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Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), of which annual number has rapidly increased in Japan, and it can be divided into two categories based on the amino acid at position 233 in the Tax protein. Here, we conducted a nationwide surveillance of Japanese Black cattle between 2008 and 2021 in Japan. Among 237 tumor samples, 131 (55.3%) and 101 (42.6%) were harbored L233- and P233-Tax, respectively. Onset of EBL under the age of 3 years by L233-Tax-carrying BLV was frequently observed, especially in the animals born via embryo transfer. We also found that L233-Tax-carrying BLV was more prevalent in dairy areas than non-dairy areas. These findings give insight into prevention of EBL.

A recent study demonstrated the possibility that migratory birds are responsible for the global spread of avian rotavirus A (RVA). However, little is known about what types of RVAs are retained in migratory birds. In this study, to obtain information on RVA strains in migratory birds, we characterised an RVA strain, Ho374, that was detected in a faecal sample from a gull species (Larus sp.). Genetic analysis revealed that all 11 genes of this strain were classified as new genotypes (G28-P[39]-I21-R14-C14-M13-A24-N14-T16-E21-H16). This clearly indicates that the genetic diversity of avian RVAs is greater than previously recognised. Our findings highlight the need for investigations of RVA strains retained in migratory birds, including gulls.

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan., Here, Kodama et al. describe the discovery, isolation and characterization of a novel tick-borne orthonairovirus, designated Yezo virus (YEZV), from patients with an acute febrile illness in Japan. Serological testing of wildlife and molecular screening of ticks suggest an endemic circulation of YEZV in Japan.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. However, less than 5% of BLV-infected cattle will develop lymphoma, suggesting that, in addition to viral infection, host genetic polymorphisms might play a role in disease susceptibility. Bovine leukocyte antigen (BoLA)-DRB3 is a highly polymorphic gene associated with BLV proviral load (PVL) susceptibility. Due to the fact that PVL is positively associated with disease progression, it is believed that controlling PVL can prevent lymphoma development. Thus, many studies have focused on the relationship between PVL and BoLA-DRB3. Despite this, there is little information regarding the relationship between lymphoma and BoLA-DRB3. Furthermore, whether or not PVL-associated BoLA-DRB3 is linked to lymphoma-associated BoLA-DRB3 has not been clarified. Here, we investigated whether or not lymphoma-associated BoLA-DRB3 is correlated with PVL-associated BoLA-DRB3. We demonstrate that two BoLA-DRB3 alleles were specifically associated with lymphoma resistance (*010:01 and *011:01), but no lymphoma-specific susceptibility alleles were found, furthermore, two other alleles, *002:01 and *012:01, were associated with PVL resistance and susceptibility, respectively. In contrast, lymphoma and PVL shared two resistance-associated (DRB3*014:01:01 and *009:02) BoLA-DRB3 alleles. Interestingly, we found that PVL associated alleles, but not lymphoma associated alleles, are related with the anti-BLV gp51 antibody production level in cows. Overall, our study is the first to demonstrate that the BoLA-DRB3 polymorphism confers differential susceptibility to BLV-induced lymphoma and PVL.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL) and can be classified into two types based on the amino acid at position 233 in Tax protein, which probably plays crucial roles in leukemogenesis. We previously revealed that L233-Tax-expressing cells secreted chemoattractants for endothelial cells and formed significantly larger tumors accompanying neovascularization than P233-Tax-expressing cells in athymic mice. In the present study, comparative proteomic analysis of the culture medium of Tax-expressing cells revealed that annexin A3 and probably extracellular matrix protein 1 served as chemoattractants. Conversely, L233-Tax-expressing cells were impaired in the secretion of collagen alpha-1 (I) chain precursor, which participates in tissue tension homeostasis, leading to tumor mass development. The analysis also demonstrated that both L233-Tax- and P233-Tax-expressing cells had deficits in the secretion of potentially antiangiogenic molecules, including pigment epithelium-derived factor and collagen alpha-1 (VIII) chain, and they produced complement component 3, which might participate in tumor cell proliferation, metastasis, and immune evasion. These findings provided novel insights into prognostication of EBL and the function of Tax in leukemogenesis induced by BLV.

