Study of the underlying mechanisms and consequences of pathogenicity differences between two in vitro selected G1-H9N2 clones originating from a single isolate.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines.

Investigating type I feline coronaviruses (FCoVs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. This has been a perennial challenge as type I FCoV strains do not easily adapt to cell culture. Here we characterize replication kinetics and plaque formation of a model type I strain FIPV Black in Fcwf-4 cells established at Cornell University (Fcwf-4 CU). We determined that maximum virus titers (>107 pfu/mL) were recoverable from infected Fcwf-4 CU cell-free supernatant at 20 h post-infection. Type I FIPV Black and both biotypes of type II FCoV formed uniform and enumerable plaques on Fcwf-4 CU cells. Therefore, these cells were employable in a standardized plaque assay. Finally, we determined that the Fcwf-4 CU cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type I interferon than were Fcwf-4 cells purchased from ATCC.

Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection.

Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

In vitro cytotoxicity and antiviral efficacy against feline herpesvirus type 1 of famciclovir and its metabolites.

OBJECTIVES: To assess in vitro the antiviral efficacy against feline herpesvirus (FHV-1) and cytotoxicity for cultured feline cells of famciclovir and its metabolites, BRL 42359 and penciclovir. To investigate the effect of timing of penciclovir application on in vitro antiviral activity. PROCEDURES: Plaque reduction assays were used to estimate antiviral efficacy of all compounds and the effect of penciclovir exposure before or after exposure to a FHV-1 field isolate. Cytotoxicity was evaluated by assessing cell morphology and viable cell number for 72 h following exposure to each compound. RESULTS: The penciclovir concentration that inhibited FHV-1-induced plaque formation by 50% (IC50 ) was 0.86 µg/mL (3.4 µm). Famciclovir and BRL 42359 had no antiviral effect against FHV-1 at any concentration assessed. Antiviral activity was significantly enhanced when cells were exposed to 4 µm penciclovir (approximate IC50 ) for 1 h but not for 24 h before viral adsorption. Delaying exposure of cells to penciclovir for 1, 2, or 4 h after viral adsorption significantly enhanced antiviral activity. Relative to untreated control wells, >88% of cells remained viable when exposed to famciclovir (100 µm), BRL 42359 (1.06 mm), or penciclovir (40 µm) for 72 h. No morphologic evidence of cytotoxicity was noted. CONCLUSIONS: Penciclovir demonstrates potent antiviral activity against FHV-1 and may be effective at lower tissue, tear, and plasma concentrations than previously targeted. The duration of in vitro antiviral effect of penciclovir suggests that frequent famciclovir administration may be necessary in vivo. Famciclovir and BRL 42359 showed no signs of in vitro cytotoxicity.

A previously unknown mechanism in viral pathogenesis leading to effective new vaccines and post-exposure immune treatments of viral infections.

Investigations with the rabies virus identified a previously unknown mechanism of viral pathogenesis. After ultracentrifugation of a suspension of rabid dog brain and rabies vaccine strains, the supernatant was found to contain active components, as evaluated in an in vitro plaque test. The unexpected detection of active components in non-sedimented material prompted further research and the finding that these components, and not the complete virus, were responsible for paralysis and death. Vaccination of cattle with existing rabies vaccines showed that even low titres of antibodies against these components provided protection after challenge. In a control group of non-vaccinated cows, cows that had low titres of these antibodies survived rabies challenge. These low titres could not be detected with the usual serum neutralization test in mice but only with a plaque reduction test, which is more sensitive. A hyperimmune serum raised in rabbits against active components isolated from the brain of a rabid dog was injected intracerebrally into mice that had been previously injected intramuscularly with a rabies virus. This delayed post-exposure treatment was still effective against advanced rabies (virus already in the brain, but not yet paralytic symptoms). This promising finding makes the development of a new inactivated rabies vaccine possible and opens the way for post-exposure treatment for humans, particularly in developing countries where rabies is still a major problem. The role of these active components in other viral diseases, such as human immunodeficiency virus, should be investigated.

Dispersal and survival of Flavobacterium psychrophilum phages in vivo in rainbow trout and in vitro under laboratory conditions: implications for their use in phage therapy.

Attention has been drawn to phage therapy as an alternative approach for controlling pathogenic bacteria such as Flavobacterium psychrophilum in salmonid aquaculture, which can give rise to high mortalities, especially in rainbow trout fry. Recently, phages have been isolated with a broad host range and a strong lytic potential against pathogenic F. psychrophilum under experimental conditions. However, little is known about the fate of phages at environmental conditions. Here, we quantified the dispersal and fate of F. psychrophilum phages and hosts in rainbow trout fry after intraperitoneal injection. Both phages and bacteria were isolated from the fish organs for up to 10 days after injection, and coinjection with both bacteria and phages resulted in a longer persistence of the phage in the fish organs, than when the fish had been injected with the phages only. The occurrence of both phage and bacterium was most prevalent in the kidney and spleen, with only minor occurrence in the brain. The experiment showed that injected phages were rapidly spread in the internal organs of the fish, also in the absence of bacteria. Parallel examination of the regulation of bacteriophage infectivity in controlled laboratory experiments at various environmental conditions showed that pH had only minor effects on long-term (3 months) phage infectivity within a pH range of 4.5 to 7.5, whereas phage infectivity was immediately lost at pH 3. In the absence of host cells, phage infectivity decreased by a factor of 10,000 over 55 days in untreated pond water, while the sterilization and removal of particles caused a 100-fold increase in phage survival relative to the control. In addition, F. psychrophilum-specific phages maintained their infectivity for ∼2 months in glycerol at -80°C, whereas infectivity decreased by a factor 10 when kept in a buffer at 20°C. Only a very small degradation in infectivity was seen when bacteriophages were added and dried on fish feed pellets. Together, these results indicate that application of bacteriophages represents a promising approach for the control of F. psychrophilum infections in trout and suggest fish feed as a potential delivery method.

Comparative susceptibility among three stocks of yellow perch, Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes.

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV-IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate-to-severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28-day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.

Review of diagnostic plaque reduction neutralization tests for flavivirus infection.

Flavivirus infections (including Japanese encephalitis, West Nile encephalitis and dengue fever/severe dengue) present a worldwide public health problem. Recent climate change may affect the geographical distribution of the arthropod vectors for these viruses and so the risk of flavivirus epidemics may increase. Many methods have been developed for the serological diagnosis of flavivirus infections, such as haemagglutination inhibition assay, enzyme-linked immunosorbent assay, and immunofluorescence in staining. However, the specificity of these assays varies. The plaque reduction neutralizing test (PRNT) using live viruses is currently the 'gold standard' for the differential serodiagnosis of flaviviruses. The specificity of results obtained with PRNT is better than that for other protocols and many laboratories apply the PRNT protocol to the differential serodiagnosis of flaviviruses. Here, recent refinements to the PRNT protocols with genetically modified recombinant viruses or reporter-harbouring virus-like particles are reviewed. Further, the problems associated with the differential serodiagnosis of flaviviruses using PRNT are discussed.

The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding.

Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to α4ß1 or α4ß7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that α4ß1 and α4ß7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.

Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus.

A faster semi-automated 96-well microtiter plate assay to determine viral infectivity titers, or viral focal units (vfu), of equine infectious anemia virus (EIAV) stocks is described. Optimization of the existing method modernizes a classic virological technique for viral titer determination by quantitating EIAV in experimentally infected cells via a cell-based ELISA. To allow for automation, multiple parameters of the current assay procedures were modified resulting in an assay that required only one quarter the original amount of virus and/or serum for infectivity or neutralization assays, respectively. Equivalent reductions in the required volumes of tissue culture, cell processing, and protein detection reagents were also achieved. Additionally, the new assay decreased the time required from start to finish from 10 days to 6 days (viral titer) or 7 days (viral neutralization), while increasing the number of samples that can be processed concurrently by transition to a 96-well microtiter plate format and by automated counting.

Swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment.

In order to assess the dynamics of influenza virus infection in pigs, serological and virological follow-ups were conducted in two whole batches of pigs from two different farms (F1 and F2), from 3 weeks of age until market age. Anti-swine influenza virus (SIV) antibodies (measured by ELISA and hemagglutination inhibition) and nasal virus shedding (measured by RRT-PCR and isolation in embryonated chicken eggs and MDCK cells) were carried out periodically. SIV isolates were subtyped and hemagglutinin and neuraminidase genes were partially sequenced and analyzed phylogenetically. In F1, four waves of viral circulation were detected, and globally, 62/121 pigs (51.2%) were positive by RRT-PCR at least once. All F1 isolates corresponded to H1N1 subtype although hemagglutination inhibition results also revealed the presence of antibodies against H3N2. The first viral wave took place in the presence of colostral-derived antibodies. Nine pigs were positive in two non-consecutive sampling weeks, with two of the animals being positive with the same isolate. Phylogenetic analyses showed that different H1N1 variants circulated in that farm. In F2, only one isolate, H1N2, was detected and all infections were concentrated in a very short period of time, as assumed for a classic influenza outbreak. These findings led us to propose that influenza virus infection in pigs might present different patterns, from an epidemic outbreak to an endemic form with different waves of infections with a lower incidence.

