Genome analysis of hepatitis C virus strain 274933RU isolated in Russian Federation

Five overlapping cDNA fragments of hepatitis C virus (HCV) isolate 274933RU, obtained by RT-PCR, were amplified and cloned. Complete nucleotide RNA sequence has been determined. The genomic organization of 274933RU was, from 5' to 3' terminals, 5' UTR (341 nt), polyprotein ORF (9033 nt), 3' UTR (40 nt except for the poly(U-UC) and polypyrimidine stretch), and X-tail (98 nt). Phylogenetic analysis of the core and NS5 genes showed that the isolated strain belonged to HCV 1b subtype.     

Identification and characterization of a natural inter-genotypic (2b/1b) recombinant hepatitis C virus in Japan

A hepatitis C virus (HCV) strain (HC10-0804) recovered from a 12-year-old Japanese female with chronic hepatitis C segregated into discordant genotypes, 2b and 1b, in the 5'UTR/core and NS5B regions, respectively, thus suggesting an inter-genotypic recombination. The HC10-0804 isolate had a genomic length of 9,423 nucleotides (nt), excluding the poly(U) tract at the 3' terminus, and encoded a single open reading frame (ORF) for a polyprotein of 3,014 amino acids (aa). Based on Simplot and Bootscan analyses, the crossover point from 2b to 1b was estimated at nt 3443/3444 (aa 1034/1035), just after the beginning of the NS3 region. Comparison of the entire genomic sequence showed that the HC10-0804 strain was only 90.2% identical to the previously reported 2b/1b recombinant strain (SE-03-07-1689) from the Philippines, whose putative crossover point was 24 nt downstream of that of HC10-0804. These results indicate the circulation of a novel inter-genotypic (2b/1b) recombinant HCV in Japan.     

Molecular cloning of an Australian isolate of hepatitis C virus

Summary.  The genomic sequence of an Australian isolate of hepatitis C virus (HCV) was determined from overlapping cDNA clones obtained from a small amount (1.2 ml) of serum from a single individual with hepatitis C. The isolate (HCV-A) comprises 9 379 nucleotides (nt) including 324 nt of a 5′ untranslated region (5′UTR), a single long open reading frame of 9 033 nt encoding a polyprotein of 3 010 amino acids (aa), and 22 nt of a 3′ untranslated region (3′UTR). Sequence analysis of a 251 nt region within the 5′UTR and a 222 nt region within NS5B showed the genotype of HCV-A to be subtype 1b. A striking difference in the amino acid sequence of the hypervariable region 1 (HVR-1), and not in the surrounding sequence, was seen in cDNA clones synthesised from serum taken 52 weeks after the initial sample, indicating a significant population diversity of HCV genomes. Accepted October 17, 1977 Received July 7, 1997     

Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9

Echovirus 18 (EV18) is one of the echovirus serotypes associated with human diseases and in particular aseptic meningitis. To facilitate studies of the molecular epidemiology of EV18 and the evolution of enteroviruses in general, the complete nucleotide (nt) sequence was determined for the echovirus 18 prototype strain (Metcalf, EV18M). Excluding the poly A sequence, the genome consists of 7410 nt divided into a 740 nt 5' untranslated region (5' UTR), a 6567 nt long open reading frame coding for a 2189 amino acid (aa) polyprotein and a 103 nt 3' UTR. Molecular analysis of the EV18M genome showed a typical enterovirus-like organization. Phylogenetic analysis of the structural and non-structural genes revealed a pattern of different relationships to other echo- and coxsackieviruses. Similarity analysis demonstrated that the Hill strain of echovirus 9 is most likely the result of a previous recombination event between ancestors of the echovirus 9 strain Barty (5' half of the genome) and EV18M (3' half). Using a maximum likelihood approach, the recombination point was mapped to the 2C gene.     

The Complete Nucleotide Sequence of a Pakistani Isolate of Watermelon Mosaic Virus Provides Further Insights into the Taxonomic Status in the Bean Common Mosaic Virus Subgroup

Watermelon mosaic virus (WMV) is a potyvirus with a worldwide distribution, but is mostly found in temperate and Mediterranean regions. The complete nucleotide (nt) sequence of a Pakistani isolate of WMV (WMV-Pk) was determined and compared with French isolate (WMV-Fr) and other closely related potyviruses. WMV-Pk showed overall identities of 94.4% (nt) and 96% (amino acid; aa) with the WMV-Fr. However, variability was observed in the 5′ UTR and P1 region. Although sequence identities over most of the genome were well above 90% at both the nt and aa levels, reaching 99.6% (aa) in the CP and 100% (aa) in the 6K1 and 6K2, thereby suggesting that WMV-Pk and WMV-Fr are identical strains, but the sequence identities in the P1 region were only 80.6% (aa) and 82.8% (nt), while that in the 5′ UTR was 82%. These differences may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in two different agro-ecological zones. The sequence of WMV-Pk is very close to that of Soybean mosaic virus (SMV) over most of the genome, except for the N-terminal region, which is subject to recombination between SMV and Peanut stripe virus (PSV)/Bean common mosaic virus (BCMV), as revealed by Simplot and phylogenetic analyses of N- and C-terminal P1, HC-Pro, and 5′ UTR regions of the genome.     

The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA.

The complete nucleotide sequence of hepatitis C virus (HCV) cloned from the liver tissue of a Taiwanese patient with post-transfusion type C hepatitis was determined. The 5' end of HCV genomic RNA was located 341 nucleotides upstream from the initiation codon for the viral polyprotein open reading frame. The 5' end of the viral antigenomic RNA was shown to have 13 consecutive As. Thus the 3' terminus of the viral genome is a stretch of U which ends about 50 nucleotides downstream from the stop codon of the large open reading frame. The nucleotide sequence homology between this HCV strain and two Japanese isolates was 90.5 and 90.7%, respectively. Homology with the United States strain, however, was only 77.8%. Accordingly, the indigenous Taiwanese HCV strain is of the same subtype as the Japanese isolates. Novel features of the viral genome termini are possibly relevant to HCV genome replication.     

Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome

UV cross-linking analyses were performed in an attempt to determine cellular protein-viral RNA interactions with the 3' untranslated region (3' UTR) of the hepatitis C virus RNA genome. Two cellular proteins, with estimated molecular masses of 58 kDa (p58) and 35 kDa (p35), respectively, were found to specifically bind to the 3' UTR. The p58 protein was determined to be the polypyrimidine tract-binding protein. In addition to binding to the conserved 98 nucleotides (nt) of the 3' UTR, p58 also binds to the poly(U) tract of the 3' UTR. The p35 protein was found to interact only with the poly(U) tract of the 3' UTR. These conclusions are supported by the following findings: (1) p58, and not p35, binds to the 3' end conserved 98 nt, (2) both p58 and p35 bind to a 3' UTR RNA with a deletion of the conserved 98 nt, (3) the 98-nt deletion mutant 3' UTR competed out both p58 and p35 binding, (4) a poly(U) homopolymer competed out both p58 and p35 binding, (5) a 3' UTR RNA with deletion of the poly(U) tract competed out only p58 binding but not p35 binding, and (6) an RNA containing the variable region of the 3' UTR with a deletion of both poly(U) tract and 98 nt failed to compete for binding of either p58 or p35. Interaction of these cellular proteins with the HCV 3' UTR is probably involved in regulation of translation and/or replication of the HCV RNA genome.     

Complete sequence and phylogenetic analysis of a porcine bocavirus strain swBoV CH437

Porcine bocavirus (PBoV), a member of genus Bocavirus, family Parvoviridae, was first identified in 2009 in Swedish swine herds suffering from postweaning multisystemic wasting syndrome. Up to date, the different species of PBoVs have been reported in different countries. Especially, the virus isolated in China was complicated. In this study, we detected a novel PBoV strain swBoV CH437 from clinical samples collected in Gansu Province, Northwest China. The complete genome of swBoV CH437 was 5,275 nucleotides (nt) in length and contains three ORFs: ORF1 encodes NS1 (2,004 nt, 667 aa), ORF3 encodes NP1 (681 nt, 226 aa), and ORF2 encodes VP1 (2,049 nt, 682 aa) and VP2 (1,641 nt, 546 aa). Sequence analysis demonstrated that the NS1 gene shared 24.2–88.6 % nucleotide sequence identity, the NP1 shared 21.3–89.9 %, less than 95 % nucleotide sequence identity with other PBoV strains. Therefore, we propose that swBoV CH437 should be classified as a novel PBoV species.     

Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals.

A hepatitis C virus (HCV) genome was isolated and sequenced from a single Japanese patient with chronic non-A, non-B hepatitis. The genome (HCV-JT), which was constructed with 23 cDNA clones, consisted of 9436 nucleotides with a long open reading frame which could encode a sequence of 3010 amino acid residues. To study the sequence variation of the HCV genome in an individual, we analyzed another sequence of the HCV genome (HCV-JT') constructed with different cDNA clones derived from the same patient. The nucleotide variation between HCV-JT and -JT' was less than 1%, and was distributed throughout the genome except in the 5' non-coding region, where no variation was observed. The diversity was higher (1.6%) in the putative envelope protein region than in other regions. The nucleotide and deduced amino acid sequences of HCV-JT showed homologies of about 91 and 95%, respectively, with those of other Japanese HCV isolates. The nucleotide diversity was high in the gp 70 region (corresponding to the NS 1 region of flaviviruses) and low in the 5' non-coding and p22 (putative core protein) regions. A similar pattern of distribution of nucleotide changes was observed on comparison of HCV-JT with an American isolate HCV-US, where the homologies in nucleotide and amino acid sequences were about 79 and 85%, respectively. Base transversions contributed about 50% of the total base exchanges between the Japanese and American HCV sequences, but only 20% or less of those among Japanese HCV or among American HCV sequences. Thus, the Japanese and American HCVs are genetically distinguishable, supporting our earlier prediction that these two HCVs could be classified as different subtypes.     

