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Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2.IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

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.

Bats serve as natural hosts of Pteropine orthoreovirus (PRV), an emerging group of bat-borne, zoonotic viruses. Bats appear to possess unique innate immune system responses that can inhibit viral replication, thus reducing clinical symptoms. We examined the innate immune response against PRV and assessed viral replication in cell lines derived from four bat species (Miniopterus fuliginosus, Pteropus dasymallus, Rhinolophus ferrumequinum, and Rousettus leschenaultii), one rodent (Mesocricetous auratus), and human (Homo sapiens). The expression levels of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferons (IFNB1 and IFNL1) were higher and PRV replication was lower in cell lines derived from M. fuliginosus, R. ferrumequinum, and R. leschenaultii. Reduction of IFNB1 expression by the knockdown of PRRs in the cell line derived from R. ferrumequinum was associated with increased PRV replication. The knockdown of RIG-I led to the most significant reduction in viral replication for all cell lines. These results suggest that RIG-I production is important for antiviral response against PRV in R. ferrumequinum. Supplementary Information The online version contains supplementary material available at 10.1007/s11262-021-01865-6.

To investigate the seroprevalence of severe fever with thrombocytopenia syndrome (SFTS) among wild and companion animals on Tsushima Island, Japan, SFTS virus (SFTSV)-specific ELISA and virus-neutralizing tests were conducted on 50 wild boars, 71 Sika deer, 84 dogs, 323 domestic cats, and 6 Tsushima leopard cats. In total, 1 wild boar (1.8%), 2 dogs (2.4%), 7 domestic cats (2.2%), and 1 Tsushima leopard cat (16.7%) were positive for anti-SFTSV antibodies. Among the 11 positive animals, 10 were collected after 2019, and all were found on the southern part of the island. SFTSV, thus far, seems to be circulating within a limited area of Tsushima Island. To protect humans and animals, including endangered Tsushima leopard cats, from SFTSV infection, countermeasures are needed to prevent the spread of SFTSV on Tsushima Island.

A 61-d-old fennec fox ( Vulpes zerda), 11 d after receiving a multivalent, modified-live virus vaccine containing canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), parainfluenza virus, parvovirus, and canine coronavirus, developed oculonasal discharge, and subsequently convulsions, and hemoptysis, and died. Microscopic changes in the cerebrum were evident, including neuronal degeneration and necrosis; intracytoplasmic eosinophilic inclusion bodies were observed in astrocytes. CDV was detected in the brain tissue by immunohistochemistry. Pulmonary lesions of multifocal necrotizing bronchopneumonia had Cowdry type A intranuclear inclusions in the bronchial epithelial cells. Electron microscopy revealed crystalline arrays of adenovirus-like particles within the intranuclear inclusions. Additionally, the hemagglutinin gene of CDV and the CAdV-2 DNA polymerase gene were detected in the fennec fox; sequence analysis showed 100% identity with those of the vaccine strain viruses. To our knowledge, vaccine-induced CDV and CAdV-2 coinfections using molecular analysis have not been reported previously. Therefore, vaccine strains should be considered prior to CDV vaccination in nondomestic carnivores.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September–November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.

Ferrets are animals that are known to be susceptible to influenza A virus (IAV) infection. To evaluate the risk of IAV transmission from diseased ferrets to humans, we performed a serosurvey to detect specific antibodies against the H1, H3, H5, and H7 subtypes of IAV. We found a high positive rate of the H1 (24.1%) and H3 (5.2%) subtypes in pet ferrets by using an enzyme-linked immunosorbent assay for hemagglutinin proteins. The results were confirmed by the virus-neutralization test for representative antibody-positive serum samples. We also detected hemagglutinin and neuraminidase genes in two ferrets showing acute respiratory illness and whose owner was diagnosed with IAV infection; a human H1N1pdm virus was isolated from one of these ferrets. Our findings suggest that attention should be paid for IAV infection from humans to ferrets, and vice versa.

A 32-year-old man, who visited Japan from the Philippines in 2020, was diagnosed with rabies, the first reported case in Japan since 2006. This is the fourth imported case of rabies since 1957; one case in 1970 was imported from Nepal and two in 2006 were imported from the Philippines.

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

During 2016–2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Mosquitoes transmit many kinds of arboviruses (arthropod-borne viruses), and numerous arboviral diseases have become serious problems in Indonesia. In this study, we conducted surveillance of mosquito-borne viruses at several sites in Indonesia during 2016-2018 for risk assessment of arbovirus infection and analysis of virus biodiversity in mosquito populations. We collected 10,015 mosquitoes comprising at least 11 species from 4 genera. Major collected mosquito species were Culex quinquefasciatus, Aedes albopictus, Culex tritaeniorhynchus, Aedes aegypti, and Armigeres subalbatus. The collected mosquitoes were divided into 285 pools and used for virus isolation using two mammalian cell lines, Vero and BHK-21, and one mosquito cell line, C6/36. Seventy-two pools showed clear cytopathic effects only in C6/36 cells. Using RT-PCR and next-generation sequencing approaches, these isolates were identified as insect flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), new permutotetravirus (designed as Bogor virus) (family Permutotetraviridae, genus Alphapermutotetravirus), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus). We believed that this large surveillance of mosquitoes and mosquito-borne viruses provides basic information for the prevention and control of emerging and re-emerging arboviral diseases.

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by the SFTS phlebovirus (SFTSV). SFTS patients were first reported in China, followed by Japan and South Korea. In 2017, cats were diagnosed with SFTS for the first time, suggesting that these animals are susceptible to SFTSV. To confirm whether or not cats were indeed susceptible to SFTSV, animal subjects were experimentally infected with SFTSV. Four of the six cats infected with the SPL010 strain of SFTSV died, all showing similar or more severe symptoms than human SFTS patients, such as a fever, leukocytopenia, thrombocytopenia, weight loss, anorexia, jaundice and depression. High levels of SFTSV RNA loads were detected in the serum, eye swab, saliva, rectal swab and urine, indicating a risk of direct human infection from SFTS-infected animals. Histopathologically, acute necrotizing lymphadenitis and hemophagocytosis were prominent in the lymph nodes and spleen. Severe hemorrhaging was observed throughout the gastrointestinal tract. B cell lineage cells with MUM-1 and CD20, but not Pax-5 in the lesions were predominantly infected with SFTSV. The present study demonstrated that cats were highly susceptible to SFTSV. The risk of direct infection from SFTS-infected cats to humans should therefore be considered.

Tick-borne encephalitis virus (TBEV), a flavivirus that causes severe neurological symptoms in humans, has been found in Hokkaido, Japan. In the present study, we detected sequences from a novel tick-borne flavivirus, designated Yamaguchi virus (YGV), in liver and serum samples obtained from a wild boar in the Yamaguchi prefecture, Japan. Phylogenetic analysis revealed that YGV belongs to the TBEV complex and is closely related to Langat virus (LGTV). YGV was also detected by specific RT-PCR from 20 in 378 pools of ticks (2923 ticks) collected in Yamaguchi and Wakayama prefectures and from seven in 46 wild boar captured in Wakayama. The major ticks infected with YGV belong to the genus Haemaphysalis. Unfortunately, YGV could not be isolated from any samples from the RT-PCR positive wild boar or ticks. Therefore, ELISA for detection of antibodies against YGV was established using LGTV, and surveillance was performed among wild boar in 10 different prefectures on Honshu Island, the main island of Japan. The results showed that the seroprevalence of tick-borne flavivirus infection in the Wakayama and Hyogo prefectures of western Japan was significantly higher than that in the other prefectures, while antibodies against tick-borne flavivirus were not detected in any wild boar in the Tochigi prefecture in the eastern part of Japan. In addition, wild raccoons or masked palm civets in the Hyogo prefecture did not possess detectable antibodies against tick-borne flaviviruses. In conclusion, YGV appears to be maintained primarily among wild boar and ticks in the western part of Japan. YGV is the second flavivirus (after Japanese encephalitis virus) shown to be circulating on Honshu Island in Japan.

