRM16) as previously described in detail (20).

#### **STATISTICAL ANALYSIS**

The proportion seropositive and 95% CI was calculated. For both human and rabbit data, seropositivity was first compared across age group, location (state/region), sex (human only), and time period in univariate analyses using the chi-squared test or a univariate logistic regression. Where a significant relationship with seropositivity was observed for two or more variables, these variables were entered into a logistic regression model [with seropositivity (reactive or non-reactive) as the dependent variable

(non-reactive as the reference) and the other variables as factors]. Microsoft Excel (Microsoft Pty. Ltd., North Ryde, NSW, Australia) and the Statistical Package for the Social Sciences (SPSS; IBM Australia Ltd., St. Leonards, NSW, Australia) were used for data management and analyses.

## **RESULTS**

.

## **PROFILE OF THE HUMAN STUDY POPULATIONS**

A total of 316 individuals were included in this study, consisting of 148 in the pre-2011 cohort and 168 in the post-2011 cohort (**Table 1**). Just over half (52.2%) of the samples were from male donors. The median age of donors was 43 years (IQR 26–56) for the pre-2011 sample group and 51 years (IQR 40.5–61) for the post-2011 sample group, with a slightly skewed distribution toward older donors in the post-2011 sample group. The majority of samples were collected from regional NSW, west of the Great Dividing Range (**Table 1**).

## **SEROPREVALENCE OF FLAVIVIRUSES, WNVKUN, AND MVEV IN HUMAN BEINGS IN COASTAL NSW**

Flavivirus total antibody was detected in 15 of the 148 samples in the pre-2011 group (10.1%), while 13 of 168 (7.7%) samples were observed to be seropositive for flavivirus antibody in the post-2011 group (**Table 2**). Seroprevalence of WNVKUN total antibody in the pre-2011 samples was 0.7% with just one sample testing seropositive, which is similar to the seroprevalence observed in the post-2011 samples (0.6%) where again, just one sample was observed to be seropositive to WNVKUN (**Table 2**). These two WNVKUN seropositive samples were both from males, with ages of 68 and 65, respectively, both of whom were residents of Tamworth in the North West Slopes subregion of regional NSW. Both WNVKUN seropositive samples were also tested in the neutralization assay

**Table 1 | Characteristics of the human study population**.

rabbits during 2011–2012.

and confirmed to have neutralizing antibodies against WNVKUN with titers of 40 and 80, respectively. None of the total flavivirus seropositive samples tested positive for antibodies against MVEV.

No significant difference was observed between pre- and post-2011 time points in either total flavivirus seropositivity (*p* = 0.454) or in WNVKUN seropositivity (*p* = 0.928). Overall, the proportion of males seropositive for total flavivirus antibody was significantly higher than females (*p* = 0.001); however, age group (*p* = 0.146) and region (*p* = 0.889) did not significantly influence seropositivity (**Table 3**).

## **SEROPREVALENCE OF FLAVIVIRUSES IN RABBITS**

Out of a total of 675 rabbits sampled between April 2011 (Southern Hemisphere Autumn) and November 2012 (Southern Hemisphere Spring) 86 animals (12.7%) had antibodies specific for WNVKUN as determined in the 3.111G bELISA (**Table 4**). Of these, 28 (32.5% of bELISA positive) rabbits had WNVKUN neutralizing antibodies with titers varying from 20 to 160, as determined in the microneutralization assay (data not shown). By univariate analysis, there was a significant association between state (*p* < 0.001) and time period (*p* = 0.050), while age had no apparent effect (*p* = 0.876). By multivariate logistic regression, both state (*p* < 0.001) and time period (*p* = 0.018) were still associated with seropositivity (**Table 5**). Specifically, the seroprevalence was higher in the states of Queensland and Victoria compared to Tasmania, and also in summer 2012 and winter 2012 compared to spring 2012 (**Tables 4** and **5**; **Figures 2** and **3**).

**Table 2 | Flavivirus seroprevalence in blood donors from eastern NSW collected in the months prior to and soon after the equine 2011WNVKUN epidemic**.

**Table 3 | Breakdown of total flavivirus seropositivity by sex, age group, and region.**

†Not applicable.

**Table 4 | KUN seropositivity in Australian rabbits, from April 2011 to November 2012**.

**Table 5 | Multivariate logistic regression analysis: influence of factors on KUN seropositivity in Australian rabbits**.

## **DISCUSSION**

West Nile virus is an on-going global public health concern, causing large outbreaks in the USA, Europe, and more recently Australia. Following extensive flooding across Eastern Australia in 2011 promoting ideal conditions for mosquito breeding, an unprecedented outbreak of equine encephalitis occurred, leading to the isolation of the first virulent strain of WNV in Australia. However, a number of unusual epidemiological features associated with this outbreak, including the lack of severe human cases, previously unseen transmission of WNVKUN in coastal regions and a number of cases reported outside the areas affected by heavy rainfall, instigated further studies to try to explain the 2011 outbreak.

Historically, WNVKUN has been associated with only mild symptoms in humans and horses. The emergence of the WNVNSW2011 strain suggested the virus may have undergone changes and evolved to become more virulent and able to cause fatal encephalitis in horses. However, since no severe human cases were seen during the outbreak, this study aimed to determine whether the lack of human involvement in the outbreak was simply due to pre-existing antibody against WNVKUN. The very low seroprevalence of WNVKUN in humans observed in samples collected both before (0.7%) and after (0.6%) the 2011 outbreak of equine flavivirus encephalitis suggest that, in contrast to what was seen during the WNV incursion into the USA, humans residing in easternmost NSW were not exposed to the new WNVNSW2011. However, these seroprevalences are considerably lower than seen in previous serosurveys conducted in the Murray Valley region of NSW between 1991 and 2011, where WNVKUN seroprevalences had been observed to range between 2.2 and 2.5% in the human population (21, 22). Higher WNVKUN seroprevalence rates, rangingfrom 2.1 to 3.1%, have also been observed inVictorian residents (21, 23). It is possible that our results are biased because of the small sample size relative to previous studies and the fact that we studied a seemingly health conscious subset of the population, i.e., blood donors, who may take measures to limit their exposure to mosquitoes and thus arbovirus disease in accordance with Public Health recommendations. This suggestion is also supported by the absence of antibodies to MVEV despite 16 confirmed cases of MVEV in humans during 2011, as reported by the National Notifiable Diseases Network (12). However, it should be noted that the coastal regions of NSW are not typically affected by flavivirus diseases. Rather, MVEV tends to occur in the Murray Valley basin and occurrence is governed by rainfall and the resulting migration of birds. Previous VIC-based studies observed a MVEV seroprevalence of 3.7% (*n* = 2,783) in 1991–1992 and 2.2% (*n* = 1115) in 2011 (21, 23).

