Development of a vesicular stomatitis virus pseudotyped with herpes B virus glycoproteins and its application in a neutralizing antibody detection assay.

Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.

Construction of Vero cell-adapted rabies vaccine strain by five amino acid substitutions in HEP-Flury strain.

Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Cross-Neutralization Activities of Antibodies against 18 Lyssavirus Glycoproteins.

Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.

Establishment of serological neutralizing tests using pseudotyped viruses for comprehensive detection of antibodies against all 18 lyssaviruses.

Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

High Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Infection among the Dog Population in Thailand.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). In Thailand, three human cases of SFTS were reported in 2019 and 2020, but there was no report of SFTSV infection in animals. Our study revealed that at least 16.6% of dogs in Thailand were seropositive for SFTSV infection, and the SFTSV-positive dogs were found in several districts in Thailand. Additionally, more than 70% of the serum samples collected at one shelter possessed virus-neutralization antibodies against SFTSV and the near-complete genome sequences of the SFTSV were determined from one dog in the shelter. The dog SFTSV was genetically close to those from Thailand and Chinese patients and belonged to genotype J3. These results indicated that SFTSV has already spread among animals in Thailand.

Pet Animals Were Infected with SARS-CoV-2 from Their Owners Who Developed COVID-19: Case Series Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among pets owned by coronavirus disease 2019 (COVID-19) patients has been reported around the world. However, how often the animals are exposed to SARS-CoV-2 by their owners is still unclear. We have collected swab samples from COVID-19 patients' pets and performed real-time RT-PCR to detect the viral genome. In total, 8 of 53 dogs (15.1%) and 5 of 34 cats (14.7%) tested positive for the SARS-CoV-2 N gene. The result of a virus neutralization (VN) test also showed VN antibodies in four cats and six dogs. Our results indicate that the virus often passed from infected owners to their pets, which then excreted the virus despite having no or mild clinical signs.

Simple and rapid detection of severe fever with thrombocytopenia syndrome virus in cats by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay using a dried reagent.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes lethal hemorrhagic diseases in human, cats, and dogs. Several human cases involving direct transmission of SFTSV from diseased animals have been reported. Therefore, rapid diagnosis in veterinary clinics is important for preventing animal-to-human transmission. Previously, we developed a simplified reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for human that does not require RNA extraction for detecting the SFTSV genome. In this study, we improved the simplified RT-LAMP assay for cats by introducing a dried reaction reagent and investigated the applicability of this method for diagnosing SFTS in cats. SFTSV RNA was detected in 11 of 12 cats naturally infected with SFTSV by RT-LAMP assay using both liquid and dried reagents. The RT-LAMP assay using liquid and dried reagents was also applicable to the detection of SFTSV genes 3-4 days after challenge in cats experimentally infected with SFTSV. The minimum copy number of SFTSV genes for 100% detection using the RT-LAMP assay with liquid and dried reagents was 4.3 × 104 and 9.6 × 104 copies/mL, respectively. Although the RT-LAMP assay using the dried reagent was less sensitive than that using the liquid reagent, it was sufficiently sensitive to detect SFTSV genes in cats with acute-phase SFTS. As the simplified RT-LAMP assay using a dried reagent enables detection of SFTSV genes more readily than the assay using a liquid reagent, it is applicable for use in veterinary clinics.

Increased Risk of Infection with Severe Fever with Thrombocytopenia Virus among Animal Populations on Tsushima Island, Japan, Including an Endangered Species, Tsushima Leopard Cats.

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.

Lethal Disease in Dogs Naturally Infected with Severe Fever with Thrombocytopenia Syndrome Virus.

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.

Risk assessment of infection with severe fever with thrombocytopenia syndrome virus based on a 10-year serosurveillance in Yamaguchi Prefecture.

In Japan, the first patient with severe fever with thrombocytopenia syndrome was reported in Yamaguchi in 2012. To understand the severe fever with thrombocytopenia syndrome virus (SFTSV) infection in this region, a retrospective surveillance in sika deer and wild boars in Yamaguchi was conducted using a virus-neutralizing (VN) test. The result revealed that 510 of the 789 sika deer and 199 of the 517 wild boars were positive for anti-SFTSV antibodies. Interestingly, seroprevalence in sika deer increased significantly from 2010-2013 to 2015-2020. The SFTSV gene was detected in one of the 229 serum samples collected from sika deer, but not from wild boars. In conclusion, SFTSV had spread among wild animals before 2012 and expanded gradually around 2013-2015 in Yamaguchi.

Roles of raccoons in the transmission cycle of severe fever with thrombocytopenia syndrome virus.

The present study investigated severe fever with thrombocytopenia syndrome virus (SFTSV) infection in raccoons in Wakayama Prefecture from 2007 to 2019. To perform surveillance, an enzyme-linked immunosorbent assay (ELISA) was established, and the sensitivity and specificity of the ELISA were 100% in comparison with a 50% focus-reduction neutralization assay. Using the established ELISA, we performed serosurveillance of SFTSV infection in 2,299 raccoons in Tanabe region, Wakayama Prefecture from 2007 to 2019. The first anti-SFTSV-positive raccoon was captured in October 2009. The seroprevalence of SFTSV infection was <10% between April 2009 and March 2013, 23.9% between April 2013 and March 2014, 37.5% between April, 2014 and March 2015, and over 50% from April 2015. Next, we performed detection of SFTSV genes in sera of raccoons captured in Wakayama Prefecture after April 2013. The results indicated that 2.4% of raccoons were positive for SFTSV genes and that the frequency of SFTSV infection among raccoons between January and March (0.7%) was lower than that between April and June (3.4%). In addition, virus genes were detected from many specimens, including sera and feces of two raccoons, and viral antigens were detected in lymphoid cells in lymphoid follicles in the colon by immunohistochemical staining. In conclusion, SFTSV had recently invaded the area and had rapidly spread among wild animals. The first patient in this area was reported in June 2014, indicating that raccoons are good sentinels for assessing the risk of SFTSV in humans.

