title: Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa

text: Background: Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. Objective: We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Design: Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg(−1) · h(−1)) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg(−1) · h(−1)). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis–based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student’s t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Results: Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl–transfer RNA synthetase, glutaminyl–transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Conclusions: Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. This trial was registered at clinicaltrials.gov as NCT01254110.


title: Serodiagnosis of Lyme Disease by Kinetic Enzyme-Linked Immunosorbent Assay Using Recombinant VlsE1 or Peptide Antigens of Borrelia burgdorferi Compared with 2-Tiered Testing Using Whole-Cell Lysates

text: In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent


title: Computer simulations of protein–membrane systems

text: The interactions between proteins and membranes play critical roles in signal transduction, cell motility, and transport, and they are involved in many types of diseases. Molecular dynamics (MD) simulations have greatly contributed to our understanding of protein–membrane interactions, promoted by a dramatic development of MD-related software, increasingly accurate force fields, and available computer power. In this chapter, we present available methods for studying protein–membrane systems with MD simulations, including an overview about the various all-atom and coarse-grained force fields for lipids, and useful software for membrane simulation setup and analysis. A large set of case studies is discussed.


Viroses Causing Common Respiratory Infections in Man. III. Respiratory Syncytial Viroses and Coronavimses


title: Pleomorphic, Enveloped, Virus-Like Particles Associated with Gastrointestinal Illness in Neonates

text: Pleomorphic, enveloped, virus-like particles were detected by electron microscopy in the stools of symptomatic infants during an outbreak of gastrointestinal illness in a neonatal intensive-care unit. To determine the incidence of virus-like particles in the stool and their relation to gastrointestinal symptoms, eight surveys of stools for the particles were conducted over 40 weeks. The incidence of virus-like particles in the stool decreased from 69% to <10% over the study period. Most infants surveyed were premature; overall, 32 (36%) of 88 neonates were positive for virus-like particles. Statistically significant associations were found between virus-like particles in the stool and gastrointestinal symptoms within one week of each survey. These symptoms included water-loss stools, blood in the stool, gastric retention, bilious gastric aspirates, and abdominal distention. Several infants with virus-like particles whose mothers had gastrointestinal or “flu-like” symptoms before delivery were identified in the community (not part of the survey study).


title: Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide

text: Inflammation is a prominent feature of several disorders characterized by primary demyelination, but it is not clear whether a relationship exists between inflammation and myelin damage. We have found that substantial demyelination results from the focal inflammatory lesion caused by the injection of lipopolysaccharide (LPS; 200 ng) directly into the rat dorsal funiculus. Within 24 h, such injections caused a focal inflammatory response consisting of a substantial number of polymorphonuclear cells and ED1-positive and inducible nitric oxide synthase (iNOS)-positive macrophages/microglia. The number of inflammatory cells was substantially reduced by day 7. OX-52-positive T-cells were less frequently observed but were present in the meninges at 8 h, reached a maximum in the dorsal funiculus at 7 days, and were rare at 14 days. The inflammation was followed by the appearance of a large lesion of primary demyelination that encompassed up to ∼75% of the cross-sectional area of the dorsal funiculus. Treatment with dexamethasone significantly reduced the number of cells expressing iNOS, but did not prevent the demyelination. By 28 days the lesions were largely remyelinated, usually by Schwann cells. These changes were not observed in control, saline-injected animals. We conclude that the intraspinal injection of LPS results in inflammation and subsequently in prominent demyelination. The mechanisms underlying the demyelination are not clear, but it is notable that it typically begins with disruption of the adaxonal myelin. Indeed, there is an early loss of myelin-associated glycoprotein within the lesion, despite the persistence of proteolipid protein. This combination is a feature of the pattern III lesion recently described in multiple sclerosis (Lucchinetti et al., 2000), and we therefore suggest that LPS-induced demyelination may serve as the first experimental model available for the study of this type of multiple sclerosis lesion.


title: The utility and perspectives of NGS-based methods in BSL-3 and BSL-4 laboratory – sequencing and analysis strategies

text: Modern diagnostics is in general based on molecular biology methods. Nowadays sequencing-based methods, especially whole genome sequencing, are becoming increasingly important. Implementation of such methods into routine diagnostic of highly dangerous pathogens, like Bacillus anthracis, Francisella tularensis, Yersinia pestis, Ebola virus, MERS, Lassa virus etc. would be very helpful. The best diagnostic strategy would be the metagenomic sequencing directly from the clinical sample. Implementation of majority of currently available WGS platforms inside the BSL-3 or 4 laboratory is impractical because of the size of the equipment and time consuming wet lab part (e.g. library preparation). Nowadays there is a possibility to implement pocket size MinION - real time whole genome sequencer into BSL-3 and 4 laboratory for rapid and precise diagnostic purposes.


title: Enteric Lesions and Diarrhea in Gnotobiotic Calves Monoinfected with Cryptosporidium Species

text: The pathogenicity of Cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. Two control calves were inoculated with similar material from which the oocysts had been removed by filtration. Oocyst-inoculated animals shed Cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. At necropsy five days after inoculation, endogeneous stages of Cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. The parasites were most numerous in the lower small intestine. Atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. Control animals remained normal. Extraneous agents were not detected in any of the calves. The results indicate that Cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves.


title: Polyadenylate in the Virion RNA of Mouse Hepatitis Virus

text: Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.


title: Rhinovirus-Associated Hospitalizations in Young Children

text: Background. Rhinoviruses frequently cause the common cold but have not been considered important causes of acute respiratory hospitalizations in children. Methods. A population-based surveillance study was performed among children <5 years of age who were hospitalized with respiratory symptoms or fever and who resided within counties encompassing Nashville, Tennessee, or Rochester, New York, from October 2000 through September 2001. Data collected included questionnaires, nasal and throat swabs for viral culture and polymerase chain reaction testing, and chart review. Rates of rhinovirus-associated hospitalizations were calculated. Results. Of 592 children enrolled, 156 (26%) were rhinovirus positive, representing 4.8 (95% confidence interval [CI], 4.3–5.2) rhinovirus-associated hospitalizations/1000 children. Age-specific rates per 1000 children were 17.6 (95% CI, 14.9–20.6) for 0–5-month-olds, 6.0 (95% CI, 5.0–7.0) for 6–23-month-olds, and 2.0 (95% CI, 1.6, 2.4) for 24–59-month-olds (P<.01) Children with a history of wheezing/asthma had significantly more rhinovirusassociated hospitalizations than those without a history (25.3/1000 children [95% CI, 21.6–29.5/1000 children] vs. 3.1/1000 children [95% CI, 2.7–3.5/1000 children]). Conclusions. Rhinoviruses were associated with nearly 5 hospitalizations/1000 children <5 years of age and were highest in children with a history of wheezing/asthma.


title: Membrane-Disruptive Effect of Human Milk: Inactivation of Enveloped Viruses

text: Fresh human milk treated with antibody to secretory IgA had no effect on viral infectivity but became antiviral after 1 hr in the stomachs of suckling infants. Antiviral activity also appeared in fresh milk stored at 4 C for at least two days. The antiviral activity, which reduced titers of virus by as much as 10,000-fold, only affected enveloped viruses and was localized in the milk lipid fraction. Its appearance in stored milk was apparently due to fatty acids released by the activity of milk lipases, particularly lipoprotein lipase. Antiviral activity in the infants' stomach, however, most likely resulted from the activity of gastric and linguallipases on milk triglycerides and caused the release of antiviral fatty acids. Milk and stomach contents that were antiviral also lysed cultured cells by disruption of their plasma membrane. Cell lysis was also caused by purified linoleic acid, which is a normal constituent of human milk triglycerides.


Human Bocavirus: Multisystem Detection Raises Questions about Infection


title: A Prospective Study of Rotavirus Infection in Infants and Young Children

text: Diarrhea in neonates, followed as a cohort, and their families wasstudied prospectively. The families were followed for an average of 16.3 months. Stool and serum specimens were obtained at least every three months. Stool specimens were examined for viruses by electron microscopy and cultured for enteropathogens, and serum specimens were tested for antibodies to rotavirus and Norwalkvirus. During the study, 237episodes of gastroenteritis were observed in 104 infants and their 62 siblings. Rotavirus, detected 82 timesin 72 children, wasby far the most common enteropathogen. It was associated with gastrointestinal symptoms in 72% (with diarrhea in 65%). Rotavirus diarrhea occurred mostly in winter months and was significantly more frequently associated with respiratory symptoms than were diarrheas with other etiologies. Rotavirus infection was uncommon in the first six months of life, but by two years of age, 62% of the infants had had at least one infection. Neither breast feeding nor the presence of antibody to rotavirus in cord blood appeared to be protective.