Another factor governing transmission of arboviruses is vector competence, which is the intrinsic ability for an arthropod vector to become infected with and transmit an arbovirus (24). Studies are currently being undertaken to determine whether the NSW2011 strain of WNVKUN exhibits increased fitness in the primary vector, *Cx. annulirostris*, compared to non-pathogenic strains (van den Hurk et al., personal communication). If the NSW2011 strain is transmitted more efficiently by mosquitoes, then increased vector competence of *Cx. annulirostris* may have contributed to the equine epidemic, making it even more intriguing that corresponding increases in human exposures and clinical cases were not seen.

A large scale serological survey was conducted in the NSW equine population in late 2011 following cessation of the equine epidemic (25). This study had two notable findings, firstly that the overall seroprevalence of WNVKUN in horses was low (3.9% of 1054 horses across the state) and lower than in previous studies. Secondly, almost all the seropositive horses came from far-western

districts of NSW (25),while the majority of clinical cases were seen in the eastern half of NSW (14). This also suggests that infection with this new strain of WNVKUN was associated with very high morbidity in the equine population.

2012.

In considering the distribution of equine encephalitis cases in the 2011 epidemic (14), we hypothesized that animal species other than birds might be involved in the spread of WNVKUN along the eastern coastal areas of NSW. Notably, this area largely avoided the flooding events of early 2011 and dry conditions prevailed in the region during the summer and autumn of 2011. One species with wide distribution across Australia is the feral rabbit (www.invasiveanimals.com). Rabbits have been shown experimentally to develop a viremia in the absence of clinical signs following infection with WNV (16, 17). Interestingly, the present study revealed a higher seroprevalence in rabbits caught in Queensland andVictoria, compared to NSW. However, this finding may be biased due to the sampling areas, with the NSW sampling area being west of the main regionfor equine cases [(14); **Figure 1**]. In contrast, the high seroprevalence in Victorian rabbits is in concordance with the equine epidemic extending into this state, and the fact that the rabbit sampling area (Hattah-Kulkyne National Park) was within the region with many equine cases. The findings for Queensland are particularly notable, as this state avoided the equine encephalitis epidemic despite major flooding (14) and despite the presence of a susceptible equine population (20). The lack of correlation between equine clinical cases and seroprevalence in rabbits in some areas may be taken to suggest that rabbits have no role in the transmission cycle. Nevertheless, the finding that seroprevalence in this species was independent of age may suggest that they could be used as sentinels for arbovirus activity in a particular region. As the rabbit is a pest species in Australia and various programs are in place to control them, culled animals could be sampled for serological surveys prior to destruction and in this way inform Public Health authorities about potential arbovirus activity in a particular region.

WNV is estimated to have infected approximately four million humans in North America, causing over 37,000 clinical infections and 1443 deaths between 1999 and 2012 (4). On-going surveillance of currently circulating WNV strains in North America has indicated that the virus is continually changing with at least three different genotypes identified to date. Given that North American and Australian strains of WNV are very close genetically, sharing ~99% amino acid identity, and all WNV strains share a common transmission cycle, the possibility of emerging virulent strains of WNV in Australia, able to induce severe human disease, remains a definite possibility. In light of the observations from this study, the Australian population is still vulnerable to these emerging virulent strains, as very few people appear to have levels of WNVKUNspecific antibodies sufficient to afford protective immunity.

## **AUTHOR CONTRIBUTIONS**

Conceived and designed the experiments: Natalie A. Prow, Helen M. Faddy, Roy A. Hall, Helle Bielefeldt-Ohmann. Performed the experiments: Natalie A. Prow, Elise K. Hewlett, Flaminia Coiacetto, Wenqi Wang, Tarnya Cox. Analyzed the data: Helen M. Faddy, Natalie A. Prow, Tarnya Cox, Helle Bielefeldt-Ohmann. Drafted the manuscript: Natalie A. Prow, Helen M. Faddy, Helle Bielefeldt-Ohmann. Revisions and final approval of the submitted version: all authors.

### **ACKNOWLEDGMENTS**

We wish to thank the donors and staff of the Australian Red Cross Blood Service (Blood Service), who have assisted in provision of specimens for testing in this study. The Blood Service is fully funded by the Australian Government for the provision of blood products and services to the Australian community. We thank Joshua Deerain for creating Figure 1, Dr. Peter Kirkland for facilitating access to sera from feral rabbits, and Willy Suen for providing positive rabbit control sera. The studies were funded by the Australian Research Council (ARC-LP1210686 to Roy A. Hall, Helle Bielefeldt-Ohmann, Peter D. Kirkland) in conjunction with the Hunter Valley Equine Research Centre Ltd., the Queensland Health Forensic and Scientific Services, and the NSW Department of Primary Industries. Rabbit sampling was undertaken as part of an Invasive Animals Cooperative Research Centre national rabbit management program. Animal ethics approval was obtained through the Orange Agricultural Institute Animal Ethics Committee (Animal Research Authority ORA 11/14/001).

#### **REFERENCES**

*Encephalitis Viruses*. Sydney: Department of Agriculture, Fisheries and Forestry, NSW Government (2012).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 July 2014; accepted: 02 September 2014; published online: 17 September 2014.*

*Citation: Prow NA, Hewlett EK, Faddy HM, Coiacetto F, Wang W, Cox T, Hall RA and Bielefeldt-Ohmann H (2014) The Australian public is still vulnerable* *to emerging virulent strains of West Nile virus. Front. Public Health 2:146. doi: 10.3389/fpubh.2014.00146*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Prow, Hewlett, Faddy, Coiacetto, Wang , Cox, Hall and Bielefeldt-Ohmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Emergence of West Nile virus lineage 2 in Europe: a review on the introduction and spread of a mosquito-borne disease

#### **Luis M. Hernández-Triana1 , Claire L. Jeffries 1 , Karen L. Mansfield1 , George Carnell 2 , Anthony R. Fooks 1,3 and Nicholas Johnson1\***

1 Wildlife Zoonoses and Vector-Borne Diseases Research Group, Animal and Plant Health Agency, Addlestone, UK

2 London School of Hygiene and Tropical Medicine, London, UK

3 Department of Clinical Infection, University of Liverpool, Liverpool, UK

#### **Edited by:**

A. Paulo Gouveia Almeida, Universidade Nova de Lisboa, Portugal

#### **Reviewed by:**

Yingchen Wang, University of North Carolina Greensboro, USA Lin Wang, The University of Hong Kong, China A. Paulo Gouveia Almeida, Universidade Nova de Lisboa, Portugal

#### **\*Correspondence:**

Nicholas Johnson, Animal and Plant Health Agency (APHA), Woodham Lane, Addlestone, Surrey KT15 3NB, UK e-mail<EMAIL_ADDRESS>West Nile virus (WNV) is transmitted by mosquitoes and causes fever and encephalitis in humans, equines, and occasionally wild birds. The virus was first isolated in sub-Saharan Africa where it is endemic. WNV lineage 1 has been responsible for repeated disease outbreaks in the countries of the Mediterranean basin over the past 50 years. This lineage was also introduced into North America in 1999 causing widespread human, equine, and avian mortality. WNV lineage 2, the first WNV lineage to be isolated, was believed to be restricted to sub-Saharan Africa causing a relatively mild fever in humans. However, in 2004, an investigation in Hungary of a case of encephalitis in a wild goshawk (Accipiter gentiles) resulted in the isolation of WNV lineage 2. During the summer of 2004, and in subsequent years, the virus appeared to spread locally throughout Hungary and into neighboring Austria. Subsequently, WNV lineage 2 emerged in Greece in 2010 and in Italy in 2011, involving outbreaks on the Italian mainland and Sardinia. Further spread through the Balkan countries is also suspected. Whole genome sequencing has confirmed that the virus responsible for the outbreaks in Greece and Italy was almost identical to that isolated in Hungary. However, unlike the outbreaks in Hungary, the burden of disease in Mediterranean countries has fallen upon the human population with numerous cases of West Nile fever and a relatively higher mortality rate than in previous outbreaks. The emergence of WNV lineage 2 in Europe, its over-wintering and subsequent spread over large distances illustrates the repeated threat of emerging mosquito-borne diseases.This article will review the emergence of WNV lineage 2 in Europe; consider the pathways for virus spread and the public health implications for the continent.

**Keywords:West Nile virus, lineage, emergence, encephalitis, Europe**

## **INTRODUCTION**

In recent years, arthropod-borne viruses have shown an increasing ability to spread beyond the areas, which had been considered to be their established geographic ranges. A number of factors are driving this process including bird migration, increasing global trade, and the movement of vector species (1). This range expansion threatens public and livestock health. Examples of viruses that have emerged in Europe and which are pathogenic for livestock include bluetongue virus (2) and most recently Schmallenberg virus (3). Such disease outbreaks incur both economic and animal health costs that threaten the livestock industry. Other emerging viruses are zoonotic, and the repeated emergence of West Nile virus (WNV) in Europe is a particular example of one such range expansion (4). The ability to detect and respond to emerging disease outbreaks, through rapid pathogen testing and host-specific serological assays, is a key component for disease response. This review considers the emergence of WNV lineage 2 in Europe as an example of the threat to public and livestock health from emerging zoonoses.

West Nile virus is classified within the genus *Flavivirus*, family *Flaviviridae*, and is phylogenetically and antigenically related to Japanese encephalitis virus. The virus was first isolated from the blood of a woman suffering a febrile illness in the West Nile district of Uganda (5). This first isolate is now believed to belong to lineage 2 suggesting its early zoonotic potential. Subsequent studies made further isolations of WNV from human sera in Egypt (6), and from birds and mosquitoes (7). This established that mosquitoes were the likely virus vector and through blood-feeding on birds,the virus was maintained in an endemic cycle. Early phylogenetic studies (discussed in more detail in subsequent sections) demonstrated that there are two major lineages, both present in Africa (8). Subsequent events including the emergence of WNV in North America, its spread throughout the western hemisphere, and repeated outbreaks in Europe suggest that WNV has the largest distribution of any arthropod-borne virus. In Africa, WNV is endemic and widely distributed. Human cases have been sporadic, but environmental conditions favoring mosquitoes, such as high diurnal temperatures or frequent rainfall, have led to large epidemics (9). One such

episode in the Karoo region of South Africa in 1974 involved tens of thousands of human infections. Currently, approximately 5–15 human cases are confirmed each year in South Africa, however, only a small proportion of cases undergo laboratory investigation, so this may be a considerable underestimate of the actual number of infections (9). In horses and birds, serology studies have demonstrated a high seroprevalence of WNV infection in southern Africa (10, 11).

Annual late-summer outbreaks of WNV are now a regular occurrence in European countries that border the Mediterranean Sea and the virus is now considered endemic in some regions (12, 13). Most outbreaks have been identified as WNV lineage 1 and were closely related to outbreaks in Israel and North America (14). This lineage has also been responsible for deaths in humans,horses, and avian species. Strikingly, in Africa, where lineage 2 predominates, relatively few cases of neurological disease are reported, whereas in North America and Europe, numerous human and equine cases have occurred, leading to the suggestion that lineage 1 strains had increased pathogenicity, while lineage 2 strains were of low virulence (9). However, experimental studies in mice (15) and case reports (16, 17), have demonstrated that both WNV lineages have the ability to cause zoonotic disease, with the potential for fatal neuroinvasive disease. This has been realized fully with repeated outbreaks of West Nile fever in Greece since 2010, caused by WNV lineage 2, resulting in hundreds of human cases of West Nile neurological disease (WNND) (18).

## **EMERGENCE OF WNV LINEAGE 2 IN EUROPE**

The first WNV isolated in 1937 in Uganda has since been shown to be a lineage 2 isolate, and for many years this lineage was believed to be restricted to sub-Saharan Africa (5). Until the early 2000s,WNV infections beyond Africa, including its emergence in North America, and Kunjin virus in Australia, were caused by viruses within lineage 1 (19). This included a number of outbreaks in Europe and countries around the Mediterranean Basin (12). Outbreaks of West Nile disease were recorded in Algeria (1994), Morocco and Romania (1996), Tunisia (1997), Italy (1998), Israel and Russia (1999), and France (2000). Detailed phylogenetic analysis of viruses isolated from these outbreaks suggested that those around the western Mediterranean were caused by a single strain, referred to as the WMed subtype, and that this was a single introduction of virus that overwintered over a number of years (20). It was conjectured that this sub-lineage was transferred between Mediterranean countries by viremic birds, leading to the initiation of new outbreaks. A second closely related sub-lineage included viruses isolated from Romania and Russia, and a more divergent sublineage was responsible for outbreaks in Israel and North America. Each sub-lineage likely represents a separate introduction of WNV into Europe from Africa.

West Nile lineage 2 was first detected in Europe in 2004 with its isolation from the brain of a goshawk (*Accipiter gentiles*) in Hungary (21). A human case of WNV lineage 2 infection was retrospectively confirmed to have occurred in Russia in the same year (22). Subsequent surveillance between 2004 and 2009 of dead birds of prey, especially in goshawks, led to repeated isolations of the virus across Hungary (23). The species nests and thrives in areas of deciduous and coniferous forests and so would be targeted by ornithophilic *Culex pipiens* complex mosquitoes, the main transmitters of WNV in Europe. The prevalence of infection in the northern goshawk could also result from oral transmission as this species preys on other birds, particularly pigeons. Local spread resulted in WNV infection of raptors in Austria (24). Subsequent outbreaks have occurred in a number of other European countries including Austria, Greece, Romania, Serbia, and Italy (**Table 1**; **Figure 1**).

The outbreak in Greece has been particularly severe. The virus was first detected in 2010 (32) in Northern Greece and developed, in contrast to the emergence in Hungary, into a large human epidemic. The majority of cases were reported west of the city of Thessaloniki, between the rivers Axios and Aliakmonas. Retrospective serology suggests that this virus, or a related one, had been circulating in Greece for some years prior to the first human cases of WN fever (WNF) (33). However, this was at a low level, <1% seropositivity, and was not accompanied by reports of disease. The first case in 2010 occurred in early July and incidence peaked in mid-August. The last cases occurred in early October. In total, 262 patients were recorded, with 65 classified as West Nile fever and 197 suffered neurological disease, of which 33 died (34). Age profiling demonstrated that the elderly were most at risk of disease, particularly those over 70 years of age, and risk was further increased if the individual had an existing medical condition such as hypertension, heart disease, or diabetes. Subsequent surveillance isolated WNV lineage 2 from mosquitoes (35), giving rise to the Nea Santa-Greece 2010 strain, and from wild resident birds (36). Epidemics of WNV lineage 2 have occurred during the late summer in Greece in both 2011 and 2012. Further sequencing of viruses detected in humans has confirmed that the same virus strain is present in both humans and wildlife and has been present in each subsequent year, suggesting endemic persistence in Greece (37). This strain appears to cause disease in humans and wild avian species with relatively few confirmed reports of disease in equine species.

West Nile lineage 2 emerged in Italy in 2011, the year after the first detection in Greece. The first reported case occurred in a man in his late 50s in the coastal town of Ancona on the Adriatic Sea (27). The patient reported symptoms of malaise and

### **Table 1 | Information on confirmed outbreaks of WNV lineage 2 in Europe between 2004 and 2013**.

**FIGURE 1 | West Nile virus lineage 2 outbreaks in Europe**. The outbreaks occurred in Hungary (1), Austria (2), Greece (3), Italy (4), Sardinia (5), Russia (6), Serbia (7), and Romania (8). Map data: Google maps.

fever early in September, and was admitted to hospital. With the patient reporting no history of travel outside of Italy, this was considered an autochthonous case. Shortly after this, six cases of neurological disease due to WNV lineage 2 were reported in Sardinia between September and October, 2011 (28). Mosquito surveillance for WNV detected lineage 2 in *Cx. pipiens* mosquito pools and in a collared dove (*Streptopelia decaocto*) in northern Italy, where lineage 1 has been endemic since 2008 (38). Detections of WNV lineage 2 in Italy have continued in subsequent years, and have included further human cases (29, 31). As in Greece, instances of WNV infection in horses in Italy are rare.

West Nile fever was detected in humans in Romania in 2010 and Serbia in 2012. In Romania 57 cases were reported, 54 with neuroinvasive disease (26). In Serbia, 58 patients were confirmed infected (30). Of these, 52 developed neuroinvasive disease and 9 died. Virus isolation and phylogenetic analysis confirmed that both outbreaks were due to WNV lineage 2 (26, 39).

There is currently no vaccine against WNV licensed for human use, and although WNV lineage 2 has not been detected in countries in northern Europe, travelers to affected areas during the periods of vector activity are at risk of infection. This was illustrated by a human case of WNV infection in a 73 years-old Belgian woman who was visiting Greece in the summer of 2012 (40). Following her return to Belgium, samples of serum and cerebrospinal fluid were both positive for WNV IgM and IgG. The serum sample, taken 29 days after development of fever, was positive for WNV by real-time RT-PCR. A 116 base pair sequence derived from the amplicon was highly suggestive of the presence of WNV lineage 2.

Bird migration has been considered one of the major drivers for translocation of WNV (41–43). The emergence of WNV lineage 2 in Hungary followed by dissemination, both locally and to countries to the south could have resulted from translocation through infected bird movements. Two of the regions where the virus has emerged are dominated by wetland areas, namely the Po Delta in north-east Italy and the Aliakmonas Delta in northern Greece (44). Such areas attract migrating birds moving north from Africa and then returning south again from breeding grounds in central Europe. These areas are also associated with abundant populations of *Cx. pipiens* complex mosquitoes. Surveillance in these areas has detected WNV in pools of *Cx. pipiens*, and to a lesser extent *Cx. modestus* (45). This could drive the spread of WNV to indigenous bird species and eventually lead to spill-over infection in humans and horses. This coalescence of events, bird migration, landing periods in wetland areas, and peak vector abundance are needed to stimulate emergence, hence human cases tend to occur in late summer. The repeated emergence of WNV lineage 2 over subsequent summers suggests that over-wintering is occurring, supported mainly by phylogenetic evidence (20), although this is by no means conclusive as local re-introductions could give the same result. If over-wintering is occurring then this would likely be through infected adult females, the primary means of survival of *Culex* mosquitoes from 1 year to the next. The prevalence of

virus within the population would gradually increase through the subsequent summer, although always remaining at low levels relative to the total population of mosquitoes, but triggering spill-over infection in humans during late summer.

## **DISEASE CAUSED BY WNV LINEAGE 2 IN EUROPE**

West Nile virus infection is typically asymptomatic. However, a febrile self-limiting illness is reported in around 20% of infected humans and is associated with headaches, myalgia, nausea, vomiting, and chills. A papular rash is reported in some cases, but generally symptoms resolve within 7 days (46–48). In approximately 1% of cases, WNV will enter the central nervous system, infecting neurons and cause neuroinvasive disease (49, 50). Neurological forms are varied and can include encephalitis, meningitis, meningoencephalitis, or acute flaccid paralysis (49). Symptoms are exacerbated by old age and immunosuppression. A follow-up study from the 2010 Greece epidemic reported anorexia, muscle weakness, memory loss, and depression to be the most common sequelae in a group of elderly patients who suffered from WNND. Only 31.8% (7/22) patients recovered fully (48–50).

Equines have been reported to be susceptible to lineage 2 strains, with an increased risk of developing WNND. Clinical signs in horses include ataxia, weakened limbs, paresis, complete paralysis, seizures, chewing, partial blindness, and jaundice/hepatitis (16).

Lineage 2 has been reported with varying mortality across the affected regions. The reasons for this may be linked to the previously characterized threonine 249 to proline (T249P) substitution within the NS3 gene, which was present in WNV isolates responsible for the Greek outbreak but not the Hungarian or Italian isolates (16, 51). However, an Italian strain identical to the Nea Santa-Greece 2010 strain (with T249P) was reported from the Veneto region of Italy, although there has been an absence of human disease as had been observed in Greece (25, 52). The Romanian outbreak of WNV lineage 2 in 2010 had an 8.8% fatality rate with 57 cases of WNND compared to a previous outbreak of WNV lineage 1 in 1996, which had a 4.4% fatality rate.

## **PHYLOGENY OF WNV LINEAGE 2 AND STRAIN VARIATION**

West Nile virus has a single-stranded RNA genome of approximately 11 kb in length. The genome encodes three structural proteins and seven non-structural proteins. A large number of complete WNV genomes have now been sequenced and can be used for phylogenetic comparison with emerging viruses. This has greatly assisted in the investigation of the likely origins of WNV emergence. The 2004 lineage 2 WNV strain was isolated from a wild goshawk (*Accipiter gentiles*) in Hungary, and the genomic sequence of this isolate demonstrated closest homology with a group of southern African strains (53). The introduction of WNV lineage 2 into the wetlands of Hungary could have occurred through migratory birds that had become infected in Africa and remained sufficiently viremic during migration to infect mosquitoes in Europe on arrival (23). However, the goshawk is not considered a migratory bird, suggesting that this African lineage 2 strain must have been transmitted by local mosquitoes prior to detection in 2004.

Genetic characterization of the lineage 2 strain detected in Greece confirmed that it was most closely related to the strain that had previously emerged in Hungary (25). Similarly, strains detected in Serbia in 2012 were most closely related to strains previously identified in Greece, Hungary, and Italy (39). The strains detected in Italy in 2013 show closest homology with lineage 2 strains isolated in Italy in 2011 and Austria in 2008 (27). These observations suggest that the spread of lineage 2 throughout Europe was due to spread of the 2004 Hungarian strain, rather than from separate incursions via migratory birds from Africa. Migratory birds could also have played a role in the spread of lineage 2 WNV in Europe during the southerly migration. During autumn, turtle doves (*Streptopelia turtur*) migrate from breeding areas in central Europe to wintering areas in Africa, and serological studies on individual birds on their arrival in resting areas of Greece suggest that they were exposed to WNV lineage 2 in the area of origin in central Europe, and may constitute a candidate for introduction of lineage 2 strains from central Europe to Greece (37).

The mutation T249P within non-structural protein 3 (NS3) of Greek lineage 2 strains is similar to that observed in neuroinvasive lineage 1 isolates (51), while analysis of Italian strains isolated in 2013 has identified a number of amino acid substitutions including E638K and D831G within the NS5 protein (51). **Figure 2** highlights the relationship between lineage 2 strains detected in Europe, and the southern African strains from which they may have originated. The isolates used for phylogenetic analysis are detailed in **Table 2**. The figure demonstrates the close homology between strains from Italy, Austria, Greece, Serbia, and Hungary (marked by a bracket), with clear divergence between these strains and strains from Africa and Russia, suggesting that lineage 2 WNV might now circulate in a separate enzootic cycle within Europe. A number of these viruses

MAD-78 is used as an outgroup.

**Table 2 | Details of WNV lineage 2 sequences used for phylogenetic analysis in Figure 2**.

were derived from human cases, confirming the zoonotic nature of these outbreaks.

## **MOSQUITO SPECIES ASSOCIATED WITH DISEASE TRANSMISSION: THE CASE FOR EMERGENCE IN THE UNITED KINGDOM**

Outbreaks of WNV are associated with abundant populations of mosquitoes, which can occur as a result of flooding and subsequent dry and warm weather, or formation of suitable larval breeding habitats (54). Although WNV has been isolated from over 40 species of mosquitoes, the principal mosquitoes involved in WNV transmission belong to the genus *Culex*, particularly species in the *Cx. pipiens* complex (55, 56). Two species are formally recognized in the complex, *Cx. pipiens* (northern, temperate regions) and *Cx. quinquefasciatus* (southern, tropical regions) (57). In the UK, the subgenus *Culex* is represented by the nominotypical *Cx. pipiens* (58), which include two forms, *Cx. pipiens* f. *pipiens* and *Cx. pipiens* f. *molestus*. In addition, *Cx. torrentium* and *Cx. europaeus* are also recorded in the UK (59). These species are fairly common and widespread, with the exception of *Cx. europaeus*, for which few records are available. A further species, *Cx. modestus,* was also believed to be rare in the UK (59). However, recent inventories of UK mosquito fauna have revealed that populations of *Cx. modestus* are well established and commonly found in the North Kent marshes (60, 61).

The occurrence and abundance of potential vector species are a prerequisite for enzootic transmission of mosquito-borne viruses in the UK. Thirty-four species of mosquitoes have been recorded in the British Isles, of which nine species have been implicated in WNV transmission elsewhere (62). Thirteen species are likely to be bridge vectors as they readily bite both birds and humans. In Britain, the ecology of, and the potential risk of WNV transmission by, mosquito species have been detailed by several authors (62–64).

*Cx. pipiens sensu stricto* (*s.s.*) bite both humans and birds; the two forms within this species are morphologically indistinguishable, but they are physiologically different. *Cx. pipiens* f. *pipiens* is mostly ornithophagic and rarely bite humans, the immature stages are found in permanent water, it overwinters in the adult stage and is multivoltine (62). By contrast, *Cx. pipiens* f. *molestus* is highly anthropophilic (though it may bite birds), the immature stages live underground (e.g., flooded basements, sewer tunnels, underground railway systems), and all life stages occur throughout the year (63). The females can be nuisance biters in winter. Medlock et al. (64) stated that the form *molestus* might pose a threat for WNV transmission in suburban and rural areas. Where both forms are sympatric, hybridization can result (65, 66). This leads to increased numbers of mammophilic mosquitoes that can affect transmission of WNV (67). In the UK, the increased use of water containers in private gardens has also been cited as a possible factor that could lead to increases in mosquito abundance in urban areas that in turn could lead to more nuisance biting and increase the risk of WNV maintenance should it be introduced (68).

Golding et al. (60) showed that *Cx. modestus* accounted for 73% of all mosquito species from all sites sampled in the county of Kent and was collected in strong association with *Anopheles maculipennis sensu lato (s.l.)*. Although the risk of WNV transmission to humans in the UK is still low due to limited human exposure to bridge vectors, the risk for transmission was higher in Kent because of the presence of other bridge vector species such as *Cx. pipiens s.l.* and both migratory and resident birds. The authors highlighted the potential risk for horses, which are often grazed in this part of country. At present, screening of specimens of *Cx. modestus* for WNV and other flaviviruses such as Usutu virus is being undertaken by cross-governmental groups in the UK under a "One Health" initiative.

