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.     

Complete nucleotide sequence analysis of a Korean strain of hepatopancreatic parvovirus (HPV) from Fenneropenaeus chinensis

Hepatopancreatic parvovirus (HPV) of shrimp is distributed worldwide and the entire genome of Thailand and Indian strains (PmDNV) and one Australian strain (PmergDNV) have now been reported. The complete nucleotide sequence of a HPV strain isolated from the fleshy prawn Fenneropenaeus chinensis in Korea (FcDNV) was determined and compared to previously reported sequences. The entire genome of FcDNV contains 6,336 nucleotides, with 40% G+C content, which is the biggest of the known HPV strains. The HPV genome has three open reading frames (ORFs) with a slight overlap between the first and second ORFs. The three ORFs encode the NS2 and NS1 proteins and VP that consist of 425, 578, and 820 amino acids, respectively. Among the three proteins, the NS1 protein shows the highest sequence similarity to the NS1 protein of other known HPV strains, followed by the NS2 protein and the VP protein. Phylogenetic analyses showed that HPV can be grouped into three genotypes, as previously reported, and FcDNV can be grouped as genotype I, with HPV strains isolated in Madagascar and Tanzania. The nucleotide sequences of the noncoding regions at the 5′- and 3′-ends of the plus-strand genome showed a Y-shaped hairpin structure and simple hairpin structure, respectively.     

Genomic characterization of an enterovirus 97 strain isolated in Shandong,China

The genomic characterization of human enterovirus 97 (EV97) strain isolated from an acute flaccid paralysis case in Shandong province, China in 1999, is described. The strain, designated as 99188/SD/CHN/1999/EV97 (abbreviated as 99188), had a genome of 7394 nucleotides. Compared with other EV97 strains, it had 81.3–83.3% nucleotide similarity and 94.0–95.4% amino acid similarity in VP1 coding region, and it had 81.4% complete genomic similarity with prototype strain BAN99-10355. The most striking feature was the deletion of 18 nucleotides in the 3′ end of VP1 coding region, combined with two deletions and one insertion in 5′ and 3′ untranslated regions. All these findings demonstrated the strain 99188 had a distant genetic relationship with other EV97 strains. In the phylogenetic trees generated from VP1 and 3D sequences of human enterovirus species B (HEV-B), the lineages of strain 99188 were not congruent, suggesting the event of recombination. Similarity plot analysis further provided the evidence of recombination with other strains of HEV-B in P2 and P3 coding region. This is the first finding of EV97 in China and the third genomic sequence of EV97 reported.     

Genome organisation and sequence comparison suggest intraspecies incongruence in M RNA of Watermelon bud necrosis virus

Watermelon bud necrosis virus (WBNV), a member of the genus Tospovirus, family Bunyaviridae is an important viral pathogen in watermelon cultivation in India. The complete genome sequence properties of WBNV are not available. In the present study, the complete M RNA sequence and the genome organisation of a WBNV isolate infecting watermelon in Delhi (WBNV-wDel) were determined. The M RNA was 4,794 nucleotides (nt) long and potentially coded for a movement protein (NSm) of 34.22 kDa (307 amino acids) on the viral sense strand and a Gn/Gc glycoprotein precursor of 127.15 kDa (1,121 amino acids) on the complementary strand. The two open reading frames were separated by an intergenic region of 402 nt. The 5′ and 3′ untranslated regions were 55 and 47 nt long, respectively, containing complementary termini typical of tospoviruses. WBNV-wDel was most closely related (79.1% identity) to Groundnut bud necrosis virus, an important tospovirus that occurs in several crops in India, and was different (63.3–75.2% identity) from the other cucurbit-infecting tospoviruses known to occur in Taiwan and Japan. Sequence analysis of NSm and Gn/Gc revealed phylogenetic incongruence between WBNV-wDel and another isolate originating from central India (WBNV-Wm-Som isolate). The Wm-Som isolate showed evolutionary divergence from the wDel isolate in the Gn/Gc protein (74.6% identity) potentially due to recombination with the other tospoviruses that are known to occur in India. This is the first report of a comparison of complete sequences of M RNA of WBNV.     

The complete genome sequence of an El Amar isolate of plum pox virus (PPV) and its phylogenetic relationship to other PPV strains

Summary. The genomic sequence of an El Amar isolate of plum pox virus (PPV) from Egypt was determined by sequencing overlapping cDNA fragments. This is the first complete sequence of a member of the El Amar (EA) strain of PPV. The genome consists of 9791 nt, excluding a poly(A) tail at the 3′ terminus. The complete nt sequence of PPV EA is 79–80%, 80%, 77%, and 77% homologous with isolates of strains D/M, Rec (BOR3), C, and W, respectively. The polyprotein identity ranged from 87–91%. Phylogenetic analysis using the complete genome sequence of PPV EA confirmed its strain status. No significant recombination signals were identified using PhylPro and SimPlot scans of the PPV EA sequence, however an interesting recombination signal was identified in the P1/HC-Pro region of PPV W3174.     

The genomic constellation of a novel<Emphasis Type="Italic"> avian orthoreovirus</Emphasis> strain associated with runting-stunting syndrome in broilers

Avian orthoreoviruses (ARVs) are responsible for considerable economic losses in broiler chickens; yet, the genetic characterization of most ARV strains is limited to a few genes, and the full coding region has been determined for only S1133 and 138, two ARV strains associated with tenosynovitis. Recently, in parts of the United States, ARVs with novel neutralization antigen type were isolated from chickens afflicted with runting-stunting syndrome. One such strain, AVS-B, was selected for full genome sequencing and phylogenetic analysis. The complete genome was 23,494 bp in size and included 12 open reading frames. The lengths of the coding regions, as well as those of the 5′ and 3′ ends, were fairly well conserved between AVS-B and other reference strains. In pairwise comparisons to the S1133 and 138 strains, the AVS-B strain shared a wide range of sequence identities along each genome segment, i.e., a range of 54–55% for the σC coding region of S1 genome segment and 91–93% for the S2 genome segment. Phylogenetic analyses of individual genes of AVS-B did not identify any single common ancestor among more completely characterized ARV strains for which sequence data are available. One exception to this lack of identity was strain 138, which shared 90–93% nt identity with AVS-B along seven of ten genome segments; only M2, M3, and S1 segments of these strains shared lower sequence identities. Collectively, our analyses indicated that multiple reassortment events and strong divergence caused by the accumulation of point mutations could have led to the observed assortment and genetic heterogeneity of the AVS-B genome.     

Complete genome sequence of attenuated low-temperature Thiverval strain of classical swine fever virus

The Thiverval vaccine strain of classical swine fever virus (CSFV) was derived from virulent Alfort strain through the serial passages in cells at 29–30°C. In this study, we determined the complete genome sequence of this strain and found that its genome contains one open reading frame (ORF) that encodes a polyprotein with 3,898 amino acids. The 5′-UTR of Thiverval is 373 nt long with only one mutation at position 220. In contrast, the length of 3′-UTR is highly heterogeneous ranging from 233 to 259 bp. The heterogeneity of length of the 3′-UTR was due to an insertion of a variable length of T-rich sequence ranging from 6 to 32 nt. The insertion may change the structure and free energy of the 3′-UTR, resulting in a destabilization of the 3′-UTR. Sequence alignment of Thiverval and other CSFV strains showed 85.2–99.6% identities at the nucleotide level and 92.5–99.5% at the amino acid level. The phylogenetic tree analysis of the complete ORF, partial region of E2, and NS5B suggests that the CSFV Thiverval strain belongs to genetic group 1 and subgroup 1.1. The results from this study provide insight into the molecular mechanism of the attenuation of Thiverval vaccine strain.     

Complete genome sequence of a virulent Newcastle disease virus isolated from an outbreak in chickens in Egypt

The complete genome sequence of a virulent Newcastle disease virus (NDV) isolated from chickens in Egypt was determined and compared to the sequence of NDV strains isolated from different parts of the world. The genome is 15,186 nucleotides (nt) long and consists of 6 genes in the order of 3′-N-P-M-F-HN-L-5′. The genome contains a 55-nt leader region at the 3′ end and a 114-nt trailer region at the 5′ end. Interestingly, the phylogenetic analysis showed that strain Egypt is closely related with the NDV strains isolated in China. In addition, the sequence of the fusion protein cleavage site of strain Egypt was identical to that of the NDV strain recently isolated in Mali. Determination of complete genome sequences of additional NDV strains from Africa is necessary to understand the epidemiology of currently circulating viruses in Africa.     

Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China

Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3’ untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3’UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.     

The complete genome sequence of a member of a new species of tobamovirus (rattail cactus necrosis-associated virus) isolated from Aporcactus flagelliformis

In this study, we identified a new tobamovirus from diseased Aporcactus flagelliformis cactus plants, named it rattail cactus necrosis-associated virus (RCNaV), and determined its complete genome sequence. The full RCNaV genome consisted of 6,506 nucleotides and contained four open reading frames coding for proteins of M(r) 128 kDa (3,441 nt), 185 kDa (4,929 nt), 55 kDa (1452 nt), 36 kDa (1,005 nt) and 19 kDa (513 nt) from the 5′ to 3′ end, respectively. The overall similarities for the four ORFs of RCNaV were from 32.5% to 64.1% and from 17.0% to 67.3% to those of the other tobamoviruses, at the nucleotide and amino acid level, respectively. Comparison of the coding and non-coding regions of the virus with those of other tobamoviruses showed that RCNaV is the most closely related to cactus mild mottle virus.     

Genomic characterization of two avian paramyxovirus type 2 isolates from chickens in China

The complete genome sequences were determined for avian paramyxovirus type 2 (APMV-2) strains F8 and NK isolated from chickens in China. Both strains had a genome of 14,904 nucleotides (nt) in length, which followed the “rule of six”. Each genome consisted of six genes in the order 3′-N-P-M-F-HN-L-5′, with a 55-nt leader at the 3′ end and a 154-nt trailer at the 5′ end. Sequence alignment and phylogenetic analysis showed that APMV-2 strains F8 and NK shared the highest sequence identity with APMV-2 prototype strain Yucaipa, being classified in the same subgroup as strains Yucaipa, England and Kenya, while strain Bangor represented another subgroup of APMV-2. Among the APMVs, APMV-2 strains F8 and NK exhibited a closer evolutionary relationship with APMV-7 and APMV-8 representative strains.     

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     

Evidence for inter- and intra-genotypic variations in dengue serotype 4 viruses representing predominant and non-predominant genotypes co-circulating in Thailand from 1977 to 2001

In order to characterize viral genetic variation among predominant and non-predominant genotypes of Thai dengue serotype 4 viruses (DENV-4) and follow mutations that occur during virus evolution, we performed a comparative analysis of the complete genomic sequences of six DENV-4 isolates representing three genotypes (I, IIA, and III) co-circulating in Thailand over a 24-year period. The results revealed [1] remarkable genetic variation in the viral genome between predominant and non-predominant genotypes; [2] inter-genotype-specific amino acid and nucleotide mutations in most regions of the viral genome; [3] more amino acid and nucleotide substitutions in later as compared to earlier isolates for predominant genotype I strains; [4] a single nucleotide substitution at nucleotide position 77 of the 5-′NTR of two non-predominant genotype III strains that disrupted a small conserved 3′stem–loop (SL) in the cyclization sequence required for virus replication; [5] a high degree of conservation of PrM/M and NS2B proteins, and the 5′-NTR in predominant genotype I strains with no mutations observed over the 24-year period of observation; and [6] no molecular markers that appeared to correlate with disease severity. Several mutations identified in this study might have a significant impact on the persistence of virus in the population, including one in the 5′-NTR that disrupted a small, highly conserved 3′SL2 structure at the terminus of the cyclized 5′–3′ RNA sequences in two genotype III strains, and three amino acid (aa) charge change mutations in the E and NS5 proteins of genotype I strains. The conserved 3′-SL structure may be a target for antiviral drug development.     