Loss of retinoblastoma protein, but not p53, is associated with the presence of papillomaviral DNA in feline viral plaques, Bowenoid in situ carcinomas, and squamous cell carcinomas.

Although papillomaviral (PV) DNA is frequently present in feline cutaneous squamous cell carcinomas (SCCs), a causative association cannot be proven. Oncogenic human PVs cause neoplastic transformation by inhibiting retinoblastoma (pRb) and p53 activity. Therefore, absence of pRb and p53 immunostaining, along with increased p16 immunostaining, indicates a PV cause in some human SCCs. If PVs cause cutaneous feline SCCs, it was hypothesized that a similar immunohistochemistry profile, along with PV DNA, would be detectable. This was investigated using 5 feline viral plaques, 10 Bowenoid in situ carcinomas, 19 SCCs from ultraviolet-exposed (UV-exposed) skin, and 11 SCCs from UV-protected skin. Papillomaviral DNA was amplified by polymerase chain reaction from 30 of 45 lesions. Reduced pRb immunostaining was present in 26 of 45; increased p16 immunostaining was in 30; and p53 immunostaining was in 19. Both reduced pRb immunostaining and increased p16 immunostaining were more frequent in lesions containing PV DNA. In contrast, no association was observed between p53 immunostaining and the presence of PV DNA. SCCs from UV-protected skin more frequently contained PV DNA, reduced pRb, and increased p16 than UV-exposed SCCs. UV exposure was not associated with p53 immunostaining within the SCCs. These results suggest that feline PVs alter cell regulation by degrading pRb. Unlike oncogenic human PVs, there was no evidence that feline PVs degrade p53. These results provide further evidence that PVs may cause feline cutaneous SCCs, especially those in UV-protected skin, and they suggest a possible mechanism of this oncogenic action.

Antibody response and virus shedding of chickens inoculated with left end deleted fowl adenovirus 9-based recombinant viruses.

The nonpathogenic fowl adenoviruses (FAdVs) are suitable recombinant virus vectors. Two different replication-competent FAdV-9-based recombinant viruses carrying the enhanced green fluorescent protein (EGFP) gene within a nonessential DNA sequence at the left end genomic region were tested in chickens to study the antibody response by enzyme-linked immunosorbent assay to both the foreign proteins, EGFP and FAdV-9, and virus shedding through the feces. All inoculations were done intramuscularly: groups 1 and 2 with the recombinant viruses and group 3 with the wild-type FAdV-9 virus. Group 4 was mock inoculated. Sentinel birds also were included in groups 1-3 to study virus transmission. Boosting inoculations were done in all groups at 2, 3, and 4 wk after the first inoculation. Antibodies to EGFP were detected at 3-7 wk postinoculation in groups 1 and 2 only. Antibody response to FAdV-9 in groups 1-3 did not differ significantly (P > 0.06). Virus was not detected in the feces of chickens in groups 1 and 2, including the sentinel birds, but virus was present in the feces of chickens in group 3, including the sentinel birds. These results further supported our previous findings regarding the suitability of the nonessential region at the left end of the viral genome as an insertion site for foreign genes and its importance in in vivo replication. In this work, we demonstrated the potential of FAdV-9-based recombinant viruses as vaccines for poultry.

Equine alphaherpesviruses (EHV-1 and EHV-4) differ in their efficiency to infect mononuclear cells during early steps of infection in nasal mucosal explants.

Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.

A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa.

Several alphaherpesviruses breach the basement membrane during mucosal invasion. In the present study, the role of proteases in this process was examined. The serine protease-specific inhibitor AEBSF inhibited penetration of the basement membrane by the porcine alphaherpesvirus pseudorabies virus (PRV) by 88.1% without affecting lateral spread. Inhibitors of aspartic-, cysteine-, and metalloproteases did not inhibit viral penetration of the basement membrane. Further analysis using the Soybean Type I-S trypsin inhibitor for the serine protease subcategory of trypsin-like serine proteases resulted in a 96.9% reduction in plaque depth underneath the basement membrane. These data reveal a role of a trypsin-like serine protease in PRV penetration of the basement membrane.

Comparative analysis of replication characteristics of BoHV-1 subtypes in bovine respiratory and genital mucosa explants: a phylogenetic enlightenment.

