Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants.

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.

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.

Impact of Reinfection with SARS-CoV-2 Omicron Variants in Previously Infected Hamsters.

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. IMPORTANCE The 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.

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.

Successful Treatment with Letermovir in a Heart Transplant Recipient with UL97 Mutation Ganciclovir-Resistant Cytomegalovirus Colitis and Viremia.

Currently available anti-cytomegalovirus (CMV) agents are sometimes poorly tolerated, owing to their side effects. Letermovir is a novel anti-CMV drug that is only approved for CMV prophylaxis in hematopoietic stem cell transplant recipients, with fewer side effects. We report the case of a heart transplant recipient with UL97 mutation (L595F) ganciclovir-resistant cytomegalovirus colitis who was successfully treated with off-label use of letermovir. In treating CMV infection or disease with letermovir, a transient rise or lag in the clearance of CMV-DNA polymerase chain reaction levels has been observed. Our case suggests that CMV-pp65 antigenemia can be an additional marker of treatment efficacy.

Heparan sulfate 3-O-sulfotransferase 4 is genetically associated with herpes zoster and enhances varicella-zoster virus-mediated fusogenic activity.

Acute pain that is associated with herpes zoster (HZ) can become long-lasting neuropathic pain, known as chronic post-herpetic neuralgia (PHN), especially in the elderly. HZ is caused by the reactivation of latent varicella-zoster virus (VZV), whereas PHN is not attributed to ongoing viral replication. Although VZV infection reportedly induces neuronal cell fusion in humans, the pathogenesis of PHN is not fully understood. A genome-wide association study (GWAS) revealed significant associations between PHN and the rs12596324 single-nucleotide polymorphism (SNP) of the heparan sulfate 3-O-sulfotransferase 4 (HS3ST4) gene in a previous study. To further examine whether this SNP is associated with both PHN and VZV reactivation, associations between rs12596324 and a history of HZ were statistically analyzed using GWAS data. HZ was significantly associated with the rs12596324 SNP of HS3ST4, indicating that HS3ST4 is related to viral replication. We investigated the influence of HS3ST4 expression on VZV infection in cultured cells. Fusogenic activity after VZV infection was enhanced in cells with HS3ST4 expression by microscopy. To quantitatively evaluate the fusogenic activity, we applied cytotoxicity assay and revealed that HS3ST4 expression enhanced cytotoxicity after VZV infection. Expression of the VZV glycoproteins gB, gH, and gL significantly increased cytotoxicity in cells with HS3ST4 expression by cytotoxicity assay, consistent with the fusogenic activity as visualized by fluorescence microscopy. HS3ST4 had little influence on viral genome replication, revealed by quantitative real-time polymerase chain reaction. These results suggest that HS3ST4 enhances cytotoxicity including fusogenic activity in the presence of VZV glycoproteins without enhancing viral genome replication.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo.

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.

Association of human cytomegalovirus (HCMV) neutralizing antibodies with antibodies to the HCMV glycoprotein complexes.

BACKGROUND: Human cytomegalovirus (HCMV) causes asymptomatic infections, but also causes congenital infections when women were infected with HCMV during pregnancy, and life-threatening diseases in immunocompromised patients. To better understand the mechanism of the neutralization activity against HCMV, the association of HCMV NT antibody titers was assessed with the antibody titers against each glycoprotein complex (gc) of HCMV. METHODS: Sera collected from 78 healthy adult volunteers were used. HCMV Merlin strain and HCMV clinical isolate strain 1612 were used in the NT assay with the plaque reduction assay, in which both the MRC-5 fibroblasts cells and the RPE-1 epithelial cells were used. Glycoprotein complex of gB, gH/gL complexes (gH/gL/gO and gH/gL/UL128-131A [PC]) and gM/gN were selected as target glycoproteins. 293FT cells expressed with gB, gM/gN, gH/gL/gO, or PC, were prepared and used for the measurement of the antibody titers against each gc in an indirect immunofluorescence assay (IIFA). The correlation between the IIFA titers to each gc and the HCMV-NT titers was evaluated. RESULTS: There were no significant correlations between gB-specific IIFA titers and the HCMV-NT titers in epithelial cells or between gM/gN complex-specific IIFA titers and the HCMV-NT titers. On the other hand, there was a statistically significant positive correlation between the IIFA titers to gH/gL complexes and HCMV-NT titers. CONCLUSIONS: The data suggest that the gH/gL complexes might be the major target to induce NT activity against HCMV.

Differences in the Likelihood of Acyclovir Resistance-Associated Mutations in the Thymidine Kinase Genes of Herpes Simplex Virus 1 and Varicella-Zoster Virus.

Acyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene.

Differences in the effects of mutations in GP131, a guinea pig cytomegalovirus homologue of pentameric complex component UL130, on macrophage and epithelial cell infection.

As congenital cytomegalovirus (CMV) infection is the major cause of developmental abnormalities in children, the development of effective vaccines is critical to public health. Recent studies have demonstrated that the pentameric complex (Pentamer) of glycoproteins, which is required for human CMV infection of endothelial and epithelial cells, could be a potent vaccine antigen. As guinea pig CMV (GPCMV) infects congenitally and encodes homologues of all Pentamer components, GPCMV models are considered to be useful for the development of vaccine strategies. Here, to clarify the precise requirement of GP131, one of the GPCMV Pentamer components, for the infection of epithelial cells and macrophages, we prepared several mutants with a charged amino acid-to-alanine alteration in GP131 and found some differences in the effects of the mutations on the infection of the two cell types, suggesting the existence of cell type-dependent recognition or function of Pentamer in GPCMV infection.

Vaccine Development for Cytomegalovirus.

The development of a cytomegalovirus (CMV) vaccine has become a top priority due to its potential cost-effectiveness and associated public health benefits. However, there are a number of challenges facing vaccine development including the following: (1) CMV has many mechanisms for evading immune responses , and natural immunity is not perfect, (2) the immune correlates for protection are unclear, (3) a narrow range of CMV hosts limits the value of animal models, and (4) the placenta is a specialized organ formed transiently and its immunological status changes with time. In spite of these limitations, several types of CMV vaccine candidate, including live-attenuated, DISC , subunit, DNA, vectored, and peptide vaccines, have been developed or are currently under development. The recognition of the pentameric complex as the major neutralization target and identification of various strategies to block viral immune response evasion mechanisms have opened new avenues to CMV vaccine development. Here, we discuss the immune correlates for protection, the characteristics of the various vaccine candidates and their clinical trials, and the relevant animal models.

Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients.

Background: Antiviral-resistant herpes simplex virus type 1 (HSV-1) has been recognized as an emerging clinical problem among patients undergoing hematopoietic stem cell transplantation (HSCT). Methods: A prospective observational study was conducted at a hematological center over a 2-year period. Oropharyngeal swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation. The HSV-1 isolates were tested for sensitivity to acyclovir (ACV). The prognosis of patients with ACV-resistant (ACVr) HSV-1 and the genetic background of the ACVr HSV-1 isolates were assessed. Results: Herpes simplex virus type 1 was isolated in 39 of 268 (15%) HSCT patients within 100 days after transplantation. Acyclovir-resistant HSV-1 emerged in 11 of these 39 patients (28%). The 100-day death rates of HSCT patients without HSV-1 shedding, those with only ACV-sensitive HSV-1 shedding, and those with ACVr HSV-1 shedding were 31%, 39%, and 64%, respectively. Patients with HSV-1, including ACVr HSV-1, shedding showed a significantly higher mortality rate. Relapsed malignancies were a significant risk factor for the emergence of ACVr HSV-1. Acyclovir resistance was attributable to viral thymidine kinase and DNA polymerase mutations in 6 and 5 patients, respectively. Conclusions: Herpes simplex virus type 1, including ACVr HSV-1, shedding was associated with poorer outcome in HSCT patients, even if HSV disease did not always occur. Patients with relapsed malignancies were at especially high risk for the emergence of ACVr HSV-1.

