Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.     

A nationwide survey of hepatitis E virus (HEV) infection in wild boars in Japan: identification of boar HEV strains of genotypes 3 and 4 and unrecognized genotypes

To investigate the nationwide prevalence of hepatitis E virus (HEV) infection and to characterize HEV genomes among Japanese wild boars (Sus scrofa leucomystax), 578 boars captured in 25 prefectures from 2003 to 2010 were studied. Anti-HEV IgG was detected in 8.1%, and HEV RNA in 3.3% of boars. Among the 19 boar HEV isolates obtained from infected boars, 14 isolates (74%) were classified as genotype 3, 4 isolates (21%) as genotype 4, and the remaining isolate (wbJOY_06) was distantly related to all known HEV isolates of genotypes 1-4, differing by 18.4-25.0% and 18.0-24.3% within the 412-nucleotide sequence of ORF1 and ORF2, respectively. A genotype 4 boar HEV isolate (wbJGF_08-1) obtained herein shared 98.6% identity over the entire genome with a human HEV isolate obtained from a patient who developed acute hepatitis after consuming undercooked wild boar meat, suggesting that wild boars are also reservoirs for genotype 4 HEV in humans.     

A near-complete genome sequence of a distinct isolate of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> from China,representing a sixth new genotype

The 5803 nt genomic sequence of a Sugarcane yellow leaf virus (SCYLV) isolate (SCYLV-chn1) from China was determined. It covered more than 98% of the complete viral genome and contained all the six ORFs and the entire intergenic untranslated region. This isolate was most closely related to SCYLV genotype CUB (isolates CUB-YL1 and CB86010) with identities of 95.2–97.4% (nt) (93.2–97.2% aa) in ORF0, ORF1, and ORF2. Sequence comparison and phylogenetic analyses supported the view that this isolate represents a new genotype; SCYLV CHN1 was suggested as the name for this new genotype.     

Analysis of the full-length genome of hepatitis A virus isolated in South America: heterogeneity and evolutionary constraints

Hepatitis A virus (HAV) is a hepatotropic member of the family Picornaviridae. Currently, the entire nucleotide sequence is available for only 26 HAV isolates. The complete genome sequence of genotype IA HAV from strains isolated in South America, where genotype IA is the most prevalent genotype, remains unknown. In this study, the complete nucleotide sequence was determined for a genotype IA HAV isolate recovered from a Uruguayan patient (HAV5). Phylogenetic analysis performed using HAV5 and all available full-length IA genotype HAV strains revealed a high synonymous substitution rate throughout the HAV polyprotein. The results of these studies revealed strong selection against amino acid replacements along the HAV polyprotein and may explain, at least in part, the presence of a single HAV serotype.     

The entire nucleotide sequences of two distinct TT virus (TTV) isolates (TJN01 and TJN02) remotely related to the original TTV isolates

Summary.  TT virus (TTV) has a wide range of sequence divergence by which it is classified into at least 16 genotypes. A TTV isolate of genotype 12 (TJN01) and another of genotype 13 (TJN02) were sequenced in the entire genome, and compared with the reported TTV isolates. TJN01 and TJN02 had genomic lengths of 3787 and 3794 nucleotides (nt), respectively, which were shorter by 66 and 59 nt than the prototype TTV isolate of genotype 1 (TA278). TJN01 and TJN02 shared the nucleotide sequence with TA278 merely in 53.9% and 55.2%, respectively. They possessed two major open reading frames (ORFs) and the noncoding region with a GC-rich region forming stem-loop structures, which are characteristic of TTV. However, their amino acid sequences in ORF1 were similar to that of TA278 in only 35.4 and 34.0%, respectively; TJN01 was 45.4% similar to TJN02. Comparison with TTV isolates of the same genotype identified hypervariable regions in ORF1 of TJN01 and TJN02, as in the prototype TTV of genotype 1. However, quasispecies were barely observed in them. Furthermore, sequences of hypervariable regions scarcely changed during 2–5.5 years in both TJN01 and TJN02. These results indicate that TTV of genotypes 12 and 13 are much different from the prototype TTV of genotype 1. Received January 24, 2000 Accepted March 22, 2000     

