Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques.

HTLV-1 infection occurs by cell-to-cell transmission and can induce fatal adult T-cell leukemia (ATL). Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of ten vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the two vaccinated macaques without NAb induction and ten unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

HTLV-1 Proliferation after CD8+ Cell Depletion by Monoclonal Anti-CD8 Antibody Administration in Latently HTLV-1-Infected Cynomolgus Macaques.

Human T-cell leukemia virus type 1 (HTLV-1) induces chronic asymptomatic latent infection with a substantial proviral load but without significant viral replication in vivo. Cumulative studies have indicated involvement of CD8-positive (CD8+) cells, including virus-specific CD8+ T cells in the control of HTLV-1 replication. However, whether HTLV-1 expression from latently infected cells in vivo occurs in the absence of CD8+ cells remains unclear. Here, we examined the impact of CD8+ cell depletion by monoclonal anti-CD8 antibody administration on proviral load in HTLV-1-infected cynomolgus macaques. Five cynomolgus macaques were infected with HTLV-1 by inoculation with HTLV-1-producing cells. Administration of monoclonal anti-CD8 antibody in the chronic phase resulted in complete depletion of peripheral CD8+ T cells for approximately 2 months. All five macaques showed an increase in proviral load following CD8+ cell depletion, which peaked just before the reappearance of peripheral CD8+ T cells. Tax-specific CD8+ T-cell responses were detected in these recovered CD8+ T cells. Importantly, anti-HTLV-1 antibodies also increased after CD8+ cell depletion, indicating HTLV-1 antigen expression. These results provide evidence indicating that HTLV-1 can proliferate from the latent phase in the absence of CD8+ cells and suggest that CD8+ cells are responsible for the control of HTLV-1 replication. IMPORTANCE HTLV-1 can cause serious diseases such as adult T-cell leukemia (ATL) in humans after chronic asymptomatic latent infection with substantial proviral load. Proviruses are detectable in peripheral lymphocytes in HTLV-1 carriers, and the association of a higher proviral load with a higher risk of disease progression has been observed. However, neither substantial viral structural protein expression nor viral replication was detectable in vivo. Cumulative studies have indicated involvement of CD8+ cells, including virus-specific CD8+ T cells in the control of HTLV-1 replication. In the present study, we showed that CD8+ cell depletion by monoclonal anti-CD8 antibody administration results in HTLV-1 expression and an increase in proviral load in HTLV-1-infected cynomolgus macaques. Our results indicate that HTLV-1 can proliferate in the absence of CD8+ cells, suggesting that CD8+ cells are responsible for the control of HTLV-1 replication. This study provides insights into the mechanism of virus-host immune interaction in latent HTLV-1 infection.

Low Replication Efficiency of a Japanese Rabbit Hepatitis E Virus Strain in the Human Hepatocarcinoma Cell Line PLC/PRF/5.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus.

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Mongolia Gerbils Are Broadly Susceptible to Hepatitis E Virus.

Although cell culture systems for hepatitis E virus (HEV) have been established by using cell lines such as PLC/PRF/5 and A549, small-animal models for this virus are limited. Since Mongolia gerbils are susceptible to genotype 1, 3 and 4 HEV (HEV-1, HEV-3 and HEV4), we intraperitoneally inoculated Mongolia gerbils with HEV-5, HEV-7, HEV-8, rabbit HEV or rat HEV in addition to the above three genotypes to investigate the infectivity and to assess whether Mongolia gerbil is an appropriate animal model for HEV infection. The results indicated that (i) HEV-5 and rat HEV were effectively replicated in the Mongolia gerbils in the same manner as HEV-4: large amounts of the viral RNA were detected in the feces and livers, and high titers of the serum anti-HEV IgG antibodies were induced in all animals. The feces were shown to contain HEV that is infectious to naïve gerbils. Furthermore, HEV-4, HEV-5 and rat HEV were successfully transmitted to the gerbils by oral inoculation. (ii) Although the viral RNA and serum anti-HEV IgG antibodies were detected in all animals inoculated with HEV-1 and HEV-8, both titers were low. The viral RNA was detected in the feces collected from two of three HEV-3-inoculated, and one of three HEV-7-inoculated gerbils, but the titers were low. The serum antibody titers were also low. The viruses excreted into the feces of HEV-1-, HEV-3-, HEV-7- and HEV-8-inoculated gerbils failed to infect naïve Mongolia gerbils. (iii) No infection sign was observed in the rabbit HEV-inoculated gerbils. These results demonstrated that Mongolia gerbils are broadly susceptible to HEV, and their degree of sensitivity was dependent on the genotype. Mongolia gerbils were observed to be susceptible to not only HEVs belonging to HEV-A but also to rat HEV belonging to HEV-C1, and thus Mongolia gerbil could be useful as a small-animal model for cross-protection experiments between HEV-A and HEV-C1. Mongolia gerbils may also be useful for the evaluation of the efficacy of vaccines against HEV.