Severe fever with the thrombocytopenia syndrome virus (SFTSV) causes fatal disease in humans, cats, and cheetahs. In this study, the information on seven dogs with SFTS was summarized. All dogs showed anorexia, high fever, leukopenia, and thrombocytopenia, two dogs showed vomiting and loose stool, and five dogs had tick parasites. All dogs also had a history of outdoor activity. The SFTSV gene was detected in all dogs. Remarkably, three dogs (43%) died. SFTSV was isolated from six dogs and the complete genomes were determined. A significant increase in anti-SFTSV-IgG antibodies was observed in two dogs after recovery, and anti-SFTSV-IgM antibodies were detected in four dogs in the acute phase. Using an ELISA cut-off value of 0.410 to discriminate between SFTSV-negative and positive dogs, the detection of anti-SFTSV-IgM antibodies was useful for the diagnosis of dogs with acute-phase SFTS. Four out of the ninety-eight SFTSV-negative dogs possessed high anti-SFTSV IgG antibody titers, indicating that some dogs can recover from SFTSV infection. In conclusion, SFTSV is lethal in some dogs, but many dogs recover from SFTSV infection.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergence’s impact on choosing optimal drug treatment.

Significance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are of concern, with the P.1 variants dominating in Brazil. Brazil is now seeing a record number of deaths. Here, we report that the pathogenicity in hamsters of a P.1 variant is similar to that of nonvariant SARS-CoV-2. However, it has an expanded host range as shown by its replication in mice. Prior infection with nonvariant SARS-CoV-2 strains efficiently prevented replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. Convalescent sera from patients infected with nonvariants or sera from messenger RNA vaccinees showed comparable neutralization titers among the P.1 and previously circulating strains. These results suggest that previous SARS-CoV-2 infection and vaccines based on the original SARS-CoV-2 will provide some protection against P.1 infection., The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10–17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure., Author summary SARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

Antibody titers against SARS-CoV-2 slowly wane over time. Here, we examined how time affects antibody potency. To assess the impact of antibody maturation on durable neutralizing activity against original SARS-CoV-2 and emerging variants of concern (VOCs), we analyzed receptor binding domain (RBD)-specific IgG antibodies in convalescent plasma taken 1–10 months after SARS-CoV-2 infection. Longitudinal evaluation of total RBD IgG and neutralizing antibody revealed declining total antibody titers but improved neutralization potency per antibody to original SARS-CoV-2, indicative of antibody response maturation. Neutralization assays with authentic viruses revealed that early antibodies capable of neutralizing original SARS-CoV-2 had limited reactivity toward B.1.351 (501Y.V2) and P.1 (501Y.V3) variants. Antibodies from late convalescents exhibited increased neutralization potency to VOCs, suggesting persistence of cross-neutralizing antibodies in plasma. Thus, maturation of the antibody response to SARS-CoV-2 potentiates cross-neutralizing ability to circulating variants, suggesting that declining antibody titers may not be indicative of declining protection., Graphical abstract, Antigenic drifts in SARS-CoV-2 variants permit escape from neutralizing antibody in COVID-19 convalescent plasma. Moriyama et al. reveal the evolution of serological immunity with time that counters SARS-CoV-2 variants via affinity maturation and durable elicitation of IgG antibodies that are resistant to viral escape.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging tick-borne disease in East Asia, and is maintained in enzootic cycles involving ticks and a range of wild animal hosts. Direct transmission of SFTSV from cats and dogs to humans has been identified in Japan, suggesting that veterinarians and veterinary nurses involved in small-animal practice are at occupational risk of SFTSV infection. To characterize this risk, we performed a sero-epidemiological survey in small-animal-practice workers and healthy blood donors in Miyazaki prefecture, which is the prefecture with the highest per capita number of recorded cases of SFTS in Japan. Three small-animal-practice workers were identified as seropositive by ELISA, but one had a negative neutralization-test result and so was finally determined to be seronegative, giving a seropositive rate of 2.2% (2 of 90), which was significantly higher than that in healthy blood donors (0%, 0 of 1000, p &lt, 0.05). The seroprevalence identified here in small-animal-practice workers was slightly higher than that previously reported in other high-risk workers engaged in agriculture and forestry in Japan. Thus, enhancement of small-animal-practice workers’ awareness of biosafety at animal hospitals is necessary for control of SFTSV.

