Comparative analysis of disease severity between genotypes IA and IIIA of hepatitis A virus

Although hepatitis A is a major health problem worldwide, it has not yet been clarified whether or not viral factors affect the clinical characteristics. This study aimed to investigate if a genotype of hepatitis A virus (HAV) affects disease severity among adolescent and adult populations. Clinical data and specimens were collected from patients ≥16‐years‐of‐age with acute hepatitis A at two university hospitals in Korea during the two study periods: 1998 and 1999 (n = 45), and 2009 (n = 66). Nucleotide sequencing of the complete VP1 region of the HAV isolates was performed for phylogenetic analysis and genotyping. Clinical parameters related to disease severity were compared by HAV genotype to determine its clinical relevance. Of the 87 patients, 47 were male and the mean age was 29.8 ± 8.1 years. The genotype IIIA (93.0%, 53/57) was predominant in the year 2009, whereas IA (93.3%, 28/30) was the major genotype in 1998 and 1999. When comparing disease severity between the two HAV genotypes, the patients with genotype IIIA were older and had higher alanine aminotransferase (ALT) levels, prolonged prothrombin times and lower serum albumin levels. In a multivariate logistic regression model, higher ALT levels ≥ 1,000 IU/L (odds ratio [OR] 11.7, 95% confidence interval [CI] 2.5–54.0) and longer hospitalization (OR 22.49, 95%CI 4.6–132.5) were associated independently with genotype IIIA. In conclusion, this study indicates that HAV genotype might be one of the viral factors responsible for the disease severity of hepatitis A. J. Med. Virol. 83:1308–1314, 2011. © 2011 Wiley‐Liss, Inc.     

Genetic analysis of HAV strains in Tunisia reveals two new antigenic variants

To evaluate the genetic variability of hepatitis A virus (HAV) isolates in Tunisia, serum samples were collected from 99 patients in different Tunisian areas in 2003 containing 92 cases with acute hepatitis, five with severe acute hepatitis and two with fulminant hepatitis. The entire VP1 gene was amplified and sequenced. Sequences were then aligned and a phylogenetic analysis was performed. Additionally, the amino acid (aa) sequence of the VP1 was determined. The analysis of Tunisian HAV isolates revealed that all the isolates were sub-genotype IA with 96.4%–99.8% of identity and showed the emergence of two novel antigenic variants. The Tun31-03 antigenic variant, with a 38 aa deletion containing Met156, Val171, Leu174 and Ala176 and located between 150 and 187 aa of the VP1 protein where neutralization escape mutations, was found. The second antigenic variant, Tun36-03, was isolated from a patient with fulminant hepatitis and presented a substitution of Thr by Pro at position 10 of the VP1 protein. This amino acid is located in a peptide presenting an antigenically reactive epitope of the VP1 protein. This substitution has never been described previously.     

Genetic analysis of hepatitis A virus isolates from Brazil

A limited number of hepatitis A virus (HAV) isolates from South America have been characterised at the genomic level. IgM anti-HAV positive serum samples collected from patients with hepatitis A living in the five geographical regions of Brazil (North, Northeast, Central, South, and Southeast) were used to obtain HAV isolates and determine their genetic relatedness. Of the 232 case isolates, sequence data were obtained from the VP1/2A junction region of the HAV genome. All isolates were classified in genotype I; 231 belonged to subgenotype IA, and one to subgenotype IB. HAV isolates from four States formed distinct clusters of highly related sequences. However, isolates from other states did not cluster and the sequences from those states were intermingled with sequences found in the other states. The amino acid sequences of all but two isolates showed a Leu --> Ile substitution at position 42 in the 2A protein. This substitution appeared to be a characteristic geographic fingerprint of HAV sequences within Brazil.     

Hepatitis A viral load in relation to severity of the infection

A correlation between hepatitis A virus (HAV) genomes and the clinical severity of hepatitis A has not been established. The viral load in sera of hepatitis A patients was examined to determine the possible association between hepatitis A severity and HAV replication. One hundred sixty‐four serum samples from 91 Japanese patients with sporadic hepatitis A, comprising 11 patients with fulminant hepatitis, 10 with severe acute hepatitis, and 70 with self‐limited acute hepatitis, were tested for HAV RNA. The sera included 83 serial samples from 20 patients. Viral load was measured by real‐time RT‐PCR. The detection rates of HAV RNA from fulminant, severe acute, and acute hepatitis were 10/11 (91%), 10/10 (100%), and 55/70 (79%), respectively. Mean values of HAV RNA at admission were 3.48 ± 1.30 logcopies/ml in fulminant, 4.19 ± 1.03 in severe acute, and 2.65 ± 1.64 in acute hepatitis. Patients with severe infection such as fulminant hepatitis and severe acute hepatitis had higher initial viral load than patients with less severe infection (P < 0.001). Viremia persisted for 14.2 ± 5.8 days in patients with severe infection and 21.4 ± 10.6 days in those with acute hepatitis after clinical onset (P = 0.19). HAV RNA was detectable quantitatively in the majority of the sera of hepatitis A cases during the early convalescent phase by real‐time PCR. Higher initial viral replication was found in severely infected patients. An excessive host immune response might follow, reducing the viral load rapidly as a result of the destruction of large numbers of HAV‐infected hepatocytes, and in turn severe disease might be induced. J. Med. Virol. 83:201–207, 2011. © 2010 Wiley‐Liss, Inc.     

Genetic relatedness of hepatitis A virus isolates during a community-wide outbreak

In 1993-94, a community-wide outbreak of hepatitis A occurred in Stanislaus County, California. Stool specimens collected from a sample of 33 case patients were used to evaluate the duration of hepatitis A virus (HAV) excretion and the genetic relatedness of HAV isolates. Twenty-four percent of the patients had a stool sample positive for HAV antigen by enzyme immunoassay, whereas 91% had at least one stool positive for HAV RNA by RT-PCR amplification. Children were found to excrete low levels of HAV RNA for up to 10 weeks after the onset of symptoms. Analysis of the HAV VP1 amino terminus and VP1/P2A regions showed that a limited number of HAV isolates circulated during the epidemic and the majority of the cases were infected with the same strain.     

Spontaneous hepatitis in the crab-eating macaque exposed to immunodepressants

A case of spontaneous hepatitis was detected in experiments aimed at working out the conditions for reproduction of the immunosuppressed state in Macaca fascicularis with the purpose of subsequent infection of these monkeys with non A non B hepatitis virus transmitted by the fecal-oral route. One of 6 monkeys at the 8th day of the experiment was found to have developed an increase in the level of serum aminotransferases which grew progressively reaching high values by day 14. Fecal specimens from this monkey collected on the 5th day and later contained spherical virus-like structures 27 nm in diameter, antigenically identical with hepatitis A (HAV) virus. In the other 5 monkeys, no similar structures were found in fecal specimens throughout the experiment. The monkey with the signs of hepatitis was sacrificed on the 16th day of experiment, i. e. on the 8th day from the onset of hyperenzymemia. Immune electron microscopy of extracts of hepatic tissue and fecal specimens collected from this monkey has revealed 27 nm particles antigenically identical with HAV. The bulk of viral particles from the liver sedimented in cesium chloride buoyant density zone of 1.32 g/cm3, and from fecal specimens in the zone of 1.36 g/cm3. In the liver of this monkey, histological changes were found which are observed in acute hepatitis, and HAV antigen in hepatocyte cytoplasm was detected by the fluorescent antibody technique. It is suggested that the spontaneous disease of this monkey was due to natural infection with HAV which could be provoked by experimental immunosuppression.     

Genetic analysis of HAV strains isolated from patients with acute hepatitis in Korea, 2005-2006

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis, which represents a significant public health problem. HAV is usually transmitted by oral-fecal route and prevalent not only in developing countries but also in developed countries worldwide. To characterize the HAV wild type strains circulating in Korea, the VP3/VP1 and VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives during 2005 and 2006. Among 160 HAV IgM positive sera, 30% (n = 48) were positive for HAV RNA. Additionally, the VP3/VP1 junction regions were detected all six stools, which collected from outbreak in Gyeonggi province. Phylogenetic analysis of the sequences obtained from 54 distinct HAV isolates revealed that most of the strains (n = 45) belonged to genotype IA and the others including nine strains belonged to genotype IIIA. Interestingly, a Q --> S amino acid change was dominantly observed at position 810 of the VP1/P2A junction region in 14 isolates. The molecular epidemiology of HAV infection in Korea has changed with the co-circulation of at least two genotypes and 810Q --> S amino acid substitutions were found to be prevalent. These results strongly suggest that various HAV strains, including genotype IIIA, might be imported from high-endemic countries into Korea.     

Molecular epidemiology of an outbreak of hepatitis A in Italy

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/ PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived rom the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB. © Wiley-Liss, Inc.     

Acute hepatitis E virus infection in Taiwan 2002–2006 revisited: PCR shows frequent co‐infection with multiple hepatitis viruses

Sporadic cases of acute hepatitis E virus (HEV) infection with production of anti‐HEV IgM have been reported occasionally in Taiwan despite no reported outbreaks in the past. This study was undertaken to determine whether serological markers correlated with virus detection. From 2002 to 2006, 72 reported cases of acute hepatitis E seropositive for anti‐HEV IgM in Taiwan were enrolled for investigation. Acute phase serum samples were collected for detection of HEV RNA, HBV DNA, HCV RNA, and GBV‐C RNA by PCR. The results showed that viral sequences of HEV, HBV, HCV and GBV‐C were detected in 54 (75%), 21 (29.2%), 9 (12.5%), and 22 (30.6%) of cases, respectively. Acute hepatitis A co‐infection was excluded in all patients because none were seropositive for anti‐HAV IgM and, nine patients (12.5%) did not seroconvert to anti‐HEV IgG. These results suggest that serum markers did not correlate completely with viremia in the diagnosis of acute HEV infection. Multiple viruses may co‐infect with acute hepatitis E virus in Taiwan. Detection of hepatitis E viremia together with seropositivity for anti‐HEV IgM and followed by seroconversion to anti‐HEV IgG should be included in the diagnostic criteria for HEV infection. J. Med. Virol. 81:1734–1742, 2009. © 2009 Wiley‐Liss, Inc.     

Epidemiological and virological characteristics of symptomatic acute hepatitis E in Greater Cairo,Egypt

The aim of the study was to describe the characteristics of acute hepatitis E in Greater Cairo. Patients with acute hepatitis E were identified through a surveillance of acute hepatitis using the following definition: recent (<3 weeks) onset of fever or jaundice, alanine aminotransferase at least three times the upper limit of normal (uln), negative markers for other causes of viral hepatitis and detectable hepatitis E virus (HEV) RNA. Comparison of the liver tests between acute hepatitis E and hepatitis A virus (HAV), case–control analysis (four sex-matched and age-matched (±1 year) HAV controls per case) to explore risk factors and phylogenetic analyses were performed. Of the 17 acute HEV patients identified between 2002 and 2007, 14 were male. Median age was 16 years (interquartile range 13-22). Compared with HAV (n = 68 sex-matched and ±1 year age-matched), HEV patients had higher bilirubin (mean (SD) 10.9 (5.7) uln versus 7.5 (4.4) uln, p 0.05) and aspartate aminotransferase levels (38.6 (27.1) uln versus 18.3 (18.1) uln, p 0.02). Co-infection (hepatitis C virus RNA or hepatitis B surface (HBs) -antigen positive/IgM anti-hepatitis B core (HBc) anitgen negative) was diagnosed in four patients. In univariate matched analysis (17 cases, 68 matched controls), HEV cases were more likely to live in a rural area than HAV controls (matched OR 7.9; 95% CI 2.0-30.4). Of the 16 isolates confirmed as genotype 1, 15 belonged to the same cluster with 94-98.5% identity in the open-reading frame 2 region. Our findings documented the sporadic nature of HEV in Greater Cairo, characterized a large number of Egyptian HEV genotype 1 strains and identified living in a rural area as a potential risk factor for infection.     

检测病毒性肝炎患者血清中SEN病毒及其临床意义

目的：检测病毒性肝炎患者血清中SEN病毒D和H(SENV-D、SENV-H),并探讨其临床意义。方法：采用巢式聚合酶链反应法（nPCR）检测甲、乙、丙、戊型肝炎和非甲－戊型肝炎患者血清中SENV-D和SENV-H DNA。结果：在180例病毒性肝炎患者血清中，SENV-D和SENV-H检出率分别为17.2%(31/180)和5.6%(10/180)，总检出率为18.3%(33/180)、甲、乙、丙、戊型肝炎患者的SENV-D/H检出率高于非甲－戊型肝炎患者。从甲、乙、丙、戊型肝炎和非甲－戊型肝炎患者分离的SENV-D/H核苷酸序列，与SENV-D/H原型株比较，其同源性在94％以上。甲、乙、丙和戊型肝炎患者有无SENV-D/H合并感染，其血清生化学指标无明显差异。结论：SENV-D/H可能不是非甲-戊型肝炎的病原，甲、乙、丙和戊型肝炎患者合并感染SENV-D/H并不加重病情。     

Analysis of genetic and serology of hepatitis A virus infection during and after outbreak in two junior high schools in Surabaya,Indonesia

Outbreaks of hepatitis A have occurred in some cities in Indonesia. In Surabaya, the capital city of East Java province, Indonesia, hepatitis A outbreaks have been reported since2013, with a marked increase in the number of cases in 2015. The aim of the present study was to analyze the genetic and serology of acute symptomatic cases (early infection) during a hepatitis A outbreak and asymptomatic cases after the outbreak in two junior high schools in Surabaya in 2015 to 2016. Students with acute symptomatic hepatitis A during the outbreak and other students who were asymptomatic 3 to 4 months after the outbreak were enrolled. Asymptomatic students had no symptoms from the outbreak until they were enrolled. Sera were collected to identify anti-hepatitis A virus (HAV) IgM (by enzyme-linked immunosorbent assay) and HAV genetic variations/genotypes (using polymerase chain reaction [PCR]-sequencing and phylogenetic analysis). A total of 33 (97.1%) out of 34 sera of students with acute symptoms were positive for anti-HAV IgM and 18% of them were positive by PCR, identified as HAV subgenotype IA. No prominent amino acid variations were observed from reported HAV sequences from Indonesia. Among 38 sera of asymptomatic students, most (55.3%) were positive for anti-HAV IgM, while none were positive by PCR. In conclusion, HAV-IA was the only subgenotype identified in acute symptomatic cases during the outbreak. The percentage of HAV-specific IgM-positive cases was very high among acute symptomatic students, but that was also high among asymptomatic students, which might contribute as the important source of infection during the outbreak.     

Exposure to multiple subgenotypes of hepatitis A virus during an outbreak using matched serum and saliva specimens

Matched serum and saliva samples were collected simultaneously from 124 subjects exposed during a hepatitis A virus (HAV) outbreak at a daycare center in Rio de Janeiro, Brazil. All samples were tested for IgM and total anti-HAV antibodies by enzyme immunoassay (EIA). HAV was detected by nested PCR in serum, saliva, and water samples employing primers for the VP1/2A region of the viral RNA; all positive products were then sequenced. The viral load of the matched samples was determined by real-time PCR using the TaqMan system. HAV-RNA was identified by nested PCR in 37.7% of the saliva samples, 29% of the serum samples, and one drinking water sample. The mean HAV viral load was similar in the serum and saliva specimens (10(3) copies/ml). HAV genotypes IA and IB were detected in both specimen types, and the water sample isolate was classified as genotype IB, indicating the existence of more than one source of infection at the daycare center. In six infected patients, a different HAV subgenotype was found in their serum than in their saliva, and this unusual pattern of mixed HAV infection was investigated further by molecular cloning followed by nucleotide sequencing. All clones derived from the saliva samples belonged to subgenotype IB and shared 96.5-100% identity. However, clones derived from their corresponding serum sample belonged to subgenotype IA and shared 90.5-100% identity. This study showed the important role that non-invasive saliva samples can play in the molecular epidemiological analysis of a hepatitis A outbreak.     

Acute viral hepatitis types E,A, and B singly and in combination in acute liver failure in children in North India

The aetiological agents responsible for, and the outcome of, acute liver failure were investigated prospectively in 44 children (29 males, 15 females) attending a tertiary health care facility in India. The children were between the ages of 2 months and 13 years. Studies for viral infections and other etiologies could be carried out in 40 patients. Specific aetiological labels were possible in 35 (87.5%) patients. Thirty (75%) had evidence of acute viral hepatitis. Acute hepatitis E virus (HEV) infection was found in a total of 18 children, with hepatitis A (HAV) in 16, hepatitis B in 5, and C in 1. Seven had isolated infection with hepatitis E, five with A, and four with B. Nine had both E and A infection. Superinfection of HEV was observed in a child with Indian childhood cirrhosis (ICC). Acute HEV infection was confirmed by immunoblot assay in all the patients and in eight of these, HEV-RNA was also detected in the serum. HAV was involved in 37.5% of cases with isolated infection in 10% (4 of 40). The aetiological factors associated with acute liver failure, apart from HAV and HEV, were other hepatotropic viruses (22.5%), Wilson's disease (5%), ICC (5%), and hepatotoxic drugs (7.5%). In five patients, no serological evidence of acute viral hepatitis could be found, neither did the metabolic screen yield any result. It was observed that enterically transmitted hepatitis viruses (HAV and HEV) were associated with 60% of acute hepatic failure in children. Mixed infection of HAV and HEV formed the single largest aetiological subgroup. In developing countries, where hepatitis A and E infections are endemic, severe complications can arise in the case of mixed infection. This may contribute to most of the mortality from acute liver failure during childhood. © 1996 Wiley-Liss, Inc.