Bovine leukemia virus (BLV) can be divided into two categories based on the amino acid at position 233 in the Tax protein, which probably plays a crucial role in leukemogenesis. We show here that a rat fibroblast cell line stably expressing L233-Tax formed significantly larger tumors than P233-Tax-expressing cells in a murine xenograft study. Although the microvessel density was comparable in both tumors, visible blood vessel invasion was observed only on tumors from L233-Tax-expressing cells. Endothelial cell tube formation assays using human umbilical vein endothelial cells showed no significant difference in angiogenic activity between conditioned medium from L233- and P233-Tax-expressing cells, whereas in vitro chemotaxis assays revealed that only L233-Tax-expressing cells produced a chemoattractant for endothelial cells. Since pathological neovascularization can occur from the recruitment of endothelial progenitor cells, these results suggest that L233-Tax-expressing cells recruit murine endothelial progenitor cells and promote neovascularization to support tumor growth. BLV-infected lymphoma cells may also recruit bovine endothelial progenitor cells to promote neovascularization. The findings of this study are consistent with our previous observation that BLV carrying P233-Tax has a significantly longer incubation period for developing tumors than the virus carrying L233-Tax and provide insight into the function of Tax in leukemogenesis by BLV.

Recent discoveries of tick-borne pathogens have raised public health concerns on tick-borne infectious diseases and emphasize the need to assess potential risks of unrecognized tick-borne pathogens. First, to determine the existence of tick-borne phleboviruses (TBPVs), genetic surveillance of phleboviruses in ticks was conducted mainly in Hokkaido, the northernmost island in Japan from 2013 to 2015. Genes of two TBPVs, previously reported as Mukawa virus (MKWV) and a newly identified relative of MKWV, Kuriyama virus (KURV), were detected and the viruses were isolated from Ixodes persulcatus collected in Hokkaido, but not in I. persulcatus collected from other areas of Japan. These viruses were phylogenetically and antigenically similar to each other. Next, to investigate the infection of MKWV in mammals, serum samples from wildlife captured in Hokkaido from 2007 to 2011 were used for serological screening. Neutralizing antibodies against MKWV were detected in both Yezo-deer (Cervus nippon yesoensis) (2/50) and raccoons (Procyon lotor) (16/64). However, no infectious MKWV was recovered from laboratory mice in experimental infections, though viral RNAs were detected in their tissues. Thus, MKWV and KURV may maintain tick-mammalian life cycles in Hokkaido, suggesting their potential as causative agents of tick-borne diseases in mammals.

The bovine leukemia virus (BLV) Tax protein is believed to play a crucial role in leukemogenesis by the virus. BLV usually causes asymptomatic infections in cattle, but only one-third develop persistent lymphocytosis that rarely progress after a long incubation period to lymphoid tumors, namely enzootic bovine leucosis (EBL). In the present study, we demonstrated that the BLV tax genes could be divided into two alleles and developed multiplex PCR detecting an L233P mutation of the Tax protein. Then, in order to define the relationship between the Tax protein and leukemogenicity, we examined 360 tumor samples randomly collected from dairy or breeding cattle in Japan, of which Tax proteins were categorized, for age at the time of diagnosis of EBL. The ages of 288 animals (80.0%) associated with L233-Tax and those of 70 animals (19.4%) with P233-Tax individually followed log-normal distributions. Only the two earliest cases (0.6%) with L233-Tax disobeyed the log-normal distribution. These findings suggest that the animals affected by EBL were infected with the virus at a particular point in life, probably less than a few months after birth. Median age of those with P233-Tax was 22 months older than that with L233-Tax and geometric means exhibited a significant difference (P