title: CD4(+) T Helper Cell Responses against Human Bocavirus Viral Protein 2 Viruslike Particles in Healthy Adults

text: Background. Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. Methods. HBoV viruslike particles (VLPs) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. HBoV viral protein 2 (VP2)-specific antibodies and CD4(+) T helper cell responses were analyzed by enzyme-linked immunsorbent assay and enzyme-linked immunospot assay. Results. VP2 capsid proteins of HBoV were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral VLPs (diameter, 21–25 nm; sedimentation density, 1.33 g/cm(3)) was demonstrated. A significant increase in secretion of VP2-specific interferon-γ was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for HBoV-specific immunoglobulin G antibodies, compared with control stimulations. In parallel, T cell responses against identically expressed parvovirus B19 VP2 VLPs were frequently observed in the individuals studied, without there being obvious cross-reactions between HBoV and parvovirus B19. Conclusions. Data suggest the presence of HBoV-specific immune responses in adults and strongly support a high prevalence of HBoV among humans.


title: Gastrointestinal Viral Infections in Homosexual Men Who were Symptomatic and Seropositive for Human Immunodeficiency Virus

text: Gastrointestinal viruses, predominantly rotaviruses and adenoviruses, were detected by enzyme-linked immunosorbent assay, electron microscopy, or cell culture in >50% of two groups of homosexual men with symptomatic human immunodeficiency virus (HIV) infection, who did (54%) or did not (50%) have diarrhea. Lower detection rates were observed in HIV-seronegative (15%) and asymptomatic HIV-seropositive (16%) men. In the patients with diarrhea, 95% of the isolates of virus were found in the most immuno suppressed patients, those patients with AIDS-related complex or opportunistic infections associated with AIDS. High excretion rates of these viruses are probably associated with both anal-oral transmission and immunosuppression. These viruses apparently cause acute episodes or relapses of diarrhea in some patients but may be co-pathogens or noncontributory to chronic diarrhea in others.


title: Review of computational methods for virus–host protein interaction prediction: a case study on novel Ebola–human interactions

text: Identification of potential virus–host interactions is useful and vital to control the highly infectious virus-caused diseases. This may contribute toward development of new drugs to treat the viral infections. Recently, database records of clinically and experimentally validated interactions between a small set of human proteins and Ebola virus (EBOV) have been published. Using the information of the known human interaction partners of EBOV, our main objective is to identify a set of proteins that may interact with EBOV proteins. Here, we first review the state-of-the-art, computational methods used for prediction of novel virus–host interactions for infectious diseases followed by a case study on EBOV–human interactions. The assessment result shows that the predicted human host proteins are highly similar with known human interaction partners of EBOV in the context of structure and semantics and are responsible for similar biochemical activities, pathways and host–pathogen relationships.


title: Chemokine CC Receptor 2 Is Important for Acute Control of Cardiac Parasitism but Does Not Contribute to Cardiac Inflammation after Infection with Trypanosoma cruzi

text: The CC chemokine ligand 2 (CCL2) and CC chemokine receptor 2 (CCR2) are expressed in the heart after infection with Trypanosoma cruzi suggesting that they play an important role in host defense. Infection of CCR2-deficient (CCR2(−/−)) mice with T. cruzi resulted in increased cardiac parasitism, yet the severity of cardiac inflammation was not affected. In addition, expression of interferon-γ and inducible NO synthase in the heart, which are associated with effective killing of trypomastigotes, was not affected in CCR2(−/−) mice. These observations reveal that CCR2 signaling plays a distinct role that is separate from that of influencing either chemotaxis or previously defined anti-trypomastigote mechanisms for the control of T. cruzi’s replication in the heart


Human Immune Response to Giardia lamblia Infection


title: Serum and plasma zinc, copper and iron concentrations in Aboriginal communities of North Western Australia

text: Two Aboriginal communities situated in the tropical north-west of the Australian continent have been investigated in regard to trace metal status (zinc, copper, and iron) and other laboratory and epidemiological information. A total of 364 persons, ranging in age from 5 to 77 years were studied. The incidence of hypozincemia (serum or plasma zinc concentration <0.71 µg/ml) of the two communities when combined was 24.4%, while hypercupremia (defined as serum or plasma copper levels greater than 1.38 µg/ml) was 47.9%. Depressed serum iron levels were demonstrated in more than 50% of the Aborigines studied. Hypozincemia was most prevalent (incidence 31 to 67%) in children at the time of the important pre- and postadolescent growth period (10 to 15 years) and in women beyond 60 years of age (incidence 33 to 64%). Serum total protein and vitamin B(12) levels tended to be increased. Mild anemia was seen in approximately one in five persons aged less than 20 years. Intestinal parasites and pathogenic enterobacteria were frequently isolated in fecal specimens. In one community, half of the persons examined had positive isolates of enteric pathogens. Intestinal parasites predominated and were more frequently isolated from persons aged less than 20 years. Ancylostoma duodenale accounted for 32% of the pathogens isolated. Evidence is presented that suggests that both communities are exposed to numerous bacterial, viral, and parasitic infections. The diet consumed in these communities is predominately white flour and refined sugar. Geophagia is practiced in this area of Australia. It is emphasised that all the etiological prerequisites and many of the laboratory findings ascribed to the zinc deficiency syndrome appear to be operating in the two Aboriginal communities studied.


title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

text: Aberrant activation of hedgehog (HH) pathway has been implicated in the development of human malignancies. This study aimed at investigating the role of HH molecules in human ovarian carcinogenesis. The expression profiles of HH molecules were examined in ovarian tumor samples and ovarian cancer cell lines and the in vitro effects of HH molecules on cell proliferation, apoptosis, migration, invasion and cell differentiation as well as related downstream target genes were assessed. Overexpression of Patched and Gli1 protein in ovarian cancers correlated with poor survival of the patients (P = 0.008; P = 0.004). Significantly elevated expression of Sonic hedgehog messenger RNA was observed in ovarian cancers compared with normal tissues and benign ovarian tumors and such differential expression was specific to histological types (P < 0.05). Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness and change in differentiation in association with increased expression of E-cadherin, vimentin, Bcl-2, caspases as well as β1 integrin, membrane type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF). Treatment with 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine induced cancer cell apoptosis, suppressed cell growth, mobility and invasiveness and induced cancer cell dedifferentiation with decreased expression of E-cadherin, cytokeratin 7, Snail, calretinin, vimentin, Bcl-2, caspases, β1 integrin, MT1-MMP and VEGF. Our data suggested that abnormal HH signaling activation plays important roles in the development and progression of ovarian cancers. Gli1 expression is an independent prognostic marker. Inhibition of the HH pathway molecules might be a valid therapeutic strategy for ovarian cancers.


title: Intranasal Interferon-α2b for Seasonal Prophylaxis of Respiratory Infection

text: Efficacy of intranasal recombinant alpha interferon (IFN-α2b) was evaluated over a four-week period. The first 400 participants received either 1,500,000 IU of IFN-α2b or placebo twice daily. Rhinovirus infections were prevented (protective efficacy, 76%). Parainfluenza infections were not prevented, but symptoms in associated episodes of disease were significantly reduced. The medication was generally well tolerated, but side effects were often observed. The most commonly reported symptom was blood-tinged mucus. A pilot study of IFN-α2b or placebo administered on a once-daily dose schedule was also carried out in 150 participants. There was a suggestion of continued efficacy with reduced side effects. Overall, these findings would limit the use of IFN-α2b on the twice-daily schedule to shorter time periods or to special situations in which the efficacy clearly outweighs side effects, and they encourage further examination of other dosage schedules.


The Host Control of Lytic and Latent Infection with Human Herpesvirus–8


Intranasal Interferon-α(2) Prophylaxis of Natural Respiratory Virus Infection


title: Molecular Genetic Insights on Cheetah (Acinonyx jubatus) Ecology and Conservation in Namibia

text: The extent and geographic patterns of molecular genetic diversity of the largest remaining free-ranging cheetah population were described in a survey of 313 individuals from throughout Namibia. Levels of relatedness, including paternity/maternity (parentage), were assessed across all individuals using 19 polymorphic microsatellite loci, and unrelated cheetahs (n = 89) from 7 regions were genotyped at 38 loci to document broad geographical patterns. There was limited differentiation among regions, evidence that this is a generally panmictic population. Measures of genetic variation were similar among all regions and were comparable with Eastern African cheetah populations. Parentage analyses confirmed several observations based on field studies, including 21 of 23 previously hypothesized family groups, 40 probable parent/offspring pairs, and 8 sibling groups. These results also verified the successful integration and reproduction of several cheetahs following natural dispersal or translocation. Animals within social groups (family groups, male coalitions, or sibling groups) were generally related. Within the main study area, radio-collared female cheetahs were more closely interrelated than similarly compared males, a pattern consistent with greater male dispersal. The long-term maintenance of current patterns of genetic variation in Namibia depends on retaining habitat characteristics that promote natural dispersal and gene flow of cheetahs.


title: Factors Affecting the Stability of Transmissible Enteritis Virus of Turkeys

text: The effects of environmental factors on the stability of transmissible enteritis virus of turkeys were studied, using an assay system of poult inoculation. Viral infectivity persisted for less than 6 hr at 37 C in nutrient broth. Survival of virus was enhanced in the presence of cysteine and nicotinamide-adenine dinucleotide, and at pH 5.5. Survival was also enhanced in a growing culture of intestinal microflora and could be observed as well in a culture of Streptococcus fecalis in medium with reduced oxygen tension. These results were compared with findings in experiments with several other intestinal viruses, and it is suggested that some intestinal viruses may be adapted to the conditions of low pH and redox potential that are normal in the intestine.


