Integrin α3 is involved in non-enveloped hepatitis E virus infection.

Hepatitis E virus (HEV) causes acute and fulminant hepatitis worldwide. Although enveloped (e) and non-enveloped (ne) forms of HEV have been discovered, host factors involved in infection, including receptors, remain to be elucidated. Here, we identified integrin α3 (encoded by ITGA3), a protein that binds and responds to the extracellular matrix, as an essential host factor for HEV infection. Integrin α3 expression was lower in four HEV-non-permissive cell subclones than in an HEV-permissive subclone. ITGA3 knockout cells lost HEV permissibility, suggesting that integrin α3 is critical for HEV infection. Stable expression of integrin α3 in an HEV-non-permissive subclone provided permissibility only to infection by neHEV; expression of integrin α3 lacking the ectodomain did not. Direct interaction between neHEV and the integrin α3 ectodomain was confirmed by co-precipitation using a soluble integrin α3-Fc. These results strongly suggest that integrin α3 is a key molecule for cellular attachment and entry of neHEV.

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.

Multiple antiviral activities of the antimalarial and anti-hepatitis C drug candidates N-89 and N-251.

The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses.

Evaluation of Heating Conditions for Inactivation of Hepatitis E Virus Genotypes 3 and 4.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >105 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 108 HEV genome RNA copies (103 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products.

Determination of Ferret Enteric Coronavirus Genome in Laboratory Ferrets.

Ferret enteric coronavirus (FRECV) RNA was detected in laboratory ferrets. Analysis of the complete genome sequence of 2 strains, FRCoV4370 and FRCoV063, revealed that FRECV shared 49.9%-68.9% nucleotide sequence identity with known coronaviruses. These results suggest that FRECV might be classified as a new species in the genus Alphacoronavirus.

Serological evidence of hepatitis E virus infection in dromedary camels in Ethiopia.

The genome of dromedary camel hepatitis E virus (DcHEV) has been detected in stool and serum samples from dromedary camels, but the sero-epidemiological information of DcHEV infection remains unclear. A total of 246 serum samples collected from dromedary camels (Camelus dromedarius) in Ethiopia, and 40 serum samples from Bactrian camels (Camelus ferus) in Mongolia were examined for the detection of anti-DcHEV IgG antibody by a newly developed enzyme-linked immunosorbent assay (ELISA) by using DcHEV-like particles (DcHEV-LPs) as the antigen. The results revealed that 55 of the 246 (22.4%) dromedary camels were positive for anti-DcHEV IgG, whereas all 40 samples from the Bactrian camels were negative for DcHEV IgG antibody. A total of 98 serum samples from dromedary camels, including 25 anti-DcHEV-IgG positive samples, were used for the detection of DcHEV RNA by reverse transcription-polymerase chain reaction (RT-PCR), however, no positive samples were identified. These results suggested that the DcHEV infection occurred in the dromedary camels in Ethiopia. Further studies are required to determine whether Bactrian camels are susceptible to DcHEV infection. In addition, not only DcHEV-LPs, but also virus-like particles (VLPs) delivered from G1, G3 and G5 HEV are likely applicable for the detection of the anti-DcHEV IgG antibody.

Genetic and physicochemical analyses of a novel ferret hepatitis E virus, and clinical signs of infection after birth.

A novel cluster of five ferret hepatitis E virus (HEV) strains was detected from nine laboratory ferrets (Mustela putorius furo) imported from a ferret farm in the U.S. Our detection of ferret HEV RNA and anti-HEV antibodies, and alanine aminotransferase (ALT) value assessment indicated that all of the 9 ferrets were infected with ferret HEV, and that the infection exhibited three patterns: sub-clinical infection (n=2), acute hepatitis (n=6) and persistent infection (n=1). Next-generation sequence analyses of the entire genome sequences of the five strains revealed that their nucleotide sequence identities ranged from 99.5% to 99.9%, indicating that genetically similar ferret HEVs had been circulating at this the U.S. ferret farm. In contrast, the strains shared 82% and 89% nucleotide sequence identities with other ferret HEV that isolated from the Netherlands (JN998607) and the U.S. (AB890374), suggesting that these strains form a novel cluster of ferret HEV with diverse genomes depending on the region where their host. Particles with a diameter of ~35nm at a density of 1.201g/cm3 were observed in the fecal specimens by electron microscopy. There was no evidence that the particles were associated with the cell membrane. The ferret HEV RNA was not constantly detected in urine, suggesting that the excretion of ferret HEV into urine is not a common feature of HEV infection.

No Evidence of Rat Hepatitis E Virus Excretion in Urine Samples of Rats.

To investigate whether rat hepatitis E virus (rat HEV) is excreted in the urine of HEV-infected rats, we infected 3 Wistar and 6 nude rats with rat HEV and examined the rat-HEV RNA in serum, fecal, and urine samples. We detected rat-HEV RNA in the serum and fecal samples of all 9 rats but not in any of the urine samples. Our results suggest that in rats, rat HEV is not transmitted via urine.