Neutralizing-antibody-independent SARS-CoV-2 control correlated with intranasal-vaccine-induced CD8+ T cell responses.

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (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 investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is 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.

Experimental Cross-Species Transmission of Rat Hepatitis E Virus to Rhesus and Cynomolgus Monkeys.

Rat hepatitis E virus (rat HEV) was first identified in wild rats and was classified as the species Orthohepevirus C in the genera Orthohepevirus, which is genetically different from the genotypes HEV-1 to HEV-8, which are classified as the species Orthohepevirus A. Although recent reports suggest that rat HEV transmits to humans and causes hepatitis, the infectivity of rat HEV to non-human primates such as cynomolgus and rhesus monkeys remains controversial. To investigate whether rat HEV infects non-human primates, we inoculated one cynomolgus monkey and five rhesus monkeys with a V-105 strain of rat HEV via an intravenous injection. Although no significant elevation of alanine aminotransferase (ALT) was observed, rat HEV RNA was detected in fecal specimens, and seroconversion was observed in all six monkeys. The partial nucleotide sequences of the rat HEV recovered from the rat HEV-infected monkeys were identical to those of the V-105 strain, indicating that the infection was caused by the rat HEV. The rat HEV recovered from the cynomolgus and rhesus monkeys successfully infected both nude and Sprague-Dawley rats. The entire rat HEV genome recovered from nude rats was identical to that of the V-105 strain, suggesting that the rat HEV replicates in monkeys and infectious viruses were released into the fecal specimens. These results demonstrated that cynomolgus and rhesus monkeys are susceptible to rat HEV, and they indicate the possibility of a zoonotic infection of rat HEV. Cynomolgus and rhesus monkeys might be useful as animal models for vaccine development.

A Human Intestinal Cell Line Suitable for the Propagation of Human Parechovirus Type 1 to 6 with a Clear Cytopathic Effect.

Human parechoviruses (HPeVs) are being increasingly recognized as pathogens that cause mild-to-life-threatening diseases in children and adults. Recently, nucleic acid detection has become the mainstream method for pathogen detection. However, virus isolation is important for virus detection and further virologic characterization studies, and securing human pathogenic virus bioresources. We recently explored conventional cell lines suitable for human sapovirus isolation and accidentally identified a human duodenal cell line, HuTu80, that supported efficient growth of human parechovirus type 3 (HPeV-3) with clear cytopathic effects (CPE). Subsequently, we confirmed that all representative prototype HPeV type 1-6 strains were propagated efficiently in HuTu80 cells with clear CPE within 4 days. Another human ileocecal cell line, HCT-8 (HRT-18), also supports HPeV propagation except for HPeV-3. Titer values in HuTu80 and HCT-8 reached approximately 6.83-8.83 and 6.50-8.17 log10 50% tissue culture infectious dose/50 µL, respectively, when inoculated with multiplicity of infection of 0.0025. Previously reported cell lines likely support HPeV types 1-6 with different efficiency, especially for HPeV-3. In summary, HuTu80 can be used as an additional cell line for HPeV isolation, propagation with a clear CPE to produce a high titer value and for the virus neutralization assays.

A Cross-Species Transmission of a Camel-Derived Genotype 8 Hepatitis E Virus to Rabbits.

Novel genotypes of hepatitis E virus (HEV), i.e., HEV-5, HEV-7, and HEV-8, have been identified in wild boar, dromedary camels, and Bactrian camels, respectively, and they transmit to cynomolgus monkeys in a trans-species manner, raising the potential for zoonotic infection. Rabbits are the natural reservoir for rabbit HEV, but they are also susceptible to HEV-3 and HEV-4. It has been unknown whether rabbits are susceptible to HEV-5, HEV-7, and HEV-8. To investigate the infectivity of novel HEVs in rabbits and to assess whether rabbits are appropriate animal models for these HEVs, we inoculated Japanese white rabbits with HEV-5, HEV-7, and HEV-8, respectively. We observed that viral RNA was present in the fecal specimens of the HEV-8-inoculated rabbits and anti-HEV IgG antibodies were present in its sera, although anti-HEV IgM was undetectable and no significant elevation of ALT was observed. These results indicated that HEV-8 crossed species and infected the rabbits. No evidence for replication was observed in HEV-5 and HEV-7, suggesting that rabbits are not susceptible to these genotypes. The antibodies elicited in the HEV-8-infected rabbits did not protect them from the rabbit HEV challenge, suggesting that the antigenicity differs between HEV-8 and rabbit HEV. Antigenic analyses demonstrated that anti-HEV-8 antibodies reacted more strongly with homologous HEV-8 virus-like particles (VLPs) compared to heterologous rabbit HEV VLPs, but anti-rabbit HEV antibody had similar reactivity to the VLPs of rabbit HEV and HEV-8, suggesting that HEV-8 lacks some epitope(s) that exist in rabbit HEV and induced the neutralizing antibodies against rabbit HEV.

Genotype-Dependent Immunogenicity of Dengue Virus Type 2 Asian I and Asian/American Genotypes in Common Marmoset (Callithrix jacchus): Discrepancy in Neutralizing and Infection-Enhancing Antibody Levels between Genotypes.

Owing to genotype-specific neutralizing antibodies, analyzing differences in the immunogenic variation among dengue virus (DENV) genotypes is central to effective vaccine development. Herein, we characterized the viral kinetics and antibody response induced by DENV type 2 Asian I (AI) and Asian/American (AA) genotypes using marmosets (Callithrix jacchus) as models. Two groups of marmosets were inoculated with AI and AA genotypes, and serial plasma samples were collected. Viremia levels were determined using quantitative reverse transcription-PCR, plaque assays, and antigen enzyme-linked immunosorbent assay (ELISA). Anti-DENV immunoglobulin M and G antibodies, neutralizing antibody titer, and antibody-dependent enhancement (ADE) activity were determined using ELISA, plaque reduction neutralization test, and ADE assay, respectively. The AI genotype induced viremia for a longer duration, but the AA genotype induced higher levels of viremia. After four months, the neutralizing antibody titer induced by the AA genotype remained high, but that induced by the AI genotype waned. ADE activity toward Cosmopolitan genotypes was detected in marmosets inoculated with the AI genotype. These findings indicate discrepancies between heterologous genotypes that influence neutralizing antibodies and viremia in marmosets, a critical issue in vaccine development.

Immunogenicity and Antigenicity of Rabbit Hepatitis E Virus-Like Particles Produced by Recombinant Baculoviruses.

Rabbit hepatitis E virus (HEV) is a novel HEV belonging to genotype 3 (HEV-3) in the Orthohepevirus A species of the genus Hepevirus, family Hepeviridae. Rabbit HEV was originally isolated from rabbits and found to cause zoonotic infection. Although rabbit HEV can be successfully grown in culture with several cell lines, including the human carcinoma cell line PLC/PRF/5, it is difficult to obtain the large amounts of viral antigen required for diagnosis and vaccine development. In this study, we expressed N-terminal 13 and 111 aa-truncated rabbit HEV ORF2 proteins using recombinant baculoviruses and obtained two types of virus-like particles (VLPs), RnVLPs and RsVLPs with ~35 and 24 nm diameter, respectively. Anti-rabbit HEV IgG antibodies were induced in high titer by immunizing rabbits with RnVLPs or RsVLPs. The antibody secretion in the serum persisted more than three years. RsVLPs showed stronger antigenic cross-reactivity against HEV-1, HEV-3 and HEV-4 than rat HEV. Moreover, anti-RsVLPs antibodies neutralized not only the cognate virus but also HEV-1, HEV-3 and HEV-4 ex vivo, indicating that rabbit HEV had the same serotype as human HEVs. In contrast, the antibody did not block rat HEV infection, demonstrating that rat HEV belonged to a different serotype. Animal experiments indicated that immunization with either RnVLPs or RsVLPs completely protected the rabbits from challenge by rabbit HEV, suggesting that the VLPs are candidates for rabbit HEV vaccine development.