Epidemiological and genetic analysis of a sustained community-wide outbreak of hepatitis A in the Republic of Korea, 2008: A hospital-based case–control study

BackgroundThe epidemiological shift of hepatitis A has contributed to a sustained community-wide outbreak in Korea during 2008.ObjectivesTo assess the risk factors associated with hepatitis A virus (HAV) propagation, and to analyze the circulating genotype in the sustained community-wide outbreak.Study designThe hospital-based case–control study was conducted in an 850-bed university hospital in Seoul from April to August, 2008. For molecular analysis of HAV isolates, a 488-bp gene fragment of the VP1 region was amplified and sequenced.ResultsIn the multivariated logistic regression model, the risk factors of HAV infection adjusted by age were contacts with hepatitis A case (OR 3.98, 95% CI: 1.36–11.66), residence with child aged ≤5 years (OR 3.43, 95% CI: 1.32–8.87), consuming uncooked lettuce (OR 3.98, 95% CI: 1.83–8.68) or carrot (OR 2.38, 95% CI: 2.38–5.09), drinking tap water (OR 3.68, 95% CI: 1.62–8.37) or portable spring water (OR 2.71, 95% CI: 1.11–6.62) supplied by water purifiers, and eating out (OR 3.87, 95% CI: 1.53–9.78). All isolates analyzed belonged to genotype IIIA. There were 42 nucleotide differences in the sequenced VP1 region among the isolates. Amino acid sequences were identical with each other.ConclusionsOur study suggests that sporadically contaminated food- or water-borne sources as well as person-to-person transmission might lead a sustained community-wide HAV outbreak and pre-existing dominant genotype IA might be replaced with genotype IIIA as a major epidemic strain in Korea. Our findings urge the health authority to make public guidelines for HAV vaccination and outbreak control.     

Consensus RT-nested PCR detection of yellow head complex genotypes in penaeid shrimp

A consensus RT-nested (n)PCR is described that detects the six distinct genotypic variants in the yellow head virus (YHV) complex. The PCR primers targeted ORF1b gene regions more highly conserved amongst the reference strains of YHV (genotype 1) and gill-associated virus (GAV, genotype 2) and a set of 57 field isolates containing multiple representatives of each genotype. The test employed short PCR (359 bp) and nPCR (147 bp) amplicons to minimise the effects of RNA degradation. To ensure < or = 8-primer degeneracy, two primers were designed to each site, one accommodating sequence variations amongst genotype 1 isolates and the other variations amongst isolates of the other genotypes. The analytical sensitivity limits of the PCR and nPCR were estimated to be approximately 1250 and approximately 1.25 RNA copies, respectively. The superior group-specificity of the consensus RT-nPCR compared to other OIE-recommended PCR tests for YHV/GAV was demonstrated using RNA from 17 Penaeus monodon shrimp infected with representatives of each of the six genotypes. Phylogenetic analysis using the 94 nt ORF1b gene sequence spanned by the nPCR primers generated genotype assignments that were consistent with those obtained using the extended 671 nt sequence used for the initial identification of genotypes.     

Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR

The genomic cloning and sequence of hepatitis E virus (HEV) from an epidemic in North India is reported. We describe here a simple method wherein the viral RNA was reverse transcribed and then amplified in a single step using an extra long polymerase chain reaction procedure. The full genome nucleotide sequence of this HEV isolate (called Yam-67) was made up of 7191 nucleotides, excepting the poly(A) tail and had three open reading frames: ORF1 coding for 1693 amino acids (aa), ORF2 coding for 659 aa and ORF3 coding for 122 aa. This North Indian isolate of HEV showed close sequence homology to other HEV isolates from India and Asia, but was distant from the Chinese genotype 4, Japanese, Mexican and US isolates. There is no indication from sequence analysis that this may be an atypical strain of HEV, as reported earlier.     

Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif ¹¹²E-Q-Q-E-R-L¹¹⁷ at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.     

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.     

Genome segment RNA-1 of a flat apple isolate of <Emphasis Type="Italic">Cherry rasp leaf virus</Emphasis>: nucleotide sequence analysis and RT-PCR detection

Summary. The sequence of the RNA-1 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was determined using overlapping cDNA fragments. CRLV-FA RNA-1 consists of 6992 nucleotides (nt), excluding a 3′ poly (A) tail. A single open reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a putative polyprotein consisting of 2235 amino acid (aa) residues, approximately 249.6 kDa. When compared to CRLV-pot (potato isolate) RNA-1 ORF, 2 deletions of 5 aa and 10 aa (total 15 aa) were observed at the variable N-terminus of the protease cofactor of CRLV-FA. Non-coding regions were identified at the 5′-(142 nt) and 3′-end (145 nt). CRLV-FA and CRLV-pot are isolates of the same virus with identity levels for the RNA-1 associated nt and deduced aa of 94% and 95%, respectively. RT-PCR targeting CRLV-FA RNA-1 appear to be of similar sensitivity and just as reliable as RT-PCR targeting RNA-2.     

Genetic diversity of beak and feather disease virus detected in psittacine species in Australia

The complete nucleotide (nt) sequence of eight isolates of beak and feather disease virus (BFDV) obtained from a range of psittacine species with psittacine beak and feather disease (PBFD) from throughout Australia were compared with the sequences of two BFDV isolates previously reported from Australia (BFDV-AUS) and America (BFDV-USA), respectively. All isolates had the same basic structure including the position of the open reading frames, the hairpin structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the three motifs of Rep protein encoded from ORF1 and involved in rolling circle replication, and the P-loop motif previously described, but the genome size of the eight isolates ranged from 1992 to 2018 nt. Overall nt identity of the isolates compared to BFDV-AUS ranged from 84 to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both coding and noncoding regions. The identity of the nt sequence of ORF2 compared to BFDV-AUS varied from 80 to 99%, while the identity of the deduced amino acid sequences varied from 73 to 99%. Phylogenetic analysis grouped the isolates into four clusters but there were no apparent regional differences or differences related to the psittacine species of origin. While seven ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the BFDV-AUS isolate described previously, only three of these ORFs were detected in all 10 BFDV isolates for which sequence data were available. The three ORFs were ORF1 that presumably encodes the Rep protein, ORF2 presumably the major capsid protein, and the ORF previously designated ORF5. The ORF5 was of two size classes in different isolates, 303 and 474 nt, and only the first 303 nt of the viruses with an ORF of 474 nt were common to the other isolates.     

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.     

Erratum to: Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif ¹¹²E-Q-Q-E-R-L¹¹⁷ at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.     

北京地区TT病毒分离株全基因的克隆及序列测定

目的 克隆和测定北京地区ＴＴ病毒（ＴＴＶ）分离株全基因序列。方法 采用聚合酶链反应（ＰＣＲ）技术从１例临床非甲￣庚型肝炎病人血清中分段扩增ＴＴＶ全基因,获得５个互相重叠的片段,分别长１９９ｂｐ（１－１９９）,１２６７ｂｐ（９０－１３５６,８７８ｂｐ（１３５４－２２３１,１１２９ｂｐ（２１７８－３３０６,４３４ｂｐ（３３０６－３７３９）,用标准的分子生物学方法测定了这５个序列,从而得到全长ＴＴＶ基因     

Sequential changes in hepatitis A virus genotype distribution in Estonia during 1994 to 2001

Hepatitis A virus (HAV) isolates from a large outbreak and from non-outbreak cases in Estonia were characterized by sequencing the aminoterminal VP1 region. From January 1998 to December 1999, a total of 1084 cases of hepatitis A were reported to the Harjumaa-Tallinn and Ida-Virumaa Health Protection Services in Estonia. The attack rate was highest among males aged 15-29. Initial cases were noted to be associated with injecting drug use. IgM anti-HAV positive sera were available from 107 hospitalized outbreak cases and from 68 patients sampled during 1994 to 2001. HAV RNA was detected in 42% of sera from 1994-1996 and in 88% of sera from 1998-2001. It was possible to obtain HAV sequences from 83 outbreak and 29 background cases. The outbreak strain was represented by five different sequences, all belonging to subtype IIIA. During the outbreak, this IIIA strain also spread into the general population. All available non-outbreak isolates from 1994 to 2001 but one belonged to genotype IA and formed distinct clusters as compared to isolates from other parts of the world. One subtype IIIA isolate from 1995 was unrelated to the outbreak strain. Subtype IA had been dominating in Estonia during 1994-2001, but the outbreak strain from 1998 to 1999 was IIIA. This subtype was encountered previously in addicts in Sweden during the 1980s and in Norway at the end of the 1990s. This study supports the use of limited sequencing within the aminoterminal VP1 region for studying the molecular epidemiology of hepatitis A.     

Hepatitis A virus genotype and its correlation with the clinical outcome of acute hepatitis A in Korea: 2006-2008

Korea has recently experienced a nationwide outbreak of hepatitis A. This study aimed to investigate hepatitis A virus (HAV) genotypes and to compare clinical features between patients infected with HAV genotype IA and those with genotype IIIA. From September 2006 to August 2008, 595 patients with symptomatic hepatitis A were enrolled prospectively in four hospitals in Korea. Among them, 556 patients participated in this study by providing serum or stool samples for genotypic analysis. HAV RNA was detected in 499 patients (89.7%). Major genotypes included IA (n = 244, 48.9%) and IIIA (n = 244, 48.9%), and the remaining genotype was IB (n = 11, 2.2%). From September 2006 to August 2007, the distribution of genotypes IA and IIIA were 64.6% and 35.6%, respectively, which changed to 42.3% and 54.6%, respectively, from September 2007 to August 2008, indicating change of circulating HAV genotypes in the study period from IA to IIIA. Major patterns of amino acid substitution in the VP3/VP1 junction region were observed at position 512 (P → L) in genotype IA and at 520 (R → K) in genotype IIIA. Patients with genotype IIIA infection showed significantly higher aminotransferase levels, prothrombin time, and leukocyte count, with more severe symptoms than those with genotype IA at the time of admission. These results suggest the occurrence of a change of circulating HAV genotypes in recent community‐wide outbreaks of hepatitis A in Korea, and genotype IIIA infection, compared with genotype IA infection, might show more severe clinical manifestations. J. Med. Virol. 83:2073–2081, 2011. © 2011 Wiley Periodicals, Inc.     

猪戊型肝炎病毒株swGX32全基因组序列分析

目的 分析从广西地区分离的1株猪戊型肝炎病毒swGX32全基因组序列并比较其与其他分离株的差异.方法 设计PCR引物,用巢式反转录聚合酶链反应法(RT-nPCR)分段扩增戊型肝炎病毒(HEV)株swGX32全基因序列,用cDNA末端快速扩增法(RACE)扩增其末端序列,对扩增产物进行克隆和测序,并对拼接后的基因组进行序列和进化分析.结果 除3′polyA尾巴外,swGX32全长7240 nt, ORF1与ORF2重叠4 nt, ORF3包含在ORF2序列中.swGX32全基因序列与HEV1～4型核苷酸序列的同源性分别为:73%～74%、73%、74%～75%,83%～94%,其中与中国人源HEV株JKO-ChiSai98C同源性最高,达94%.全基因序列进化分析显示,swGX32位于HEV基因4型分枝上,ORF2部分核苷酸序列进化分析显示,swGX32与JKO-ChiSai98C同在HEV 4a亚型分枝上.结论 猪HEV swGX32在全基因组结构及分子进化上均与人HEV JKO-ChiSai98C有密切关系,为揭示戊型肝炎是一种人兽共患病提供了分子生物学依据.