Clinical Features and Transmission Pattern of Hepatitis A: An Experience from a Hepatitis A Outbreak Caused by Two Cocirculating Genotypes in Sri Lanka.

Sri Lanka is one of the intermediate-endemic areas for hepatitis A virus (HAV), and concerns exist about the increasing HAV-susceptible population. In fact, Sri Lanka recorded a large hepatitis outbreak, possibly hepatitis A, around the end of the Sri Lankan war. It included more than 14,000 patients consisting of local residents, internally displaced personnel, and military personnel in the main combat zone. The outbreak had slowed down by October 2009; however, acute viral hepatitis continued to occur sequentially among military personnel. We obtained clinical information and serum samples from 222 patients with acute hepatitis who visited the Military Hospital Anuradhapura between January and September 2010. Samples were subjected to laboratory testing including HAV-immunoglobulin M and genotyping. Most patients (98.2%) were confirmed as having hepatitis A belonging to two subgenotypes: IA and IIIA. We did not observe any differences in clinical or biochemical features among patients with subgenotypes IA and IIIA except for pale stools and upper abdominal discomfort. During the investigation period, we observed a serial outbreak caused by identical HAV strains with an interval in line with that of typical HAV incubation periods. Most patients in the first outbreak were found in the training center, and patients in the second outbreak were found in multiple places where soldiers were assigned after the training center. These findings indicate that a strain of HAV diffused from one place to another along with movement of infected persons among the HAV-susceptible population. HAV vaccination for high-risk groups, such as young soldiers, is necessary.

Production of infectious dromedary camel hepatitis E virus by a reverse genetic system: Potential for zoonotic infection.

BACKGROUND & AIMS: The pathogenicity, epidemiology and replication mechanism of dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus (HEV), has been unclear. Here we used a reverse genetic system to produce DcHEV and examined the possibility of zoonotic infection. METHODS: Capped genomic RNA derived from a synthetic DcHEV cDNA was transfected into human hepatocarcinoma cells PLC/PRF/5. The DcHEV capsid protein and RNA were detected by an enzyme-linked immunosorbent assay (ELISA) or RT-qPCR. A neutralization test for DcHEV was carried out by using antisera against HEV-like particles. DcHEV was used to inoculate two cynomolgus monkeys to examine the potential for cross-species infection. RESULTS: The transfection of PLC/PRF/5 cells with capped DcHEV RNA resulted in the production of infectious DcHEV. The genome sequence analysis demonstrated that both nucleotide and amino acid changes accumulated during the passages in PLC/PRF/5 cells. The cynomolgus monkeys showed serological signs of infection when DcHEV was intravenously inoculated. DcHEV was neutralized by not only anti-DcHEV-LPs antibody, but also anti-genotype 1 (G1), G3 and G4 HEV-LPs antibodies. Moreover, the monkeys immunized with DcHEV escaped the G3 HEV challenge, indicating that the serotype of DcHEV is similar to those of other human HEVs. CONCLUSIONS: Infectious DcHEV was produced using a reverse genetic system and propagated in PLC/PRF/5 cells. The antigenicity and immunogenicity of DcHEV are similar to those of G1, G3 and G4 HEV. DcHEV was experimentally transmitted to primates, demonstrating the possibility of a zoonotic infection by DcHEV. LAY SUMMARY: Dromedary camel hepatitis E virus (DcHEV) was produced by a reverse genetic system and grows well in PLC/PRF/5 cells. Cynomolgus monkeys experimentally infected with DcHEV indicated serological signs of infection, suggesting that DcHEV has the potential to cause zoonotic HEV infection.

Ferret hepatitis E virus infection induces acute hepatitis and persistent infection in ferrets.

Ferret hepatitis E virus (HEV), a novel hepatitis E virus, has been identified in ferrets. However, the pathogenicity of ferret HEV remains unclear. In the present study, we compared the HEV RNA-positivity rates and alanine aminotransferase (ALT) levels of 63 ferrets between before and after import from the US to Japan. We found that the ferret HEV-RNA positivity rates were increased from 12.7% (8/63) to 60.3% (38/63), and ALT elevation was observed in 65.8% (25/38) of the ferret HEV RNA-positive ferrets, indicating that ferret HEV infection is responsible for liver damage. From long term-monitoring of ferret HEV infection we determined that this infection in ferrets exhibits three patterns: sub-clinical infection, acute hepatitis, and persistent infection. The ALT elevation was also observed in ferret HEV-infected ferrets in a primary infection experiment. These results indicate that the ferret HEV infection induced acute hepatitis and persistent infection in ferrets, suggesting that the ferrets are a candidate animal model for immunological as well as pathological studies of hepatitis E.

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV.

An Outbreak of Acute Hepatitis Caused by Genotype IB Hepatitis A Viruses Contaminating the Water Supply in Thailand.