A woman in her fifties showed symptoms of fever, loss of appetite, vomiting, and general fatigue 2 days after she was bitten by a sick cat, which had later died, in Yamaguchi prefecture, western Japan, in June 2016. She subsequently died of multiorgan failure, and an autopsy was performed to determine the cause of death. However, the etiological pathogens were not quickly identified. The pathological features of the patient were retrospectively re-examined, and the pathology of the regional lymph node at the site of the cat bite was found to show necrotizing lymphadenitis with hemophagocytosis. The pathological features were noted to be similar to those of patients reported to have severe fever with thrombocytopenia syndrome (SFTS). Therefore, the lymph node section was retrospectively tested immunohistochemically, revealing the presence of the SFTS virus (SFTSV) antigen. The sick cat showed similar symptoms and laboratory findings similar to those shown in human SFTS cases. The patient had no history of tick bites, and did not have skin lesions suggestive of these. She had not undertaken any outdoor activities. It is highly possible that the patient was infected with SFTSV through the sick cat&rsquo, s bite. If a patient gets sick in an SFTS-endemic region after being bitten by a cat, SFTS should be considered in the differential diagnosis.

SARS-CoV-2 variants have raised concerns regarding resistance to neutralizing antibodies elicited by the parental virus. In the present study, we monitored multiple parameters of neutralizing antibodies in convalescent plasma up to 10 months after the symptom onset. In contrast to marked decline of neutralizing antibody titers, the neutralization potency per antibody increased with time. The antigenic drift of B.1.351 (501Y.V2) and P.1 (501Y.V3) variants conferred resistance to neutralization by convalescent plasma in the early phase; however, the resistance of these variants was attenuated by progressive increase of neutralization potency and breadth over time. Furthermore, this increase in the neutralization potency/breadth were highly correlated with the affinity maturation of IgG antibodies that target virus conserved sites with extended half-lives. Despite a quantitative decline in the neutralizing antibody titers, the serological immunity shapes the neutralization potency and breadth with time, thereby acquiring persistent neutralizing activity toward SARS-CoV-2 variants. Declaration of Interest: All authors declare that they have no competing interests. Ethical Approval: All studies were approved by the Institutional Review Board of the National Institute of Infectious Diseases (#1127, #1132, and #1197). This study was performed in accordance with the Declaration of Helsinki. All volunteers provided written informed consent prior to enrollment.

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigated the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Nine vaccinated macaques exhibited significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs was significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment. Funding: This work was supported by Japan Agency for Medical Research and Development (AMED [JP19fk0108104 to A.K.-T. and JP20nk0101601, JP20jm0110012, JP21fk0410035, JP21fk0108125, and JP21jk0210002 to T.M.]) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (JSPS Grants-in-Aid for Scientific Research [21H02745 to T.M.]). Declaration of Interests: H.I., K.K., R.S., and T.M are the inventors on Patent Cooperation Treaty (PCT) application for SeV-NME vaccine. Authors have no other conflicts of interest to declare. Ethics Approval Statement: Approval by the Committee on the Ethics of Animal Experiments in NIID (permission number: 520001) under the guidelines for animal experiments in accordance with the Guidelines for Proper Conduct of Animal Experiments established by the Science Council of Japan.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain&rsquo, s chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to &beta, lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

BackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble to those of SFTS patients and SFTS-contracted cats shows high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals.Methodology/Principle findingsFour primer sets were newly designed from consensus sequences constructed by 108 strains of SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets were successfully and specifically detected several clades of SFTSV. Their limits of detection are 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.Conclusion/SignificanceThis newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.Author summaryThis study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing antibodies. SFTS cat cases were distributed in the area where SFTS patients have been reported highly, indicating the establishment of the circulation of SFTSV in the environment. These diagnostic assays could be helpful tools to detect and not to miss SFTS animal cases.

Hepatitis E virus (HEV) is known to cause zoonotic infections from pigs, wild boars and deer. Domestic pigs have been used as an experimental animal model in medical research and training; however, the risks of HEV infection from pigs during animal experiments are largely unknown. Here, we retrospectively investigated the seroprevalence and detection rates of viral RNA in 73 domestic pigs (average 34.5 kg) introduced into an animal experimental facility in a medical school during 2012–2016. We detected anti-HEV immunoglobulin G antibodies in 24 of 73 plasma samples (32.9%), though none of the samples were positive for viral RNA. Plasma samples of 18 pigs were sequentially monitored and were classified into four patterns: sustained positive (5 pigs), sustained negative (5 pigs), conversion to positive (6 pigs) and conversion to negative (2 pigs). HEV genomes were detected in 2 of 4 liver samples from pigs that were transported from the same farm during 2016–2017. Two viral sequences of the overlapping open reading frame (ORF) 2/3 region (97 bp) were identical and phylogenetically fell into genotype 3. A 459-bp length of the ORF2 region of an amplified fragment from a pig transported in 2017 was clustered with the wbJYG1 isolate (subgenotype 3b) with 91.5% (420/459 bp) nucleotide identity. Based on our results, we suggest that domestic pigs introduced into animal facilities carry a potential risk of HEV infection to researchers, trainees and facility staff. Continuous surveillance and precautions are important to prevent HEV infection in animal facilities.

Wild boars are a reservoir for many zoonotic pathogens and a good sentinel for surveillance of zoonotic viral infections, but collection of serum samples from wild boars in the field is sometimes difficult and requires special equipment and techniques. In this study, ELISA using meat juices extracted from the heart and diaphragm of wild boars, instead of serum samples, was performed to detect antibodies against zoonotic pathogens, Japanese encephalitis virus and hepatitis E virus. The results of ELISA using meat juice samples were significantly correlated with those using serum samples and meat juice contained one-fifth the antibodies of serum samples. As meat juice is easily collected from wild animals in the field without special equipment and techniques, ELISA using meat juice is a simple and superior method for serological survey of zoonosis among wild animals.

Highlights • EMCV genome was widely found in fecal guanos in Taiwanese, Korean, and Japanese caves. • Miniopterus fuliginosus is the main source of the fecal guano. • It is possible that Miniopterus fuliginosus is one of the natural hosts of EMCV in East Asia., Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

Flying foxes belonging to the genus Pteropus are known to be reservoirs of zoonotic viruses. In this study, we describe the isolation of Pteropine orthoreovirus (PRV) from rectal swab samples of Pteropus vampyrus in Indonesia. PRV is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. Rectal swabs (n = 91) were screened by PCR for PRV and 10 (11%) were positive. Phylogenetic analysis based on nucleotide sequences indicated that the S2, S3, S4, M3, L2, and L3 segments of one isolate (Garut-69) were closely related to previously isolated strains in Indonesia. The remaining gene segments showed both similarity and genetic divergence with other PRV strains, suggesting that re-assortment events had occurred. This is the first report of PRV infection to P. vampyrus in West Java, Indonesia.

Two captive cheetahs from a zoo in Japan died of a severe fever with thrombocytopenia syndrome-like illness. Severe fever with thrombocytopenia syndrome virus, an endemic tickborne phlebovirus, was detected systemically with secretion of infectious viruses into the saliva. These cases highlight the risk for exposure of captive animals to endemic arthropodborne pathogens.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

Several recent studies have reported that various bat species harbor bat hepatitis E viruses (BatHEV) belonging to the family Hepeviridae, which also contains human hepatitis E virus (HEV). The distribution and ecology of BatHEV are not well known. Here, we collected and screened 81 bat fecal samples from nine bat species in Japan to detect BatHEV RNA by RT-PCR using HEV-specific primers, and detected three positive samples. Sequence and phylogenetic analyses indicated that these three viruses were BatHEVs belonging to genus Orthohepevirus D like other BatHEV strains reported earlier in various countries. These data support the first detection of BatHEVs in Japanese microbats, indicating their wide geographical distribution among multiple bat species.

A 67-year-old male veterinarian presented with fatigue, anorexia, and diarrhea. Although there were no tick bite marks, we suspected severe fever with thrombocytopenia syndrome (SFTS) due to bicytopenia, mild disturbance of consciousness, and a history of outdoor activities. Thus, we started immunoglobulin therapy immediately. A serum reverse transcription-polymerase chain reaction (RT-PCR) test for SFTS virus (SFTSV) was positive. The patient had treated a cat with thrombocytopenia 10 days prior to admission. The cat’s serum SFTSV RT-PCR test result was positive, and the whole genome sequences of the patient’s and cat’s SFTSV were identical, suggesting the possibility of transmission from the cat to the patient. Other cases of direct cat-to-human SFTV transmission have been reported recently. Mucous membranes should be protected, including eye protection, in addition to standard precautions, when in contact with any cat with suspected SFTS.

Rabbit hepatitis E virus (HEV) has been detected among rabbits and recently isolated from immunocompromised patients, suggesting zoonotic transmission. In this study, HEV infection among feral rabbits (Oryctolagus cuniculus) was assessed by detection of anti-HEV antibodies and HEV RNA. The prevalence of anti-HEV antibodies in sera was of 33 % (20/60) and HEV RNA was detected from only one of fecal swabs (1.7 %, 1/58). Furthermore, one naive rabbit was intravenously inoculated with the suspension of the HEV-positive fecal specimen, exhibiting persistent HEV shedding in feces, intermittent viremia, seroconversion to anti-HEV IgM and IgG, and high alanine aminotransferase (ALT) values, indicating persistent HEV infection. The isolate JP-59 had a length of 7,282 bp excluding a poly (A) tail and possessed the characteristic 93 bp-insertion in ORF1. Phylogenetic analysis indicated that JP-59 formed a cluster with other rabbit HEV isolates from rabbits and human origin. The JP-59 shared the nucleotide sequence identities less than 87 % with other rabbit HEVs, suggesting that a novel rabbit HEV strain was circulating in Japan.

In Japan, indigenous tick-borne phleboviruses (TBPVs) and their associated diseases first became evident in 2013 by reported human cases of severe fever with thrombocytopenia syndrome (SFTS). In this study, we report a novel member of the genus Phlebovirus designated as Kabuto Mountain virus (KAMV), which was isolated from the ixodid tick Haemaphysalis flava in Hyogo, Japan. A complete viral genome sequencing and phylogenetic analyses showed that KAMV is a novel member of TBPVs, which is closely related to the Uukuniemi and Kaisodi group viruses. However, unlike the Uukuniemi group viruses, the 165-nt intergenic region (IGR) in the KAMV S segment was highly C-rich in the genomic sense and not predicted to form a secondary structure, which are rather similar to those of the Kaisodi group viruses and most mosquito/sandfly-borne phleboviruses. Furthermore, the NSs protein of KAMV was highly divergent from those of other TBPVs. These results provided further insights into the genetic diversity and evolutionary relationships of TBPVs. KAMV could infect and replicate in some rodent and primate cell lines. We evaluated the infectivity and pathogenicity of KAMV in suckling mice, where we obtained a virulent strain after two passages via intracerebral inoculation. This is the first report showing the existence of a previously unrecognized TBPV in Japan, other than the SFTS virus.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne zoonosis, has been rapidly spread in many Asian counties since 2010, which raises the great concern in East Asia. Nevertheless, the infection status of SFTS in Taiwan remains unclear. To investigate the existence of SFTSV in Taiwan, a total of 151 serum samples collected from 31 sheep, 63 bovine and 57 dogs were enrolled this study. Furthermore, 360 adult female Rhipicephalus microplus were also included. One-step RT-nested PCR and IgG ELISA were conducted to test SFTSV specific RNA and antibodies, respectively. The result provided the first evidence of the existence of SFTSV RNA and antibodies in ruminants and ticks in Taiwan.

Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed growth kinetics similar to those shown by the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h postinfection. Furthermore, we found that A72 cells derived from canine fibroblasts permitted FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.IMPORTANCE Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is a lethal disease in cats. Tissue culture-adapted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.

Ticks are one of the arthropods that play an important role in the transmission of numerous pathogens to livestock and humans. We investigated the presence of tick-borne bacteria in 23 Amblyomma varanense that fed on a water monitor (Varanus salvator) in Indonesia. Anaplasmataceae and borreliae were detected by PCR in 17.4% and 95.7% of ticks, respectively. "Candidatus Rickettsia sepangensis", spotted fever group of Rickettsia, was detected in 21.7% of ticks. The water monitor is a common reptile that is widely encountered in city areas in Asian countries. Our results suggested that Am. varanense on water monitor in Indonesia harbored several kinds of bacteria.

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of 35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

Canine coronavirus (CCoV) is an important pathogen that causes enteritis in dogs, but there is no information on CCoV infection in Vietnam. To examine the prevalence of CCoV infection among Vietnamese dogs, 201 serum samples were analyzed by virus-neutralization (VN) test. The results showed that antibody against CCoV-II was present in 87 dogs (43.3%). To detect genes of CCoV, fecal samples collected from 30 diarrheic and 50 healthy dogs were examinated by RT-PCR, confirming that 2 diarrheic dogs and 5 healthy dogs were positive for CCoV. Nucleotide sequences of N-terminal region of spike (S) gene indicated that CCoV strains were divided into two subgenotypes, CCoV-IIa and -IIb, respectively. Furthemore, we succeeded in isolating CCoV/dog/HCM47/2015, the isolate was plaque-purified three times, and 3'-terminal one-third of the genome was analyzed. Interestingly, the plaque-purified virus had a large deletion in ORF3abc and E genes (1,165 nt), and a short deletion in ORF7b gene (60 nt), suggesting that these regions are not necessary for in vitro replication of CCoV. Next, the antigenicity between the isolated CCoV-IIb and the other CCoV-IIa was compared by VN test, revealing that antigenicty of the isolated CCoV is equal or higher than that of the other CCoV. In summary, two subgenotypes of CCoV-II are spreading among Vietnamese dogs. The isolated virus with a large deletion after in vitro passage may be useful for the development of vaccine, owing to its antigenicity and efficient viral growth in vitro.

Serum samples were collected from 385 wild boars between 2010 and 2013 to examine the seroprevalence of influenza A virus (IAV) in Japan. Antibodies against IAV were identified using a commercial kit in 13 wild boars (3.4%). To identify the serotypes, positive sera were examined by virus-neutralization test using representative serotypes and strains. Three wild boars in Yamaguchi and four in Tochigi showed the highest antibody titers against the pandemic H1N1 2009 virus and classical swine H1N1 virus strains, respectively. These data indicate that wild boars may have close contact with humans and domestic pigs and therefore that there is potential for IAVs to reassort in wild boars as they have been shown to do in pigs.

Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a food- and water-borne disease. In developed countries, consumption of meats from pigs, wild boars and deer is a major source of infection. Although HEV and HEV-related viruses have been detected in many animal species, their zoonotic potential and prevalence has not been completely understood. To detect anti-HEV antibody in mammalian species, a simple enzyme-linked immunosorbent assay (ELISA) was established using extract from cells expressing HEV capsid protein and protein A/G as an antigen and a reagent for detection of antibody. Absorbance in the ELISA was compared with those in our previous ELISA using VLPs and anti-swine antibody, suggesting that newly established ELISA was similarly specific and sensitive as the previous ELISA. Seroprevalence of HEV infection among wild boars was examined in Yamaguchi Prefecture, confirming that 111 of 364 wild boars (30.5%) were positive for anti-HEV antibody. Next, this ELISA was applied to humans, dogs, cats, ferrets, raccoons and masked palm civets in Japan, and anti-HEV antibodies were detected in humans, ferrets, dogs and cats. This ELISA is thus useful for serological surveys and comparison of HEV infection among various mammals, including humans.

Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans.