Genetic characterization of low-pathogenic avian influenza viruses isolated on the Izumi plain in Japan: possible association of dynamic movements of wild birds with AIV evolution.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site of endangered cranes (hooded cranes and white-naped cranes) and of many other migratory birds (including wild ducks) that are considered carriers of avian influenza viruses (AIVs). To assess the risks of a highly pathogenic avian influenza outbreak in the crane populations, we tested various environmental samples for AIVs in this area. In the 2014-2015 winter season, we isolated one AIV of the H6N2 subtype from the cranes' roost water and two AIVs of the H11N9 subtype from a crane fecal sample and a cloacal swab of a dead spot-billed duck. Genetic analysis of these AIV isolates indicated that our H6N2 isolate is genetically close to AIVs isolated from wild birds in Southeast Asian countries, except that the PB1 and NS genes belong to the North American virus lineage. All genes of the two H11N9 isolates are related to AIVs belonging to the Eurasian virus lineage. Notably, in our phylogenetic trees, H11 HA and N9 NA genes showing high sequence similarity to the corresponding genes of isolates from wild birds in South Africa and Spain, respectively, did not cluster in the major groups with recent wild-bird isolates from East Asia. These results suggest that AIVs with viral gene segments derived from various locations and bird species have been brought to the Izumi plain. These findings imply a possible association of dynamic movements of wild birds with AIV evolution.

Full genome sequences of torque teno sus virus strains that coinfected a pig with postweaning multisystemic wasting syndrome in Japan: implications for genetic diversity.

We determined the complete genome sequences of torque teno sus viruses (TTSuVs) detected in pigs with postweaning multisystemic wasting syndrome (PMWS) and in healthy pigs in Japan. Unexpectedly, we found coinfection of a PMWS-affected pig in Japan with one strain of TTSuV1, five strains of TTSuV2, and one strain of PCV2. Full-genome sequencing of each of these strains, followed by phylogenetic analysis, revealed broad genetic diversity in the TTSuV2 strains infecting the PMWS-affected pig. These results suggest that the geographical bias in the available genetic information about TTSuVs has a limited impact on the evaluation of their genetic diversity.

Isolation and characterization of influenza A viruses from environmental water at an overwintering site of migratory birds in Japan.

The Izumi plain in Kagoshima prefecture, Japan, is an overwintering site of more than 10,000 cranes. The wet paddy areas are artificially created to provide roosting sites for the cranes every winter. Since wild ducks, known to be a natural reservoir of influenza A viruses, also overwinter in this area, the cranes' roost water likely serves as a source of influenza A virus infection. To assess this potential risk, we collected 126 water samples from the cranes' roost in the 2012/2013 winter season for virus isolation. We isolated six influenza viruses of three subtypes (H3N8, H4N6, and H4N8) from the water samples collected in the months of November and December. Genetic analysis of our isolates indicated that these viruses were genetically similar to the low-pathogenic avian influenza viruses circulating among Eurasian waterfowl. These findings suggest the possibility of the cranes becoming infected with the avian influenza viruses that are present in their roost water.

Efficient isolation of Swine influenza viruses by age-targeted specimen collection.

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

Genomic polymorphisms in 3ß-hydroxysterol Δ24-reductase promoter sequences.

It was recently reported by the present team that 3ß-hydroxysterol Δ24-reductase (DHCR24) is induced by hepatitis C virus (HCV) infection. In addition, upregulation of DHCR24 impairs p53 activity. In human hepatoma HuH-7 cells, the degree of DHCR24 expression is higher than in normal hepatic cell lines (WRL68) at the transcriptional level. The genomic promoter sequence of DHCR24 was characterized and nucleotide substitutions were observed in HuH-7 cells at nucleotide numbers -1453 (G to A), -1420 (G to T), -488 (A to C) and -200 (G to C). The mutations of these sequences from HuH-7 cell types to WRL68 cell types suppressed DHCR24 gene promoter activity. The sequences were further characterized in hepatocytes from patient tissues. Four tissues from HCV-positive patients with cirrhosis or hepatocellular carcinoma (#1, 2, 3, 5) possessed HuH-7 cell type sequences. Interestingly, one patient with liver cirrhosis (#4) possessed WRL68 cell-type sequences; this patient had been infected with HCV and was HCV negative for 17 years after interferon therapy. Next, the effect of HCV infection on these polymorphisms was examined in humanized chimeric mouse liver and HuH-7 cells. The human hepatocytes possess WRL68 cell type and did not show the nucleotide substitution after HCV infection. The HCV-replicon was removed by interferon treatment and established the cured K4 cells. These cells possess HuH-7 cell type sequences. Thus, this study showed the genomic polymorphism in DHCR24 promoter is not directly influenced by HCV infection.