title: Predicting proteolytic sites in extracellular proteins: only halfway there

text: Motivation: Many secretory proteins are synthesized as inactive precursors that must undergo post-translational proteolysis in order to mature and become active. In the current study, we address the challenge of sequence-based discovery of proteolytic sites in secreted proteins using machine learning. Results: The results revealed that only half of the extracellular proteolytic sites are currently annotated, leaving over 3600 unannotated ones. Furthermore, we have found that only 6% of the unannotated sites are similar to known proteolytic sites, whereas the remaining 94% do not share significant similarity with any annotated proteolytic site. The computational challenges in these two cases are very different. While the precision in detecting the former group is close to perfect, only a mere 22% of the latter group were detected with a precision of 80%. The applicability of the classifier is demonstrated through members of the FGF family, in which we verified the conservation of physiologically-relevant proteolytic sites in homologous proteins. Contact: kliger@compugen.co.il; yossef.kliger@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.


title: Up-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 in gastrointestinal cancer and its clinical relevance

text: Serum carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is dysregulated in various malignant tumors and has been associated with tumor progression. However, the expression and regulatory mechanisms of serum CEACAM1 in gastrointestinal cancer are still unclear. The expression ratio of the CEACAM1-L and CEACAM1-S isoforms has seldom been investigated in gastrointestinal cancer. In this study, we intended to explore the expression and diagnostic value of CEACAM1 in gastrointestinal cancer. Serum CEACAM1 levels were measured by enzyme-linked immunosorbent assay. The protein expression and distribution of CEACAM1 in tumors were examined by immunohistochemical staining. The expression patterns and ratio of CEACAM1-L/S were analyzed by reverse transcription-polymerase chain reaction. The results showed that serum CEACAM1 levels were significantly higher in cancer patients than in healthy controls. CEACAM1 was found in secreted forms within the neoplastic glands, and its expression was more intense at the tumor invasion front. The CEACAM1-L/S (L:S) ratios were up-regulated during tumorigenesis. Our data suggest that the serum level of CEACAM1 may be used to discriminate gastrointestinal cancer patients from health controls.


title: Diarrhea in Young Red Deer Associated with Infection with Cryptosporidium

text: In an outbreak of diarrhea among 82 artificially reared red deer calves, 56 developed the disease and 20 subsequently died. During the outbreak 80% of diarrheal and 50% of apparently healthy calves excreted cryptosporidial oocysts in feces. The coincidence of infection with Cryptosporidium and clinical diarrhea suggested a causal relationship. Histologic examination of intestinal sections from a necropsied deer calf showed lesions consistent with field and experimental cryptosporidiosis in other species. The deer Cryptosporidium subclinically infected newborn mice; in indirect immunofluorescence tests, it could not be distinguished from a calf Cryptosporidium.


title: Important Roles of the C-Terminal Portion of HPC-1/Syntaxin 1A in Membrane Anchoring and Intracellular Localization(1)

text: HPC-1/syntaxin 1A (HPC-1), which plays an important role in vesicular transport to the plasma membrane, possesses a hydrophobic sequence at its C terminus. When expressed from cDNA in COS cells, wild-type HPC-1 was localized in the Golgi complex and the plasma membrane. Truncation of the hydrophobic domain resulted in the cytoplasmic localization of the mutant, thus indicating that the domain indeed functions as a membrane anchor. A fusion protein with the C-terminal glycosylation sites was glycosylated in transfected cells, providing evidence that HPC-1 has a transmembrane structure, and that the protein is first inserted into the endoplasmic reticulum and then transported to the plasma membrane. A chimeric protein consisting of Escherichia coli maltose-binding protein with the last 24 amino acids of HPC-1 was inserted into the endoplasmic reticulum in a transmembrane topology and localized along the exocytic pathway of transfected cells similar to HPC-1. These results indicate that the portion is important for intracellular localization of HPC-1.


title: Posttransfusion Non-A, Non-B Hepatitis: Physicochemical Properties of Two Distinct Agents

text: Two separate and distinct episodes of non-At non-B hepatitis were induced in each of two chimpanzees by two inocula: one containing a chloroform-resistant agent and the other containing a chloroform-sensitive agent. Both agents were recovered from liver tissue and plasma obtained from a single chimpanzee during the acute and chronic phases of infection with a factor VIII concentrate, respectively. The chloroform-resistant agent did not cause unique changes in hepatocytes; in contrast, the chloroform-sensitive agent did induce the formation of cytoplasmic tubules, convoluted endoplasmic reticulum, and dense reticular inclusion bodies. The latter changes are similar in character to those induced in infected cells by some enveloped mammalian RNA viruses.


title: Neutralizing Antibodies to Interferon-α: Relative Frequency in Patients Treated with Different Interferon Preparations

text: The frequencies of antibody development so far reported in patients treated with different interferons (IFNs) are not readily comparable because of differences in treatment regimens and assay methods. Thus the frequency of neutralizing antibody development was analyzed in a large sample of sera derived from a relatively homogeneous group of patients treated with different IFN-α preparations. The frequency of developing neutralizing antibody to IFN varied according to the IFN given. Particularly, the seroconversion frequency was significantly higher in patients treated with recombinant IFN-α2a (20.2%) than in patients treated with either recombinant IFN-α2b (69%) or IFN-αN1 (1.2%), a lymphoblastoid IFN-α. Furthermore, sera obtained from patients treated with either recombinant IFN neutralized both types of recombinant IFNs but failed to neutralize IFN-αN1.


title: Tissue Culture in the Laboratory Diagnosis of Viral Infections

text: The use of both rhesus monkey kidney and human fetal diploid cell cultures for attempts to isolate virus from human clinical specimens recovered approximately 30% more isolates from fecal, throat, skin lesion, and tissue specimens than were recovered in a single cell culture system. The sensitivities of the two cell types for isolation of viruses in the major groups infecting man are compared. The use of both BS-C-1 and RK-13 cells yielded approximately 25% more rubella virus isolates than were recovered in a single cell system. Procedures such as the establishment of cell cultures from tissues suspected of harboring viruses, cocultivation of cells from biopsy or autopsy specimens with other cell types, and the use of organ cultures, have in certain situations resulted in greater sensitivity for recovery of viruses. Micro cell culture tests have been devised for virus identification and antibody assays; these are far more economical than conventional tests in terms of reagents, space requirements, and personnel time. Methods have been developed for the production of higher-titered viral serologic antigens in cell culture systems.


title: Bcl-2-associated transcription factor 1 interacts with fragile X-related protein 1

text: The absence of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), which is the leading cause of hereditary mental retardation. Fragile X-related protein 1 (FXR1P), which plays an important role in normal muscle development, is one of the two autosomal paralogs of FMRP. To understand the functions of FXR1P, we screened FXR1P-interacting proteins by using a yeast two-hybrid system. The fragile X-related gene 1 (FXR1) was fused to pGBKT7 and then used as the bait to screen the human fetal brain cDNA library. The screening results revealed 10 FXR1P-interacting proteins including Bcl-2-associated transcription factor 1 (BTF). The interaction between FXR1P and BTF was confirmed by using both β-galactosidase assay and growth test in selective media. Co-immunoprecipitation assay in mammalian cells was also carried out to confirm the FXR1P/BTF interaction. Moreover, we confirmed that BTF co-localized with FXR1P in the cytoplasm around the nucleus in rat vascular smooth muscle cells by using confocal fluorescence microscopy. These results provide clues to elucidate the relationship between FXR1P and FXS.


title: Demonstration of a Glycoprotein Derived From the Ceacam10 Gene in Mouse Seminal Vesicle Secretions(1)

text: CEACAM10 was purified from mouse seminal vesicle secretions by a series of purification steps that included ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was shown to be a 36-kDa glycoprotein with an N-linked carbohydrate moiety. The circular dichromoism spectrum of CEACAM10 in 50 mM phosphate buffer at pH 7.4 appeared as one negative band arising from the β form at 217 nm. CEACAM10 was expressed predominantly in seminal vesicles of adult mice. Both CEACAM10 and its mRNA were demonstrated on the luminal epithelium of the mucosal folds in the seminal vesicle. The amount of Ceacam10 mRNA in the seminal vesicle was correlated with the stage of animal maturation. Castration of adult mice resulted in cessation of Ceacam10 expression, while treatment of castrated mice with testosterone propionate in corn oil restored Ceacam10 expression in the seminal vesicle. During the entire course of pregnancy, Ceacam10 might be silent in the embryo. A cytochemical study illustrated the presence of the CEACAM10 binding region on the entire surface of mouse sperm. CEACAM10-sperm binding greatly enhanced sperm motility in vitro.


title: A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

text: Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.