Complete nucleotide sequence and genome organization of a Chinese isolate of tobacco bushy top virus<Superscript>*</Superscript>

Summary.  The complete nucleotide sequence of a Chinese isolate of tobacco bushy top virus (TBTV), designated TBTV-Ch, was determined from cDNA generated from double-stranded RNA extracted from diseased tobacco. The genome is 4152 nucleotides (nt) in size, contains four putative open reading frames (ORFs) and untranslated regions of 10 nt and 645 nt at the 5′ and 3′ ends, respectively. In genome organization and in the amino acid sequence of its potential products, the RNA of TBTV-Ch is similar to other umbraviruses sequenced to date. The results suggested that TBTV should be regarded as a definitive species of the genus Umbravirus. Received May 15, 2002; accepted September 6, 2002     

Complete genome sequence of highly virulent neurotropic Newcastle disease virus strain Texas GB

Newcastle disease virus (NDV) strain Texas GB is a highly virulent neurotropic virus that is used as a standard vaccine challenge virus in the U.S. In this study, the complete genome sequence of strain Texas GB was determined and compared with the complete genome sequences of other NDV strains. The genome is 15,186 nucleotides (nt) long and consists of six genes in the order of 3′leader-N-P-M-F-HN-L-5′trailer. The genome contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. The intergenic sequences are 2, 1, 1, 31, and 47 nt between N/P, P/M, M/F, F/HN, and HN/L genes, respectively. The putative cleavage site of fusion protein showed amino acid sequence of R-R-Q-K-R↓F in position 112 to 117, which corresponds to those of virulent NDV strains. The phylogenetic analysis showed that strain Texas GB is closely related to the neurovirulent mesogenic strain Beaudette C (BC) and to NDV viruses isolated in China and Egypt than to other strains of NDV.     

Nucleotide sequences of four segments of chrysovirus in Korean <Emphasis Type="Italic">Cryphonectria nitschkei</Emphasis> BS122 strain

The near full-length genome consisting of four segments of dsRNA from a chrysovirus infecting Korean Cryphonectria nitschkei BS122 strain (CnV1-BS122) was sequenced. The open reading frames of segments 1, 2, 3, and 4 were 2,889, 2,721, 2,475, and 2,232 nucleotides (nt) in length, respectively. Sequence analysis and homology searches of the amino acid sequences deduced from the ORFs of each segment revealed that segments 1, 2, 3, and 4 encoded RNA-dependent RNA polymerase, capsid protein, a putative cysteine protease, and replication-associated protein, respectively. The entire 5′ ends of segments 1, 2, and 4 were 82, 242, and 698 nt in length, respectively; the sequence of the 5′ end of segment 3 was not determined because of difficulty in amplification. The entire 3′ end of segment 3 was 77 nt in length. Partial amplification of the 3′ ends of segments 1, 2, and 4 yielded amplimers of 7, 17, and 30 nt, respectively.     

Evidence of genetic diversity generated by recombination among avian coronavirus IBV

Summary.  Previously, we demonstrated that the DE072 strain of IBV is a recombinant which has an IBV strain D1466-like sequence in the S gene. Herein, we analyzed the remaining 3.8 kb 3′ end of the genome, which includes Gene 3, Gene 4, Gene 5, Gene 6, and the 3′ non-coding region of the DE072 and D1466 strains. Those two viruses had high nucleotide similarity in Gene 4. However, the other individual genes had a much different level of sequence similarity with the same gene of the other IBV strains. The genome of five IBV strains, of which the complete sequence of the 3′ end of the genome has been determined, were divided at an intergenic (IG) consensus sequence (CTGAACAA or CTTAACAA) and compared phylogenetically. Phylogenetic trees of different topology indicated that the consensus IG sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. Phylogenetic analysis of selected regions of the genome of the DE072 serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the S gene. Presumably this occurs because the consensus IG sequence serves as the template switching site for the viral encoded polymerase. Received December 1, 1999 Accepted March 24, 2000     

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.     

Determination of the nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus genome

Summary.  The nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3′ and 5′ non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5′ NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5′ very end, and the extra 9 nucleotides in the US-2 strain. In the 3′ NCR, swine HEV shared 90–91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58–65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV. Received April 20, 2001 Accepted July 10, 2001