Effects of host-cell adaptation of the hemagglutinin (HA) protein were evaluated by the analyses of four pairs of recent influenza B field isolates, each pair consisting of an Madin Darby canine kidney (MDCK)- and an embryonated chicken egg-derived isolates from the same clinical specimen. Among the isolates examined, all of the MDCK-derived isolates retained glycosylation site at amino acid 197 on the HA1 molecule, whereas three egg-derived isolates lost it. Antigenic difference in the HA molecule between an MDCK- and an egg-derived isolates of three of these pairs was demonstrated to be associated with the glycosylation 197. Replication of the MDCK-derived isolates was suppressed in eggs, suggesting that the presence of the glycosylation 197 was disadvantageous to replication in eggs. Virus-binding affinity assay revealed that the loss of carbohydrate chain did not significantly alter the preferential recognition of sialic acid linkage. Immunogenicity and vaccine efficacy of an MDCK- and an egg-derived clones of B/Akita/27/2001, the former retained the glycosylation 197 and the latter lost it, were compared in a hamster model. When formalin-inactivated whole virion vaccines prepared from the paired isolates were administered into hamsters, no significant difference between them was observed in protective ability against challenges by the homologous and heterologous clones. Implication of the egg adaptation of influenza virus to antigenic surveillance of the field isolates as well as the selection of vaccine strains, and possibility of the involvement of the viral protein(s) other than the HA in the egg adaptation were discussed.

To characterize Japanese encephalitis virus (JEV) strains recently prevalent in Japan, JEV surveillance was performed in pigs from 2002 to 2004. Eleven new JEV isolates were obtained and compared with previous isolates from Japan and other Asian countries. All of the isolates were classified into genotype 1 by nucleotide sequence analysis of the E gene. Two new isolates with different levels of neurovirulence and neuroinvasiveness, but with only one nucleotide difference in the E gene, Sw/Mie/34/2004 and Sw/Mie/40/2004, were isolated at the same farm on the same day. Sw/Mie/40/2004 displayed higher neurovirulence and neuroinvasiveness in mice than the other four new isolates. Another new isolate, Sw/Hiroshima/25/2002, was neutralized by antiserum to Beijing-1 at a level similar to the homologous Beijing-1 strain, whilst seven other new isolates were neutralized at 10-fold-lower titres. However, there were no amino acid differences in the E protein among these eight isolates. The present study indicated that the 11 new JEV isolates were genetically similar, but biologically and serologically heterogeneous. The GenBank/EMBL/DDBJ accession numbers for the E gene and 39 NTR sequences of the 14 JEV isolates determined in this study (-indicates 'data not available'

Influenza A and B viruses possess a neuraminidase protein that shows sialidase activity. Influenza virus-specific neuraminidase inhibitors (NAIs) are commonly used for clinical treatment of influenza. However, some influenza A and B viruses that are resistant to NAIs have emerged in nature. NAI-resistant viruses have been monitored in public hygiene surveys and the mechanism underlying the resistance has been studied. Here, we describe a new assay for selective detection and isolation of an NAI-resistant virus in a speedy and easy manner by live fluorescence imaging of viral sialidase activity, which we previously developed, in order to achieve high-efficiency capture of an NAI-resistant virus. An NAI-resistant virus maintains sialidase activity even at a concentration of NAI that leads to complete deactivation of the virus. Infected cells and focuses (infected cell populations) of an oseltamivir-resistant virus were selectively visualized by live fluorescence sialidase imaging in the presence of oseltamivir, resulting in high-efficiency isolation of the resistant viruses. The use of a combination of other NAIs (zanamivir, peramivir, and laninamivir) in the imaging showed that the oseltamivir-resistant virus isolated in 2008 was sensitive to zanamivir and laninamivir but resistant to peramivir. Fluorescence imaging in the presence of zanamivir also succeeded in selective live-cell visualization of cells that expressed zanamivir-resistant NA. Fluorescence imaging of NAI-resistant sialidase activity will be a powerful method for study of the NAI resistance mechanism, for public monitoring of NAI-resistant viruses, and for development of a new NAI that shows an effect on various NAI-resistant mutations.

1) Seroepidemiological analysis of influenza pandemics (1986 -2003) in Shizuoka Prefecture and all Japan revealed differences in geographical, annual, seasonal, and age distributions.(2) For 17 years, the pandemics generally began at the 50th week every year showing over 1.0 patient/clinic, reached the peak at 5th week the following year, and ended over 10 -15th week. Two big A/H3N2 pandemics were seen in 1989/1990 and 1997/1998 seasons, claiming over 1 million patients in Japan. (3) As herald strains, A/H3N2 strains (A/Sydney-like) were found in October 1999, and B strains (B/Victoria-and B/Yamagata-like) were detected in July and November 1998 and, in August and December 2000 in Shizuoka. B/Shizuoka/1/98 strain was registered internationally as a vaccine-recommended strain. (4) A/H3N2 and B viruses were detected in 55 -78% of flu patients (almost under 10 years) with encephalopathy in 1999/2000 and 78 -91% in 2000/ 2001 by MDCK and reverse transcription polymerase chain reaction (RT-PCR) methods. (5) High hemagglutination inhibition (HI) titers over 40 in 250 persons were shown against A/Sydney/5/97 (H3N2), A/Yokohama/8/98 (H3N2), A/Panama/2007/99 (H3N2) and A/Moscow/10/99 (H1N1) strains, while low titers showed against A/Beijing/262/95 (H1N1) and A/New Caledonia/20/99 (H1N1), and B/Beijing/243/97, B/Shangdong/7/97 and B/Yamanashi/106/98 strains in 1998 -2000.(6) In anti-HA titers against A/H3N2, A/H1N1 and B subtypes, clear generation gaps were observed between children (0 -19 years), adults (20 -59 years) and old men (over 60 years). (7) The pandemics are dependent on host immunity (acquired and vaccinated) and climatic conditions (low temperature, low humidity and limited rainfall), considering highly pathogenic avian influenza (HPAI) viruses (A/H5N1, A/H7N7) like severe acute respiratory syndrome (SARS) corona virus in 2002 -

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