Nationwide survey of hepatitis E virus infection among wildlife in Japan.

In Japan, hepatitis E virus (HEV) causes hepatitis in humans through the consumption of raw or undercooked meat, including game meat. In the present study, nationwide surveillance of HEV infection among a total of 5,557 wild animals, including 15 species, was conducted in Japan. The prevalence of anti-HEV antibodies in wild boar was 12.4%, with higher positive rates in big boars (over 50 kg, 18.4%) than in small individuals (less than 30 kg, 5.3%). Furthermore, HEV RNA was more frequently detected in piglets than in older boars. Interestingly, the detection of HEV among wildlife by ELISA and RT-PCR suggested that HEV infection in Sika deer was a very rare event, and that there was no HEV infection among wild animals except for wild boar, Sika deer and Japanese monkeys. In conclusion, wild boar, especially piglets, are at high risk of HEV infection, while other wild animals showed less risk or no risk of HEV transmission.

A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals.

Introduction: Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. Material and Methods: The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Results: Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. Conclusion: The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

A Patient with Severe Fever with Thrombocytopenia Syndrome (SFTS) Infected from a Sick Dog with SFTS Virus Infection.

Severe fever with thrombocytopenia syndrome (SFTS) caused by Dabie bandavirus (formerly SFTS virus, SFTSV), which belongs to the Bandavirus genus (formerly Phlebovirus genus) of the Phenuiviridae family (formerly Bunyaviridae family), is a tick-borne novel bunyavirus infection with high rates of mortality. SFTSV infection was diagnosed virologically in a 4-year-old dog with symptoms of lethargy and anorexia in western Japan in June 2017. The dog's owner, a man in his 40s, had taken care of the sick dog and became sick 10 days after disease onset in the dog, showing symptoms, such as fever, arthralgia, headache, nausea, and vomiting. Total blood cell counts revealed leukocytopenia and thrombocytopenia. He was treated as an outpatient. He had no scars suggesting that he had not been bitten by ticks. He was diagnosed as having SFTS via the detection of IgM and neutralizing antibodies to SFTSV. The patient was directly infected with SFTSV from the SFTSV-infected dog. In conclusion, humans can be at a risk of SFTSV infection through direct contact with sick dogs infected with SFTSV.

Vaccination-infection interval determines cross-neutralization potency to SARS-CoV-2 Omicron after breakthrough infection by other variants.

Background: The immune profile against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by Omicron in individuals with various immune histories. Methods: The neutralization susceptibility of the variants, including Omicron and their ancestors, was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections of Alpha/Delta with multiple time intervals following vaccination. Findings: Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against Omicron was induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions: Immune histories with breakthrough infections can overcome the resistance to infection by Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against Omicron and future variants. Funding: This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).

Retrospective study on the possibility of an SFTS outbreak associated with undiagnosed febrile illness in veterinary professionals and a family with sick dogs in 2003.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-born disease and its animal-to-human transmission has come to attention recently. During our sero-survey of SFTS virus (SFTSV) among veterinary professionals in 2018, a veterinarian and his assistant working in an animal hospital were tested positive by enzyme-linked immunosorbent assay (ELISA). An additional survey implied a cluster of SFTS cases in which four more people, a family who brought two sick dogs to the animal hospital in 2003, were involved. This study aimed at assessing the possibility of animal-to-human transmission of SFTSV in this cluster. METHODS: Retrospective interviews were performed with the owner family of the dogs and their clinical records were obtained from each hospital. SFTSV-IgG were tested by ELISA and virus neutralization test using the sera collected from them in 2018. RESULTS: The interviews revealed that a total of six people, the two veterinary professionals and the owner family who took care of the sick dogs, suffered from SFTS-like symptoms in the same period of time in 2003. All patients did not have tick bite before the onset and all suspected causative agents were excluded by laboratory tests. The serological tests in this study revealed the four owner family members were all positive for SFTSV antibodies. CONCLUSIONS: Considering the extremely low seroprevalence of SFTSV antibodies among inhabitants of the region, the existence of SFTSV antibodies in all these six people presents a possibility that they were involved in an SFTS outbreak originated in the sick dogs in 2003.

Suspected Transmission of Severe Fever with Thrombocytopenia Syndrome Virus from a Cat to a Veterinarian by a Single Contact: A Case Report.

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.

Zoonotic Infection with Oz Virus, a Novel Thogotovirus.

Oz virus is a novel thogotovirus isolated from ticks that causes lethal infection in mice. We conducted serosurveillance of Oz virus infection among humans and wild mammals in Japan using virus-neutralization tests and ELISAs. Results showed that Oz virus may be naturally infecting humans and other mammalian hosts.