Low host immune pressure may be associated with the development of hepatocellular carcinoma: a longitudinal analysis of complete genomes of the HBV 1762T, 1764A mutant.

Background: It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Materials and methods: Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). Results: The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). Conclusions: The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC.

A novel subgenotype I3 of hepatitis B virus in Guangxi, China: a 15-year follow-up study.

Genotype I of hepatitis B virus (HBV) was proposed recently following sequencing of complete HBV genomes from Vietnam and Laos. However, its long-term molecular evolution is unknown. The objectives of this study were to study the molecular evolution of this genotype from an asymptomatic HBsAg carrier from the Long An cohort over a 15-year period was studied using both NGS and clone-based sequencing. The number of complete genome sequences obtained in 2004, 2007, 2013, and 2019 are 17, 20, 19, and 10, respectively. All strains belong to subgenotype I1, except for six (five from 2007 and one from 2019) and 8 further strains from 2007 which form a cluster branching out from other subgenotype I sequences, supported by a 100% bootstrap value. Based on complete genome sequences, all of the estimated intragroup nucleotide divergence values between these strains and HBV subgenotypes I1-I2 exceed 4%. These strains are recombinants between genotype I1 and subgenotype C but the breakpoints vary. The median intrahost viral evolutionary rate in this carrier was 3.88E-4 substitutions per site per year. The Shannon entropy (Sn) ranged from 0.55 to 0.88 and the genetic diversity, D, ranged from 0.0022 to 0.0041. In conclusion, our data provide evidence of novel subgenotypes. Considering that the 8 strains disappeared after 2007, while one of the 6 strains appears again in 2019, we propose these 6 strains as a new subgenotype, provisionally designated HBV subgenotype I3 and the 8 strains as aberrant genotype.

Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15 year follow-up study.

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A â†’ T) and 1764(G â†’ A) and a stop mutation at nt1896(G â†’ A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations.

Infection with Hepatitis B Virus May Increase the Serum Concentrations of Osteopontin.

BACKGROUND: Serum osteopontin (OPN) concentrations were found to be significantly increased in patients infected with hepatitis B virus (HBV) and patients with hepatocellular carcinoma (HCC). OBJECTIVE: The aim of this study was to determine the association among HCC, OPN, and HBV. METHODS: Two hundred and forty-one subjects were recruited and divided into 6 groups: healthy controls, asymptomatic HBsAg carriers, HBsAg (-) patients with other tumors, HBsAg (+) chronic liver disease patients, HBsAg (+) patients with HCC, and HBsAg (-) patients with HCC or liver cirrhosis (LC). Serum concentrations of OPN and HBsAg were measured and analyzed. RESULTS: OPN concentrations in the HBsAg (+) HCC group were significantly higher than the healthy control group and the HBsAg (-) patients with other cancers (both p = 0.0001). The OPN concentrations of the HBsAg (-) patients with HCC or LC also did not differ significantly from those of the healthy control group (p = 0.075). There is a correlation between the titer of HBsAg and concentrations of OPN in all 3 HBsAg (+) groups (all p values <0.05). CONCLUSIONS: Infection with HBV may increase the serum concentrations of OPN. The association of OPN and HCC may be not attributable to tumor development per se but, rather, to HBV infection.

Changing risk factors for hepatocellular carcinoma in hyperendemic regions in the era of universal hepatitis B vaccination.

BACKGROUND: LongAn, Guangxi, was the first county in China to implement universal childhood hepatitis B virus (HBV) immunization. We aimed to determine its long-term effects in preventing hepatocellular carcinoma (HCC) 32 years after the immunization programme was launched. METHODS: Information on HCC deaths for LongAn and its neighbouring county, BinYang (where universal hepatitis B vaccination was not started till 2002), were obtained from the national mortality surveillance system. The data were analysed using Poisson regression. RESULTS: The overall age-adjusted mortalities of HCC in LongAn and BinYang during 2017-2018 were 53.3/100,000 and 45.4/100,000, respectively. The mortality of males aged 20-29 years in LongAn, who were vaccinated at birth, was lower (2.7/100,000, 95%CI 0.8-4.5) than that of males in BinYang, who were not vaccinated (4.7/100,000, 95%CI 3.2-6.3). In LongAn, the HCC mortality in adults aged 20-29 years declined significantly from 7.9/100,000 (95%CI 4.4-11.4) in 2004 to 1.4/100,000 (95%CI 0.4-2.4) in 2017-2018 (χ2 = 5.554, p = 0.018). Among those vaccinated at birth, the HCC mortality in mountainous areas, where dietary exposure to aflatoxins is more common, is higher (9.0/100,000, 95%CI 4.5-13.5) than in low-lying areas (6.5/100,000, 95%CI 3.6-9.4) (χ2 = 0.2393, p = 0.618). CONCLUSION: Immunization of infants against HBV has reduced their risk of developing HCC as children and young adults but could not prevent all cases of HCC, suggesting that the major risk factor for HCC in hyperendemic regions is shifting from HBV to other factors. Additional prevention strategies for HCC will be needed in the future.

Locus 5p13.1 may be associated with the selection of cancer-related HBV core promoter mutations.

Background: The basal core promoter (BCP) double mutations (A1762T and G1764A) of hepatitis B virus (HBV) have been reported to be an aetiological factor of hepatocellular carcinoma (HCC). What distinguishes the subset of HBV carriers in whom these mutations are selected? Methods: A genome-wide association study (GWAS) was carried out on 218 asymptomatic HBsAg carriers infected with HBV with BCP double mutations and 191 controls infected with HBV with the wild type BCP. The highest ranking nucleotide polymorphisms (SNPs) were validated with other study subjects, 203 cases and 181 controls. The expression of the gene nearest a SNP found to be significant was examined using RT-PCR. Results: Forty-five candidate SNPs were identified in the GWAS. Three SNPs were found to be associated with the selection of HBV BCP double mutations in the replication stage, including rs7717457 at 5p13.1, rs670011 at 17q21.2, rs2071611 at 6p22.2. Especially, rs7717457 (P= 4.57×10-5 combined P) reached the potential GWAS significance level. The expression of gene complement component 7 (C7), nearest to SNP rs7717457, differed significantly between the case and control groups (t=2.045, P=0.04), suggesting that SNP rs7717457 was associated with the expression of its nearest gene. Conclusions: SNP rs7717457 is associated with the selection of HBV BCP double mutations, providing an important clue to understanding the mechanisms of oncogenesis of HBV BCP double mutations.

Novel subgenotype D11 of hepatitis B virus in NaPo County, Guangxi, bordering Vietnam.

Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1-D10 exceed 4 %. Compared to the other subgenotypes (D1-D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.

Asymptomatic hepatitis B carriers who were vaccinated at birth.

The long-term persistence of immunity following universal infant immunization against hepatitis B virus (HBV) and the need for a subsequent booster dose in adolescence remain under debate. With data derived from Long'an County, Guangxi, China, we reported previously that the prevalence of hepatitis B surface antigen (HBsAg) among adults born from 1987 to 1993 increases with age, although these individuals had received a first dose of the vaccine within 24 hours of birth. Here, we sought the source of transmission by comparison of genotypes among their family members using phylogenetic analysis of complete HBV S gene sequences. For comparison, we screened 2199 vaccinated individuals aged 5 to 17 in Cang Wu County and 1592 vaccinated individuals aged 3 to 7 in Ling Shan County in Guangxi for HBsAg carriers and investigate their family members. In total, 50 asymptomatic HBsAg carriers who were vaccinated at birth and 152 family members were analyzed. The results showed that 25% (95% CI: 6.0-44.0) of the HBsAg-positive children had not acquired their HBV infection from their mothers. This phenomenon showed a trend that increases with age. Antibody escape mutations were detected in 22.9% (95% CI: 11.0-34.8) of the isolates. In conclusion, a booster dose may be necessary for adolescence who were vaccinated at birth in highly endemic countries.

High Prevalence of HBV Lamivudine-Resistant Mutations in HBV/HIV Co-Infected Patients on Antiretroviral Therapy in the Area with the Highest Prevalence of HIV/HBV Co-Infection in China.

OBJECTIVES: We aimed to determine the prevalence of hepatitis B virus (HBV) drug-resistant mutations in patients co- infected with HBV/human immunodeficiency virus (HIV), including both drug-naïve subjects and those who received antiretroviral therapy (ART) in Guangxi, where the prevalence of HIV/HBV co-infection is highest in China. METHODS: Two hundred and three subjects co-infected with HBV/HIV were recruited, including 123 drug-naïve patients (group 1) and 80 who received ART (group 2). The polymerase gene of HBV in the serum of all study subjects was analysed. RESULTS: The results showed that the prevalence of HBV drug-resistant mutations in group 2 (76.5%, 95% CI 56.3-96.7) was significantly higher than that in group 1 (1.4%, 95% CI -1.4 to 4.2; χ2 = 50.955, p < 0.05). The major pattern of lamivudine (3TC)-resistant mutations is L180M+M204I+L80I (35.7%). In total, 95% of subjects with resistant mutations had cross-resistance to telbivudine and entecavir. No putative tenofovir disoproxil fumarate (TDF) resistance change was found. Five subjects (6.5%) in group 2 had HBV viral loads over 10 × 106 copies/mL. Four of them had 3TC-resistant mutations. Multivariate analysis showed that ART was the only factor associated with the development of drug-resistant mutations. CONCLUSION: Treating HIV in HIV/HBV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistant mutations. TDF could not completely suppress HBV replication.

ICTV Virus Taxonomy Profile: Hepeviridae.

The family Hepeviridae includes enterically transmitted small non-enveloped positive-sense RNA viruses. It includes the genera Piscihepevirus, whose members infect fish, and Orthohepevirus, whose members infect mammals and birds. Members of the genus Orthohepevirus include hepatitis E virus, which is responsible for self-limiting acute hepatitis in humans and several mammalian species; the infection may become chronic in immunocompromised individuals. Extrahepatic manifestations of Guillain-Barré syndrome, neuralgic amyotrophy, glomerulonephritis and pancreatitis have been described in humans. Avian hepatitis E virus causes hepatitis-splenomegaly syndrome in chickens. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Hepeviridae, which is available at www.ictv.global/report/hepeviridae.

Des-γ carboxyprothrombin may not be a good biomarker for hepatocellular carcinoma in those chronically infected with hepatitis B virus with basal core promoter double mutations (T

BACKGROUND: The accuracy of des-γ -carboxyprothrombin (DCP) in the detection of hepatocellular carcinoma (HCC) in those infected hepatitis B virus (HBV) from cross-sectional or case-control studies is contradictory. OBJECTIVE: To resolve this contradiction using a prospective study. METHODS: Three hundred male individuals persistently infected with HBV were recruited from the Chinese cohort and followed up once per year from 2012 to 2015. Each subject was screened for HCC by measurements of serum alpha-fetoprotein (AFP), lectin-bound α -fetoprotein (AFP-L3), DCP concentrations and ultrasonographic examinations. RESULTS: Nineteen HCC cases were identified. The area under receiver operating characteristic (AUROC) at first, second and third visit for AFP, AFP-L3 and DCP ranges from 0.710-0.897, 0.566-0.637 and 0.520-0.595, respectively. The rate of elevated DCP is not significantly different between the HCC cases and controls (52.6% vs. 47.4%) (P > 0.05). The incidence of HCC in subjects with elevated DCP is not significantly higher than that of those with normal DCP (9.5% vs. 4.6%) (P > 0.05). The AUROC of combinations of these biomarkers was higher than that of AFP alone at the first visit. However, it was reduced at the second visit. At the third visit, the AUROCs of AFP + DCP and AFP + AFP-L3 + DCP, but not that of AFP + AFP-L3, were higher than that of AFP alone. CONCLUSIONS: AFP but DCP or AFP-L3 remains a valuable biomarker for HCC in those chronically infected with HBV. The combination with AFP-L3 and DCP may not increase the accuracy of AFP in differentiating HCC cases from controls, among those infected with HBV.

Hepatitis B Virus Core Promoter Double Mutations (A1762T, G1764A) Are Associated with Lower Levels of Serum Dihydrolipoyl Dehydrogenase.

