Yuta Kanai

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30–110 days old, and 17 developed viremia at 40–100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real‐time reverse transcriptase‐polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 106.0 copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans. J. Med. Virol. 82:69–76, 2010.

Hepatitis E virus in Norway rats (Rattus norvegicus) captured around a pig farm

BackgroundHepatitis E virus (HEV) transmitted via the oral route through the consumption of contaminated water or uncooked or undercooked contaminated meat has been implicated in major outbreaks. Rats may play a critical role in HEV outbreaks, considering their negative effects on environmental hygiene and food sanitation. Although the serological evidence of HEV infection in wild rodents has been reported worldwide, the infectivity and propagation of HEV in wild rats remain unknown. To investigate if rats are a possible carrier of HEV, we studied wild Norway rats (Rattus norvegicus) that were caught near a pig farm, where HEV was prevalent among the pigs.MethodsWe examined 56 Norway rats for HEV. RNA from internal organs was examined for RT-PCR and positive samples were sequenced. Positive tissue samples were incubated with A549 cell line to isolate HEV. Anti-HEV antibodies were detected by ELISA.ResultsSixteen rats were seropositive, and the HEV RNA was detected in 10 of the 56 rats. Sequencing of the partial ORF1 gene from 7 samples resulted in partially sequenced HEV, belonging to genotype 3, which was genetically identical to the HEV prevalent in the swine from the source farm. The infectious HEVs were isolated from the Norway rats by using the human A549 cell line.ConclusionsThere was a relatively high prevalence (17.9%) of the HEV genome in wild Norway rats. The virus was mainly detected in the liver and spleen. The results indicate that these animals might be possible carrier of swine HEV in endemic regions. The HEV contamination risk due to rats needs to be examined in human habitats.

Imported Case of Acute Respiratory Tract Infection Associated with a Member of Species Nelson Bay Orthoreovirus

A Japanese man suffered from acute respiratory tract infection after returning to Japan from Bali, Indonesia in 2007. Miyazaki-Bali/2007, a strain of the species of Nelson Bay orthoreovirus, was isolated from the patients throat swab using Vero cells, in which syncytium formation was observed. This is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of Nelson Bay orthoreovirus. Given the possibility that all of the patients were infected in Malaysia and Indonesia, prospective surveillance on orthoreovirus infections should be carried out in Southeast Asia. Furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of Nelson Bay orthoreovirus would seem to be low.

Recovery of African horse sickness virus from synthetic RNA

African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe and effective vaccine is sought urgently. As part of a programme to generate a designed highly attenuated vaccine, we report here the recovery of AHSV from a complete set of RNA transcripts synthesized in vitro from cDNA clones. We have demonstrated the generation of mutant and reassortant AHSV genomes, their recovery, stable passage, and characterization. Our findings provide a new approach to investigate AHSV replication, to design AHSV vaccines and to aid diagnosis.

AMANTADINE- AND OSELTAMIVIR-RESISTANT VARIANTS OF INFLUENZA A VIRUSES IN THAILAND

Amantadine and oseltamivir are used to treat influenza A virus infections; however, resistance to these drugs has been widely reported throughout the world. In this study, the frequency and genetic characteristics of the drug-resistant influenza A viruses that circulated in Thailand from 2006 to 2008 were investigated. The nucleotide sequences of the NA and M2 genes were elucidated in order to identify mutations that confer oseltamivir- and amantadine-resistant phenotypes, respectively. A total of 66 influenza A viruses including 44 H1N1 and 22 H3N2 subtypes isolated in Bangkok and 13 provinces of Thailand from 2006 to 2008 were analyzed. Our results demonstrated that seven out of 32 (22%) of the H1N1 viruses isolated in 2006 in Thailand carried the amino acid S31N substitution, which confers amantadine-resistance, although no isolates in 2007 or 2008 possessed the mutation. In the cases of oseltamivir-resistance, four of 10 (40%) of the H1N1 viruses isolated in 2008 were predicted to be resistant to the drug, although none of the 34 viruses isolated in 2006 or 2007 were predicted to be resistant. Surprisingly, all 9 H3N2 viruses isolated in 2008 appeared to be resistant to the amantadine and none were resistant in 2006 or 2007. Phylogenetic analysis based on the HA, M, and NA genes demonstrated that the amantadine-resistant H1N1 isolates had been produced by genetic reassortment. All of the amantadine-resistant H3N2 viruses were clustered in one of these three genes and possessed double mutations of S193F and D225N in the HA gene.

Entirely plasmid-based reverse genetics system for rotaviruses.

Significance Rotaviruses (RVs) are a group of viruses that cause severe gastroenteritis in infants and young children. Until now, no strategy has been developed to generate infectious RVs entirely from cloned cDNAs. The absence of a reliable reverse genetics platform has been a major roadblock in the RV field, precluding numerous studies of RV replication and pathogenesis and hampering efforts to develop the next generation of RV vaccines. Here, we developed a plasmid-based reverse genetics system that is free from helper viruses and independent of any selection for RV. This technology will accelerate studies of RV pathobiology, allow rational design of RV vaccines, and yield RVs suitable for screening small molecules as potential antivirals. Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Immunogenicity of recombinant VP2 proteins of all nine serotypes of African horse sickness virus

African horse sickness (AHS) is an equine disease with a mortality of up to 90% for susceptible horses. The causative agent AHS virus (AHSV) is transmitted by species of Culicoides. AHSV serogroup within the genus Orbivirus of the Reoviridae family consists of nine serotypes that show no or very limited cross-neutralization. Of the seven structural proteins (VP1-VP7) of AHSV, VP2 is the serotype specific protein, and the major target for neutralizing antibodies. In this report, recombinant VP2 proteins of all nine serotypes were expressed individually by the baculovirus expression system and the immunogenicity of each was studied by immunization of guinea pigs with single VP2 as well as with cocktails of VP2 proteins. Homologous neutralizing antibodies measured by 50% plaque reduction assay showed varying degrees (from 37 to 1365) of titers for different VP2 proteins. A low cross-neutralizing antibody titer was found for genetically related AHSV serotypes. Immunization with VP2 cocktails containing equal amounts of each of the VP2 proteins also triggered neutralizing antibodies albeit to lower titers (4-117) to each of the serotypes in the cocktail. This study is a first step to develop a VP2 subunit vaccine for AHS and our results indicate that VP2 subunit vaccines are feasible individually or in a multi-serotype cocktail.

Reliability of a Newly-Developed Immunochromatography Diagnostic Kit for Pandemic Influenza A/H1N1pdm Virus: Implications for Drug Administration

Background For the diagnosis of seasonal influenza, clinicians rely on point-of-care testing (POCT) using commercially available kits developed against seasonal influenza viruses. However, POCT has not yet been established for the diagnosis of pandemic influenza A virus (H1N1pdm) infection due to the low sensitivity of the existing kits for H1N1pdm. Methodology/Principal Findings An immunochromatography (IC) test kit was developed based on a monoclonal antibody against H1N1pdm, which does not cross-react with seasonal influenza A or B viruses. The efficacy of this kit (PDM-IC kit) for the diagnosis of H1N1pdm infection was compared with that of an existing kit for the detection of seasonal influenza viruses (SEA-IC kit). Nasal swabs (n = 542) were obtained from patients with flu-like syndrome at 13 clinics in Osaka, Japan during the winter of 2010/2011. Among the 542 samples, randomly selected 332 were further evaluated for viral presence by reverse transcriptase polymerase chain reaction (RT-PCR). The PDM-IC kit versus the SEA-IC kit showed higher sensitivity to and specificity for H1N1pdm, despite several inconsistencies between the two kits or between the kits and RT-PCR. Consequently, greater numbers of false-negative and false-positive cases were documented when the SEA-IC kit was employed. Significant correlation coefficients for sensitivity, specificity, and negative prediction values between the two kits were observed at individual clinics, indicating that the results could be affected by clinic-related techniques for sampling and kit handling. Importantly, many patients (especially influenza-negative cases) were prescribed anti-influenza drugs that were incongruous with their condition, largely due to physician preference for patient responses to questionnaires and patient symptomology, as opposed to actual viral presence. Conclusions/Significance Concomitant use of SEA-IC and PDM-IC kits increased the likelihood of correct influenza diagnosis. Increasing the credibility of POCT is anticipated to decrease the inappropriate dispensing of anti-influenza drugs, thereby minimizing the emergence of drug-resistant H1N1pdm strains.

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was >or=4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than approximately 15 nm in diameter).

Fermentation metabolites from Lactobacillus gasseri and Propionibacterium freudenreichii exert bacteriocidal effects in mice.

Lactobacillus gasseri OLL 2716 promotes the elimination of Helicobacter pylori and is utilized in yogurts that are specifically labeled as health foods. On the other hand, milk whey fermented by Propionibacterium freudenreichii ET-3, which increases the numbers of Bifidobacterium, is effective for intestinal disorders. We previously demonstrated that oral administration of L. gasseri and P. freudenreichii fermentation metabolites (LP-FM) improved calf intestinal microflora and reduced the incidence of diarrhea. However, the detailed immunological mechanisms responsible for these effects remain to be fully understood. In this study, we investigated whether LP-FM stimulates the innate immune response and promotes the elimination of Listeria monocytogenes in mice. The C57BL/6 female mice that were treated with LP-FM or L. gasseri fermentation metabolites alone for 4 weeks had more peripheral white blood cells than the untreated control mice. In particular, LP-FM-treated mice had higher CD4- and CD8-positive T-cell counts. The levels of reactive oxygen and nitrogen species produced by peritoneal macrophages were also higher in LP-FM-treated mice. Furthermore, LP-FM-treated mice that were infected with L. monocytogenes exhibited significant enhancement of the elimination of Listeria from the spleen and the liver in comparison with untreated control mice infected with Listeria. The activation of innate immunity by LP-FM was increased by the combination of fermentation metabolites from P. freudenreichii. These results suggest that LP-FM, which contains metabolites from L. gasseri and P. freudenreichii, stimulates the function of the innate immune system, thereby significantly promoting the elimination of L. monocytogenes in mice.