BACKGROUND: In 2000, an outbreak of acute hepatitis A was reported in a province adjacent to Bangkok, Thailand. AIMS: To investigate the cause of the 2000 hepatitis A outbreaks in Thailand using molecular epidemiological analysis. METHODS: Serum and stool specimens were collected from patients who were clinically diagnosed with acute viral hepatitis. Water samples from drinking water and deep-drilled wells were also collected. These specimens were subjected to polymerase chain reaction (PCR) amplification and sequencing of the VP1/2A region of the hepatitis A virus (HAV) genome. The entire genome sequence of one of the fecal specimens was determined and phylogenetically analyzed with those of known HAV sequences. RESULTS AND CONCLUSIONS: Eleven of 24 fecal specimens collected from acute viral hepatitis patients were positive as determined by semi- nested reverse transcription PCR targeting the VP1/2A region of HAV. The nucleotide sequence of these samples had an identical genotype IB sequence, suggesting that the same causative agent was present. The complete nucleotide sequence derived from one of the samples indicated that the Thai genotype IB strain should be classified in a unique phylogenetic cluster. The analysis using an adjusted odds ratio showed that the consumption of groundwater was the most likely risk factor associated with the disease.

Epidemiological and genetic analysis of a 2014 outbreak of hepatitis A in Japan.

Hepatitis A virus (HAV) is one of the most common causes of feces-transmitted acute hepatitis worldwide. In Japan, most of HAV infections have been sporadic cases and a relatively low number of cases (approximately 100-150) of acute hepatitis A were reported in 2012 and 2013. However, in 2014, 342 cases were reported as of week 22. In order to characterize the viral agents causing this outbreak, we collected stool or sera (and both for three case) from patients with hepatitis A from many regions throughout Japan and performed genotyping of the VP1/P2A regions of HAV. We then used a multiple-alignment algorithm to compare the nucleotide sequences with those of reference strains. Phylogenetic tree analyses revealed that the 159 HAV isolates were divided into three subgenotypes: IA (137 cases), IB (4 cases), and IIIA (18 cases). The most unique feature of this outbreak was that for most subgenotype IA cases (103 out of 137 IA cases) the sequences analyzed shared 100% homology. Interestingly, the peak week for these IA infections was almost the same nationwide, suggesting that the epidemic of hepatitis A caused by this subgenotype IA strain may have expanded from a single source possibly because of one food-borne or waterborne source that was distributed nationwide at once.

Monkeys and Rats Are Not Susceptible to Ferret Hepatitis E Virus Infection.

Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in the Netherlands, Japan, and the US. To determine whether ferret HEV transmits to other animals, we inoculated laboratory rats (Wistar), nude rats (Long-Evans-rnu/rnu), and cynomolgus monkeys with ferret HEV (F4351) by intravenous injection. None of the animals demonstrated a positive sign for virus replication, indicating that rats and monkeys are not susceptible to ferret HEV.

Generation of hepatitis E virus-like particles of two new genotypes G5 and G6 and comparison of antigenic properties with those of known genotypes.

In addition to the four major genotypes (G1 through G4) known for human hepatitis E virus (HEV), two new genotypes (G5 and G6) were suggested, based on unique viral nucleotide sequences derived from wild boars in Japan. It has been unknown whether the virus of these new genotypes can cause hepatitis in humans; neither G5 nor G6 HEV has been found in patients to date. To study the antigenic properties of G5 and G6 HEV, we expressed N-terminus-truncated HEV ORF2 protein by a recombinant baculovirus system, and we obtained virus-like particles (VLPs) for both G5 and G6. The VLPs showed antigenic cross-reactivity against G1, G3 and G4 HEV more strongly than against ferret or rat HEV. Moreover, both anti-G5 and anti-G6 VLPs antibodies could neutralize G3 HEV's ability to infect PLC/PRF/5 cells, suggesting that G5 and G6 HEV have the same serotype as human HEV.

Construction and characterization of an infectious cDNA clone of rat hepatitis E virus.

Rat hepatitis E virus (HEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of rat HEV strain R63/DEU/2009 was cloned downstream of the T7 RNA polymerase promoter and capped genomic RNA generated by in vitro transcription was injected into nude rats. Rat HEV RNA could be detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. Rat HEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to rat HEV RNA detection in serum and faeces of nude rats, and to seroconversion in Wistar rats. In addition, rat HEV was isolated in PLC/PRF/5 cells from the rat HEV RNA-positive faecal suspension of nude rats and then passaged. The cell culture supernatant was infectious for nude rats. Genome analysis identified nine point mutations of the cell-culture-passaged virus in comparison with the originally cloned rat HEV genome. The results indicated that infectious rat HEV could be generated from the cDNA clone. As rats are widely used and well-characterized laboratory animals, studies on genetically engineered rat HEV may provide novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses.