Complete genome sequence of a novel porcine enterovirus strain in China

The porcine enteroviruses (PEVs) belong to the family Picornaviridae. We report a complete genome sequence of a novel PEV strain that is widely prevalent in pigs at least in central and eastern China. The complete genome consists of 7,390 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 812 and 7318 and encodes a 2,168-amino-acid polyprotein. Phylogenetic analysis based on the 3CD and VP1 regions reveals that this PEV strain belongs to a species of PEV9 but may represent a novel sero-/genotype in CPE group III. We also report the major findings from bootscan analysis based on the whole genomes of PEVs in the present study and those available in GenBank.     

Complete Genome Sequence of a Novel Human Parechovirus

Human parechoviruses (HPeVs) belonging to the family Picornaviridae are widely spread pathogens among young children. We report the complete genome sequence of a novel HPeV isolated from the stool sample of a hospitalized child with diarrhea in China. The genome consists of 7,305 nucleotides, excluding the 3′ poly(A) tail, and has an open reading frame that maps between nucleotide positions 675 and 7217 and encodes a 2,180-amino-acid polyprotein. The genome sequence of the virus was sufficiently distinct from the 8 known HPeV types. Phylogenetic analysis based on the complete genome indicated that the HPeV strain represents a new genotype.     

Full Genome Sequence of a Novel Coxsackievirus B5 Strain Isolated from Neurological Hand,Foot, and Mouth Disease Patients in China

Coxsackievirus B5 (CVB5) belongs to the human enterovirus B species within the family Picornaviridae. We report the complete genome sequence of a novel CVB5 strain, CVB5/SD/09, that is associated with neurological hand, foot, and mouth disease in China. The complete genome consists of 7,399 nucleotides, excluding the 3′ poly(A) tail, and has an open reading frame that maps between nucleotide positions 744 and 7301 and encodes a 2,185-amino-acid polyprotein. Phylogenetic analysis based on different genome region regions reveals that CVB5/SD/09 belongs to a novel CVB5 lineage, and similarity plotting and bootscanning analysis based on the whole genome of CVB5 in the present study and those available in GenBank indicate that the genome of CVB5/SD/09 has a mosaic-like structure, suggesting that recombination between different CVB5 strains may occur.     

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.     

Complete genome sequence of a novel type of human parechovirus strain reveals natural recombination events

Human parechoviruses (HPeVs) are a species in the Parechovirus genus of the Picornaviridae family. We report a complete genome sequence of a novel HPeV strain, CH-ZJ1, that was found in an infant with gastroenteritis in Zhenjiang City, China. The complete genome consists of 7,298 nucleotides (nt), excluding the 3' poly(A) tail; the open reading frame is mapped between nucleotide positions 654 and 7211 and encodes a 2,185-amino acid (aa) polyprotein. The phylogenetic tree obtained for the complete genome of this HPeV strain and the other HpeV strains available in GenBank indicated that CH-ZJ1 is intervenient between HpeV type 4 (HpeV4) and HpeV5. Phylogenetic analysis based on the 3D and VP1 genes reveals two incongruent trees. Recombination detection indicated that CH-ZJ1 might be a recombinant which was produced by more than one genomic recombination event that occurred among HPeV1, HPeV4, and HPeV3 strains.     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.     

Complete genome sequence of a recombinant coxsackievirus b4 from a patient with a fatal case of hand, foot, and mouth disease in guangxi, china

The coxsackievirus B4 (CVB4) belongs to human enterovirus B species within the family Picornaviridae. Here we report a novel complete genome sequence of a recombinant CVB4 strain, CVB4/GX/10, which was isolated from a patient with a fatal case of hand, foot, and mouth disease in China. The complete genome consists of 7,293 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 742 and 7293 and encodes a 2,183-amino-acid polyprotein. Phylogenetic analysis based on different genome regions reveals that CVB4/GX/10 is closest to a CVB4 strain, EPIHFMD-CLOSE CONTACT-16, in the 5' half (VP4～2B) of the genome, although it is closer to a Chinese CVB5 strain, CVB5/Henan/2010, in the 3' half (2C～3D) of the genome. Furthermore, similar bootscan analysis based on the whole genomes demonstrates that recombination has possibly occurred within the 2C domain and that CVB4/GX/10 is a possible progeny of intertypic recombination of the CVB4 strain EPIHFMD-CLOSE CONTACT-16 and CVB5/Henan/2010 that occurred during their cocirculation and evolution, which is a relatively common phenomenon in enteroviruses.     

Complete nucleotide sequence of the S10 genome segment of grass carp reovirus (GCRV)

Hemorrhagic disease, caused by the grass carp reovirus (GCRV), is one of the major diseases of grass carp in China. Little is known about the structure and function of the gene segments of this reovirus. The S10 genome segment of GCRV was cloned and the complete nucleotide sequence is reported here. The S10 is 909 nucleotides long and contains a large open reading frame (ORF) encoding a protein of 276 amino acids with a deduced molecular weight of approximately 29.7 kDa. Comparisons of the deduced amino acid sequence of GCRV S10 with those of other reoviruses revealed no significant homologies. However, GCRV S10 shared a putative zinc-finger sequence and a similar distribution of hydrophilic motifs with the outer capsid proteins encoded by Coho salmon aquareovirus (SCSV) S10, striped bass reovirus (SBRV) S10, and mammalian reovirus (MRV) S4. It was predicted that this segment gene encodes an outer capsid protein.     

Molecular Characterization of a Bovine Enteric Calicivirus: Relationship to the Norwalk-Like Viruses

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.     

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.     

Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.     

Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae

In this study, we report the characterization of a novel calicivirus (CV), the Tulane virus (TV), which was isolated from stool samples of captive juvenile rhesus macaques (Macaca mulatta) of the Tulane National Primate Research Center. The complete genome of TV contains 6,714 nucleotides plus a poly(A) tail and is organized into three open reading frames (ORFs) that encode the nonstructural (NS) polyprotein (ORF1); the capsid protein (ORF2), with an estimated molecular mass of 57.9 kDa; and a possible minor structural protein (ORF3), with an isoelectric point (pI) of 10.0 and a calculated molecular mass of 22.8 kDa. The NS polyprotein revealed all typical CV amino acid motifs, including GXXGXGKT (NTPase), EYXEX (Vpg), GDCG (protease), and GLPSG and YGDD (polymerase). Phylogenetic trees constructed for the NS polyprotein, NTPase, protease, polymerase, and capsid protein sequences consistently placed the TV on a branch rooted with Norovirus, but with distances equal to those between other genera. The TV can be cultured in a monkey kidney cell line (LLC-MK2) with the appearance of typical cytopathic effect. TV exhibits a typical CV morphology, with a diameter of 36 nm, and has a buoyant density of 1.37 g/ml. According to these physicochemical and genetic characteristics, TV represents a new CV genus for which we propose the name "Recovirus" (rhesus enteric CV). Although the pathogenicity of TV in rhesus macaques remains to be elucidated, the likelihood of TV causing intestinal infection and the availability of a tissue culture system make this virus a valuable surrogate for human CVs.     

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.     

水稻南方黑条矮缩病毒湖南鼎城株系S10片段的基因组序列分析

运用RT-PCR技术克隆了水稻南方黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)湖南鼎城株系的基因组S10片段(SRBSDV-HuNDCS10),并对其全序列进行了测定和生物信息学分析。结果显示,SRBSDV-HuNDC S10片段全长为1797bp(登录号:JQ337964),含有1个ORF,编码557个氨基酸残基的衣壳蛋白,推测分子量约62.6kD,推测等电点为7.62,与已报道的广东、海南和云南分离物病毒的S10作比较,它们的核苷酸相似性分别为99.7%、99.0%和98.4%,氨基酸相似性分别为100.0%、99.5%和99.3%。对SRBSDV-HuNDCS10及部分Fijiviruses病毒对应片段在5’URT与3’URT存在的保守序列和互补序列进行了归纳,对其ORF编码的氨基酸序列进行了motif查找,得到该属(Fijiviruses)氨基酸序列的10个保守区段。此外,进行了糖基化位点、磷酸化位点及B细胞抗原表位预测,发现了3个可能的N端豆蔻酰基化位点,可能与病毒的侵染机制有关。