OBJECTIVES: The aim of this study was to identify serum proteins with differential concentrations between hepatocellular carcinoma (HCC) patients and HBsAg asymptomatic carriers among individuals infected with hepatitis B virus (HBV) with basal core promoter (BCP) double mutations (A1762T, G1764A). METHODS: iTRAQ and liquid chromatography-tandem mass spectrometry were used to identify differentially expressed protein, and an ELISA test was used for the validation test. RESULTS: The total number of proteins identified was 1,125, of which 239 showed statistically significant differences in their expression. The relative concentrations of serum dihydrolipoyl dehydrogenase (DLD), which showed the most significant correlation with liver diseases and infection, were significantly lower in HCC patients than asymptomatic HBsAg carriers and individuals negative for HBsAg. However, only the difference between HCC patients with BCP double mutations and HBsAg-negative individuals could be confirmed by ELISA. Meanwhile, we found that the concentrations of serum DLD in those infected with HBV with BCP double mutations were significantly lower than in individuals with the wild-type BCP. However, the difference in the concentrations of serum DLD between individuals with wild-type BCP and those negative for HBsAg was not significant. CONCLUSIONS: HBV with BCP double mutations are associated with lower concentrations of serum DLD.

Proposed reference sequences for hepatitis E virus subtypes.

The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website (http://talk.ictvonline.org/r.ashx?C). The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

Detection and assessment of infectivity of hepatitis E virus in urine.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is known to be excreted in the stool but there has been no report of its presence in urine. This study investigated the presence of HEV RNA and antigen (HEV-Ag) in urine and its possible transmission. METHODS: Serum and urine samples from patients with chronic or acute HEV infection and HEV infected monkeys were tested for viral and biochemical markers. Liver and kidney biopsies from the infected monkeys were analyzed by histopathology and immunohistochemistry. The infectivity of HEV from urine was assessed by inoculation into monkeys. RESULTS: HEV RNA and HEV-Ag were detected persistently in the urine of a patient with chronic HEV infection. Subsequently, HEV RNA was detected in the urine of three of the eight (37.5%) acute patients, all of whom had detectable HEV-Ag in their urine. HEV RNA and HEV-Ag were also detectable in the urine of HEV infected monkeys. The ratio of HEV-Ag to RNA in the urine of the infected monkeys was significantly higher than in their sera and feces. The parameters of routine urinalysis remained within the normal ranges in the hepatitis E patients and infected monkeys, however, pathological changes and HEV-Ag were observed in the kidneys of the infected monkeys. Furthermore, one of two monkeys became infected with HEV after inoculation with urine from another infected monkey. CONCLUSIONS: HEV infection may result in kidney injury and the urine may pose a risk of transmission. HEV-Ag detection in urine may be valuable for diagnosis of ongoing HEV infection.

Occult HBV Infection May Be Transmitted through Close Contact and Manifest as an Overt Infection.

The importance of transmission of occult HBV infection (OBI) via transfusion, organ transplantation and hemodialysis has been widely recognized. However, data regarding the transmission of OBI through close contact remain limited. In this study, serum samples were obtained from a child and his parents. The child had received the standard vaccination regimen at birth and produced protective antibody. Sera were tested for HBV serological markers. Nested PCR assays were used to detect HBV DNA and the amplicons were cloned and their sequences subjected to phylogenetic analysis. The results showed that both parents had occult infections while the child had an overt infection. Twelve, eleven and nine clones, from the father, mother and son, respectively, were sequenced. Serotypes adrq+, ayw1, ayw and ayr were found in the father and ayw1, adw2 and adwq+ in the mother; adrq+ was the only serotype in son. Genotype B, subgenotype C2 and a recombinant were identified in the father and genotype B, subgenotype C5 and three recombinants were found in the mother. Subgenotype C2 was the only genotype identified in the child. A phylogenetic tree showed that all of the child's sequences and most of the father's sequences clustered together. However, none of mother's sequences clustered with those of the child. The surface gene from the child and his father had the same amino acid substitution pattern (T118K, T123N and G145A). We concluded that the father was the source of the son's HBV infection, suggesting that occult HBV infection may be transmitted through close contact and manifest as an overt infection.

The incidence rate over 10 years of naturally occurring, cancer related mutations in the basal core promoter of hepatitis B virus.

Cross-sectional analyses showed that the prevalence of basal core promoter (BCP) double mutations (nt 1762T, 1764A) of hepatitis B virus (HBV) gradually increases with age. We aimed to determine the incidence rate of the mutations over 10 years. Study subjects were selected from the Long An cohort established in 2004, including 59 with HBV with single mutations at nt 1762 or 1764 in the BCP and 342 with wild type BCP sequences at baseline. Their serum samples for analysis were obtained at the 3rd and 10th annual visits, respectively. The results showed that the annual incidence rate of BCP double mutations is 3.8% (95% confidence interval [CI]: 1.4-6.2) and tends to decrease with age. The peak incidence is in the 30-34 years age-group. The incidence rate in HBeAg positive individuals (5.5%) is significantly higher than in those without HBeAg (3.4%) (P<0.05). The incidence rate is significantly higher in genotype C (4.8%) than in genotype B (2.8%) or I (3.1%). The incidence rate of the mutations (6.8%) developing from a single mutation at nt 1762 or 1764 is significantly higher than that (3.8%) from the wild type sequence (P<0.005). The difference in incidence of single mutations between nt 1762 (0.7%) and 1764 (0.03%) is significant (P<0.05). In conclusion, the incidence rate of BCP double mutations tends to decrease with age after the age of 35 years. Viruses with a single mutation at nt 1762 or 1764 are more prone to develop double mutations. Nt 1762 is the more common site of the first mutation.

Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

The correlation between serum HBsAg levels and viral loads depends upon wild-type and mutated HBV sequences rather than the HBeAg/anti-HBe status.

Despite several studies regarding the correlation between serum HBsAg titers and viral loads, the association remains uncertain. Eighty-nine individuals were selected randomly from a Chinese cohort of 2,258 subjects infected persistently with hepatitis B virus (HBV) for cross-sectional and longitudinal analysis. Viral loads of mutant HBV are lower than those of wild type HBV. The serum HBsAg titers correlate positively with viral loads in both HBeAg positive and negative subjects (r = 0.449, P = 0.013; r = 0.300, P = 0.018, respectively). No correlation between serum HBsAg titer and viral loads was found in any of the four phases of chronic HBV infection. The serum HBsAg titers correlate positively with viral loads in the group with wild type sequences of the PreS/S, basal core promoter (BCP), and preC regions of HBV(r = 0.502, P = 0.040). However, the correlation was not seen in the group with mutations in these regions (r = 0.165, P = 0.257). The correlation between HBsAg titers and viral loads was seen in individuals with wild type PreS/S sequences but not in the subgroup with BCP double mutations or PreC stop mutation, although their sequences in the preS/S regions were wild type. All these findings were confirmed by the longitudinal analysis. In conclusion, the correlation between serum HBsAg levels and viral loads may not differ between HBeAg positive and negative individuals but may depend on wild-type or mutated genomic sequences. Therefore, HBsAg quantitation may be used as a surrogate for viral loads in only wild-type HBV infections.

Hepatitis E genotype 4 virus from feces of monkeys infected experimentally can be cultured in PLC/PRF/5 cells and upregulate host interferon-inducible genes.

The understanding of the interaction between hepatitis E virus (HEV) and its host cells has been impeded greatly by the absence of a cell culture system. In this study, an efficient cultivation method was developed in PLC/PRF/5 cells for HEV genotype 4 from the feces of monkeys infected experimentally. Compared to minimal essential medium (MEM), mixed Dulbecco's Modified Eagle's Medium (DMEM)/M199 improved the infection efficiency of HEV in PLC/PRF/5 cells. The incubation time and temperature were set at 6 hr and 40°C, respectively. Compared to a 100% ELISA positive ratio (EPR) of 1 × 10(6) copies/ml HEV inoculated flasks, the ELISA positive ratio was 100%, 75%, 37.5%, and 100% for flasks inoculated with HEV incubated for 30 min under the conditions of pH 3.0, pH 11.0, 56°C and delipidation treatment, respectively. Gene expression profiles of HEV inoculated and control PLC/PRF/5 cells were assayed using a microarray. Four interferon-inducible genes, IFI27, IFI6, Mx1, and CMPK2, were up-regulated during HEV-infection. Furthermore, the replication of HEV was inhibited at 3-14 days after treatment with 500 IU/ml IFN-α2b.