Heterogeneity and evolution rates of delta virus RNA sequences.

To investigate the geographical divergence of delta virus RNA sequences, 868 nucleotides (nt), including the delta antigen-coding region, were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct sequencing and compared with three previously reported nucleotide sequences. The sequence obtained for hepatitis delta virus RNA from patient M was approximately 92% identical to sequences previously obtained for two other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 81% identical to the previously sequenced strains. This suggests that delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed. To study the evolution rate of delta virus RNA, viral isolates obtained 3 and 4 years apart from each of two patients were also sequenced. It was estimated that the substitution rate of viral RNA was 0.57 x 10(-3) nt per site per year in patient M and 0.64 x 10(-3) nt per site per year in patient S for the delta antigen gene.     

Complete Genomic Sequence of a Chinese Isolate of Duck Hepatitis Virus

The complete genomic sequence of Duck hepatitis virus 1(DHV-1) ZJ-V isolate was sequenced and determined to be 7 691 nucleotides(nt) in length with a 5'-terminal un-translated region(UTR) of 626 nt and a 3'-terminal UTR of 315 nt(not including the poly(A) tail).One large open reading frame(ORF) was found within the genome(nt 627 to 7 373) coding for a polypeptide of 2 249 amino acids.Our data also showed that the poly(A) tail of DHV-1 has at least 22 A's.Sequence comparison revealed significant homology(from 91.9% to 95.7%) between the protein sequences of the ZJ-V isolate and those of 21 reference isolates.Although DHV-1 has been classified as an unassigned virus in the Picornaviridae family,its genome showed some unique characteristics.DHV-1 contains 3 copies of the 2A gene and only 1 copy of the 3B gene,and its 3'-NCR is longer than those of other picornaviruses.Phylogenetic analysis to do sequence homology based on the VP1 protein sequences showed that the ZJ-V isolate shares high sequence homology with the reported DHV-1 isolates(from 92.9% to 99.2%),indicating that DHV-1 is genetically stable.     

Complete genomic sequence of a Chinese isolate of Duck hepatitis virus

The complete genomic sequence of Duck hepatitis virus 1 (DHV-1) ZJ-V isolate was sequenced and determined to be 7 691 nucleotides (nt) in length with a 5'-terminal un-translated region (UTR) of 626 nt and a 3'-terminal UTR of 315 nt (not including the poly(A) tail). One large open reading frame (ORF) was found within the genome (nt 627 to 7 373) coding for a polypeptide of 2 249amino acids. Our data also showed that the poly (A) tail of DHV-1 has at least 22 A's. Sequence comparison revealed significant homology (from 91.9% to 95.7%) between the protein sequences of the virus in the Picornaviridae family, its genome showed some unique characteristics. DHV-1 contains 3copies of the 2A gene and only 1 copy of the 3B gene, and its 3'-NCR is longer than those of other picornaviruses. Phylogenetic analysis to do sequence homology based on the VP1 protein sequences showed that the ZJ-V isolate shares high sequence homology with the reported DHV-1 isolates (from 92.9% to 99.2%), indicating that DHV-1 is genetically stable.     

Molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in China

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3'-terminal sequences of VP1 (115 nt) and the 5' half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3' half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of approximately 3.7 x 10(-2) substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.     

水稻条纹病毒RNA4基因间隔区序列分析——混合侵染及基因组变异证据

克隆和测定了我国水稻条纹病毒（RSV）22个分离物 RNA4基因间隔区（Intergenic region,IR）序列,序列比较结果表明,我国RSV RNA4 IR在长度上存在634bp、654bp及732bp 3种不同的类型,其中3种不同类型的序列在云南省均有存在,而在其它省份仅存在654bp 类型的序列,云南省还存在不同片段类型序列在同一分离物中混合侵染的现象。序列内部具有两个重要的结构特征,一是具有插入序列;二是有两处反向重复序列,可形成两个明显的发夹结构,其中一个序列比较保守,形成的发夹结构稳定;但另一个发夹结构由于碱基变异导致其稳定性在各个分离物中差异较大。本论文还讨论了插入片段特性、不稳定的发夹结构的形成最低自由能及病毒致病性分化的关系。     

Genomic typing of hepatitis C viruses present in China.

Hepatitis C virus (HCV) genomic clones were obtained from the serum of Chinese HCV carriers using a polymerase chain reaction-based approach. Consensus sequences were derived from (1) the structural region (nt 1-1543) for one carrier, (2) the hypervariable region V (nt 1156-1233) from four carriers and (3) region V3 from four carriers. Region V3, located in the nonstructural domain NS5 (nt 7066-7137), has been previously shown to be a particularly good marker for the genomic typing of HCV isolates [Inchauspe et al., Proc. Natl. Acad. Sci. USA 88 (1991) 10292-10296]. Comparison of these sequences with sequences from geographically distinct HCV isolates indicates that Chinese HCV strains are closely related to, though distinguishable from, Japanese prototype strains. One amino acid motif, GGAA, located in region V, was found to be conserved only among Chinese isolates. This may define a new subgroup among HCV isolates.     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

Emergence of Viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.     

Population Structure and Genetic Diversity within Peach latent mosaic viroid Field Isolates from Peach Showing three Symptoms

To gain insight into the molecular basis of field isolates inducing the symptoms of leaf yellowing, discolouration along leaf sides and leaf mosaic, six isolates from peach showing the three different symptoms in the field were studied by single-strand conformation polymorphism analysis and nucleotide sequence analysis. Results revealed that each Peach latent mosaic viroid (PLMVd) isolate is composed of a population of genetically related variants (haplotypes), one being predominant with frequencies from 32% to 57%, and most of others having a low frequency of 4–5%. Each predominant haplotype was sequenced, as well as some non-predominant haplotypes selected randomly for comparative purposes. In each isolate, sequence alignment among the predominant and non-predominant haplotypes demonstrated that the predominant haplotype had the least variation with others among them, and its sequence was identical to the consensus sequence, which inflected that the predominant haplotype displayed a wide representative of sequence for others in a PLMVd isolate. The similarities and genetic distance between the predominant sequences from peach showing the same symptoms were higher and smaller, respectively, than that with different symptoms; they were more than 98.8% and <1%, respectively, between the predominant sequences with same symptomatic source, and were <98.5% and more than 1%, respectively, between the predominant sequences with different symptomatic source. Some particular variations were indicated for these isolates, and it revealed that the isolates with the symptom of discolouration along leaf sides on their source peach trees had a G or U in position 169 nt, and the isolates with the symptom of leaf yellowing had U and C in 115 and 116 nt, respectively, and the isolates with the symptom of leaf mosaic showed diversity at (3 nt: delete C; 5 nt: A and 54 nt: U).     

Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages

A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined. Approximately 95-99% of all L. lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment. Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns. The complete nt sequence for a 1.6-kb region from nine independent L. lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp. This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein. Three out of the five changes occur in a 187-bp region, 5' to this large ORF. The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein. The encoded protein is extremely charged and shares some homology with yeast translation initiation factor. In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.     

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.     

从重症肝炎病人血清分离的丁型肝炎病毒核酶区的克隆与序列分析

从我国一例四川丁型肝炎病毒HDVRNA,丁型肝炎抗原均阳性的重症肝炎患者的血清中,用异硫氰酸胍法提取RNA经过逆转录PCR扩增后获得一长289bp的HDVcDNA片段,将PCR产物回收后直接克隆到PCRTMⅡ质粒,挑出阳性克隆并亚克隆入M13mp19的EcoRⅠ区位点,提取单链进行序列分析,结果显示与已知的中国河南、美国、日本、秘鲁和中国台湾5株HDVcDNA相同片段比较,同源性分别为对97.2％、93％、94％、79％、96％。     

Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain.

