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 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.

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.

Full genome-based characterization of G4P[6] rotavirus strains from diarrheic patients in Thailand: Evidence for independent porcine-to-human interspecies transmission events.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

Isolation and Characterization of Hepatitis E Virus Subtype 4b Using a PLC/PRF/5 Cell-Derived Cell Line Resistant to Porcine Sapelovirus Infection.

The human hepatocarcinoma cell line PLC/PRF/5 is susceptible to hepatitis E virus (HEV) infection and is used for HEV isolation. It is difficult to use the cell line for this purpose directly from fecal specimens of swine or wild boar contaminated with porcine sapelovirus (PSV) because PSV infection results in rapid and extensive cytopathic effects in PLC/PRF/5 cells, interrupting the growth of HEV. Herein, we used a PSV infection-resistant cell line, N1380, derived from PLC/PRF/5 cells, and successfully isolated a HEV-4b strain from a PSV-positive swine fecal specimen. Our results indicated that N1380 cells are a useful tool for the isolation of HEV from swine or wild boar fecal specimens, even when the cells are co-infected with PSV.

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.

Hepatitis E virus infection in 6-month-old pigs in Taiwan.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Genotype 3 (G3) and 4 (G4) HEV have recently been identified in and isolated from swine as the main HEV genotypes worldwide. However, there is limited information on HEV infection status among pigs in Taiwan, especially pigs in the stage before transportation to the slaughterhouse. To determine the frequency of HEV infection among pigs in Taiwan, we detected and quantified HEV RNA contained in 295 fecal specimens collected from 6-month-old pigs bred in 30 pig farms located in 8 counties. We found that 25.1% (74/295) of the fecal specimens were positive for HEV RNA by a quantitative real-time reverse transcription-polymerase chain reaction, and the copy number ranged from 2.3 × 103 to 2.08 × 107 copies/g. Amplification of a 338 bp sequence in ORF2 was achieved in 16 of 74 HEV RNA-positive samples, and their nucleotide sequences were determined. Two HEV sequences appeared to belong to subtype 3a of G3 and the remaining 14 HEV sequences belonged to subtype 4b of G4 (G4b). The entire genome sequence of two G4b HEVs was obtained by next-generation sequence analyses, and the phylogenetic analyses indicated that unique G4b HEVs were circulating in pig farms in Taiwan. In the present study, we found that both G3 and G4 HEVs were circulating in Taiwanese pig farms and G4b was the predominant subtype. In addition, the relatively high detection frequency of HEV RNA in the 6-month-old pigs indicated that Taiwanese pigs just before transportation to the slaughterhouse are at risk of carrying HEVs, and thus thorough cooking or heating of pork meat or organs is needed before consumption in Taiwan and possibly in other countries as well.

Immunization of human hepatitis E viruses conferred protection against challenge by a camel hepatitis E virus.

Dromedary camel hepatitis E virus is a novel HEV that belongs to the family Hepeviridae, and is classified as genotype 7 HEV (HEV-7). Since HEV-7 is transmitted from camels to humans and causes acute hepatitis E, this virus is a non-negligible pathogen for zoonosis, and a vaccine against HEV-7 infection is urgently needed. Here, we first intravenously inoculated HEV-7 to rhesus monkeys to explore the susceptibility, and we established an animal model. We then used virus-like particles (VLPs) of HEV-1 (HEV-1 VLPs) and HEV-3 (HEV-3 VLPs), a candidate hepatitis E vaccine, to intramuscularly inoculate rhesus monkeys. The monkeys elicited IgG antibody titers as high as >1:102,400 against heterologous HEV-7 without any adjuvants. The HEV-1 VLPs and HEV-3 VLPs-immunized monkeys were challenged intravenously with HEV-7, and they were protected completely from the infection, demonstrating that these VLPs could be a usable vaccine against HEV-7 infection. We also observed that HEV-7-infected rhesus monkeys did not show any liver damage during these experiments. Further efforts are necessary to establish an animal model for investigation of the pathogenesis of hepatitis E caused by HEV-7 infection.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

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.

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.

Full genome characterization of novel DS-1-like G9P[8] rotavirus strains that have emerged in Thailand.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand.

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.

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.

The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection.

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV.

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.

Current status of hepatitis E virus infection at a rhesus monkey farm in China.

Rhesus and several other species of monkeys are susceptible to genotypes of hepatitis E virus (HEV), and these species are thus commonly used as animal models for experimental HEV infection. However, information regarding HEV infection in monkeys in nature or at monkey farms is limited. To investigate the status of HEV infection in rhesus monkeys at farms, we collected 548 serum and 48 fecal samples from a rhesus monkey farm in China, and analyzed their levels of anti-HEV IgG antibodies and HEV RNAs. An enzyme-linked immunosorbent assay using genotype 3 HEV-like particles as antigen revealed anti-HEV IgG-positivity in 388 (70.8%) monkeys. The antibody-positive rates in the 1-year-old and 2-year-old monkeys were significantly lower than those in monkeys >3 years old. The antibody-positive rate was greatly increased from 7.4% in the 2-year-old monkeys to 100% in the 3-year-olds, suggesting that the latter received HEV infection at a high frequency. HEV RNA was detected in one of 88 sera from 1- and 2-year-old monkeys and 10 of 48 fecal specimens from 3-year-old monkeys by reverse transcription-polymerase chain reaction. Phylogenetic analyses revealed that the HEV strain RmKM15 was present in a serum sample that belonged to subtype 4b in genotype 4, whereas 10 strains detected in the fecal specimens belonged to subtype 4 h, suggesting that two genetically different strains were circulating at the farm. However, no significant clinical signs were observed in these monkeys. Further studies are required to identify the source of infection and to evaluate the pathogenicity of HEV in rhesus monkeys.

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.

Detection of Subgenotype IA and IIIA Hepatitis A Viruses in Rivers Flowing through Metro Manila, the Philippines.

Hepatitis A virus (HAV) is a common infectious etiology of acute hepatitis worldwide. The Philippines remains highly endemic for hepatitis A, but there is still a lack of information about HAV in the country. To evaluate the HAV contamination in environmental water in the Philippines, we conducted the detection and genetic analyses of HAV RNA in samples from river water. Twelve water samples were collected at 6 sampling sites of 3 rivers in Metro Manila, in both the dry and wet seasons in 2012 and 2013. The HAV RNA was detected in all the 6 samples collected in the dry season, and in one sample from the wet season. Phylogenetic analysis confirmed that the HAV strains detected in the river water included multiple sequences belonging to subgenotypes IA and IIIA. This indicates that at least 2 genotypes of the HAV strains are circulating in the environment in the Philippines, posing a risk of HAV infection to not only residents, but also tourists, especially in the dry season.