 nucleotide sequences of IHHNV isolated from Peru (A, C, D, E, F, and G), Ecuador (E1), and other known IHHNV isolates deposited in the GenBank database.
Nucleotide sequences were aligned using MAFFT (Auto: according to data size, 200 PAM, k = 2), and the best model for the phylogenetic tree was estimated by jmodel Test 2.1.10 [20]. The phylogenetic tree was built by using MrBayes (http://www.phylogeny.fr/one_task.cgi?task_type=mrbayes) (KY85, 4by4, invariable+Gramma, number of generation = 10,000). The numbers on the tree branches indicate posterior probability. The nucleotide sequences from IHHNV isolated from Peru and Ecuador are shown within a blue box.
Fig 5. Phylogenetic tree based on the predicted amino acid sequence of capsid protein gene of IHHNV isolates originating in Peru (A, C, D, E, F, and G), Ecuador (E1), and other known homologous IHHNV sequences deposited in the GenBank database.
Amino acid sequences were aligned using MAFFT (Auto: according to data size, blossom), and the best model for the phylogenetic tree was estimated by ProtTest 3.4.2 [21]. The phylogenetic tree was built by using MrBayes (http://www.phylogeny.fr/one_task.cgi?task_type=mrbayes) (GTR, VT, no rate variation, number of generations: 10,000). The number on the tree branches indicates posterior probability. The IHHNV isolates from Ecuador and Peru are marked by a blue box.
Two out of seven IHNNV genome sequences obtained for this study, isolates C and D originating in Peru, contained DTR at the 5′ and 3′ ends of the viral genome. The DTR1 in isolate D was 197 nt long, with a 99% nucleotide sequence similarity between the 5′- and 3′-end sequences of this repeat (Fig 6A). In IHHNV isolate C, the DTR1 was incomplete and was 114 nt long, with a similarity of 98% between the 5′ and 3′ ends of this sequence. The similarity between the DTR1 of the two IHHNV isolates was 98.4%. Unlike the DTR1, the DTR 2 palindromic sequence in both strains showed a similarity of 100%. The DTR2 in IHHNV isolate D was 12 nt longer (92 nt) than that in IHHNV isolate C (80 nt) (Fig 6A). In addition, repeat 2 formed a well-supported hairpin structure (Fig 6B).
Fig 6. Terminal structures of IHHNV isolates C and D.
(A) Direct terminal repeats (repeat 1) located at the 5′ and 3′ ends and the palindromic sequences (repeat 2) located at the 3′ end of the genomes of IHHNV strains D and C. (B) Hairpin model of the palindromic sequences (repeat 2) located at the 3′ end of IHHNV isolate D.
Two IHHNV challenges were performed using an isolate from Peru and another isolate from Ecuador. At the end of the IHHNV-challenge, that is, at 39 d.p.i (Peru) and 30 d.p.i (Ecuador), the final survival was high in both experimental challenges. Survival in IHHNV-challenged shrimp was similar to that in the unchallenged group (Table 4).
Table 4. Final survival rate in Penaeus vannamei, P. monodon, and P. stylirostris following IHHNV challenge via injection at 39 (Peru) and 30 (Ecuador) days post-challenge.
The final survival rate of P. vannamei in Challenges 1 and 2 was high (Table 4). In the IHHNV-challenged shrimp, IHHNV infection was analyzed by real-time PCR and histopathology and further confirmed by ISH. Two additional penaeid species, P. monodon and P. stylirostris were included in experimental challenge 2. The final survival rates of these two species were also as high as P. vannamei (Table 4). At the end of the challenge, there was no evidence of RDS or other clinical signs in the IHHNV-challenged shrimp. Real-time PCR confirmed IHHNV in all three species: P. vannamei, P. monodon and P. stylirostris. The IHHNV copy number was highest in P. vannamei population, followed by P. monodon and P. stylirostris (P<0.001) (Fig 7).