## **INVASIVE MOSQUITO SPECIES**

The increase in human population, expansion of trading routes and tourism, deforestation, and climate change are some of the factors that have facilitated the rapid dispersal of vector species into new geographical areas (69). Recent emergence of bluetongue virus and outbreaks of WN fever and Chikungunya fever in Europe are just a few examples of the risk of exotic vector-borne pathogens being transported to a new region (69).

For invasive mosquitoes in Europe, six exotic species have been identified (*Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, Ae. koreicus, and Ae. triseriatus*). These have mainly been imported through the international trade in used tires and Lucky bamboo, although public and/or private ground transport have also been implicated. Of these, *Ae. albopictus* presents the greatest threat. Female *Ae. albopictus* lay desiccation-resistant eggs above the surface of the water in tree holes, tires, or other water-holding containers (70). Its ability to breed in artificial containers has facilitated its spread in recent decades along major transportation routes. Currently, *Ae. albopictus* is considered one of the top 100 invasive species of mosquitoes worldwide (71).

*Aedes albopictus* is a known vector of CHIKV, and laboratory infectious studies have shown that this species is a competent vector of WNV (72). It has been recorded in 20 European countries including the Netherlands and Belgium (its northernmost latitude) (73), although it has not been recorded in the UK. Surveillance for this and other invasive mosquito species should be a priority as their presence would radically change the risk of virus emergence.

## **CONCLUSION**

West Nile virus lineage 2 was introduced into Europe in 2004 and subsequently emerged in a number of countries. It has overwintered in these countries and in the case of Italy is co-circulating with WNV lineage 1 and Usutu virus. Migratory birds are likely to be the main vehicle of movement for the virus, both as a source of introduction and the means by which it has spread within Europe. However, there is little direct evidence to support this. The disease manifestations appear to vary in different countries. In Hungary, raptors appear to be affected while in Greece the main burden of disease has fallen on the human population. The reasons for this are not clear, but the variation could be caused by mutations within particular virus strains that persist in different locations. The abundance and composition of mosquito populations, particularly *Culex s*pecies, are critical for the spread of disease. Further study of factors such as mosquito distribution, host biting preference, and species hybridization will improve understanding of WNV persistence and assessment of the risk to human populations.

Following detection of WNV, there is a need to implement public health measures to protect at risk populations, particularly the elderly. This includes measures to reduce mosquito biting such as destruction of larval habitats and applications of larvicides. Measures that protect the individual include the application of mosquito repellents and clothing that reduces the exposure of bare skin. Currently, there is no human vaccine available. The risk of further spread of WNV lineage 2 to countries around the Mediterranean Sea is high. Countries in northern Europe appear to be at lower risk due to reduced mosquito abundance and lower winter temperatures. However, constant vigilance is needed to monitor for any change in environmental or ecological conditions that could make the introduction and persistence of WNV in northerly latitudes possible.

## **AUTHOR CONTRIBUTIONS**

Nicholas Johnson conceived the idea for this review. Luis M. Hernández-Triana, Claire Jeffries, Karen Mansfield, George Carnell, Anthony Fooks, and Nicholas Johnson prepared the manuscript. All authors read and approved the final manuscript.

## **ACKNOWLEDGMENTS**

This study was funded by Defra, UK, through project SE4112 (Development of research tools to support arthropod-borne virus investigation) and European Union FP7 project ANTIGONE (Anticipating the Global Onset of Novel Epidemics) project number 278978.

## **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 June 2014; accepted: 23 November 2014; published online: 08 December 2014.*

*Citation: Hernández-Triana LM, Jeffries CL, Mansfield KL, Carnell G, Fooks AR and Johnson N (2014) Emergence of West Nile virus lineage 2 in Europe: a review on the introduction and spread of a mosquito-borne disease. Front. Public Health 2:271. doi: 10.3389/fpubh.2014.00271*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Hernández-Triana, Jeffries, Mansfield, Carnell, Fooks and Johnson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# PUBLIC HEALTH

# Recent outbreaks of Rift Valley fever in East Africa and the Middle East

#### **Yousif E. Himeidan1,2\*, Eliningaya J. Kweka3,4, Mostafa M. Mahgoub5 , El Amin El Rayah6 and Johnson O. Ouma2,7**

1 Entomology Unit, Faculty of Agriculture and Natural Resources, University of Kassala, New Halfa, Sudan

2 Africa Technical Research Centre, Vector Health International, Arusha, Tanzania

3 Division of Livestock and Human Diseases Vector Control, Tropical Pesticides Research Institute, Arusha, Tanzania

4 Department of Medical Parasitology and Entomology, Catholic University of Health and Allied Sciences, Mwanza, Tanzania

5 Blue Nile National Institute for Communicable Diseases, University of Gezira, Madani, Sudan

6 Department of Zoology, University of Khartoum, Khartoum, Sudan

7 Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, Kikuyu, Kenya

#### **Edited by:**

Juan-Carlos Navarro, Universidad Central de Venezuela, Venezuela

#### **Reviewed by:**

Tetsuro Ikegami, University of Texas Medical Branch, USA Jordi Figuerola, Estacion Biologica de Doñana – CSIC, Spain

#### **\*Correspondence:**

Yousif E. Himeidan, Vector Health International, Africa Technical Research Centre, Vector Control Unit, Dodoma Road, P.O. Box 15500, Arusha, Tanzania e-mail<EMAIL_ADDRESS>
Rift Valley fever (RVF) is an important neglected, emerging, mosquito-borne disease with severe negative impact on human and animal health. Mosquitoes in the Aedes genus have been considered as the reservoir, as well as vectors, since their transovarially infected eggs withstand desiccation and larvae hatch when in contact with water. However, different mosquito species serve as epizootic/epidemic vectors of RVF, creating a complex epidemiologic pattern in East Africa. The recent RVF outbreaks in Somalia (2006–2007), Kenya (2006–2007), Tanzania (2007), and Sudan (2007–2008) showed extension to districts, which were not involved before. These outbreaks also demonstrated the changing epidemiology of the disease from being originally associated with livestock, to a seemingly highly virulent form infecting humans and causing considerably high-fatality rates. The amount of rainfall is considered to be the main factor initiating RVF outbreaks. The interaction between rainfall and local environment, i.e., type of soil, livestock, and human determine the space-time clustering of RVF outbreaks. Contact with animals or their products was the most dominant risk factor to transfer the infection to humans. Uncontrolled movement of livestock during an outbreak is responsible for introducing RVF to new areas. For example, the virus that caused the Saudi Arabia outbreak in 2000 was found to be the same strain that caused the 1997–98 outbreaks in East Africa. A strategy that involves active surveillance with effective case management and diagnosis for humans and identifying target areas for animal vaccination, restriction on animal movements outside the affected areas, identifying breeding sites, and targeted intensive mosquito control programs has been shown to succeed in limiting the effect of RVF outbreak and curb the spread of the disease from the onset.

#### **Keywords: RVFV outbreaks, Aedes mosquitoes, rainfall, East Africa**

## **INTRODUCTION**

Rift Valley fever (RVF) is an important neglected, emerging, mosquito-borne disease with severe negative economic impact as it affects human and animal health. The disease is caused by RVF virus (RVFV) an acute febrile arbovirus in the *Phlebovirus* genus and Bunyaviridae family. The disease was first characterized by Daubney et al. (1) while working at the Veterinary Research Laboratory at Kabete in Kenya. An earlier report by Stordy (2) had described a similar disease syndrome, which may well have been RVF, it was described as an acute and highly fatal disease in the Rift Valley in exotic wool sheep, which had been imported into East Africa from Europe (1, 3). These European stock species were more severely affected than native African stock. The disease remained a veterinary concern in East Africa until a major outbreak occurred in Egypt in 1977. A second outbreak outside East Africa occurred in 2000 when RVF moved into Saudi Arabia and

Yemen in the Arabian Peninsula (4). This was the first time the disease was being detected outside of Africa – where it had been confined so far – becoming a threat to the Middle East.

From the most recent outbreaks that occurred in Kenya, Somalia, Tanzania in 2007 (5, 6), and Sudan in 2008 and 2010 (7, 8), RVF appears to have great potential for spreading into new areas and with huge impact on human and animal health. This calls for an integrated approach between different governmental sectors and organizations within and between countries and regions to address both human and animal health. Limited information is available on the evolution of RVF between East Africa and Middle East. In order to highlight the urgent need of establishing a health system for controlling RVF in the region, this review article aims to gather experiences and highlight basic information on the ecological aspects, epidemiological, and risk factors associated with the distribution of recent outbreaks in East Africa and Middle East.

## **TRANSMISSION AND IMPACT**

The virus is known to infect a range of animal hosts including sheep, cattle, goats, camels, buffaloes, and others. The incubation period in animals is between 1 and 6 days in general, 1 and 3 days in sheep, and only about 12 and 36 h in lambs (9). Sheep and to a lesser extent cattle were the principle disease hosts in both East and southern Africa (3). Sheep seemed to be the most susceptible animal as it was noted that RVF caused high rates of abortions during pregnancy and high mortalities among newborns (10, 11). Lambs can die before they acquire passive immunity and mortality and abortion rates among old sheep range from 5 to 100% (9). Infections can therefore cause severe disease and result in significant economic losses. For example, the 2007 outbreak was the most widespread affecting livestock in 11 regions in Tanzania and Kenya. A total of 16,973 cattle, 20,193 goats, and 12,124 sheep died of the disease, with spontaneous abortions reported in 15,726 cattle, 19,199 goats, and 11,085 sheep (12, 13). Considering the wide-ranging impacts of the disease on the livestock sector and other segments of the economy, the 2007 RVF outbreak in Kenya alone induced losses of over Ksh 2.1 billion (US\$32 million) on the Kenyan economy (14). The overall economic loss in East Africa is estimated to exceed \$60 million because of disruption in trade from these recent epizootics between 2006 and 2007 (15). In Saudi Arabia, during the outbreak of 2000, it was estimated that around 40,000 animals including sheep, goats, camels, and cattle died whereas 8,000–10,000 of them aborted (16). The outbreaks of 2007 in Sudan led to bans in livestock trade between Saudi Arabia and Sudan, resulting in vast economic impact on the animal market in the two countries (17).

Infection by RVF usually spreads among livestock first through mosquitoes bites. In addition, the infection can also be transmitted vertically between animals (18) (**Figure 1**). From domestic animals, the virus is transmitted to humans mainly through direct contact with blood, excreta,meat, or secretions of infected animals, consumption of raw milk (19–21), and in few cases, transmission through mosquito bites that belong to the genera *Anopheles*, *Aedes*, and *Culex* seems to occur (22, 23) (**Figure 1**). Symptoms of RVF in humans vary from a flu-like syndrome to encephalitic, ocular, or hemorrhagic syndrome. The case fatality rate of the hemorrhagic syndrome form can be as high as 50% (24). The most severe outbreaks of 1997–1998 and 2006–2007 in Tanzania, Kenya, and Somalia caused 478 human deaths in 1998 and 309 in 2007 (25–27). The outbreak of 2000 resulted in 883 human cases with 124 deaths (case fatality rate, 14%) in Saudi Arabia (28) and 1,328 human cases, with 166 deaths in neighboring north western Yemen (29–31). In Sudan, the outbreak of 2007 resulted in 698 cases, including 222 deaths (32, 33).

### **MOSQUITO VECTORS**

Mosquitoes in the *Aedes* genus have been considered the primary maintenance host and source of RVFV that initiate disease outbreaks (34, 35). RVFV is known to be carried in the eggs of *Aedes* mosquitoes, which can survive for several years in the dried mud (36). On flooding, *Aedes* mosquito species play an important role in initiation of infection and virus circulation. The survival of RVFV during inter epizootics was believed to depend on transovarial transmission of the virus in flood water by *Aedes* mosquitoes (37). Other mosquitoes in the *Culex* and *Anopheles* genus are thought to be important in amplification of virus activity during outbreaks. The virus has also been detected in phlebotomine sand flies, *Culicoides* midges, and *Amblyomma* tick species although these infections are thought not to play an important role in the life cycle of the virus or in disease outbreak settings (38–42). In the laboratory, the RVFV was also transmitted trophically or mechanically by other hematophagous flies but field relevance of these transmission routes are still unclear (43, 44).

The important RVF vectors in East Africa include *Aedes mcintoshi*,*Aedes ochraeus*,*Culex pipiens*,*Aedes dalzieli*, and *Aedes vexans* (45). Records also indicated that *A. mcintoshi* is the main vector for RVF in Kenya (41, 42, 46). Investigation on RVFV by reverse transcription-polymerase chain reaction (RT-PCR) during the recent outbreak of 2006/2007 in Kenya showed that 77 out of 3,003 pools representing 10 species, from 4 affected districts, tested positive for RVFV, including *A. mcintoshi*/*circumluteolus* (26 pools), *Aedes ochraceus* (23 pools), *Mansonia uniformis* (15 pools); *Cx. poicilipes*, *Culex bitaeniorhynchus* (3 pools each); *Anopheles squamosus*,*Mansonia africana* (2 pools each);*Culex quinquefasciatus*,*Culex univittatus*,*Aedes pembaensis* (1 pool each).*A. pembaensis*,*Cx. univittatus*, and *Cx. bitaeniorhynchus* were for the first time observed positive for the virus (42). The observation of infected *A. ochraceus* in Garissa,Kenya, represents a new RVFV-vector association in East Africa. *A. ochraceus* is a known vector of RVFV in West Africa (39), along with *A. vexans arabiensis* and *A. dalzieli*. *A. vexans arabiensis* is also a vector of RVFV in Saudi Arabia (40, 44) and although the species has not been documented in Kenya, it has been found in neighboring Somalia and Sudan (47, 48) (**Table 1**).

It has been suggested that different mosquito species serve as epizootic/epidemic vectors of RVFV in diverse ecologies, creating a complex epidemiological pattern in East Africa (42). *A. aegypti* has been found naturally infected with RVFV and seemed to be the main source of the infection during the outbreak of 2007 in Sudan. During this outbreak, RVFV was successfully detected by RT-PCR in larvae, male and females of *An. arabiensis*, *An. coustani*, *Cx. pipiens* complex, *Cx. poicilipes*, and *A. aegypti* (**Table 1**). The infections were considered as a precursor for viral circulation in these species (incriminated in dissemination or acquired the virus in its mid gut only) (21). The detection of RVFV in male and larval stages indicated transovarial (vertical) transmission of the virus within these mosquito species. It may also show possible venereal RVFV transmission when a male is infected vertically and then infects the female during mating (21).

Laboratory established colonies of *A. aegypti* from Tahiti exhibited the highest infection rates of RVFV when compared with other potential vectors in the Mediterranean region (56). *A. aegypti* has also demonstrated infection and transmission rates of the nonstructural proteins (NSs) deletion virus similar to wild-type virus, but dissemination rates were significantly reduced (35).*Cx. pipiens* was incriminated as the main RVF vector in Egypt based on field isolates and also in Maghreb and South Africa based on laboratory experiments (57–59).

#### **OCCURRENCE OF RVF OUTBREAKS**

The RVF has demonstrated capacity for emerging in new territories or for re-emerging after long periods of silence. Since the

first outbreak in 1915, epizootics occurred periodically in Kenya until the disease was recognized in South Africa in 1951 (60), when humans became ill after handling dead and infected animals (3). Further, RVF outbreaks have been confirmed in most sub-Saharan countries (61) moving through the Rift Valley from Kenya to Tanzania, Zimbabwe, Zambia, and subsequently, RVF outbreak was first recorded in 1987 in West Africa in Senegal and Mauritania (3). RVF spread northwards through the Nile Valley into Southern Sudan and toWhite Nile state in Sudan where the first outbreak was identified in 1973. The disease then spread among other neighboring states within the country (**Figure 2**) and up to the Egyptian delta where a major epidemic with 20,000–200,000 clinical illnesses and 600 deaths was reported in 1977 (3, 62, 63). The disease also spread from continental Africa to Madagascar in 1991 (64–67) and to the Arabian Peninsula in Saudi Arabia and Yemen in 2000 (4). The recent RVF outbreaks in East Africa in Somalia (2006– 2007) (5), Kenya (2006–2007) (6), Tanzania (2007) (5), and Sudan (2007–2008) (7) showed the changing epidemiology of the disease from being originally associated with livestock to infecting humans considerably and resulting in high-fatality rates (7).

Sindato and others investigated the spatial and temporal pattern of RVF outbreaks in Tanzania over the past 80 years (68). All RVF outbreaks reported during 1930–2007 were found to occur between December and June. Expansion of the disease into new geographical areas from the original documented outbreaks was observed. For example, between 1930 and 1957 only <1% of the districts in Tanzania were repeatedly involved in the outbreaks (**Figure 3**). The 1977–1978 outbreak wave had involved 3.33%

**Table 1 | Mosquito species incriminated in the transmission of RVFV during the outbreaks recorded in East Africa and the Middle East**.

districts. A relatively larger outbreak wave in 1997–1998 involved 7.70% of the districts and the widespread outbreak in 2006–2007 involved humans and domestic ruminants in 39.17% of the districts in the country (**Figure 4**). However, despite this expansion into districts, which were not involved before, RVF outbreaks still show significant spatio-temporal clustering in eastern Rift Valley during the last 80 years in Tanzania (68). The space-time clustering of livestock and human cases showed a tendency to spread from the north to east-central and western parts of the country (**Figures 3** and **4**). Uncontrolled livestock movement has been suggested as being responsible for the geographical expansion and the cumulative effect of the amount of rainfall was considered the main cause of the outbreaks (68–72). It has been suggested that the bimodal rainfall pattern experienced in this ecosystem provides an environment for *Aedes* mosquito species to emerge in large numbers at the onset of the rainy season, and therefore, resulting in extensive biting rates and transmission of the virus in animals and humans (68).

It is well established that RVFV outbreaks occur predominantly after unusual flooding events. *Aedes* mosquito species are seen as reservoir, as well as vectors, since their transovarially infected eggs withstand desiccation and larvae emerge when the eggs get into contact with water (37, 73). Transovarial transmission is assumed as the mechanism of virus persistence between epizootic events. After flooding, the infected *Aedes* mosquito eggs will hatch in the persisting water collections, and develop into infective adult mosquitoes. A study in the Ferlo region of Senegal in 2003 observed that when the rainy season began with heavy rains, the temporary ponds that serve as the breeding sites for mosquitoes were flooded to their maximum level immediately. As a result, *A. vexans arabiensis* populations were found to be abundant at the very beginning of the season, when the majority of eggs in quiescence were flooded (74). The effect of flood water on *Aedes* breeding habitats has also been studied artificially in central Kenya by sequentially flooding such habitats to determine the numbers of mosquito eggs hatching during each flooding. The authors documented that approximately 90% of the larvae sampled during four flooding events emerged during the initial one (75). This probably explains why excessive rainfall can result in high density of initial infected population. This hypothesis was supported by the study from Senegal, which found that female mosquitoes hatching from eggs laid during the previous year quickly laid eggs on the pond's wet soil (74). The study also observed that during rainless periods lasting longer than 7 days, the time needed for embryogenesis, these new eggs undergo dormancy as the water level goes down. Once, the rains fall again, large numbers of new eggs hatch resulting into an increase in population, and thereby suggesting that several generations of infected adults can exist during the same rainy season. This dynamic has been seen to also maximize the virus' chance to persist from one year to another in high-stock population, thus, facilitating endemisation of RVFV that is then amplified through feeding of infected adult female mosquitoes on wild and domestic ungulates and may reach epizootic and epidemic dimensions (42, 74).