Yoshihiro Sakoda

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Characterization of H7N9 influenza A viruses isolated from humans.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

Scrub Typhus Serologic Testing with the Indirect Immunofluorescence Method as a Diagnostic Gold Standard: A Lack of Consensus Leads to a Lot of Confusion

A review was performed to determine the evidence base for scrub typhus indirect immunofluorescence assay (IFA) methodologies and the criteria for positive results. This review included a total of 109 publications, which comprised 123 eligible studies for analysis (14 publications included 2 substudies). There was considerable underreporting of the IFA methodology and seropositivity criteria used, with most studies using a defined cutoff titer rather than an increase in the titer in paired samples. The choice of positivity cutoff titer varied by country and purpose of the IFA test. This variation limits the comparability of seroprevalence rates between studies and, more seriously, raises questions about the appropriateness of the cutoffs for positive IFA results chosen for diagnosis of acute scrub typhus infection. We suggest that the diagnosis of scrub typhus using IFA should be based on a > or =4-fold increase in the titer in paired serum samples and should only be based on a single sample titer when there is an adequate local evidence base.

Co-infection of Staphylococcus aureus or Haemophilus paragallinarum exacerbates H9N2 influenza A virus infection in chickens

SummaryH9N2 influenza viruses are frequently isolated from chicken meat and bone marrow imported from China to Japan since 2001. These isolates were experimentally inoculated into specific pathogen-free chickens intranasally. Viruses were recovered from the meat and bone marrow of birds showing no overt signs. On the other hand, chickens co-infected with H9N2 virus and either Staphylococcus aureus or Haemophilus paragallinarum showed clinical signs severer than those shown by birds infected only with the virus alone or each of the bacteria alone. In addition, H9N2 viruses were more efficiently recovered from the chickens co-infected with S. aureus or H. paragallinarum than those from the birds infected with only the virus. The present results indicate that co-infection of H9N2 influenza virus with S. aureus or H. paragallinarum enhances the replication of the virus in chickens, resulting in exacerbation of the H9N2 virus infection.

Pathogenicity of a highly pathogenic avian influenza virus, A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals

Summary.Outbreaks of highly pathogenic avian influenza (HPAI) have been occurring in domestic poultry in Asia since 1996. In the beginning of 2004, HPAI outbreaks were caused by H5N1 virus in two farms and a group of pet chickens in different areas of Japan. In the present study, the pathogenicity of A/chicken/Yamaguchi/7/04 (H5N1), which had been isolated from a dead chicken during the first outbreak in Japan, was assessed in chickens, quails, budgerigars, ducklings, mice, and miniature pigs by experimental infection. The virus was highly pathogenic to all the birds tested. Mice were susceptible to infection with a low mortality rate and miniature pigs were resistant to infection with the virus.

Pathogenicity of H5 influenza viruses for ducks

Summary.Four H5N1 highly pathogenic avian influenza (HPAI) viruses and an avirulent reassortant H5N1 virus were tested for their pathogenicity in domestic ducks. A/chicken/Yamaguchi/7/04 (H5N1) (Ck/Yamaguchi/04) isolated from a dead bird during the HPAI outbreak in Japan and A/duck/Yokohama/aq-10/03 (H5N1) (Dk/Yokohama/03) isolated from duck meat at a quarantine inspection for importation from China replicated in multiple organs including the brain of ducks. The ducks infected with Ck/Yamaguchi/04 did not show any clinical signs, while those infected with Dk/Yokohama/03 showed neurological signs. The ducks infected either with A/Hong Kong/483/97 (H5N1) or A/tern/South Africa/61 (H5N3), or with an avirulent H5N1 reassortant, did not show any clinical signs. Virus-specific antibodies were detected in the sera of the ducks infected with each of the five strains tested, indicating that all of the viral strains infected and replicated in the birds. Dk/Yokohama/03 grew in multiple organs more rapidly than did Ck/Yamaguchi/04. Considerable titers of virus were detected in the brain of the ducks infected with Dk/Yokohama/03 and these birds showed neurological signs. The present results demonstrate that the pathogenicity of influenza viruses for ducks does not correlate with that for chickens and that replication of the virus in the brain is critical for ducks to show neurological signs.

Characterization of H5N1 highly pathogenic avian influenza virus strains isolated from migratory waterfowl in Mongolia on the way back from the southern Asia to their northern territory

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.

Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010–2011 winter season in Japan

H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.

Genomic and serological diversity of bovine viral diarrhea virus in Japan.

Summary. Genomic properties of 62 field isolates of bovine viral diarrhea virus (BVDV) collected from 1974 to 1999 in Japan were investigated. The 5′ untranslated region (UTR) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and the 244 to 247 base nucleotide sequences were determined. Serological properties were also characterized by the cross-neutralization test using antisera against the representative strain of the classified genotype. Using phylogenetic tree analysis, BVDV 1 was subdivided into two major clusters, BVDV-1a (29 isolates) and BVDV-1b (27 isolates). In group 1a, 3 differed from the other viruses, and were classified in a branch assigned as 1a′. However, 4 isolates (So CP/75, 190 CP, 190 NCP and KS86-1-NCP) could not be assigned to group 1a or 1b. In comparison with the published sequence data, KS86-1-NCP, 190 CP and 190 NCP were similar to the Southern Africa isolates that have recently been proposed as BVDV 1c. The 5′ UTR sequence of So CP/75 was unique among those of BVDV 1, suggesting that the isolate should be classified into a new genotype. Only 2 out of 62 isolates collected in 1989 and 1990 were identified as BVDV 2. The results of the cross-neutralization test strongly supported these data, suggesting a close correlation between the 5′ UTR sequence and the antigenicity of BVDV.