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

Pandemic influenza H1N1 virus (A[H1N1]pdm09) emerged in 2009. To determine the phylogeography of A(H1N1)pdm09 in a single population, 70 strains of the virus were isolated from university students or trainee doctors at Tobetsu, Hokkaido, Japan, between September and December 2009. The nucleotide sequences of the HA1 region of the HA genes and described phylogenetic relationships of the strains circulating among them were analyzed. It was found that the 70 isolates could be phylogenetically separated into three groups and that two epidemics were caused by different groups of the virus. The three groups were also distinguishable from each other by three amino acid changes: A197T, S203T and Q293H. The substitution of S203T, which is located in the antigenic site, suggests antigenic drift of the virus.

Bovine leukemia virus (BLV) infection in cattle causes persistent lymphocytosis, and a few percent of infected animals develop lymphoid tumors, namely enzootic bovine leukosis (EBL). In this study, a 440-bp fragment of the env gene was amplified from 204 tumor samples collected from different regions of Japan and analyzed by restriction fragment length polymorphism (RFLP) to determine the association of BLV with EBL. Of the seven RFLP types defined, types I, II, and III were dominant and found in 12.7, 75.0, and 8.3% of tumor samples, respectively. Cattle harboring type III virus were significantly older than other animals at the time of diagnosis of EBL. Type III viruses were found in approximately 33% and 5.5% of Japanese Black and Holstein cattle, respectively, with EBL. These findings indicate that genetically distinct BLV was associated with EBL in Japan and that the genetic profile may influence the leukemogenicity of the virus.

Bovine leukemia virus (BLV) infection of cattle has been increasing yearly in Japan although several European countries have successfully eradicated the infection. In the present study, phylogenetic analysis on the env gene obtained from 64 tumor samples found in different regions in Japan was carried out in order to define the genetic background of BLV strains prevailing in the country. Most of the Japanese isolates were found to reside in the consensus cluster or genotype 1 of BLV strains (Rodriguez et al., 2009). Out of them, 21 isolates and 10 isolates exhibited the identical sequences, respectively. Only one isolate was classified into the different genotype related to the US isolates. Analysis on the deduced amino acids of gp51 demonstrated the sequence diversity in the neutralizing domain. These data may indicate that two major populations of BLV prevailed throughout Japan, whereas antigenic variants also exist. It was further proved that multiple invasion of the genetically different BLV strains have occurred in Japan.

Roosting of Asian parti-coloured bats (Vespertilio sinensis) in man-made structures is frequently results in conflict with humans. Therefore, to determine optimal alternative roosts, we studied roost preference of this species by testing the effects of roost temperature, surrounding environment and the distance from the nearest nursery colony on roost selection by V. sinensis in manipulative field experiments. We installed white (cooler) and black (warmer) bat boxes at shaded and sun-exposed sites. Although no nursery colonies were formed in the bat boxes, a few individuals (1–5) roosted during the lactation period, while many individuals (≥20) roosted during the post-lactation period. During lactation, bats used both white and black boxes, but preferred using the boxes placed at the shaded sites over those placed at the sun-exposed sites. On the other hand, during the post-lactation period, almost all individuals used the white boxes rather than the black boxes. However, adult females preferred...

Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes.

We determined the food habits of 3 sympatric species of insectivorous bats (Rhinolophus perditus, Hipposideros turpis and Miniopterus fuscus) from May 2001 to January 2003 on Ishigaki Island, in the Ryukyu archipelago, Japan, by analysing their faecal samples. We also measured wing morphology of these 3 species and assessed insect abundance near the roost. Diet composition dif- fered between the 3 bat species throughout the survey period, except between R. perditus and M. fuscus from July to November 2002. R. perditus mainly consumed Coleoptera, Diptera, Lepidoptera and Hymenoptera, and the proportion of consumption of these insect orders changed seasonally. H. turpis mainly consumed Coleoptera from spring to summer. A large amount of Anomala albopilosa in particular seemed to be consumed. M. fuscus frequently consumed Diptera, Lepidoptera and Hymenoptera throughout the study period and Coleoptera in summer. Our results indicate resource partitioning between each species of bats; this partitioning may be caused by morphological differ- ences between the 3 species of bats, which may result in different passive prey selection. Because of the variation in diet composition, habitats that maintain high insect diversity are required for bat assemblage. In the future, the preferred foraging habitats of the 3 species of bats should be studied, and the results of these studies should be combined with our results for establishing an appropriate conservation measure.

