Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

First Identification of Human Adenovirus 57 (HAdV-57) in Japan.

Neutralization tests have been routinely used for the identification of human adenovirus C species (HAdV-C) in Japan until 2007. The aim of this study was to clarify the serological cross-reactivity of antiserum that has been used exclusively in Japan and to describe the first identification of HAdV type 57 (HAdV-57) in Japan. Anti-HAdV serum to HAdV-1, 2, 5, and 6 was quantitatively evaluated for cross-reactivity to the HAdV-57 isolates. Anti-HAdV-6 serum neutralized HAdV-57 with a concentration that was 32 to 64-fold higher than what was necessary to neutralize homologous HAdV-6. HAdV-1, 2, and 5 strains were not neutralized by anti HAdV-6 serum. Furthermore, 28 HAdV-6 strains isolated from 6,476 clinical samples were re-examined for HAdVs detected in the Shimane Prefecture of Japan from 2005 to 2014. These 28 strains were re-examined by PCR-sequencing techniques using the penton, hexon, and fiber regions. 3 isolates were determined to be HAdV-57. These data show that HAdV-57 had already invaded Japan as early as 2005, and that HAdV-57 strains were misidentified as HAdV-6.

Amino acid substitution at position 95 in rabies virus matrix protein affects viral pathogenicity.

We previously reported that rabies virus strain CE(NiM), but not the parental Ni-CE strain, killed mice after intracerebral inoculation. CE(NiM) and Ni-CE are genetically identical except for two amino acids at positions 29 and 95 in the M protein. In this study, to identify which residue determines the pathogenicity, we examined pathogenicities of two Ni-CE mutants, CE(NiM29) and CE(NiM95), which were established by replacement of an amino acid residue at position 29 or 95 in the Ni-CE M protein with the corresponding residue of CE(NiM), respectively. We found that CE(NiM95), but not CE(NiM29), killed mice, indicating that the amino acid at position 95 in the M protein is the pathogenic determinant.

Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus.

The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPE is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus.

Involvement of nucleoprotein, phosphoprotein, and matrix protein genes of rabies virus in virulence for adult mice.

Rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara strain kills mice. In this study, to identify viral gene(s) related to the difference in pathogenicity between Ni-CE and Nishigahara strains, we generated chimeric viruses with respective genes of the virulent Nishigahara strain in the background of the avirulent Ni-CE genome. Since chimeric viruses, which had the N, P, or M genes of the Nishigahara strain, respectively, killed adult mice after intracerebral inoculation, it became evident that the N, P, and M genes are related to the difference in pathogenicity between Ni-CE and Nishigahara strains. Previously, we showed that the G gene is a major contributor to the difference in pathogenicity between another avirulent strain, RC-HL, and the parental Nishigahara strain. These results imply that the attenuation mechanism of the Ni-CE strain is different from that of the RC-HL strain, thus suggesting that rabies virus can be attenuated by diverse mechanisms. This is the first report of changes in viral genes other than the G gene of rabies virus causing the reversion of pathogenicity of an avirulent strain.