Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate

Summary.  The complete sequence of a North American tobacco rattle virus (TRV) isolate, ‘Oregon yellow’ (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 of TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than -SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses, ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses vis also discussed. Accepted March 21, 1998     

Hepatitis E virus genotyping based on full-length genome and partial genomic regions

Some genomic regions for hepatitis E virus (HEV) genotyping have been reported to correlate well with the results from the phylogenetic analyses on the basis of the complete genome. However, few studies have systemically investigated the genomic regions for HEV genotyping using a combined phylogenetic and statistical approach. A consensus region for HEV genotyping has not been determined. In this study the nucleotide identities and genetic distances of 24 partial genomic regions and the complete genome sequences of 37 HEV strains were compared statistically. It was demonstrated with both one-way ANOVA and two-way ANOVA that only one genomic region in RNA-dependent RNA polymerase domain (4254–4560 nt) for which there were no significant differences when compared with the full-length genome (P > 0.05). The same four genotypes were identified by phylogenetic analysis based on this statistically predicted region identified as for the complete genome. RT-PCR amplification of HEV strains from all four genotypes confirmed conservation of the flanking primer sites of this region. Serum samples from 20 patients with a clinical diagnosis of hepatitis E were further analyzed by PCR using the same primers, 13 were positive and could be classified into genotype 4. These data strongly suggested that this newly identified region could be used for future HEV genotyping.     

Molecular characterization of the full-length genome of a rabies virus isolate from India

Rabies is an important public health problem in South East Asia, with cases in this part of the world contributing to about 70% of the global burden. A large number of rabies cases occur in India, however, there is no organized system of surveillance and hence there is a lack of reliable data. Moreover, comprehensive molecular epidemiological studies have not been performed on Indian virus isolates. In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus obtained from the brain of an infected patient. Comparison of the genomic sequence with those of the ten fully sequenced rabies strains available in GenBank showed nucleotide homology ranging from 97% with AY956319 to 81% with AY705373. Amino acid homology of nucleoprotein ranged from 99.7% with AY352493 to 92% with DQ875051. In case of the glycoprotein gene, the homology ranged from 98.8% with AY956319 to 87.2 % with AY705373. An extensive nucleoprotein, glycoprotein, and full-length genome-based phylogenetic analysis was performed along with sequences available from the GenBank. Phylogenetic analysis of the complete genome sequence indicated that this isolate exhibited close homology with the ex Indian strain AY956319. Primer sequences and the scheme of amplification can be availed from the authors on request.     

Genetic characterization and sequence heterogeneity of a canadian isolate of Swine hepatitis E virus

Swine hepatitis E virus (HEV) is a newly identified potentially zoonotic agent that is possibly transmitted to humans from pigs. Swine HEV is prevalent in pig populations and does not cause abnormal clinical symptoms in infected pigs, further implicating a likelihood of a risk of transmission to humans by normal contact. To date in North America, only one strain of swine HEV (strain US swine) has been fully sequenced. In the present study, we identified a swine HEV isolate from pigs in Canada, designated the Arkell strain, and determined the full length of the genomic sequence. The genome of Canadian strain Arkell consisted of 7,242 nucleotides, excluding the poly(A) tail of at least 15 A residues. The genome contained three open reading frames (ORFs), ORF1, ORF2, and ORF3, which had coding capacities for proteins of 1,708, 660, and 122 amino acids, respectively. Comparative analysis of the full-length genomic sequence indicated that the sequence of strain Arkell was distinct from those of all other known HEV isolates by 13 to 27% and shared the highest degrees of identity with human HEV isolates US-1 and US-2, HEV isolate US swine, and the human and swine HEV isolates recently isolated in Japan. On the basis of sequence similarities and phylogenetic analyses, HEV strain Arkell was grouped into genotype 3. The sequence of the Arkell swine HEV isolate differed from those of HEV isolate US swine and HEV isolate Japan swine by 13 and 14%, respectively. To date, two isolates of swine HEV (isolates Arkell and SK3 [D. Yoo et al., Clin. Diagn. Lab. Immunol. 8:1213-1219, 2001]) have been identified in Canadian pigs, and their sequences also differ from each other by 11.8%. Our studies indicate that, as with human HEV strains, swine HEV isolates exhibit extensive genetic heterogeneity.     

Analysis of the full-length genome of hepatitis E virus isolates obtained from farm pigs in Mongolia

Although no outbreaks of hepatitis E have been reported in Mongolia, a significant proportion of the general population had antibodies to hepatitis E virus (HEV). To investigate whether pigs are possible reservoirs of HEV in Mongolia, serum samples obtained from 243 2- or 3-month-old pigs on four swine farms surrounding Ulaanbaatar, the capital city of Mongolia, were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 223 pigs (91.8%) tested positive for anti-HEV, while 89 pigs (36.6%) had detectable HEV RNA. The 89 HEV isolates obtained from the viremic pigs were 78.7-100% identical to each other, and 80.9-85.9% similar to the prototype genotype 3 HEV isolate (US1) in the 412-nucleotide (nt) sequence within open reading frame 2. They were classified into two novel phylogenetic groups within genotype 3, differing by 16.4-21.3%. The swMN06-A1288 and swMN06-C1056 isolates, representing each of the two clusters within genotype 3, had a genomic length of nucleotides (nt) 7,222 nt and 7,223 nt, respectively, excluding the poly(A) tail, and shared only 81.6% over the entire genome. Upon comparison with the 25-reported genotype 3 HEV isolates over the entire genome, swMN06-A1288 had identities of merely up to 84.9%, while swMN06-C1056 of only up to 85.9%. Phylogenetic analysis confirmed the remote relatedness of the Mongolian swine isolates to the genotype 3 HEV isolates reported thus far. These results indicate that farm pigs in Mongolia are frequently infected with presumably indigenous HEV strains of genotype 3 and could be a source of HEV infections in humans in Mongolia.     

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.     

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20–25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3′ terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.     

First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB

A hepatitis A virus (HAV) recovered in Argentina from a stool sample of a sick child in the year 2006 (HAV-Arg/06) was entirely sequenced. Phylogenetic analysis included the HAV-Arg/06 sequence in subgenotype IA, either considering the usual VP1-2A variable junction fragment or the full length nucleotide sequence. Interestingly, a recombination event with subgenotype IB, involving a portion of the 2C-3A nonstructural proteins coding region (nucleotides 4961-5140) was detected using specific software. Only subgenotype IA strains have been detected in Argentina or Uruguay, whereas subgenotype IA and IB strains have been reported to circulate in Brazil. Although recombination has been given an important role in the evolution of picornaviruses, there have been only a few reports of its involvement in the evolution of HAV, probably due to the limited number of complete HAV sequences available. This study constitutes the first report of a full-length HAV sequence in Argentina and the third in South America, after the sequence of the IA isolate HAV5 from Uruguay and the IB isolate HAF-203 from Brazil. The availability of new sequence data covering the complete HAV genome will help establish a more consistent genetic relatedness among HAV isolates and the role of recombination in its evolution.     

Prevalence of hepatitis A virus,hepatitis B virus,hepatitis C virus,hepatitis D virus and hepatitis E virus as causes of acute viral hepatitis in North India: A hospital based study

Context: Acute viral hepatitis (AVH) is a major public health problem and is an important cause of morbidity and mortality. Aim: The aim of the present study is to determine the prevalence of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV) as causes of AVH in a tertiary care hospital of North India. Settings and Design: Blood samples and clinical information was collected from cases of AVH referred to the Grade I viral diagnostic laboratory over a 1-year period. Subjects and Methods: Samples were tested for hepatitis B surface antigen, anti-HCV total antibodies, anti-HAV immunoglobulin M (IgM) and anti-HEV IgM by the enzyme-linked immunosorbent assay. PCR for nucleic acid detection of HBV and HCV was also carried out. Those positive for HBV infection were tested for anti-HDV antibodies. Statistical Analysis Used: Fisher’s exact test was used and a P < 0.05 was considered to be statistically significant. Results: Of the 267 viral hepatitis cases, 62 (23.22%) patients presented as acute hepatic failure. HAV (26.96%) was identified as the most common cause of acute hepatitis followed by HEV (17.97%), HBV (16.10%) and HCV (11.98%). Co-infections with more than one virus were present in 34 cases; HAV-HEV co-infection being the most common. HEV was the most important cause of acute hepatic failure followed by co-infection with HAV and HEV. An indication towards epidemiological shift of HAV infection from children to adults with a rise in HAV prevalence was seen. Conclusions: To the best of our knowledge, this is the first report indicating epidemiological shift of HAV in Uttar Pradesh.     

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3′ terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184). SAAS-JDY5 shared approximately 87% nucleotide similarity to human and swine strains from the United States, compared with 74–75% similarity to Asian (genotype 4) and Mexican strains (genotype 2). Alignment of the SAAS-JDY5 genomic sequence with reference sequences of the same genotype revealed one nucleotide substitution and one deletion at positions 5145 and 7189 (3′ UTR), respectively. Moreover, SAAS-JDY5 contained two additional nucleotides (AC) at the very end of the 3′-terminus preceding the poly-A tail of the genome. Comparison of the putative amino acid sequence encoded by the SAAS-JDY5 genome with sequences of other genotype 3 isolates revealed 15 unique amino acid substitutions and one deletion in ORF-1, and three substitutions in ORF-2.     

应用长链RT-PCR法扩增我国登革2、4型病毒株全长cDNA

目的：采用长链RT-PCR技术扩增登革2型及4型病毒基因组全长cDNA，为构建登革病毒全长cDNA克隆、表达，深入阐明致病机理及探索新型疫苗奠定基础。方法：根据已测定的登革2、4型病毒全基因组序列，设计上下游引物。从感染登革病毒的乳鼠脑中提取病毒基因组RNA，采用长链RT-PCR技术进行扩增。为检验扩增产物的特异性，以PCR产物为模板扩增覆盖基因组的10个片段。将含有复杂二级结构的5′非编码区扩增片段，在377A型自动测序仪进行序列分析。结果：扩增出登革2、4型病毒基因组全长近11kb cDNA分子，非编码区测序结果表明扩增产物为登革2、4型病毒所特有。结论：利用长链RT-PCR首次成功扩增出登革病毒全长cDNA分子。     

Full-length genomic sequence of a hepatitis C virus genotype 2c isolate (BEBE1) and the 2c-specific PCR primers

Summary We sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.The nucleotide sequence data reported in this paper have been deposited in the GSDB, DDBJ, EMBL, and NCBI under the accession number D50409.     