In this study, 520 serum samples from Yezo-deer in the Hidaka district, Hokkaido, Japan were examined by enzyme-linked immunosorbent assay to investigate whether the animals were infected with hepatitis E virus (HEV). The distribution of optical density values showed a bimodal pattern and 181 samples (34.8%) were deemed to be antibody-positive against HEV. At least five (2.8%) of the positive sera gave specific bands by Western blot analysis. An age-dependent increase in prevalence of the antibodies was found among the animals. These findings indicate that Yezo-deer are a possible host for HEV infection. To avoid the risk of becoming HEV infected, the consumption of raw Yezo-deer meat must be prohibited.

Glycoprotein B (gB) is the most conserved glycoprotein among herpesviruses and it plays important roles in virus infectivity. In most herpesviruses, including pseudorabies virus (PRV), gB is cleaved by a cellular protease into two disulfide-linked subunits. In the present study, I found that the PRV gB generated in human colon carcinoma LoVo cells, which lack the ubiquitous protease furin, remained in the uncleaved form and the virus replicated in these cells without cell fusion. The uncleaved gB was converted into its subunits after furin digestion. The virus also replicated in Madin–Darby bovine kidney cells without cell fusion in the presence of a furin inhibitor, whereas distinct syncytia were formed in the absence of the inhibitor. LoVo cells constitutively expressing furin showed cell fusion when they were infected with the virus. Penetration kinetics assays revealed that the virus carrying uncleaved gB penetrated the cells at the same rate as the virus carrying cleaved gB. These results indicate that PRV gB is cleaved by furin and that the cleavage is dispensable for virus replication in vitro. Furthermore, gB cleavage is involved in syncytium formation but not in penetration kinetics, suggesting that different mechanisms operate between cell–cell fusion and virus–cell fusion by PRV.

The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995-2002 were genetically analyzed. All the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage (except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.

For the prediction of future influenza pandemics, global surveillance of avian influenza has been continuing since 1991 and carried out in Russia, Mongolia, China and Japan from 2000 to 2003. Influenza virus isolates of 50 combinations of HA and NA subtypes have been identified and 3 strains selected from each of those are stocked. In addition, 47 other combinations have been generated by standard genetic reassortment procedure in the laboratory. Since we have already shown that influenza viruses have been fully adapted to ducks and cause no disease signs and are in evolutionary stasis in their natural reservoirs, virus isolates from ducks are ideal as vaccine strains. Thus, influenza viruses of 97 combinations of HA and NA subtypes are now available as vaccine strain candidates against emerging pandemic influenza in humans, domestic animals and poultry.

Glycoprotein B (gB) is the most conserved glycoprotein of herpesviruses and plays important roles in virus infectivity. Two intervening heptad repeat (HR) sequences were found in the C-terminal half of all herpesvirus gBs analysed. A synthetic peptide derived from the HR region (aa 477–510) of bovine herpesvirus type 1 (BoHV-1) gB was studied for its ability to inhibit virus replication. The peptide interfered with cell-to-cell spread and consistently inhibited replication of BoHV-1, with a 50 % effective concentration value (EC50) of 5 μM. Inhibition of replication was obtained not only with herpesviruses including pseudorabies virus and herpes simplex virus type 1 but also partly with Newcastle disease virus. Possible mechanisms of membrane fusion inhibition by the peptide are discussed.

Ten H9N2 influenza virus strains isolated from diseased chickens in different farms in China during 1995 to 1999 were antigenically and genetically characterized. The haemagglutinins of the isolates were not related to those of A/quail/Hong Kong/G1/97 (H9N2) (Qa/HK/G1/97), but were closely related to that of A/chicken/Hong Kong/G9/97 (H9N2) (Ck/HK/G9/97). The neuraminidase of these isolates had a deletion of three amino acid residues at positions 63 to 65 as compared with those of Ck/HK/G9/97, while that of Qa/HK/G1/97 lacked two amino acids at positions 38 and 39. The PB2 genes of the isolates were not related to those of Qa/HK/G1/97 or Ck/HK/G9/97, but showed some relationship to that of A/duck/Hong Kong/Y439/97 (H9N2) (Dk/HK/Y439/97). The PB1 genes of the isolates were not related to those of the three representative strains. The PA, NP, M, and NS genes of the isolates belonged to the same lineage as those of Ck/HK/G9/97, and were distinct from those of Qa/HK/G1/97 and Dk/HK/Y439/97. The present results indicate that H9N2 influenza viruses prevalent in chicken populations in China belong genetically to one lineage and are distinct from Qa/HK/G1/97, presumed to be the donor of the internal protein genes of the highly pathogenic H5N1 influenza virus in Hong Kong in 1997.

Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997–2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.

The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995–2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63–65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), to which animals are most susceptible at 4–8 years of age. In this study, we examined tumor cells associated with EBL in an 18-year-old cow to reveal that the cells carried at least two different copies of the virus, one of which was predicted to encode a reverse transcriptase (RT) lacking ribonuclease H activity and no integrase. Such a deficient enzyme may exhibit a dominant negative effect on the wild-type RT and cause insufficient viral replication, resulting in delayed tumor development in this cow.

The immediate early (IE) gene of canine herpesvirus (CHV), homologue of the infected cell protein 4 (ICP4) gene of herpes simplex virus 1, is transcribed as a 4.9kb mRNA during IE phase. The IE gene was further transcribed as a 4.8kb mRNA through early (E) and late (L) phases of productive infection. Transcription of the 4.8kb mRNA initiated from downstream of the TATA box in an intron which was spliced out during IE phase. The reverse transcription-polymerase chain reaction revealed that the IE promoter was turned off during L phase at a permissive temperature. We, thus, propose to redesignate the IE gene of CHV as CICP4 gene.

Intracerebral inoculation of field-isolates as well as established strains of equine herpesvirus-1 (EHV-1) in suckling mice results in viral replication in neurons and glial cells and induces encephalitis. By intraperitoneal (i.p.) inoculation, no histological lesion was observed in the central nervous system (CNS) in suckling mice with the EHV-1 HH1 strain (HH1), whereas a neuroadapted variant (NHH1) produced by serial passage of HH1 in the mouse brain caused severe encephalomyelitis after i.p. inoculation. The purpose of this study was to determine the route of neuroinvasion after i.p. inoculation of NHH1 and to clarify the effects of the brain passage on viral neuroinvasion. NHH1, but not HH1, targeted splenic and pulmonary macrophages and omental fat cells on days 1 and 2 post-inoculation (p.i.). From days 1 to 3 p.i., cell-associated viremia was occurred in NHH1-infected mice, but not in HH1-infected mice. On day 4 p.i., viral antigen was detected in a few endothelial cells, perivascular glial cells and neurons in the CNS in NHH1-infected mice. The number of viral antigen-positive cells increased markedly after day 5 p.i. In contrast, no viral antigen-positive cell was detected in the CNS in HH1-infected mice, except for a few nerve cells in the thoracic cord on day 4 p.i. These results suggest that NHH1 neuroinvasion is hematogenous and is correlated with enhanced extraneural virus growth.

Ebola virus causes severe hemorrhagic fever in primates, resulting in mortality rates of up to 100%, yet there are no satisfactory biologic explanations for this extreme virulence. Here we show that antisera produced by DNA immunization with a plasmid encoding the surface glycoprotein (GP) of the Zaire strain of Ebola virus enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP. Substantially weaker enhancement was observed with antiserum to the GP of the Reston strain, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. We also generated an anti-Zaire GP monoclonal antibody that enhanced viral infectivity and another that neutralized it, indicating the presence of distinct epitopes for these properties. Our findings suggest that antibody-dependent enhancement of infectivity may account for the extreme virulence of the virus. They also raise issues about the development of Ebola virus vaccines and the use of passive prophylaxis or therapy with Ebola virus GP antibodies.

The canine herpesvirus infected cell protein 0 (CICP0) gene was sequenced. The CICP0 gene was transcribed as a 1.4 kb mRNA from the end of the unique long region nearby the internal repeat during early phase of productive infection of the virus. An open reading frame of the gene encodes a polypeptide of 333 amino acids. The RING finger domain and acidic transcriptional activation domain were found at the N-terminus and within the middle region in the deduced amino acid sequence, respectively, suggesting that the CICP0, like the ICP0 of herpes simplex virus 1, is a transactivating protein.

To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101-400 and three bound to that of amino acids 518-600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein.

Heparin extensively inhibited infection of MDBK cells by equine herpesvirus 1 (EHV-1) strains adapted to bovine cells or hamsters, while the reagent merely reduced infectivity of strains passaged only in equine cells. The gC of two strains adapted to non-equine cells seemed to have higher affinity for heparin, although the reagent bound to both the gC and gB of all strains tested. Amino acid substitutions of the gC of the EHV-1 strains adapted to non-equine cells converged on the hydrophilic regions, amino acid residues 92 to 175, resulting in the glycoprotein becoming more cationic. These results indicate that these hydrophilic regions of the gC may be responsible for binding to heparin.

We identified a novel neuraminidase (NA)-deficient virus that was a 2009 pandemic influenza H1N1 virus mutant. The mutant virus had a deletion of 1,009 nt in the NA gene and lacked an enzymatic domain. Although the yield of the NA-deficient virus was limited, it formed large plaques when applied to MDCK cell cultures, indicating that the virus was able to spread to adjacent cells. Furthermore, the NA-deficient virus was eluted from chicken erythrocytes at 37 °C, even in the presence of the antiviral drug peramivir. Spread of this NA-deficient virus may pose a potential threat to anti-influenza therapies.

We constructed a recombinant baculovirus expressing feline herpesvirus type I (FHV-1) gD in insect cells (Sf9 cells). The expressed product was identified as FHV-1 gD by a panel of monoclonal antibodies specific for the FHV-1 gD, and had an apparent molecular mass of approximately 49 kDa, which was less than that of the authentic FHV-1 gD. When the FHV-1 gD protein were expressed in Sf9 cells and CRFK cells in the presence of tunicamycin, the FHV-1 gD exhibited a molecular mass of 41 kDa. It was shown that the gD protein was transported to the surface of recombinant virus-infected Sf9 cells when examined by membrane-immunofluorescence analysis, and that the gD expressed on the surface of Sf9 cells adsorbed feline erythrocytes. Mice inoculated with a lysate of Sf9 cells expressing FHV-1 gD induced antibodies with virus-neutralizing and hemagglutination-inhibition activities. Therefore, the expressed gD appears to be biologically authentic. These data suggested that recombinant FHV-1 gD produced in Sf9 cells may be a useful immunogen as a feline vaccine.

To provide information on the mechanism of perpetuation of influenza viruses among waterfowl reservoirs in nature, virological surveillance was carried out in Alaska during their breeding season in summer from 1991 to 1994. Influenza viruses were isolated mainly from fecal samples of dabbling ducks in their nesting places in central Alaska. The numbers of subtypes of 108 influenza virus isolates were 1 H2N3, 37 H3N8, 55 H4N6, 1 H7N3, 1 H8N2, 1 H10N2, 11 H10N7, and H10N9. Influenza viruses were also isolated from water samples of the lakes where they nest. Even in September of 1994 when the most ducks had left for migration to south, viruses were still isolated from the lake water. Phylogenetic analysis of the NP genes of the representative isolates showed that they belong to the North American lineage of avian influenza viruses, suggesting that the majority of the waterfowls breeding in central Alaska migrate to North America and not to Asia. The present results support the notion that influenza viruses have been maintained in waterfowl population by water-borne transmission and revealed the mechanism of year-by-year perpetuation of the viruses in the lakes where they breed.

Pandemic influenza H1N1 virus (A[H1N1]pdm09) emerged in 2009. To determine the phylogeography of A(H1N1)pdm09 in a single population, 70 strains of the virus were isolated from university students or trainee doctors at Tobetsu, Hokkaido, Japan, between September and December 2009. The nucleotide sequences of the HA1 region of the HA genes and described phylogenetic relationships of the strains circulating among them were analyzed. It was found that the 70 isolates could be phylogenetically separated into three groups and that two epidemics were caused by different groups of the virus. The three groups were also distinguishable from each other by three amino acid changes: A197T, S203T and Q293H. The substitution of S203T, which is located in the antigenic site, suggests antigenic drift of the virus.

Bovid herpesvirus 1 (BHV-1) glycoprotein gIII functions as a major virus attachment protein through binding to a heparin-like moiety on the host cells. To identify the functional domain, a panel of gIII deletion mutants was constructed, expressed in COS-7 cells, and examined for heparin-binding activity. Mutants with deletion of amino acid residues 103-173 and 324-443 bound to heparin as well as full-length gIII, whereas a mutant with residues 172-337 deleted showed no binding to heparin. In another mutant, with residues 172-211 deleted, the activity was reduced by one-third. These data suggest that the amino acid sequence between residues 172 and 323 contains the functional domain of BHV-1 gIII for heparin-binding and that especially the sequence between residues 212-323 includes a critical site for the activity.

Influenza viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1999. Phylogenetic analysis of the NP genes of the isolates in Siberia and Japan on their flyway of migration to the south indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virus in Hong Kong. Inactivated vaccine prepared from an avirulent H5N4 virus isolated from a migratory duck in Japan showed enough potency to induce protective immunity against the pathogenic H5N1 virus in mice. To expand surveillance for influenza viruses in migratory and domestic ducks and geese, chickens, quail, and pigs of the world and be informed about what influenza viruses are dominant in the animal reservoirs, we have started the “Programme of Excellence in Influenza”. Influenza virus strains isolated from animal species in the surveillance should be thoroughly characterized, stored, and be provided for the use of diagnosis and vaccine preparation for the control of future pandemics.

Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes.

The gIII glycoprotein of bovid herpesvirus 1 (BHV-1) has been shown to mediate the adsorption of the virions to cells ( K. Okazaki, E. Honda, T. Minetoma, and T. Kumagai, 1987 , Arch. Virol. 97, 297–307). In this study, the cellular receptor for BHV-1 was investigated. Addition of heparin to the virus inoculum and treatment of the cells with heparinase prevented the virus from adsorbing to and infecting the cells. Of the major glycoproteins of BHV-1 only gill was found to bind specifically to heparin. The binding of gill was inhibited by a monoclonal antibody against antigenic site la, which interferes with the adsorption of the virus. These findings indicate that the virus adsorption to cells is mediated by interaction of the gill antigenic site la with a heparinlike moiety on the cell surface, which serves as a receptor for BHV-1.

Plaque formation, replication and related cytopathic function of 9 strains of transmissible gastroenteritis (TGE) virus were examined in primary cells and cell lines such as CPK, IB-RS-2, ESK, and PK-15 originated from porcine kidney and the effects of trypsin on the replication of TGE virus were examined in CPK cells. All strains produced a cytopathic effect and grew well in CPK cells as well as in primary porcine kidney cells. The effect of trypsin on the plaque formation was different from strains. The number of plaques produced by strains TO-163, Ukiha and Niigata increased from 2.6 to 3.52 times when trypsin was present in the medium during incubation at 37 degrees C for 1 hr after adsorption of the virus at 4 degrees C for 2 hr. The plaque sizes of TO-163, h-5, Ukiha and Niigata became larger from 1.4 to 1.7 times, when trypsin was present in the agar MEM overlay.

Characteristics of CPE produced by porcine enteroviruses (PEV) were examined by Immunoperoxidase (IP) staining method. Viral antigens were detected earlier than appearance of CPE. Distinctive characteristics of the three CPE types were clearly showed by the method. In cross reactions by IP staining, the titers of the staining were high (1:6,400 to 1:25,600), even though neutralizing titers of PEV with CPE II or III were low (1:200 to 1:400). The very close relationship was detected between PEV with the same CPE type, but the very low relationships were detected between PEV with the different CPE type. The relationships in CPE I group were various. The results by IP staining were more relative to CPE type than the serotype. Thus, IP staining is one method to classify PEV clearly.

Equine herpesvirus 1 (EHV-1) shows endotheliotropism in the central nervous system (CNS) of infected horses. However, infection of endothelial cells has not been observed in the CNS of infected mice. To explore the basis for this difference in endotheliotropism, we compared the susceptibility of equine brain microvascular endothelial cells (EBMECs) and mouse brain microvascular endothelial cells (MBMECs) to EHV-1 infection. The kinetics of viral growth in EBMECs was typical of a fully productive infection whereas viral infection in MBMECs seemed to be nonproductive. Immunofluorescence microscopy using anti-EHV-1 polyclonal antibody demonstrated viral antigen in infected EBMECs, but not infected MBMECs. EHV-1 immediate early (IE), early (ICP0), and late (gB, gD and gK) transcripts were expressed in infected EBMECs. However, none of these genes was detected in infected MBMECs by reverse transcription-polymerase chain reaction. Electron microscopic examination at the stage of viral entry showed that viral particles were present within uncoated vesicles in the cytoplasm of EBMECs, but absent from those of MBMECs. These results suggest that viral entry is an important determinant of the susceptibility of EBMECs and MBMECs to EHV-1 infection.

A highly sensitive immuno-PCR method specific for the detection of antigens of and antibodies to bovine herpesvirus 1 was established. This assay attained a high sensitivity of up to 10(7.0) times higher than that of enzyme-linked immunosorbent assay (ELISA) or PCR for antigen detection and 10(5.0) times higher than that of ELISA for antibody detection.

H2 influenza virus caused a pandemic in 1957 and has the possibility to cause outbreaks in the future. To assess the evolutionary characteristics of H2 influenza viruses isolated from migratory ducks that congregate in Hokkaido, Japan, on their flyway of migration from Siberia in 2001, we investigated the phylogenetic relationships among these viruses and avian and human viruses described previously. Phylogenetic analysis showed that the PB2 gene of Dk/Hokkaido/107/01 (H2N3) and the PA gene of Dk/Hokkaido/95/01 (H2N2) belonged to the American lineage of avian virus and that the other genes of the isolates belonged to the Eurasian lineage. These results indicate that the internal protein genes might be transmitted from American to Eurasian avian host. Thus, it is further confirmed that interregional transmission of influenza viruses occurred between the North American and Eurasian birds. The fact that reassortants could be generated in the migratory ducks between North American and Eurasian avian virus lineage further stresses the importance of global surveillance among the migratory ducks.

Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997-2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.

The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995-2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63-65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.