Generation of a Bactrian camel hepatitis E virus by a reverse genetics system.

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA-transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques.

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.

Persistent infection with a rabbit hepatitis E virus created by a reverse genetics system.

Rabbit hepatitis E virus (HEV) is a novel zoonotic infectious agent. Although a cell culture system to grow the virus has been established, there is currently no reverse genetics system for generating the virus. In this study, capped genomic rabbit HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells, and the recovered viruses were subsequently passaged in the cells. The cell culture supernatant was capable of infecting rabbits negative for anti-HEV antibody by intravenous and oral inoculation, indicating that rabbit HEV generated by the reverse genetics system is infectious. Genome-wide analyses indicated that no nucleotide sequence change occurred in the virus genomes that were recovered from the cell culture supernatant after transfection and passaged one time or in the virus genomes recovered from faecal specimens of the infected rabbits. Ribavirin, a broad-spectrum anti-viral inhibitor, efficiently abrogated virus replication ex vivo and transiently suppressed the virus growth in the virus-infected rabbits, suggesting that this reagent is a candidate for therapeutic treatment. In addition, transmission of rabbit HEV to rabbits caused persistent infection, suggesting that the virus-infected rabbit could be an animal model for virus-induced hepatitis. The infectious rabbit HEV produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of viral hepatitis.

Characterization of a Novel Rat Hepatitis E Virus Isolated from an Asian Musk Shrew (Suncus murinus).

The Asian musk shrew (shrew) is a new reservoir of a rat hepatitis E virus (HEV) that has been classified into genotype HEV-C1 in the species Orthohepevirus C. However, there is no information regarding classification of the new rat HEV based on the entire genome sequences, and it remains unclear whether rat HEV transmits from shrews to humans. We herein inoculated nude rats (Long-Evans rnu/rnu) with a serum sample from a shrew trapped in China, which was positive for rat HEV RNA, to isolate and characterize the rat HEV distributed in shrews. A rat HEV strain, S1129, was recovered from feces of the infected nude rat, indicating that rat HEV was capable of replicating in rats. S1129 adapted and grew well in PLC/PRF/5 cells, and the recovered virus (S1129c1) infected Wistar rats. The entire genomes of S1129 and S1129c1 contain four open reading frames and share 78.3-81.8% of the nucleotide sequence identities with known rat HEV isolates, demonstrating that rat HEVs are genetically diverse. We proposed that genotype HEV-C1 be further classified into subtypes HEV-C1a to HEV-C1d and that the S1129 strain circulating in the shrew belonged to the new subtype HEV-C1d. Further studies should focus on whether the S1129 strain infects humans.

Characterization of a Novel Simian Sapelovirus Isolated from a Cynomolgus Monkey using PLC/PRF/5 Cells.

We isolated a novel simian sapelovirus (SSV), Cam13, from fecal specimen of a cynomolgus monkey by using PLC/PRF/5 cells. The SSV infection of the cells induced an extensive cytopathic effect. Two types of virus particles with identical diameter (~32 nm) but different densities (1.348 g/cm3 and 1.295 g/cm3) were observed in the cell culture supernatants. The RNA genome of Cam13 possesses 8,155 nucleotides and a poly(A) tail, and it has a typical sapelovirus genome organization consisting of a 5' terminal untranslated region, a large open reading frame (ORF), and a 3' terminal untranslated region. The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins (VP1, VP2, VP3, and VP4) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). We confirmed that 293 T, HepG2/C3A, Hep2C, Huh7 and primary cynomolgus monkey kidney cells were susceptible to SSV infection. In contrast, PK-15, Vero, Vero E6, RD-A, A549, and primary green monkey kidney cells were not susceptible to SSV infection. We established an ELISA for the detection of IgG antibodies against SSV by using the virus particles as the antigen. A total of 327 serum samples from cynomolgus monkeys and 61 serum samples from Japanese monkeys were examined, and the positive rates were 88.4% and 18%, respectively. These results demonstrated that SSV infection occurred frequently in the monkeys. Since Cam13 shared 76.54%-79.52% nucleotide sequence identities with other known SSVs, and constellated in a separate lineage in the phylogeny based on the entire genome sequence, we propose that Cam13 is a new genotype of the simian sapelovirus species.

High Prevalence of Hepatitis E Virus Infection in Imported Cynomolgus Monkeys in Japan.

Cynomolgus monkeys are important experimental animals for hepatitis E virus (HEV) infection. In Japan, cynomolgus monkeys are mainly imported from Asian countries for use at animal facilities and institutions. However, the status of HEV infection in cynomolgus monkeys remains unclear. Overall, 187 pairs of serum and fecal samples were collected from cynomolgus monkeys (Macaca fascicularis) imported from China and Cambodia to detect anti-HEV immunoglobulin (Ig) G and IgM antibodies, as well as HEV RNA. Based on an enzyme-linked immunosorbent assay using HEV-like particles derived from genotype 3 HEV as the antigen, 183 of 187 (97.9%) and 102 of 187 (54.5%) samples tested positive for anti-HEV IgG and IgM antibodies, respectively. In contrast, all 45 serum samples collected from cynomolgus monkeys bred and grown at the Tsukuba Primate Research Center, Japan tested negative for both antibodies. However, real-time quantitative reverse transcription polymerase chain reaction detected no HEV RNA in any of the 187 serum and fecal samples. These results strongly indicated that HEV infection is common in imported cynomolgus monkeys. A source of HEV-free monkeys for HEV studies is urgently needed.

Genotype 5 Hepatitis E Virus Produced by a Reverse Genetics System Has the Potential for Zoonotic Infection.

Neither an animal model nor a cell culture system has been established for the genotype 5 hepatitis E virus (G5 HEV), and the pathogenicity, epidemiology, and replication mechanism of the virus remain unclear. In this study, we used a reverse genetics system to generate G5 HEV and examined the possibility of zoonotic infection. Capped and uncapped genomic G5 HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells. Infectious G5 HEV was recovered from the capped G5 HEV RNA-transfected PLC/PRF/5 cells and the subsequently passaged cells. G5 HEV was also recovered from uncapped G5 HEV-transfected PLC/PRF/5 cells after a longer lag phase, suggesting that the 5'-cap structure is not essential but affected the efficiency of G5 HEV replication. G5 HEV infection was neutralized not only by anti-G5 HEV-like particles (HEV-LPs) antibody, but also by anti-G1, anti-G3, anti-G4, and anti-G7 HEV-LPs antibodies. G5 HEV was capable of infecting cynomolgus monkeys negative for anti-HEV antibody but not animals positive for anti-G7 HEV immunoglobulin G (IgG), indicating that cynomolgus monkeys were susceptible to G5 HEV, and the serotype of G5 HEV was identical to that of G7 HEV and human HEVs. Moreover, G5 HEV replication was efficiently inhibited by ribavirin and partially inhibited by sofosbuvir. Conclusion: Infectious G5 HEV was produced using a reverse genetics system, and the antigenicity was identical to that of human HEVs and G7 HEV. Transmission of G5 HEV to primates was confirmed by an experimental infection, providing evidence of the possibility of zoonotic infection by G5 HEV.

Genotype-specific and cross-reactive neutralizing antibodies induced by dengue virus infection: detection of antibodies with different levels of neutralizing activities against homologous and heterologous genotypes of dengue virus type 2 in common marmosets (Callithrix jacchus).

BACKGROUND: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus). METHODS: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay. RESULTS: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes. CONCLUSION: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.