title: Colorstock, SScolor, Ratón: RNA alignment visualization tools

text: Summary: Interactive examination of RNA multiple alignments for covariant mutations is a useful step in non-coding RNA sequence analysis. We present three parallel implementations of an RNA visualization metaphor: Colorstock, a command-line script using ANSI terminal color; SScolor, a Perl script that generates static HTML pages; and Ratón, an AJAX web application generating dynamic HTML. Each tool can be used to color RNA alignments by secondary structure and to visually highlight compensatory mutations in stems. Availability: All source code is freely available under the GPL. The source code can be downloaded and a prototype of Ratón can be accessed at http://biowiki.org/RnaAlignmentViewers Contact: ihh@berkeley.edu


title: The Roles of Individual Cysteine Residues of Sendai Virus Fusion Protein in Intracellular Transport(1)

text: The role of intramolecular disulfide bonds in the fusion (F) protein of Sendai virus was studied. The 10 cysteine residues were changed to serine residues using site-directed mutagenesis. None of the cysteine mutant F proteins reacted with a monoclonal antibody specific for the mature conformation of the F protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. The transport of these mutant proteins to the cell surface was drastically reduced. All of the cysteine mutant F proteins remained sensitive to endoglycosidase H (endo H) for 3 h after their synthesis. Moreover, cell surface transport of the hemagglutinin-neuraminidase (HN) protein co-expressed with each of these cysteine mutant F proteins was also reduced. These results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the F protein, and that interaction of the F and HN proteins takes place intracellulary.


title: INFLUENZA A/ENGLAND: AN OUTBREAK AT A MILITARY ACADEMY

text: Blouse, L E. (Epidemiology Division. USAF School of Aerospace Medicine. Brooks AFB, Texas 78236), L. N. Kolonel and V. Corrado. Influenza A/England: an outbreak at a military academy. Am J Epidemiol 100:216–221. 1974.—The first outbreak of A/England influenza in the United States during the 1972–1973 respiratory season is described. The outbreak was confined to an unvaccinated population of 3935 cadets at the U.S. Air Force Academy, Colorado. During the 10-day period of the outbreak, 778 (19.8 per cent) of the cadets were affected, and specific attack rates for each of the four cadet classes ranged from 170.8/1000 to 227.1/1000. For three days preceding the outbreak, about 400 recently vaccinated naval midshipmen from the U.S. Naval Academy shared quarters with the Air Force cadets. During the four days following the visit, a single midshipman developed possible influenza whereas, in the same period, 352 cadet cases were recognized. The highly significant difference in attack rates (2.5 versus 89.5/1000) provides evidence for the efficacy of the 1972–1973 influenza vaccine against the A/ England/42/72 variant.


title: VIRAL INFECTION IN PATIENTS WITH MULTIPLE SCLEROSIS AND HLA-DR MATCHED CONTROLS

text: Retrospective comparisons of the prevalence and age, where appropriate, of some childhood infectious illnesses and vaccinations, together with serological evidence for exposure to 16 viruses, many of which have previously been implicated in the aetiology of multiple sclerosis (MS) were made in 177 patients with acute optic neuritis, other recent isolated demyelinating episodes or established MS and 164 controls The expected high frequency of HLA-DR2 in patients with demyelinating disease was matched by preselection of normal controls with this antigen (DR2+), the remaining individuals were classified as HLA-DR2 negative/DR3 positive (DR3+) or HLA-DR2 and 3 negative (DR2/3-). Cases were compared with controls, collectively and in analyses restricted to each genetic group; these comparisons were repeated considering the three categories of patients with demyelination and two control populations separately All DR2+, DR3+ and DR2/3 individuals were compared in a single analysis to assess the effect of HLA type itself on the results Patients with demyelinating disease had rubella and measles at a later age and reported mumps infection more frequently than controls. Age of typhoid vaccination and duration of exposure to domestic dogs was higher in all cases than controls. Age of measles and mumps, but not rubella, was higher in DR2 + cases than controls, but differences were not observed in the other genetic groups Higher rubella antibody titres were present in all cases than controls and in analyses confined to DR2+ individuals in whom higher Epstein Barr virus antibody titres were also present Measles haemagglutination inhibition and parainfluenza I antibody titres were increased and influenza A antibodies detected less frequently in all patients with optic neuntis and those with DR2 compared with appropnate controls; influenza B antibody titres were lower in all DR2+ cases than controls. Higher adenovirus and varicella zoster antibody titres were present in DR2/3 patients with demyelination and other neurological diseases compared with normal controls. Overall, older age of infection and higher antibody titres were observed more often in patients with optic neuritis, in particular DR2 + cases, than other individuals with demyelination or controls. Our serological results are consistent with the presence of abnormal HLA mmunological reactivity in patients with MS but cannot be explained only by an effect of DR type itself; age at which susceptible individuals develop some common childhood infections may also influence the subsequent development of the disease.


title: Therapeutic Delivery of Ang(1–7) via Genetically Modified Probiotic: A Dosing Study

text: In recent years a number of beneficial health effects have been ascribed to the renin-angiotensin system (RAS) that extend beyond lowering blood pressure, primarily mediated via the angiotensin-converting enzyme-2 (ACE2)/angiotensin (1–7) or Ang(1–7)/MAS receptor axis. Moreover, once thought as merely a systemic effector, RAS components exist within tissues. The highest tissue concentrations of ACE2 mRNA are located in the gut making it an important target for altering RAS function. Indeed, genetically engineered recombinant probiotics are promising treatment strategies offering delivery of therapeutic proteins with precision. An Ang(1–7) secreting Lactobacillus paracasei (LP) or LP-A has been described for regulation of diabetes and hypertension; however, we are the first to the best of our knowledge to propose this paradigm as it relates to aging. In this Research Practice manuscript, we provide proof of concept for using this technology in a well-characterized rodent model of aging: the Fisher344 x Brown Norway Rat (F344BN). Our primary findings suggest that LP-A increases circulating levels of Ang(1–7) both acutely and chronically (after 8 or 28 treatment days) when administered 3× or 7×/week over 4 weeks. Our future preclinical studies will explore the impact of this treatment on gut and other age-sensitive distal tissues such as brain and muscle.


title: Deficient Responses of Pulmonary Macrophages from Healthy Smokers to Antiviral Lymphokines in Vitro

text: The antiviral function of pulmonary macrophages obtained by broncholavage of healthy smokers and nonsmokers was studied. Compared with nonsmokers' cells, smokers' macrophages produced significantly more virus during in vitro infection with herpes simplex virus type 1 (HSV-1). Exposure of macrophages to either antiviral macrophage-activating factor or interferon-y for 20 hr before infection resulted in diminished production of HSV-1 by both types of macrophages. However, in contrast to smokers' cells, exposure of nonsmokers' macrophages to these antiviral lymphokines totally prevented viral replication. This difference could not be attributed to diminished adsorption of virus to smokers' macrophages or to an increased proportion of extracellular to intracellular virus in smokers' cell cultures. The effect of smoking on viral infectivity did not appear to be mediated by secretion of a soluble factor by the macrophage because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not affect the growth of HSV-1.


title: Protective Immunity against Respiratory Tract Challenge with Yersinia pestis in Mice Immunized with an Adenovirus-Based Vaccine Vector Expressing V Antigen

text: The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine


Some Characteristics of Hemagglutination of Certain Strains of “IBV-like” Virus


title: Functional characterization of the partially purified Sac1p independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum

text: Several ATP-depending reactions take place in the endoplasmic reticulum (ER). Although in Saccharomyces cerevisiae ER the existence of a Sac1p-dependent ATP transport system was already known, its direct involvement in ATP transport was excluded. Here we report an extensive biochemical characterization of a partially purified adenine nucleotide transport system (ANTS) not dependent on Sac1p. Highly purified ER membranes from the wild-type and Δsac1 yeast strains reconstituted into liposomes transported ATP with the same efficiency. A chromatography on hydroxyapatite was used to partially purify ANTS from Δsac1 ER extract. The two ANTS-enriched transport activity eluted fractions showed essentially the presence of four bands, one having an apparent MW of 56 kDa, similar to that observed for ANTS identified in rat liver ER. The two fractions reconstituted into liposomes efficiently transported, by a strict counter-exchange mechanism, ATP and ADP. ATP transport was saturable with a Km of 0.28 mM. The ATP/ADP exchange mechanism and the kinetic constants suggest that the main physiological role of ANTS is to catalyse the transport of ATP into ER, where it is used in several energy-requiring reactions and to export back to the cytosol the ADP produced.


title: Antiviral Activity of Adenine Arabinoside and Iododeoxyuridine in Human Fetal Intestinal and Tracheal Organ Cultures

text: In vitro antiviral activities of two potentially clinically useful antiviral compounds, adenine arabinoside and iododeoxyuridine, were examined in human fetal intestinal and tracheal organ cultures infected with Herpesvirus hominis(types I and 2) or vaccinia virus. The two compounds were similarly active against the viruses in organ culture, and minimal inhibitory concentrations could be determined by titration of organ culture fluid harvests into tissue culture or directly in the organ cultures themselves. Minimal inhibitory concentrations were consistently lower in tracheal than in intestinal organ cultures and were consistently higher for H. hoministype 2 than for H. hoministype I. Thus the organ cultures are promising systems in which to evaluate antiviral activity against those agents that replicate in vitro only in organ culture, and they may have particular application to the study of herpetic tracheitis and esophagitis.


