Production of a monoclonal antibody specific to the El Amar strain of plum pox virus

Plum pox virus (PPV) isolates are grouped into three clusters differentiated by biological, serological, molecular and epidemiological characteristics: Marcus (M), Dideron (D) and Cherry (C). The El Amar (EA) isolate that does not fit any of the above groups is also known. Monoclonal antibodies (MAbs) that specifically recognize M, D, and C strains of PPV are already available. To complete the set of PPV strain-specific serological reagents, MAbs against the EA isolate were raised by immunizing BALB/c mice and fusing their spleen cells with NS0/1 myeloma cells. After a preliminary characterization by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), 1 of 13 selected MAbs proved to be EA strain-specific. This MAb (EA24) reacted equally well with a homologous antigen and several PPV isolates from Egyptian apricot trees, supporting the hypothesis of an additional specific PPV group. MAb EA24 did not react either with about a hundred PPV isolates belonging to the D and M groups or with PPV-SwC and PPV-SoC isolates belonging to the C group. The strain specificity of MAb EA24 was confirmed by Western blot analysis and immunoelectron microscopy. We conclude that there is now available a set of MAbs which are highly specific to the four currently known groups of PPV strains.     

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.     

Comparative sequence analysis of four complete primary structures of plum pox virus strains

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5 terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3 terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5 and 3 ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5 end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%). The sequence analysis of the coat protein region of the four complete and one partial sequence indicates that the Hungarian plum pox virus strain diverges at the larger extent, similar to the El Amar strain, from which only less than half of the sequence is available.     

The complete nucleotide sequence of Passiflora latent virus and its phylogenetic relationship to other carlaviruses

Summary. A virus identified as Passiflora latent virus (PLV) was isolated from passion fruit plants. Particle morphology, host range and serological properties suggested that this virus belongs to the genus Carlavirus. The complete genomic sequence of PLV was determined by sequencing overlapping cDNA fragments. The genome consisted of 8386 nt, excluding the poly (A) tail and contained six open reading frames, typical of carlaviruses. The overall similarities of the predicted amino acid sequence of PLV to those of other carlaviruses ranged from 25 to 73%. Phylogenetic analysis indicated that PLV was closely related to lily symptomless virus and blueberry scorch virus. This is the first report of the complete nucleotide sequence and genome structure of PLV.     

The complete nucleotide sequence of plum pox potyvirus RNA

The complete nucleotide sequence of the plum pox virus (PPV) RNA genome has been determined. The RNA sequence is 9786 nucleotides in length, excluding the 3'-terminal poly(A) tail. An AUG triplet at position 147-149 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3140 amino acid residues. The nucleotide sequence of the non-coding regions and the predicted amino acid sequence of the polyprotein of PPV were compared with those previously reported for two other potyviruses (tobacco etch virus, TEV, and tobacco vein mottling virus, TVMV), with nucleotide and amino acid sequences of other viruses, as well as with sequences from data banks. The potyvirus genomic expression is discussed in relation to the homologies observed, in particular the predicted protease recognition sequences in related viruses.     

The complete genome sequence of a Passion fruit woodiness virus isolate from Australia determined using deep sequencing, and its relationship to other potyviruses

The complete genome sequence (9,858 nucleotides) of the Passion fruit woodiness virus isolate MU-2 was determined using Illumina sequencing. The large open reading frame (ORF) encodes a polyprotein containing 3,086 amino acids, with an AUG start codon and UAA stop codon. The polyprotein yielded 11 proteins (P1, HC-Pro, P3, PIPO, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP). Putative cleavage sites between them were identified by sequence comparison to those of other known potyviruses. Accuracy of the genome sequence information was provided by 42-1691-fold sequence coverage, and viral RNA accounted for 7.38% of total polyadenylated RNA from the host plant.     