Fig 7. IHHNV copy number in three shrimp populations challenged with IHHNV.
Letters a, b, and c denote significant differences by one-way ANOVA (p<0.001).
To confirm IHHNV infection in experimentally challenged penaeid shrimp, H&E histology and ISH were performed using IHHNV-specific probes. In Table 4, the H&E histology results and ISH data are presented. IHHNV infection was confirmed by H&E histology and ISH in P. vannamei and P. monodon in several tissues and organs, including the cuticular epithelium of the gill filaments and antennal gland, nerve cord, lymphoid organ, and epicardium. In contrast, there was no evidence of IHHNV lesions either by H&E histology or ISH in P. stylirostris. In Fig 8, typical IHHNV intranuclear inclusion bodies are shown by H&E histology and confirmed by ISH in P. vannamei and P. monodon in IHHNV-challenged shrimp.
Fig 8. H&E histology and ISH of P. vannamei and P. monodon shrimp experimentally challenged with IHHNV.
Panels A, C, E, and G represent H&E histology, and Panels B, D, F and H represent ISH results using IHHNV-specific probes. A&B: P. vannamei infected with IHHNV isolate from Peru, C&D: P. vannamei infected with IHHNV isolate from Ecuador, E&F: P. monodon infected with IHHNV isolate from Ecuador, G&H: P. vannamei Specific Pathogen Free (SPF) control shrimp. A & B shows a section of the antennal gland. C-H show sections of cuticular epithelium. Arrowheads show the eosinophilic intranuclear inclusion body (Cowdry type A). Arrows show a positive reaction by ISH (dark precipitate). The black star represents a section of cuticular epithelium. The blue star shows a section of spongy connective tissue.
Here, we present data on the prevalence of IHHNV and its impact on the growth of P. vannamei in grow-out ponds in three major shrimp-producing regions in Ecuador. Furthermore, we determined the genome sequence of the virus in P. vannamei originating in Ecuador and its neighboring country, Peru, where IHHNV is prevalent. Finally, we performed an experimental bioassay using IHHNV isolates originating from Ecuador and Peru to determine the virulence of the virus on three SPF penaeid shrimp species: P. vannamei, P. monodon, and P. stylirostris.
Penaeus vannamei samples from grow-out ponds representing three different regions in Ecuador were screened for IHHNV. The virus was present in samples collected from all eight grow-out ponds located in the three different regions (Table 1). The IHHNV prevalence varies widely, from 3.3% up to 100%. In real-time PCR analysis, some shrimp showed negative results, whereas others showed a copy number of up to 2.9 × 105 copies/ng DNA (Table 2). To establish the association between shrimp weight and the presence of IHHNV, the population from each pond was divided into two groups based on real-time PCR results (IHHNV-positive and IHHNV not detected). As shown in Fig 2, there were no significant correlations between the average shrimp weight and presence of IHHNV within the same pond. Sellars et al. [4] showed that in a P. monodon farm in Australia, growth performance and survival were reduced in grow-out ponds with a high copy number of IHHNV (0.5 × 103 to 2.24 × 106 copies/ng TNA). In our study, the mean copy number of IHHNV varied between 5.9 × 100 and 1.4 × 104 copies/ng DNA, which is lower than the IHHNV load reported by Sellars et al. [4], which may partially explain the little to no influence on growth performance. There was a significant direct correlation between the IHHNV copy number and the prevalence of IHHNV in the grow-out ponds. The prevalence and copy number was high. Similar results in grow-out ponds were reported by Sellars et al. [4]. These findings are consistent with the pattern of infection with infectious agents. Walker et al. [28] suggested that when viral loads increase to acute levels, they can compromise shrimp health. In our study, histological IHHNV lesions were observed only in shrimp from grow-out ponds where the prevalence of IHHNV was 100%. In contrast, shrimp from ponds with a low prevalence of IHHNV did not display any histological lesions.
To better understand the effect of