In general, members of the Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as a preferential site for primary replication. Bovine herpesvirus type 1 (BoHV-1) may replicate at both sites and cause two major clinical entities designated as infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB) in cattle. It has been hypothesized that subtype 1.1 invades preferentially the upper respiratory mucosa whereas subtype 1.2 favors replication at the peripheral genital tract. However, some studies are in contrast with this hypothesis. A thorough study of primary replication at both mucosae could elucidate whether or not different BoHV-1 subtypes show differences in mucosa tropism. We established bovine respiratory and genital organ cultures with emphasis on maintenance of tissue morphology and viability during in vitro culture. In a next step, bovine respiratory and genital mucosa explants of the same animals were inoculated with several BoHV-1 subtypes. A quantitative analysis of viral invasion in the mucosa was performed at 0 h, 24 h, 48 h and 72 h post inoculation (pi) by measuring plaque latitude and penetration depth underneath the basement membrane. All BoHV-1 subtypes exhibited a more profound invasion capacity in respiratory tissue compared to that in genital tissue at 24 h pi. However, at 24 h pi plaque latitude was found to be larger in genital tissue compared to respiratory tissue and this for all subtypes. These similar findings among the different subtypes take the edge off the belief of the existence of specific mucosa tropisms of different BoHV-1 subtypes.

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9.

Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.

Ovine rotavirus strain LLR-85-based bovine rotavirus candidate vaccines: construction, characterization and immunogenicity evaluation.

Group A bovine rotaviruses (BRVs) are the most important cause of diarrheal diseases in neonatal calves and cause significant morbidity and mortality in the young animals, and epidemiologic surveillance of bovine rotavirus G genotypes conducted in various cattle populations throughout the world has shown that approximately 90% of the bovine rotavirus isolates belong to G6 and G10. Based on the modified Jennerian approach to immunization, we constructed and characterized a reassortant rotavirus stain, which bears a single bovine rotavirus VP7 gene encoding G genotype 6 specificity while the remaining 10 genes are derived from the ovine attenuated rotavirus LLR-85. The reassortant rotavirus strain, named as R191, and its parental virus strain LLR-85 were combined as bivalent vaccine candidates to inoculate the colostrums-deprived neonatal calves for evaluation of the immunogenicity. The calves were orally inoculated with the reassortant R191 (group 1), the parental rotavirus LLR-85 (group 2), or combined the R191 and LLR-85 (group 3), and serum specimens were detected to determine the immune response of IgG and IgA antibodies. Results showed that seroconversion to positivity for IgG and IgA antibodies occurred at postinoculation day (PID) 10 in all of the inoculated calves, and the highest titers of the serum IgG (range 1:800 to 1:6400) and IgA (range 1:800 to 1:3200) antibodies were obtained at PID 21 for all calves. Meanwhile, virus shedding was detected after inoculation, showing that the inoculated virus was positive in 2 of 77 fecal specimens (2.6%) collected from the inoculated calves during the first 7 days of oral inoculation with the rotavirus vaccine candidates. The results suggested that the rotavirus strains R191 and LLR-85 are promising bivalent vaccine candidates for the prevention of bovine G6 and G10 rotavirus infection.

Cross-reactivity of Japanese encephalitis virus-vaccinated horse sera in serodiagnosis of West Nile virus.

Flavivirus-infected sera are known to show cross-reactions in serodiagnoses of heterologous flavivirus infections. Japanese encephalitis virus (JEV) is endemic in Asia and, in Japan, many horses are vaccinated against JEV. However, the cross-reactivity level of JEV-vaccinated horse sera in the serodiagnosis of West Nile virus (WNV) has not been clarified. The antibody cross-reactivity of JEV-vaccinated horse sera in WNV serological tests, such as the plaque reduction neutralization test (PRNT), IgG indirect ELISA (IgG-ELISA) and hemagglutination inhibition (HI) test, was examined. All JEV-vaccinated horse sera were positive for JEV antibodies with JEV PRNT at both 90% and 50% plaque reductions. In WNV PRNT, 16.7% of the horses were positive at 90% plaque reduction, and 50% of the horses were positive at 50% plaque reduction. All the JEV-vaccinated horse sera showed positive-to-negative (P/N) ratios of over 2.0 with JEV IgG-ELISA, and half of them had P/N ratios of over 2.0 with WNV IgG-ELISA. There was little difference between the JEV HI and WNV HI titers in individual horses. These results indicate that in serosurveillance of WNV, JEV-vaccinated horses can produce false-positive results in WNV IgG-ELISA, HI and PRNT.