Partial sequence analysis of an atypical Turkish isolate provides further information on the evolutionary history of Plum pox virus (PPV)

A variety of techniques, such as typing with subgroup-specific monoclonal antibodies, restriction length polymorphism (RFLP) analysis or subgroup-specific RT-PCR are available for the discrimination of Plum pox virus (PPV) isolates. However, the existence of PPV isolates showing abnormal typing properties has been observed in the past [Candresse, T., Cambra, M., Dallot, S., Lanneau, M., Asensio, M., Gorris, M.T., Revers, F., Macquaire, G., Olmos, A., Boscia, D., Quiot J.B., Dunez, J., 1998. Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the D and M serotypes of Plum pox potyvirus. Phytopathology 88, 198-204.]. In an effort to understand the molecular mechanisms underlying such anomalous serological and molecular typing characteristics, partial 3' (1449 nt) and 5' (3706 nt) sequences have been determined for an atypical Turkish PPV isolate (Abricotier Turquie or Ab-Tk). The results obtained indicate that its unusual typing behaviour is caused by point mutations affecting key restriction sites and epitopes and confirm that this isolate represents a divergent member of the PPV-M subgroup. In addition, analysis of the partial Ab-Tk genomic sequences demonstrated that the 5' region of the genome of this isolate has a mosaic structure resulting from recombination event(s), shedding new light on the evolutionary history of Plum pox virus.     

The complete genome sequence of a Brazilian isolate of yam mild mosaic virus

In this study, the complete genome of an isolate of yam mild mosaic virus (YMMV) from Brazil was sequenced, and the predicted amino acid sequence was analyzed. The YMMV RNA genome consists of 9538 nt without the poly(A) tail, encoding a putative typical potyvirus polyprotein of 3084 amino acids. Furthermore, the small overlapping ORF (PIPO) in the P3 gene was also deduced, and the cleavage sites of the polyprotein were predicted. Multiple alignment with other potyviruses showed a maximum nucleotide sequence identity of 64 % to wild tomato mosaic virus. A phylogenetic tree showed that YMMV clustered with Asian potyviruses that mainly infect solanaceous plants.     

The complete genome sequence of shope (rabbit) fibroma virus

We have determined the complete DNA sequence of the Leporipoxvirus Shope fibroma virus (SFV). The SFV genome spans 159.8 kb and encodes 165 putative genes of which 13 are duplicated in the 12.4-kb terminal inverted repeats. Although most SFV genes have homologs encoded by other Chordopoxvirinae, the SFV genome lacks a key gene required for the production of extracellular enveloped virus. SFV also encodes only the smaller ribonucleotide reductase subunit and has a limited nucleotide biosynthetic capacity. SFV preserves the Chordopoxvirinae gene order from S012L near the left end of the chromosome through to S142R (homologs of vaccinia F2L and B1R, respectively). The unique right end of SFV appears to be genetically unstable because when the sequence is compared with that of myxoma virus, five myxoma homologs have been deleted (C. Cameron, S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D. Willer, D. Evans, and G. McFadden, 1999, Virology 264, 298-318). Most other differences between these two Leporipoxviruses are located in the telomeres. Leporipoxviruses encode several genes not found in other poxviruses including four small hydrophobic proteins of unknown function (S023R, S119L, S125R, and S132L), an alpha 2, 3-sialyltransferase (S143R), a protein belonging to the Ig-like protein superfamily (S141R), and a protein resembling the DNA-binding domain of proteins belonging to the HIN-200 protein family S013L). SFV also encodes a type II DNA photolyase (S127L). Melanoplus sanguinipes entomopoxvirus encodes a similar protein, but SFV is the first mammalian virus potentially capable of photoreactivating ultraviolet DNA damage.     

Complete genome sequence of the original Taiwanese isolate of sweet potato latent virus and its relationship to other potyviruses infecting sweet potato

The complete genome of sweet potato latent virus (SPLV) was determined to be 10081 nucleotides long excluding the 3’ poly (A) tail. The genome contains a single large open reading frame encoding a polyprotein of 3247 amino acids. Its genomic organization is typical of potyviruses and contains motifs conserved in members of the genus Potyvirus. Pairwise comparisons show that SPLV shares identities of 50.0 %-56.3 % to other potyviruses at the genomic sequence level. Phylogenetic analysis shows that SPLV is closely related to four other sweet potato potyviruses in the sweet potato feathery mottle virus lineage, but it lacks the unique PISPO in the P1 region of those viruses. The genome analyses confirm that SPLV is a distinct sweet potato virus in the genus Potyvirus.     

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3′ untranslated region (10 nucleotides in length) and 2 insertions in the 3′ untranslated region involving a total of 5 nucleotides. Interestingly, from the 5′ UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3′ UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus. Jin-Sheng Wen and Wen-Zhong Zhao equally contributed to this work. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number: EF011023.     

The complete genome sequence of Piper yellow mottle virus (PYMoV)

This study reports the first complete genome sequence of Piper yellow mottle virus (PYMoV, KC808712) identified in black pepper. The genome is 7,622 nucleotides long, possessing four open reading frames (ORFs). ORF1, ORF2 and ORF4 of PYMoV are reported as hypothetical proteins of unknown function with a predicted molecular mass of 15.7, 17.1 and 17.9 kDa, respectively. ORF3 of PYMoV encodes a polyprotein of 218.6 kDa and consists of a viral movement protein (MP), trimeric dUTPase, zinc finger, retropepsin, RT-LTR, and RNAse H. Detailed PYMoV genome analysis confirmed that it is a member of the family Caulimoviridae, genus Badnavirus. Fragments of two additional novel sequences resembling those found in members of the family Caulimoviridae were also identified in the black pepper sample, and the viruses from which they were derived were tentatively named Piper DNA virus 1 and 2.     

猪戊型肝炎病毒株swGX32全基因组序列分析

目的 分析从广西地区分离的1株猪戊型肝炎病毒swGX32全基因组序列并比较其与其他分离株的差异.方法 设计PCR引物,用巢式反转录聚合酶链反应法(RT-nPCR)分段扩增戊型肝炎病毒(HEV)株swGX32全基因序列,用cDNA末端快速扩增法(RACE)扩增其末端序列,对扩增产物进行克隆和测序,并对拼接后的基因组进行序列和进化分析.结果 除3′polyA尾巴外,swGX32全长7240 nt, ORF1与ORF2重叠4 nt, ORF3包含在ORF2序列中.swGX32全基因序列与HEV1～4型核苷酸序列的同源性分别为:73%～74%、73%、74%～75%,83%～94%,其中与中国人源HEV株JKO-ChiSai98C同源性最高,达94%.全基因序列进化分析显示,swGX32位于HEV基因4型分枝上,ORF2部分核苷酸序列进化分析显示,swGX32与JKO-ChiSai98C同在HEV 4a亚型分枝上.结论 猪HEV swGX32在全基因组结构及分子进化上均与人HEV JKO-ChiSai98C有密切关系,为揭示戊型肝炎是一种人兽共患病提供了分子生物学依据.     

Mayaro virus: complete nucleotide sequence and phylogenetic relationships with other alphaviruses

Mayaro (MAY) virus is a member of the genus Alphavirus in the family Togaviridae. Alphaviruses are distributed throughout the world and cause a wide range of diseases in humans and animals. Here, we determined the complete nucleotide sequence of MAY from a viral strain isolated from a French Guianese patient. The deduced MAY genome was 11,429 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. Nucleotide and amino acid homologies, as well as phylogenetic analyses of the obtained sequence confirmed that MAY is not a recombinant virus and belongs to the Semliki Forest complex according to the antigenic complex classification. Furthermore, analyses based on the E1 region revealed that MAY is closely related to Una virus, the only other South American virus clustering with the Old World viruses. On the basis of our results and of the alphaviruses diversity and pathogenicity, we suggest that alphaviruses may have an Old World origin.     

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses

Summary. The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3′ end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.