A hepatitis C virus (HCV) cDNA covering part of the nonstructural region, NS3, was amplified from the serum of 50 out of 76 French non-A, non-B hepatitis patients by the nested polymerase chain reaction (PCR). Determination of a 407-bp sequence from four such cases revealed the presence of two different virus genotypes, F1 and F2, which exhibited 19-20% sequence divergence. F1 was represented by three of the four isolates and showed a sequence homology of about 97.5% to the prototype American HCV isolate, but of only 79% to a reported Japanese isolate. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype American HCV. PCR products from the 50 samples were hybridized with labeled F1 and F2 fragments under stringent conditions; results indicated the F1-related strain(s) as the major HCV genotype. Furthermore, a total of 1477 bp of sequence has been determined for one of the isolates belonging to the F1 category. These results will have implications for the PCR detection of HCV infection and production of HCV vaccines, especially for European countries.     

Genomic Characterization of Japanese Macaque Rhadinovirus,a Novel Herpesvirus Isolated from a Nonhuman Primate with a Spontaneous Inflammatory Demyelinating Disease

Japanese macaque rhadinovirus (JMRV) is a novel gamma-2 herpesvirus that was isolated from a Japanese macaque (JM) with an inflammatory demyelinating encephalomyelitis referred to as Japanese macaque encephalomyelitis, a disease that possesses clinical and histopathological features resembling multiple sclerosis in humans. Genomic DNA sequence analysis reveals that JMRV is a gammaherpesvirus closely related to rhesus macaque rhadinovirus (RRV) and human herpesvirus 8. We describe here the complete nucleotide sequence and structure of the JMRV genome, as well as the sequence of two plaque isolates of this virus. Analysis of the JMRV genome not only demonstrates that this virus shares a number of genes with RRV that may be involved in pathogenesis but also indicates the presence of unique JMRV genes that could potentially contribute to disease development. The knowledge of the genomic sequence of JMRV, and the ability to easily propagate the virus in vitro, make JMRV infection of JM an attractive model for examining the potential role of an infectious viral agent in the development of demyelinating encephalomyelitis disease in vivo.     

The complete nucleotide sequence of RNa1 of barley mild mosaic virus (asl1) and attempts at bacterial expression of the P3 protein

The complete nucleotide sequence of RNA1 of an Aschersleben isolate of barley mild mosaic virus (BaMMV) was determined. It consists of 7263 nucleotides (nt) excluding the 3' poly (A) tail. The 5' and 3' nontranslated regions (NTR) are 148 and 338 nt in length, respectively, and flank a single large open reading frame coding for a precursor polypeptide with a calculated molecular mass of 256 kDa. Sequence comparison revealed a 96% amino acid (aa) identity to RNA1 translation products of Japanese and French BaMMV isolates. Conserved nucleotide motifs in the 3' sense and 5' complementary sense NTR of the two genomic RNAs were identified that may represent the polymerase recognition sites. A range of constructs containing various parts of the coding region of the P3 nonstructural protein was prepared for expression in Escherichia coli . A short stretch of 35 aa in the C-proximal region of P3 appeared to be highly toxic to the bacterium.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

Phenotypic and genetic characterizations of Streptococcus dysgalactiae strains isolated from fish collected in Japan and other Asian countries

Lancefield group C Streptococcus dysgalactiae is an emerging fish pathogen, which was first isolated in 2002 in Japan. Streptococcus dysgalactiae isolates collected from diseased fish in Japan ( n =12), Taiwan ( n =12), China ( n =2), Malaysia ( n =3), and Indonesia ( n =1) were characterized using biased sinusoidal field gel electrophoresis (BSFGE), sodA gene sequence analysis, and antimicrobial susceptibility. These isolates exhibited high phenotypic homogeneity irrespective of the countries from where the strains were collected. Seventeen isolates were found to be resistant to oxytetracycline and carried the tet (M) gene, except for the strains collected in Taiwan and the PP1564 strain collected in China. The sodA gene sequence analysis revealed that 23 isolates were identical, except for one Japanese isolate (KNH07902), in which a single nucleotide differed from that of the other isolates. Based on BSFGE typing by ApaI macrorestriction, the isolates – including the Japanese, Taiwanese, and Chinese isolates – could be grouped into one main cluster at a 70% similarity level. However, the macrorestriction genotypes of some isolates were apparently distinct from those of the main cluster.     

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%–94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%–81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160–176 and 306–322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.