title: A Two-Year Study of Bacterial, Viral, and Parasitic Agents Associated with Diarrhea in Rural Bangladesh

text: Enteric pathogens associated with diarrhea were studied for two years at a diarrhea treatment center in rural Bangladesh. Enterotoxigenic Escherichia coli (ETEC) was the most frequently identified pathogen for patients of all ages. Rotavirus and ETEC were isolated from ∼50% and ∼25%, respectively, of patients less than two years of age. A bacterial or viral pathogen was identified for 70% of these young children and for 56% of all patients with diarrhea. Most ETEC isolates were obtained in the hot dry months of March and April and the hot wet months of August and September. Rotavirus identification peaked in the cool dry months of December and January, but infected patients were found year-round. The low case-fatality rates for patients with watery diarrhea and substantial dehydration further document the usefulness of treating patients with diarrhea with either a glucose- or sucrose-base electrolyte solution such as those used in this treatment center.


title: Biological function through network topology: a survey of the human diseasome

text: Molecular network data are increasingly becoming available, necessitating the development of well performing computational tools for their analyses. Such tools enabled conceptually different approaches for exploring human diseases to be undertaken, in particular, those that study the relationship between a multitude of biomolecules within a cell. Hence, a new field of network biology has emerged as part of systems biology, aiming to untangle the complexity of cellular network organization. We survey current network analysis methods that aim to give insight into human disease.


title: EPIDEMIOLOGY OF RESPIRATORY SYNCYTIAL VIRUS INFECTION AMONG INFANTS AND CHILDREN IN CHICAGO

text: From January 1, 1967 to December 1971, the temporal pattern of respiratory syncytial virus infection was investigated in infants and children younger than 18 months hospitalized for acute lower respiratory tract disease. Of 4696 infants and children with acute lower respiratory tract disease admitted to the Cook County Hospital, 2530 were tested for virus infection by virus isolation or serologic procedures or both. Overall, respiratory syncytial virus infections were detected in 12% and parainfluenza 3 virus in 10.8% of individuals tested. Other respiratory viruses were less commonly identified. Respiratory syncytial virus epidemics occurred annually and were temporally synchronous with the peak periods of respiratory disease admissions. Only during epidemics of respiratory syncytial virus did admission for respiratory tract disease usually reach 40 patients or more weekly. The peak months of respiratory syncytial virus epidemics were December 1966, January 1968, February-March 1969, April 1970 and January 1971. Epidemics lasted about 17 weeks. No similar annual epidemic pattern was seen with the other myxoviruses.


title: Host Cell Proteins Bind Specifically to the Capsid-Cleaved 5' End of Leishmaniavirus RNA(1)

text: Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive- sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.


title: Hollow Hepatitis B Antigen in a Case of Viral Hepatitis

text: The serum from one case of acute hepatitis contained large numbers of hollow particles of hepatitis B antigen (HB Ag). Particles had diameters of 15–20 nm, occurred frequently in compact masses, and were sometimes connected by bridges. This form may be the precursor of normal HB Ag.


Viral diarrhea. Etiology and control


title: Ribavirin Reduces Mortality in Enterovirus 71–Infected Mice by Decreasing Viral Replication

text: Enterovirus 71 (EV71) causes fatal encephalitis in young children. However, there is no effective antiviral drug available for infected patients. Ribavirin is currently used for the treatment of several RNA virus infections clinically, so its anti-EV71 efficacy was evaluated. In vitro results showed that ribavirin effectively reduced the viral yields (with an IC(50) of 65 μg/mL) and virus-induced cytopathic effect in human and mouse cell lines. In vivo results showed that ribavirin reduced the mortality, morbidity, and subsequent paralysis sequelae in infected mice by decreasing viral loads in tissues. Thus, ribavirin could be a potential anti-EV71 drug


title: Possible Role of Histone H1 in the Regulation of Furin-dependent Proprotein Processing

text: Histone H1 and its C-terminal lysine rich fragments were recently found to be potent inhibitors of furin, a mammalian proprotein convertase. However, its role in the regulation of furin-dependent proprotein processing remains unclear. Here we report that histone H1 efficiently blocks furin-dependent pro-von Willebrand factor (pro-vWF) processing in a dose-dependent manner. Coimmunoprecipitation and immunofluorescence studies confirmed that histone H1 could interact with furin, and the interaction mainly took place on the cell surface. We noted that histone H1 was released from cells undergoing necrosis and apoptosis induced by H(2)O(2). Our findings suggested that histone H1 might be involved in extracellular and/or intracellular furin regulation.


title: Evaluation of 6-Azauridine and S-Iododeoxyuridine in the Treatment of Experimental Viral Infections

text: The potential antiviral activity of 6-azauridine and 5-iododeoxyuridine was evaluated in a coordinated study at five institutions. Experimental models in five species, the mouse, rabbit, swine, cat, and ferret, were established with use of 10 viruses: Herpesvirus hominis types 1 and 2, murine cytomegalovirus, vaccinia virus, Shope fibroma virus, transmissible gastroenteritis virus, swine influenza virus, feline viral rhinotracheitis virus, feline panleukopenia virus, and ferret distemper virus. Criteria for selection were: (1) representation from a number of major groups of viruses, (2) reproduction of natural routes of infection, and (3) simulation of potentially treatable viral infections of man. Antiviral activity was observed for 5-iododeoxyuridine in H. hominis infections in hairless mice and influenza in swine, and a slight degree of efficacy was noted in rabbits infected with Shope fibroma virus. Toxicity was also observed in most of the experimental models. There was a suggestion of antiviral activity with 6-azauridine in swine infected with transmissible gastroenteritis virus; however, enhancement of disease and some toxicity were seen in most of the other models. Efficacy of these two compounds was not well substantiated by these studies.


title: Protein degradation systems in viral myocarditis leading to dilated cardiomyopathy

text: The primary intracellular protein degradation systems, including the ubiquitin-proteasome and the lysosome pathways, have been emerging as central regulators of viral infectivity, inflammation, and viral pathogenicity. Viral myocarditis is an inflammatory disease of the myocardium caused by virus infection in the heart. The disease progression of viral myocarditis occurs in three distinct stages: acute viral infection, immune cell infiltration, and cardiac remodelling. Growing evidence suggests a crucial role for host proteolytic machineries in the regulation of the pathogenesis and progression of viral myocarditis in all three stages. Cardiotropic viruses evolve different strategies to subvert host protein degradation systems to achieve successful viral replication. In addition, these proteolytic systems play important roles in the activation of innate and adaptive immune responses during viral infection. Recent evidence also suggests a key role for the ubiquitin-proteasome and lysosome systems as the primary effectors of protein quality control in the regulation of cardiac remodelling. This review summarizes the recent advances in understanding the direct interaction between cardiotropic viruses and host proteolytic systems, with an emphasis on coxsackievirus B3, one of the primary aetiological agents causing viral myocarditis, and highlights possible roles of the host degradation systems in the pathogenesis of viral myocarditis and its progression to dilated cardiomyopathy.


title: Factors Influencing the Synonymous Codon and Amino Acid Usage Bias in AT-rich Pseudomonas aeruginosa Phage PhiKZ

text: To reveal how the AT-rich genome of bacteriophage PhiKZ has been shaped in order to carry out its growth in the GC-rich host Pseudomonas aeruginosa, synonymous codon and amino acid usage bias of PhiKZ was investigated and the data were compared with that of P. aeruginosa. It was found that synonymous codon and amino acid usage of PhiKZ was distinct from that of P. aeruginosa. In contrast to P. aeruginosa, the third codon position of the synonymous codons of PhiKZ carries mostly A or T base; codon usage bias in PhiKZ is dictated mainly by mutational bias and, to a lesser extent, by translational selection. A cluster analysis of the relative synonymous codon usage values of 16 myoviruses including PhiKZ shows that PhiKZ is evolutionary much closer to Escherichia coli phage T4. Further analysis reveals that the three factors of mean molecular weight, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in PhiKZ proteins, whereas amino acid usage of P. aeruginosa proteins is mainly governed by grand average of hydropathicity, aromaticity and cysteine content. Based on these observations, we suggest that codons of the phage-like PhiKZ have evolved to preferentially incorporate the smaller amino acid residues into their proteins during translation, thereby economizing the cost of its development in GC-rich P. aeruginosa.


Vaccination for Pandemic Influenza and Severe Acute Respiratory Syndrome: Common Issues and Concerns


title: Clinical Features and Duration of Traveler's Diarrhea in Relation to Its Etiology

text: Clinical features of traveler's diarrhea (TD) were studied among 126 adult Finnish tourists who developed this illness during or shortly after a visit to Morocco. Enteric pathogens were identified in 76 (60%) of cases, whereas the etiology remained unidentified in 50 cases (40%). Patients with an identified pathogen did not differ from those with TD of unknown etiology in terms of the time of onset of illness or the median frequency of unformed stools in the first 24 hours. In contrast, the median frequency of unformed stools between 24 and 48 hours (i.e., on the second day) was 1.0 among patients with no pathogen and 2.0 among those with enteric pathogens identified (P < .001). A similar difference was evident on the third day (1.0 vs. 2.5). Moreover, a lower proportion of patients with no pathogen identified had watery stools (28% vs. 55%). The durations of diarrhea and concomitant symptoms were significantly shorter and the recovery from TD was significantly quicker among the patients without an identified pathogen. Patients with one or more invasive pathogens had disease that was clearly more severe than that of patients with no pathogen identified; the difference in severity of disease was less marked for patients with invasive vs. noninvasive pathogens. Individuals with diarrhea due to Campylobacter species tended to have the most severe disease, whereas diarrhea caused by enterotoxigenic Escherichia coli seemed milder than that caused by other agents. Unfortunately, the clinician has only a limited opportunity to predict the etiology of TD and thus to assess the need for antimicrobial therapy at the onset of disease.


title: A Genetically Inactivated Herpes Simplex Virus Type 2 (HSV-2) Vaccine Provides Effective Protection against Primary and Recurrent HSV-2 Disease

text: A glycoprotein H (gH)-deleted herpes simplex virus type 2 (HSV-2) was evaluated as a vaccine for the prevention of HSV-induced disease. This virus, which we term a DISC (disabled infectious single cycle) virus, can only complete one replication cycle in normal cells and should thus be safe yet still able to stimulate broad humoral and cell-mediated antiviral immune responses. A gH-deleted HSV-2 virus that has been tested as a vaccine in the guinea pig model of recurrent HSV-2 infection was constructed. Animals vaccinated with DISC HSV-2 showed complete protection against primary HSV-2–induced disease, even when challenged 6 months after vaccination. In addition, the animals were almost completely protected against recurrent disease. Even at low vaccination doses, there was a high degree of protection against primary disease. A reduction in recurrent disease symptoms was also observed following therapeutic vaccination of animals already infected with wild type HSV-2.


title: A Recently Identified Rhinovirus Genotype Is Associated with Severe Respiratory-Tract Infection in Children in Germany

text: Acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. Accurate identification of causative agents is critical to case management and to prioritization in vaccine development. Sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents andmayalso facilitate the discovery of new pathogens. Recently, application of MassTag polymerase chain reaction (PCR) to undiagnosed infuenza-like illness in New York State led to the discovery of a novel rhinovirus genotype. Here we report the investigation, by MassTag PCR, of pediatric respiratory-tract infections in Germany, studying 97 cases for which no pathogen was identified through routine laboratory evaluation. Respiratory viruses were identified in 49 cases (51%); of the 55 identified viruses, 41 (75%) were rhinoviruses. The novel genotype represented 73% of rhinoviruses and 55% of all identified viruses. Infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia.


title: Interactions between Viruses and Bacteria in Patients with Chronic Bronchitis

text: The possibility that viral infections of the respiratory tract might predispose to bacterial colonization or infection was studied in 120 patients with chronic obstructive pulmonary disease and 30 control subjects; these individuals were observed for seven years. The ratio of the number of observed to the number of expected associations between viruses and bacteria was 2.43 (P = 0.037) for the pair influenza virus and Streptococcus pneumoniae and was 2.06 (P = 0.056) for influenza virus and Haemophilus influenzae. Consistently positive, but not significant, associations were detected between rhinovirus and herpes simplex virus infections and isolations of S. pneumoniae and H. influenzae. In contrast, isolations of the nonpathogenic Haemophilus parainfluenzae could not be related to prior viral infections. Significant rises in titer of antibody to H. iniluenzae were detected on 76 occasions, and 20 (26%) of these antibody rises were associated with viral or mycoplasmal infections during the preceding 120 days. The expected number of such associations was 8.34 (ratio of number observed to number expected, 2.40; P = 0.08). These results suggest that viral infections of the respiratory tract in patients with chronic obstructive pulmonary disease are associated with increased colonization by potentially pathogenic bacteria and may also predispose to infection with H. injluenxae.


title: Inhibition of hepatitis C virus replication by Monascus pigment derivatives that interfere with viral RNA polymerase activity and the mevalonate biosynthesis pathway

text: OBJECTIVES: Hepatitis C virus (HCV) infection causes chronic liver disease and is a major public health problem worldwide. The aim of this study was to evaluate the potential of Monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by Monascus spp., as HCV antiviral agents. METHODS: We performed an in vitro RNA-dependent RNA polymerase (RdRp) assay to screen for HCV RdRp inhibitors. The anti-HCV activity of RdRp inhibitors in HCV-replicating cells was evaluated by quantification of the RNA viral genome. Molecular docking analysis was performed to predict the binding sites of the selected RdRp inhibitors. RESULTS: We have identified a Monascus pigment and its derivatives as inhibitors of the HCV NS5B RdRp. A group of Monascus orange pigment (MOP) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited HCV replication. Further, combination of the MOP derivatives (Phe, Val or Leu conjugates) with interferon (IFN)-α inhibited HCV replication more than IFN-α treatment alone. Lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of NS5B. The antiviral activity of the MOP derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in HCV replication was suppressed by the MOP compounds. CONCLUSIONS: Our results identify amino acid derivatives of MOP as potential anti-HCV agents and suggest that their combination with IFN-α might offer an alternative strategy for the control of HCV replication.


title: Comparison of Tissue Distribution, Persistence, and Molecular Epidemiology of Parvovirus B19 and Novel Human Parvoviruses PARV4 and Human Bocavirus

text: Background. PARV4 and human bocavirus (HBoV) are newly discovered human parvoviruses with poorly understood epidemiologies and disease associations. We investigated the frequencies of persistence, tissue distribution, and influence of immunosuppression on replication of these viruses. Methods. At autopsy, bone marrow, lymphoid tissue, and brain tissue from human immunodeficiency virus (HIV)—infected individuals with acquired immunodeficiency syndrome (AIDS) and those without AIDS and from HIV-uninfected individuals were screened for parvovirus B19, PARV4, and HBoV DNA by means of quantitative polymerase chain reaction analyses. Results. B19 DNA was detected both in HIV-infected study subjects (13 of 24) and in HIV-uninfected study subjects (8 of 8), whereas PARV4 DNA was detected only in HIV-infected study subjects (17 of 24). HBoV DNA was not detected in any study subjects. The degree of immunosuppression with HIV infection did not influence B19 or PARV4 viral loads. B19 or PARV4 plasma viremia was not detected in any study subjects (n = 76; viral load <25 DNA copies/mL). A significantly older age distribution was found for study subjects infected with B19 genotype 2, compared with those infected with B19 genotype 1. Two genotypes of PARV4 were detected; study subjects carrying prototype PARV4 (genotype 1) were younger (all born after 1958) than those infected with genotype 2 (PARV5; study subjects born between 1949 and 1956). Conclusions. Tight immune control of replication of B19 and PARV4 was retained despite profound immunosuppression. Recent genotype replacement of PARV4, combined with absent sequence diversity among genotype 1 sequences, suggests a recent, epidemic spread in the United Kingdom, potentially through transmission routes shared by HIV.


title: Infections with Legionella pneumophila in Children

text: To learn the role of Legionella pneumophila, the agent of Legionnaires' disease, in childhood illness, a prospective study was conducted among 52 children younger than four years of age with acute disease of the lower respiratory tract. Viral, mycoplasmal, and bacterial cultures and acute- and convalescent-phase sera were obtained during 64 episodes of acute illness; additional sera were drawn annually for three to five years. On the basis of serologic evidence, none of the acute episodes appeared to be due to L. pneumophila serogroup 1 or 2. However, examination of annual serum specimens showed that 27 (52%) of the children had rises in titer of indirect immunofluorescent antibody (a fourfold or greater rise to a reciprocal titer of ⩾128). Most rises in titer were in response to the serogroup 2 antigen. These results suggest that L. pneumophila is not a common cause of acute respiratory disease in early childhood in the study area but that children are frequently exposed to the organism. Alternatively, the serologic responses might be to unrelated cross-reacting microorganisms.


title: Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

text: Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.


title: The Presence and Cleavage of Interpeptide Disulfide: Bonds in Viral Glycoproteins(1)

text: The presence and nature of interpeptide disulfide bonds in HANA (Aemagglutinin and neura-minidase) glycoprotein and F (fusion) glycoprotein of HVJ (Sendai virus) are described. In the case of HANA, subunits of the same or very similar molecular weight were inter connected with a disulfide bond(s). Cleavage of the bond(s) can easily be achieved by the addition of 1 mM dithiothreitol with concomitant loss of the biological activities of the glyco-protein. After splitting of the interconnecting bonds, all the HANA protein subunits remained bound on the viral membrane. To observe the cleavage of the interpeptide disulfide bond between the F(1) and F(2) subunits of F glycoprotein, higher concentrations of sulfhydryl compounds were required than were necessary for HANA protein. Splitting of the disulfide bond under either denaturing or non-denaturing conditions failed to release both segments of F protein from the virion. Therefore, F glycoprotein seems to have at least two membrane binding sites, one on F(1) and the other on F(2). On the other hand, the disulfide bond which connects the HA(1) and HA(2) subunits of influenza virus is hardly cleaved under non-denaturing conditions. Addition of 8 M urea or 6 M guanidine HCl, which completely inactivates HA activity, was necessary for the splitting of this disulfide bond by thiol compounds. Interestingly, the HA(1) submit was released from the virion after the cleavage. Thus, unlike F(1) and F(2) of HVJ, the HA(1) subunit seems to have no hydrophobic binding site to the membrane. A model for the arrangement of these subunits on the viral membrane is proposed.