Spectrum of hepatitis E virus infection in India

A solid phase enzyme linked immunosorbent assay (ELISA) that detects IgM and IgG to hepatitis E virus (HEV) was used to study seroepidemiology in 40 healthy subjects and 227 consecutive patients with liver diseases in an endemic area. Fifty-two of the liver diseases patients (22.9 percent) had acute hepatitis E. In contrast, none of the 40 healthy subjects were positive for IgM anti-HEV, validating the ELISA assay. Twenty-three of 25 (92%) patients with epidemic non-A, non-B hepatitis were confirmed as having acute hepatitis E. Only 1 of the 10 patients with sporadic, fulminant hepatic failuire of non-A, non-B, non-C etiology was positive for IgM anti-HEV. Five (31.2%) of the 16 patients with acute hepatitis in HBsAg carriers were positive for IgM anti-HEV. One patient with acute hepatitis B wascoinfected with acute hepatitis E. Acute hepatitis was a disease of the adult population, with peak attack rates in the second and third decades of life. This disease was seen in only 4 (16%) of the 25 patients with acute viral hepatitis occurring below 14 years of age. Cholestasis was predominant in 25% of patients, enzyme elevation was monophasic, and all patients had clinical and biochemical recovery from the disease. The data suggest that the majority of patients with acute sporadic non-A, non-B, non-C hepatitis in India have hepatitis E. However, fulminant hepatic failure to sporadic nature is rarely from hepatitis E. © 1994 Wiley-Liss, Inc.     

用杆状病毒系统表达戊型肝炎病毒全长结构基因的研究

目的：获取含有戊型肝炎病毒（HEV）全长结构基因的重组杆状病毒，表达戊型肝炎病毒结构蛋白。方法：将HEV全长结构基因（5147－7126nt）插入杆状病毒表达载体pAcUW51，与线性化杆状病毒DNA（Baculo Gold DNA）共转染Sf9昆虫细胞，挑病毒斑进一步感染Sf9细胞，用SDS－PAGE，Western Blot及免疫荧光检测戊型肝炎病毒结构蛋白的表达及活性。结果：SDS－PAGE分析表明HEV结构基因在杆状病毒系统中高效表达，有相对分子质量约为73000和63000两种形式；Western Blot及免疫荧光检测证明表达产物可与HEV阳性血清特异反应，说明具有HEV特异抗原性。结论：应用杆状病毒表达系统成功表达了HEV结构蛋白。     

Complete genome sequence analysis of an American isolate of Grapevine virus E

The complete genome sequence of Grapevine virus E (GVE) collected from a red-berried wine grape cultivar (Cabernet Sauvignon) in Washington State was determined. The 7,568 nucleotide long genome of GVE is more similar in sequence identity with a GVE isolate from a wine grape cv. Shiraz from South Africa when compared with an isolate from “Aki Queen” grape from Japan. Like GVE isolates from South Africa and Japan, the Washington isolate encodes five open reading frames (ORFs) and the overall genome organization is identical among these isolates. In addition to AlkB domain, a DExD domain, belonging to the DEAD-like helicases superfamily, was present upstream of the helicase domain in the replicase ORF of the virus.     

Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E

It was suggested that hepatitis E virus (HEV) genotype 4 is associated more closely with the severity of hepatitis E than genotype 3, although the virological basis remains unknown. The aim of this study was to examine whether genomic differences among genotype 4 HEVs are responsible for the development of fulminant hepatitis. Full-length sequences of genotype 4 HEVs from three patients with fulminant hepatitis and six patients with acute self-limited hepatitis were determined. The sequences were analyzed with those of 13 genotype 4 HEV isolates whose entire nucleotide sequence is known. Analysis of 22 full-length sequences (fulminant hepatitis, 5; acute hepatitis, 17) revealed that C at nt 1816 and U at nt 3148 (U3148), both of which do not change the amino acid sequences, were significantly associated with fulminant hepatitis (P = 0.0489, respectively). When partial nucleotide sequences containing nt 1816 or nt 3148 were determined in 16 additional HEV isolates of genotype 4, a closer association between U3148 and fulminant hepatitis (P = 0.0018) was observed. The comparison of 86 HEV isolates of all four genotypes showed that U3148 had a stronger association with fulminant hepatitis than other nucleotides at nt 3148 (P = 0.0006). Patients infected with HEV with U3148 had a significantly lower value of the lowest prothrombin activity (P = 0.0293). Nt 3148 is located within the RNA helicase domain, and 22-nt sequence including nt 3148 was well conserved among all genotypes. A silent substitution of U3148 in HEV may be associated with the development of fulminant hepatitis. Further studies are needed to clarify the underlying mechanism.