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     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

Genetic characterization of a new insect flavivirus isolated from Culex pipiens mosquito in Japan

We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.     

Complete genomic sequence analysis of a highly virulent isolate revealed a novel strain of <Emphasis Type="Italic">Sugarcane mosaic virus</Emphasis>

Sugarcane mosaic virus (SCMV) is the most prevalent virus causing maize dwarf mosaic disease in northern China. A SCMV isolate, BD8, was obtained from the maize showing dwarf and mosaic symptoms in Baoding, China. The complete genomic sequence of BD8 is 9,576 nucleotides (nt) excluding the poly(A) tail. It contains one single open reading frame of 9,192 nt and encodes a large polyprotein of 3,063 amino acids (aa), flanked by a 5′-untranslated region (UTR) of 148 nt and a 3′-UTR of 236 nt. The entire genomic sequence of BD8 shares identities of 79.1–80.8% with those of other 13 SCMV isolates available in the GenBank at nt level, while their CP genes share identities of 76.9–82.6 and 82.8–86.9% at nt and aa levels, respectively. Phylogenetic analysis of the complete genomic sequences reveals that SCMV can be clustered to four groups: group I includes isolates from maize, group II consists of isolates from sugarcane or maize, groups III and IV contain single isolate of AU-A (AJ278405) and BD8, respectively. Thus, BD8 represents a new strain of SCMV. Furthermore analysis of the CP gene sequences of more isolates shows that BD8 is clustered to a group with the isolates from Thailand and Vietnam, which implies that isolates of this strain have been distributed in South Asia. In the greenhouse, BD8 can cause severe symptoms in all the 12 maize varieties tested with high incidence, indicating that BD8 is highly virulent.     

Genotype determination of hepatitis C virus from Northern India: Identification of a new subtype

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68–79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the nonstructural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5′-noncoding (5′ NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India. © 1996 Wiley-Liss, Inc.     

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.     

Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates belonging to the European genotype 1 have recently emerged in South Korea, suggesting potential problems for disease control. In the present study, we attempted to determine the complete nucleotide sequence of the first Korean type 1 PRRSV isolate, designated KNU-07. The full-length genome of KNU-07 was found to be 15,038 nucleotides in length, which was 60 nucleotides shorter than the type 1 prototype strain Lelystad due to a notable 60-bp deletion within the nonstructural protein 2 (NSP2). The KNU-07 genome was shown to consist of a 221-nucleotide (nt) 5′ untranslated region (UTR), a 14,703-nt protein-coding region, and a 114-nt 3′ UTR, followed by a 42-73-bp poly(A) tail. A nucleotide sequence comparison of the KNU-07 genome with 20 complete PRRSV genomes revealed a 10.5–13.3% and 39.5–40.3% divergence from type 1 and type 2 strains, respectively, at the genome level, indicating a high similarity to the virus strains commonly identified as the European genotype. In order to investigate genetic variation and to understand the molecular evolution of the type 1 isolate in Korea, extensive phylogenetic analyses were performed using the ORF5 and ORF7 nucleotide sequences of published type 1 PRRSV isolates. The data further indicated that the newly emerging type 1 isolate KNU-07 belongs to the recently proposed pan-European subtype 1. Taken together, the results of this study describe the genomic characterization of the type 1 PRRSV isolated in South Korea, suggesting a recent introduction of the virus typical for this genotype that has commonly appeared worldwide.     

2a型丙型肝炎病毒5’非编码区的RACE护增及二级结构的分析

目的：获得真全长中国大陆2a型丙型肝炎病毒（HCV）5’非编码区（5’UTR）cDNA，并分析其一级结构和二级结构，为进一步研究其在HCR复制、翻译中的调控机制、开发新的抗HCV药物奠定基础。方法：利用逆转录套式聚合酶链反应（RT－PCR）联合限制性内切酶长度多态性分析（RFLP）初步筛选出1例2a型HCV感染者，采用cDNA末端快速扩增技术（RACE）扩增出一条约800bp的cDNA片段，A－T克隆，用RFLP与PCR鉴定重组子，全自动序列分析仪测定插入子序列，RNAdraw预测二级结构。结果：RACE法获得真全长2a型HCV 5’端序列。5个克隆中，3个克隆含全长5’UTR序列，与HCV－1，HC－C2，HC－J6，HC－J8相比，同源性分别为93．6％－94．4％，92．1％－93．0％，98．8％－99．7％，96．2％－96．5％，与2a型标准株HC－J6相比，21，170，222，247，339位不同，分别为G，A，C，C，T,但这些突变不影响其二级结构。另外2个克隆为5’端缺失突变株，分别缺失54bp和144bp。结论：RACE技术快速、有效、实用，可用效获得病毒基因组的末端序列；依此获得中国大陆2a型HCV的5’UTR cDNA；在感染者血液中存在5’端部分缺失的HCV基因片段。     

The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon α resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon α-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon α/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194–2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients. Ankur Goyal and Wolf P. Hofmann contributed equally to this work.