Association between sensitivity of viral thymidine kinase-associated acyclovir-resistant herpes simplex virus type 1 and virulence.

BACKGROUND: Acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) infections are concern in immunocompromised patients. Most clinical ACVr HSV-1 isolates have mutations in the viral thymidine kinase (vTK) genes. The vTK-associated ACVr HSV-1 shows reduced virulence, but the association between the level of resistance and the virulence of the vTK-associated ACVr HSV-1 is still unclear. METHODS: The virulence in mice of 5 vTK-associated ACVr HSV-1 clones with a variety of ACV sensitivities, when inoculated through intracerebral and corneal routes, was evaluated in comparison with ACV-sensitive (ACVs) parent HSV-1 TAS. RESULTS: Although all the 5 ACVr HSV-1 clones and ACVs HSV-1 TAS showed a similar single-step growth capacity in vitro, the virulence of ACVr HSV-1 clones significantly decreased. A 50% lethal dose (LD50) of each clone was closely correlated with 50% inhibitory concentrations (IC50), demonstrating that the higher the ACV-sensitvity, the the higher the virulence among the ACVr clones. One of the ACVr HSV-1 clones with a relatively low IC50 value maintained similar virulence to that of the parent TAS. The infection in mice with ACVr HSV-1 due to a single amino acid substitution in vTK induced local diseases, keratitis and dermatitis, while vTK-deficient clone did not. CONCLUSIONS: A statistically significant correlation between the virulence and susceptibility to ACV among ACVr HSV-1 clones was demonstrated.

Guinea pig cytomegalovirus GP129/131/133, homologues of human cytomegalovirus UL128/130/131A, are necessary for infection of monocytes and macrophages.

The GP129, GP131 and GP133 genes of guinea pig cytomegalovirus (GPCMV) are homologues of human cytomegalovirus UL128, UL130 and UL131A, respectively, which are essential for infection of endothelial and epithelial cells, and for viral transmission to leukocytes. Our previous study demonstrated that a GPCMV strain lacking the 1.6 kb locus that contains the GP129, GP131 and GP133 genes had a growth defect in animals. Here, we demonstrated that the WT strain, but not the 1.6 kb-deleted strain, formed capsids in macrophages prepared from the peritoneal fluid. To understand the mechanism, we prepared GPCMV strains defective in each of GP129, GP131 and GP133, and found that they were all essential for the infection of peritoneal, splenic and PBMC-derived macrophages/monocytes, and for expression of immediate-early antigens in the macrophages/monocytes, although they were dispensable for infection of fibroblasts. Monocyte/macrophage tropism could be one of the important determinants for viral dissemination in vivo.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Successful treatment of acyclovir-resistant herpes simplex virus infection with amenamevir in a patient who received umbilical cord blood transplantation for T-cell prolymphocytic leukemia.

A 34-year-old woman received umbilical cord blood transplantation for refractory T-cell prolymphocytic leukemia after salvage therapy with alemtuzumab. She developed right angular cheilitis on the 46th day after transplantation, which worsened after receiving systemic steroid therapy for extensive chronic graft versus host disease. The treatment dosage of acyclovir (ACV), ganciclovir, and vidarabine ointment was not effective due to ACV-resistant mutations of the herpes simplex virus type 1 (HSV-1) in the thymidine kinase domain. Foscarnet is expected to be effective against ACV-resistant HSV-1 infection. However, it could not be used because the patient developed renal dysfunction. Several viral thymidine kinase mutations related to ACV resistance were found in the patient's sample. Nevertheless, amenamevir, a helicase-primase complex inhibitor, was effective in our patient who was significantly immunocompromised after allogeneic hematopoietic stem cell transplantation (allo-HSCT). ACV-resistant HSV infection after allo-HSCT is an rare but important complication in the era of low-dose long-term ACV prophylaxis. To date, there is no established treatment against ACV-resistant HSV infection. This case report showed that amenamevir could be a promising treatment option for ACV-resistant HSV infection in patients with renal failure after allo-HSCT.

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.