title: Ineffectiveness of Recombinant Interferon-β(serine) Nasal Drops for Prophylaxis of Natural Colds

text: Two randomized, double-blind, placebo-controlled trials during early autumn of 1986 and 1987 evaluated the efficacy and tolerance of recombinant interferon-β(serine) (rIFN-β(ser)) nasal drops for prevention of natural rhinovirus colds. In 1986, 9 × 106 units of rIFN-β(ser) (139 subjects) or placebo (157) were adminstered once daily except Sundays for 4 w. Rhinovirus colds occurred in 2.8% of rIFN-β(ser) recipients and 6.0% of placebo recipients during the treatment period (52% reduction, P = .3). In 1987, 24 × 106 units of rIFN-β(ser) (186) or placebo (197) were given daily for 25 consecutive days. Rhinovirus colds developed in 6.3% of rIFN-β(ser) recipients and 5.3% of placebo recipients. In each study, illness frequency and number of days with subjective colds did not differ between the groups. Recipients of nasal drops of rIFN-β(ser) at either dosage did not differ in tolerance from placebo recipients. The lack of both prophylactic efficacy and nasal toxicity are in contrast to prior observations with nasal sprays of rIFN-α(2b).


Infectious enteric viruses of neonatal animals


title: Report of a Workshop on Respiratory Viral Infections: Epidemiology, Diagnosis, Treatment, and Prevention

text: An international workshop to review the epidemiology, diagnosis, treatment, and prevention of respiratory viral infections was held in Turku, Finland, in May 1991. This workshop emphasized the following points. (1) In epidemiological studies of influenza virus, serological, clinical, and gene-sequencing methods have been used to produce a full picture of genetic evolution. Less complete information exists about other viruses, although new data on respiratory syncytial virus are emerging. (2) Tools for the diagnosis of respiratory viral infections have been developed in conjunction with the use of solid-phase immunoassays. A role remains for tissue culture in surveillance and epidemiological studies. Detection of bacterial involvement in respiratory infections has been more difficult. (3) Treatment of infections due to respiratory viruses has advanced with the use of amantadine and aerosolized ribavirin. On the other hand, many viruses remain refractory to treatment. Means for preventing influenza are established, but barriers to the development of other viral vaccines—including the existence of multiple serotypes, imperfect natural immunity, and paradoxical hypersensitivity—have proven difficult to surmount.


Intracellular Targeting and Sorting: How are macromolecules delivered to specific locations?


Modernization of the Medical Sherlock Holmes


title: Neutralization of Human Cytomegalovirus by Human CD13-Specific Antibodies

text: The mechanisms of inhibition of cytomegalovirus (CMV) infection by human CD13 (aminopeptidase N)-specific antibodies were studied. These antibodies protect CD13-negative and -positive cells from CMV infection only if incubated with the virus inoculum, suggesting they bind to CMV virions. The association of a CD13-like molecule with virions was further supported by the transfer of CD13 immunoreactivity to the surface of CD13-negative cells upon binding of CMV; the binding of CD13-specific antibodies directly to the surface of CMV virions; and the presence of anti-CD13 immunoreactive bands, including one with mobility similar to that of native cellular CD13 on immunoblots of proteins of purified CMV particles. Importantly, CD13-specific antibodies neutralize CMV in urine of neonates with congenital CMV, indicating that the CD13-like molecule is associated with natural CMV and not acquired in vitro. These studies demonstrate that a CD13-like molecule is associated with CMV particles and may be important in CMV pathogenesis.


title: A beginner’s guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing

text: Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workflow proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.


title: Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

text: The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or house keeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions. qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.


title: Efficient Neutralization of Anthrax Toxin by Chimpanzee Monoclonal Antibodies against Protective Antigen

text: Four single-chain variable fragments (scFvs) against protective antigen (PA) and 2 scFvs against lethal factor (LF) of anthrax were isolated from a phage display library generated from immunized chimpanzees. Only 2 scFvs recognizing PA (W1 and W2) neutralized the cytotoxicity of lethal toxin in a macrophage lysis assay. Full-length immunoglobulin G (IgG) of W1 and W2 efficiently protected rats from anthrax toxin challenge. The epitope recognized by W1 and W2 was conformational and was formed by C-terminal amino acids 614–735 of PA. W1 and W2 each bound to PA with an equilibrium dissociation constant of 4×10(-11) mol/L to 5 × 10(−11) mol/L, which is an affinity that is 20–100-fold higher than that for the interaction of the receptor and PA. W1 and W2 inhibited the binding of PA to the receptor, suggesting that this was the mechanism of protection. These data suggest that W1 and W2 chimpanzee monoclonal antibodies may serve as PA entry inhibitors for use in the emergency prophylaxis against and treatment of anthrax


title: Effects of feeding polyclonal antibody preparations on rumen fermentation patterns, performance, and carcass characteristics of feedlot steers

text: In a previous study, preparations of polyclonal antibodies (PAP) against Fusobacterium necrophorum (PAP-Fn) or Streptococcus bovis (PAP-Sb) were successful in decreasing ruminal counts of target bacteria and increasing ruminal pH in steers fed high-grain diets. The objective of this study was to evaluate the effects of feeding PAP-Fn or PAP-Sb on performance, carcass characteristics, and ruminal fermentation variables of feedlot steers. In Exp. 1, during 2 consecutive years, 226 or 192 Angus and Angus crossbred steers were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. When measured on a BW basis, steers fed only PAP-Sb had a greater G:F (P < 0.05) than those fed no PAP. Nevertheless, when both PAP were fed, feed efficiency was similar (P > 0.10) to steers fed no PAP or only PAP-Sb. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased (P < 0.05) dressing percentage. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased severity of liver abscess (P < 0.05). No differences (P > 0.10) were observed in any other carcass characteristics. In Exp. 2, sixteen ruminally cannulated Angus crossbred steers (BW = 665 ± 86 kg) were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. Feeding only PAP-Fn or PAP-Sb for 19 d decreased (P < 0.05) ruminal counts of S. bovis when compared with steers fed both or no PAP. The ruminal counts of F. necrophorum in steers fed PAP-Fn alone or in combination with PAP-Sb were decreased by 98% (P < 0.05) after 19 d, when compared with the counts in control steers. Mean daily ruminal pH was greater (P < 0.05) in steers fed both PAP when compared with feeding either or no PAP. Ruminal pH in the first 4 h after feeding was greater (P < 0.05) for steers receiving PAP-Fn alone or in combination with PAP-Sb. Steers receiving either PAP alone or in combination had less (P < 0.05) ruminal NH(3)-N concentrations in the first 4 h after feeding when compared with those of control steers. Polyclonal antibody preparations against S. bovis were effective in enhancing G:F of steers fed high-grain diets, but dressing percentage was decreased. Mechanisms of enhancement of G:F remain unknown but may be related to changes in ruminal counts of target bacteria and associated effects on ruminal fermentation products.


title: Acute upper airway infections: Childhood respiratory infections

text: Upper respiratory tract infections are common and important. Although rarely fatal, they are a source of significant morbidity and carry a considerable economic burden. Numerous therapies for the common cold have no effect on symptoms or outcome. Complications such as cough are not improved by over-the-counter preparations, while labelling cough alone as a symptom of asthma may result in unnecessary use of inhaled steroid treatment. Clinical presentation of sore throat does not accurately predict whether the infection is viral or bacterial, while throat culture and rapid antigen tests do not significantly change prescribing practice. Antibiotics have only a limited place in the management of recurrent sore throat due to group A β-haemolytic streptococcal infection. Routine use of antibiotics in upper respiratory infection enhances parent belief in their effectiveness and increases the likelihood of future consultation in primary care for minor self-limiting illness. Respiratory viruses play a major role in the aetiology of acute otitis media (AOM); prevention includes the use of influenza or RSV vaccination, in addition to reducing other risk factors such as early exposure to respiratory viruses in day-care settings and to environmental tobacco smoke. The use of ventilation tubes (grommets) in secretory otitis media (SOM) remains controversial with conflicting data on developmental outcome and quality of life in young children. New conjugate pneumococcal vaccines appear safe in young children and prevent 6–7% of clinically diagnosed AOM.


title: Individual plasmacytoid dendritic cells are major contributors to the production of multiple innate cytokines in an organ-specific manner during viral infection

text: Plasmacytoid dendritic cells (pDCs) are an important source of IFN-α/β in response to a variety of viruses in vivo, including murine cytomegalovirus (MCMV). However, the respective contributions of various infected organs, and within these of pDCs, conventional dendritic cells and other cells, to the systemic production of IFN-α/β or other innate cytokines during viral infections in vivo is largely unknown. Whether a specialization of pDC subsets in the production of different patterns of innate cytokines exists in vivo in response to a viral infection has not been investigated. Here, by analyzing for the first time directly ex vivo, at the single-cell level, the simultaneous production of up to three cytokines in pDCs isolated from MCMV-infected mice, we show that (i) pDCs are the quasi-exclusive source of IFN-α/β, IL-12 and tumor necrosis factor (TNF)-α, early during MCMV infection, in two immunocompetent mouse lines and with two viral strains, (ii) pDC activation for IFN-α/β production is organ specific and (iii) a significant proportion of pDCs simultaneously produce IFN-α/β, TNF-α and IL-12, although TNF-α and IFN-α/β appear more often co-expressed with one another than each of them with IL-12. Altogether, these results show a broad and non-redundant role of pDCs in early innate detection of, and defense against, viral infection. The data also show differences in the responsiveness of pDCs from different tissues and suggest distinct molecular requirements for pDC production of various cytokines. These observations must be taken into account when designing new antiviral vaccination strategies aimed at harnessing pDC responses.


title: Quantification of Viral Inactivation by Photochemical Treatment with Amotosalen and UV A Light, Using a Novel Polymerase Chain Reaction Inhibition Method with Preamplification

text: Background. In evaluating a photochemical treatment process for inactivating parvovirus B19, there lacked simple culture methods to measure infectivity. The recently developed enzyme‐linked immunospot (ELISpot) infectivity assay uses late‐stage erythropoietic progenitor cells and is labor intensive and time consuming. We evaluated a novel, efficient polymerase chain reaction (PCR) inhibition assay and examined correlations with reductions in infectivity. Methods. Contaminated plasma was treated with 150 μmol/L amotosalen and 3 J/cm(2) ultraviolet A light and then tested for DNA modification using conventional PCR inhibition and a novel preamplification approach. The novel assay subjected the samples to preamplification cycles using long‐template PCR, followed by quantitative PCR (QPCR) inhibition detection. Both approaches were tested for correlations with reductions in viral infectivity by comparing ELISpot assay results of identical samples. Results. The B19 preamplification inhibition assay showed detection ranges of 2–2.5 log and demonstrated quantitative correlation with up to a 5.8‐log reduction in viral infectivity in ELISpot results. Conventional PCR detected a >5 log reduction in amplification, correlated with a 4.4‐log reduction in viral infectivity. A range of 4‐log inhibition of hepatitis B virus DNA amplification was also achieved. Conclusions. The results demonstrated that a novel preamplification QPCR assay is a useful tool for predicting reductions in infectivity after photochemical treatment. This assay was extended to show utility in circumstances where practical in vitro assays are unavailable for the determination of the efficacy of pathogen inactivation.


Travellers' diarrhoea


title: Ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses

text: Understanding viral factors that promote cross-species transmission is important for evaluating the risk of zoonotic emergence. Weconstructed a database of viruses of domestic artiodactyls and examined the correlation between traits linked in the literature to cross-species transmission and the ability of viruses to infect humans. Among these traits-genomic material, genome segmentation, and replication without nuclear entry-the last is the strongest predictor of cross-species transmission. This finding highlights nuclear entry as a barrier to transmission and suggests that the ability to complete replication in the cytoplasm may prove to be a useful indicator of the threat of cross-species transmission.


title: Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene

text: The purpose of this study was to examine the induction of unscheduled DNA synthesis (UDS) by the potent hepatocarcinogen technical grade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) using the in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344 rats were treated by gavage and hepatocytes were isolated by liver perfusion and cultured with [ (3) H]thymidine. UDS was measured by quantitative autoradiography as net grains/nucleus (NG); ≥5 NG was considered positive. Controls consistently had − 3 to − 6 NG. A dose-related increase in UDS was observed 12 h after treatment, with 200 mg/kg tgDNT producing 26 NG. A 50-fold increase in the number of cells in S-phase was observed at 48 h after treatment. This increase in S-phase cells could be suppressed in the presence of 10–20 mM hydroxyurea (HU), while the same levels of HU did not affect the level of UDS at 12 h after treatment. 2, 4-DNT produced only a weak response, in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatment of female rats with tgDNT yielded only modest increases in UDS and DNA replication relative to males. These results are consistent with the carcinogenicity studies and indicate that tgDNT is a potent genotoxic agent, with 2, 6-DNT contributing the major portion of the effect.


title: AN “OUTBREAK” OF JUVENILE DIABETES MELLITUS: CONSIDERATION OF A VIRAL ETIOLOGY

text: Huff. J. C., J. C. Hierholzer (Respiratory Virology Section, Center for Disease Control. Atlanta, Ga. 30333) and W. A. Farris. An “outbreak” of juvenile diabetes mellitus: consideration of a viral etiology. Am J Epidemiol 100:277–287. 1974.—Nine of twelve cases of juvenile diabetes mellitus, representing an unusual geographic and temporal cluster, were investigated for evidence that a specific viral infection might be etiologically related to their occurrence. Eight diabetics had experienced recent “viral-like” illnesses, predominantly repiratory, but these illnesses bore no uniform temporal relation to their onsets of diabetes. Diabetics demonstrated no serologic evidence of a recent viral illness common to all. Elevated titers to only one virus, coxsackie B-3, were more prevalent in diabetics than in controls (33% vs. 6%), but geometric mean titers of diabetics to a panel of 26 common viral antigens were similar to those of controls. These data neither support nor negate the hypothesis that infection with a specific virus precipitates juvenile diabetes mellitus.


title: Prevention of Human Rotavirus-Induced Diarrhea in Gnotobiotic Piglets using Bovine Antibody

text: The efficacy of passively administered bovine antibody for preventing human rotavirus (HRV)-induced diarrhea was investigated using a gnotobiotic pig model. Cows were immunized with inactivated HRV serotypes 1 (Wa) and 2 (S2) and simian rotavirus serotype 3 (SAIl), and immune colostrum and milk were collected. Antibody concentrates derived from these materials were fed to germ-free piglets that were subsequently inoculated with HRV Wa. Both viral shedding and diarrhea were effectively reduced or eliminated in a dose-dependent manner as a result of HRV immune antibody feeding. A quantitative virus-neutralizing (VN) antibody method permitted assessment of the functional antibody dose required to achieve a 50% reduction of disease (PD(50)) . PD(50) dose levels of 15.8 and 19.5 × 10(6) VN antibody units were determined for inhibition of diarrhea and viral shedding, respectively. Studies reported here provide new information on the quantitative relationship between protective antibody dose and diarrheal disease response.


title: Accurate prediction of human essential genes using only nucleotide composition and association information

text: MOTIVATION: Previously constructed classifiers in predicting eukaryotic essential genes integrated a variety of features including experimental ones. If we can obtain satisfactory prediction using only nucleotide (sequence) information, it would be more promising. Three groups recently identified essential genes in human cancer cell lines using wet experiments and it provided wonderful opportunity to accomplish our idea. Here we improved the Z curve method into the λ-interval form to denote nucleotide composition and association information and used it to construct the SVM classifying model. RESULTS: Our model accurately predicted human gene essentiality with an AUC higher than 0.88 both for 5-fold cross-validation and jackknife tests. These results demonstrated that the essentiality of human genes could be reliably reflected by only sequence information. We re-predicted the negative dataset by our Pheg server and 118 genes were additionally predicted as essential. Among them, 20 were found to be homologues in mouse essential genes, indicating that some of the 118 genes were indeed essential, however previous experiments overlooked them. As the first available server, Pheg could predict essentiality for anonymous gene sequences of human. It is also hoped the λ-interval Z curve method could be effectively extended to classification issues of other DNA elements. AVAILABILITY AND IMPLEMENTATION: http://cefg.uestc.edu.cn/Pheg SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


title: Existence and characterization of allelic variants of Sao, a newly identified surface protein from Streptococcus suis

text: Surface antigen one (Sao) is a newly identified protein from the major zoonotic pathogen, Streptococcus suis. In search of functional proteins related to the pathogenesis of Chinese S. suis 2 (SS2), unexpectedly, a variant of Sao protein was obtained. To test its prevalence in S. suis, PCR assay was adopted to address the coding genes systematically. It was found that there are three allelic variants of sao gene, namely sao-S, sao-M, and sao-L based on the different lengths of the genes (∼1.5, ∼1.7, and ∼2.0 kb, respectively). These differences were determined to be caused by heterogeneity within the number of C-terminal repeat sequences (R), which had been seen as a pathogenicity-related domain in the plant pathogen, Xanthomonas oryzae. Two variants (sao-M and sao-L) were only found in SS2. All three variant proteins were prepared in vitro and their biochemical and biophysical properties were characterized. A soluble form of Sao-M protein was then used as a capture antigen to develop an enzyme-linked immunosorbent assay method to detect antibodies against SS2 in convalescent pig sera. Taken together, the results exhibit the properties of Sao proteins and provide an efficient Sao-M-based method for monitoring SS2 infection.