Complete nucleotide sequence analysis of a vaccine strain and a field isolate of foot-and-mouth disease virus serotype Asia1 with an insertion in VP1 genomic region

Complete nucleotide sequences except the poly (C) tract and poly (A) tail of a vaccine strain (IND 491/97) and an atypical field isolate (IND 321/01) of Foot-and-mouth disease virus (FMDV) serotype Asia1 are described. Amino acid (aa) sequence analysis of the VP1 protein of the field isolate revealed that the latter has 212 instead of 210 or 211 aa found in the so far available sequences of other FMDV isolates of Asia1 serotype. The insertion was localized in the hypervariable region of aa 130-160 of VP1 protein. Nucleotide sequencing of the entire genome was therefore carried out to detect changes in other parts of the genome, if any, besides VP1, which could contribute to its fitness. An 8.16 kb sequence of IND 491/97 and an 8.162 kb sequence of IND 321/01 were compared with each other and also with the known sequence of IND 63/72, another vaccine strain of serotype Asia1. Comparison of the entire polyprotein coding (L to 3D) region of IND 321/01 with those of the two Asia1 vaccine strains (IND 63/72 and IND 491/97) revealed no significant differences. A similar comparison of IND 491/97 with IND 63/72 revealed variability across the entire length of the genome. In addition to the capsid-coding region, sequence variability was also observed in non-structural proteins albeit to different extent. This study shows that in the gene pool of serotype Asia1 at least three groups of isolates/strains are present with respect to the length of VP1 protein.     

Genotyping of human bocavirus using a restriction length polymorphism

Sequencing analysis of the isolates of a recently identified pathogen associated with respiratory infections, human bocavirus (HBoV), allowed for identification of two virus genotypes of the virus. In the current article a new method for a simple and fast differentiation of HBoV genotypes in clinical materials is described. The test includes an amplification of a 309 bp fragment of VP1/VP2 gene of HBoV from nasopharyngeal aspirates with a subsequent incubation of a PCR mix with the BstAPI endonuclease. Upon such a digestion, the DNA fragment derived from the genotype I HBoV isolates forms two fragments of 150 and 159 bp, while that obtained from genotype 2 isolates remains unrestricted. The developed technique may be used in epidemiological studies of HBoV infection and analysis of the potential differences in biological characteristics of HboV genotypes.     

Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data

Maize streak virus (MSV) is the most economically significant member of a diverse group of African grass-infecting Mastrevirus species in the family Geminiviridae. We designed a single set of degenerate primers which enables the PCR amplification of an approximately 1300 bp DNA fragment spanning both conserved (the RepA gene) and variable (the long intergenic region and MP gene) portions of these viruses' genomes. Using restriction fragment length polymorphism (RFLP) analysis of PCR products obtained from 39 MSV, one SSV, and two PanSV isolates, it was possible to both identify the different virus species, which differ in nucleotide sequence by up to 40%, and to differentiate between MSV isolates sharing up to 99% sequence identity. The reliability of the RFLP data for typing the MSV isolates was verified by the phylogenetic analysis of the partial genomic nucleotide sequences of a representative subset of the MSV isolates. Based on both the RFLP and sequence data, the MSV isolates could be clearly differentiated into the four groups: these were a group of predominantly maize-infecting isolates, and three groups containing grass/wheat-infecting isolates. RFLP analysis also revealed a number of mixed virus infections in which, in certain instances, it was possible to identify individual population members.     

Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.     

中国湖南株丁型肝炎病毒cDNA（443－870）片段的克隆和序列分析

取１例湖南丁型肝炎病毒（ＨＤＶ）ＲＮＡ阳性血清，经强变性剂法抽提ＨＤＶＲＮＡ，逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）扩增，扩增的ＨＤＶＣＤＮＡ片段重组到ｐＵＣ１８质粒中，获得了中国湖南株ＨＤＶＣＤＮＡ（４３３－８７０）片段克隆，ＤＮＡ测序结果显示，该片段（４３８ｈｐ长，包括一端ＰＣＲ引物２５ｈｐ）与已知的美国、意大利、法国、诺鲁和中国台湾５株ＨＤＶｃＤＮＡ相同片段比较，同源性分别为９１．３％，９４．５％，９１．３％，８４．５％和８９．３％，其中与ＨＤＶＲＮＡ复制密切相关的第一个高度保守区与美国株、意大利株和法国株完全同源。     

Phylogenetic Analysis of Canine Parvovirus VP2 Gene in Taiwan

Canine parvovirus (CPV) is a non-enveloped virus with a single-stranded DNA genome and causes infectious enteritis in dog. In this study, 36 isolates of CPV infection were obtained in Taichung, Taiwan from 2003 to 2004. Using primers that can distinguish subtypes of CPV, we amplified part of viral VP2 gene by polymerase chain reaction (PCR) and the PCR product was sequenced; results demonstrated that two isolates could be classified as type 2a of CPV and the others were type 2b. The complete coding region of VP2 gene of type 2b was also sequenced, and phylogenetic analysis of these DNA sequences revealed that our Taichung isolate was close to the V-120, FPV-314, 97-008, Taiwan 9, LCPV-T1, and T4 isolates; however, because of the degeneracy of codons, the amino acid sequences of Taichung isolate was similar to that of the 97-008 isolate from Japan. It is known that two important amino acid residues (Asn-426 in type 2a and Asp-426 in type 2b; Ile-555 in type 2a and Val-555 in type 2b) are the determinants for the discrimination of type 2a or type 2b. After scrutinizing the complete VP2 gene of our Taichung isolate, we found the VP2 protein of the Taichung isolate did possess this molecular feature of type 2b virus. Previous studies reported that type 2a virus was the major type in Taiwan; our finding showed that CPV type 2b was the predominant type in the middle part of Taiwan. Moreover, a unique Ala-489 in VP2 of our Taichung isolate was found, contrasting to a Val-489 in the VP2 of other strains.     

Sequence and phylogenetic analysis of the L and VP1 genes of foot-and-mouth disease virus serotype Asia1

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, we determine the nucleotide (nt) sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype Asia 1 isolates recovered from different outbreaks in India, and compared with each other and the vaccine strain, IND 63/72, used in the country. Independent phylogenetic analyses on both the aligned gene sequences identified two major lineages (designated A & B) in the Asia 1 isolates. Both L- and VP1-based trees were congruent with respect to the major branching pattern of the isolates. The lineage A is represented by the isolates of 1986-2000 including the vaccine strain IND 63/72, whereas, lineage B appeared to be dominant and responsible for most of the recent outbreaks. A correlation was observed between the clustering of the isolates in the phylogenetic tree and the amino acid changes at many of the positions in VP1 as well as in L protein. The annual rate of evolution in L and VP1 genes was found similar and estimated to be 4.0 x 10(-3) and 3.8 x 10(-3) substitutions per nucleotide, respectively. Our result, largely from the congruence in phylogenetic trees and the rate of evolution in both the genes, suggests the possibility for the use of L gene sequence in phylogenetic comparison of FMDV.     

Genetic mechanisms of antigenic variation in infectious bursal disease virus: Analysis of a naturally occurring variant virus

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222–334) lies a highly divergent area that we have termed thevariable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284–288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.     

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.     

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.     

Characterization of the Polyhedrin Gene of Spodoptera litura Nuclear Polyhedrosis Virus

The polyhedrin gene (polh) of a characteristically distinct Spodoptera litura nuclear polyhedrosis virus isolate (SIMNPV) is identified in the HindIII-F fragment of the viral DNA. The nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (ORF) without any intervening sequence for coding a polypeptide of 246 amino acids. Analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to known polyhedrins. The coding region is preceded by an AT rich region containing the conserved late promoter motif TAAG. The upstream promoter and coding regions of this polh gene are more similar to polh of the NPVs of Spodoptera frugiperda (Sf), Spodoptera exigua (Se) and Panolis flamea (Pf). This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence analysis of the VP2 gene hypervariable region of infectious bursal disease viruses from India

Attempts have been made to characterize infectious bursal disease virus (IBDV) isolates collected from different parts of India during 1993 to 1999. Phylogenetic analysis was performed on a sequence generated by cycle sequencing comprising the variable region of the VP2 gene of 14 isolates. Indian IBDV isolates had divergence of 0.2 to 4.3% at nucleotide and 0 to 2.2% at amino acid levels among themselves. Nine nucleotide changes were found in Indian IBDV field isolates, resulting in the four specific amino acid changes at 222P-A, 256V-I, 294L-I and 299N-S, reported regularly in very virulent isolates. One of the Indian IBDV isolates, UP1/99, had change D to N at position 212 in the first hydrophilic region. The serine-rich heptapeptide sequence 'SWSASGS' was conserved in all the isolates. Phylogenetically, all Indian field isolates were found to be closely related to very virulent IBDV isolates from Europe, Japan, China and Israel.     

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belém VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.     

Full-length sequence analysis of four IBDV strains with different pathogenicities

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.     

A BIR Motif Containing Gene of African Swine Fever Virus,4CL,Is Nonessential for Growthin Vitroand Viral Virulence

An African swine fever virus (ASFV) gene with similarity to viral and cellular inhibitor of apoptosis genes (iap) has been described in the African isolate Malawi Lil-20/1 (ORF 4CL) and a cell-culture-adapted European virus, BA71V (ORF A224L). The similarity of the ASFV gene to genes involved in inhibiting cellular apoptosis suggested the gene may regulate apoptosis in ASFV-infected cells and thus may function in ASFV virulence and/or host range. Sequence analysis of additional African and European pathogenic isolates demonstrates that this gene is highly conserved among both pig and tick ASFV isolates and that its similarity toiapgenes is limited to the presence of a single IAP repeat motif (BIR motif) in the ASFV gene. To study gene function, a4CLgene deletion mutant, Δ4CL, was constructed from the pathogenic Malawi Lil-20/1 isolate. Growth characteristics of Δ4CL in swine macrophage cell cultures were indistinguishable from those of parental virus. Infected macrophage survival time and the induction and magnitude of apoptosis in virus-infected macrophages were comparable for cells infected with either Δ4CL or parental virus. In infected swine, Δ4CL exhibited an unaltered Malawi Lil-20/1 virulence phenotype. These data indicate that, although highly conserved among ASFV isolates, the4CLgene is nonessential for growth in macrophage cell culturesin vitroand for pig virulence. Additionally, despite its limited similarity toiapgenes,4CLexhibits no anti-apoptotic function in infected macrophage cell cultures. The high degree of gene conservation among ASFV isolates, together with the apparent lack of function in the swine host, suggests4CLmay be a host range gene involved in aspects of infection in the arthropod host, ticks of the genusOrnithodoros.     

Pestiviruses isolated from pigs,cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis

Summary A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5 non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.