A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene.

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.     

The use of lumpy skin disease virus genome termini for detection and phylogenetic analysis

During 2006 and 2007 there were two outbreaks of lumpy skin disease (LSD) in Israel. An LSD virus (LSDV)-specific PCR assay was developed that can detected specifically LSDV even though the number of tested LSDV isolates were limited. Full-length sheep pox and LSDV genome sequences were aligned to find non-homologous regions, which were then used for preparing specific primers, whose specificity was tested against several LSDV DNA isolates and the system could detect all the different isolates. Specificity was tested with sheep pox, ORF and other DNA viruses such as bovine herpes 4: the primers did not support amplification of the expected-size fragments, therefore the system appears to be a valuable tool for detecting specifically LSDV. The newly developed system was activated at the first report of a possible disease outbreak. It confirmed the clinical picture, and was introduced subsequently into routine diagnosis. Phylogenetic analyses of a 466-bp fragment next to the genome ends showed that this system can distinguish between: sheep pox, goat pox and LSD, and the results revealed that the Israeli isolates from 2006 and 2007 are in the same clad and essentially identical to Ismaeliya 1989, Nigeria 1996, Senegal 1997, Cameroon 1996, the Kenya NI-2490 isolate, and the South African LD virulent isolate. In contrast the original 1958 LW Neethling vaccine appeared to be in a separate clad, suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the viruses tested suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the tested viruses or during the process of attenuating the virus by succession of egg inoculations.     

Nucleotide sequence of the thymidine kinase gene of raccoon poxvirus

Using primers directed against the thymidine kinase gene of vaccinia virus, PCR products were generated from nucleic acids extracted from raccoon poxvirus-infected Vero cells. The PCR products were consistent in size with the expected products from vaccinia virus. Nucleotide sequence determination revealed that the raccoon pox thymidine kinase gene and flanking regions were 84.3% homologous to the corresponding sequences of vaccinia virus. At the amino acid level, an open reading frame coding for a polypeptide of 177 amino acids was found with 87% homology to thymidine kinase of vaccinia.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U08228.     

Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe

Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.     

Evaluation of avidin-biotin ELISA for the detection of antibodies to goat poxvirus using noninfectious diagnostic reagent

A noninfectious soluble antigen fraction of goat poxvirus (GPV) fractionated by ammonium sulfate precipitation was tested for its suitability as coating antigen in an indirect enzyme-linked immunosorbent assay (ELISA). Accordingly, an avidin-biotin ELISA for the detection of GPV antibodies was optimized and evaluated using different groups of serum samples from goats with known or unknown immune status. A cut-off value higher by 60% than A492 reading of control negative sera gave a 91.8% specificity and a 94.1% sensitivity for the assay. Out of 90 goat pox-suspect sera obtained from the field, only 2 (2.2%) were found positive in the counter immunoelectrophoresis (CIE) test, which is so far the routinely used diagnostic test for goat pox, while 58 (64.4%) were positive in the avidin-biotin ELISA. The McNemar's analysis of these data showed that the avidin-biotin ELISA was significantly more efficient than the CIE test for the detection of GPV antibodies in goat sera.     

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.     

Cross-neutralization tests on sheep pox, goat pox and contagious pustular dermatitis viruses.

In cross-neutralization tests in cell cultures, sheep pox and contagious pustular dermatitis (CPD) hyperimmune sera did not cross-react in the heterologous system. But goat pox hyperimmune serum neutralized both sheep pox and CPD viruses revealing a one-way serological relationship of goat pox virus with both sheep pox and CPD viruses.     

Phylogenetic relationships of closely related potyviruses infecting sweet potato determined by genomic characterization of Sweet potato virus G and Sweet potato virus 2

Complete nucleotide sequences of Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2) were determined to be 10,800 and 10,731 nucleotides, respectively, excluding the 3'-poly(A) tail. Their genomic organizations are typical of potyviruses, encoding a polyprotein which is likely cleaved into 10 mature proteins by three viral proteinases. Conserved motifs of orthologous proteins of viruses in the genus Potyvirus are found in corresponding positions of both viruses. Pairwise comparisons of individual protein sequences of the two viruses with those of 78 other potyviruses show that P1 protein and coat protein (CP) of both viruses are significantly large, with the SPVG CP as the largest among the all the known species of the genus Potyvirus. The extended N-terminal region of the P1 protein is conserved in the potyviruses and ipomovirus infecting sweet potato. A novel ORF, PISPO, is identified within the P1 region of SPVG, SPV2, Sweet potato feathery mottle virus (SPFMV), and Sweet potato virus C (SPVC). The C-terminal half of CP is highly conserved among SPFMV, SPVC, SPVG, SPV2, and Sweet potato virus-Zimbabwe. Phylogenetic analysis based on the deduced CP amino acid sequences supports the view that these five viruses are grouped together in a SPFMV lineage. The analysis also reveals that Sweet potato virus Y and Ipomoea vein mosaic virus are grouped with SPV2 as one species, and these two viruses should be consolidated with SPV2.     

Isolation and phylogenetic analysis of an orf virus from sheep in Makhdoom,India

Orf (contagious ecthyma) is an exanthematic disease caused by a parapoxvirus and occurs primarily in sheep and goats with zoonotic implications. In the present investigation, an orf outbreak in the Muzzaffarnagari sheep flock at the Central Institute for Research on Goats (CIRG), Makhdoom, Mathura, Uttar Pradesh, India, was investigated. Primary goat testes cell culture was used for isolation of the orf virus (ORFV) for the first time. The identity of the virus was confirmed by amplification and sequence analysis of the major envelope glycoprotein (B2L) gene and named ORFV/sheep/India/2012/CIRG. On phylogenetic analysis of B2L protein gene, it clustered with the ORFV strains from China suggesting distinct ORFV strains are circulating in India. On comparison of nucleotide and deduced amino acid sequence analysis (n = 63), a unique 126S residue was observed in ORFV/sheep/India/2012/CIRG. On further sequence analysis (B2L) of different ORFV strains (n = 63), some conserved amino acid residues were identified as host-specific (sheep, human, camel, takin, and musk ox) and have been summarized.     

Sheep poxvirus identification by PCR in cell cultures

A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.     

Application of ELISA in the detection of goat pox antigen and antibodies

Enzyme linked immunosorbent assay (ELISA) was standardized for detection of goat pox virus (GPV) antibodies and antigen using live and inactivated antigens and hyperimmune serum (HIS), convalescent, post-vaccinal, as also post-challenge sera. The ELISA was most sensitive in detection of antibody when compared with agar gel precipitation (AGP) and counterimmunoelectrophoresis (CIE) tests. There was no complete correlation between the antibody status of vaccinated goats and protective immunity as animals having detectable seroconversion were also solidly immune to virulent challenge. The application of ELISA in pox infections of goats has been discussed.     

Identification and nucleotide sequence of the thymidine kinase gene of swinepox virus.

Using degenerative oligonucleotide probes, representing two different conserved regions of poxvirus and mammalian thymidine kinase (TK) genes, the swinepox virus (SPV) TK gene was mapped to a 1.7-kb BamHI-HindIII fragment of the viral genome. Nucleotide sequencing of this DNA piece revealed that the SPV TK gene was encoded by an open reading frame (ORF) of 177 codons. Immediately downstream of the TK gene was a second ORF with homologues at the same location in both capripoxvirus and leporipoxvirus genomes. A similar gene had translocated to near the left hand terminus of the vaccinia virus (orthopoxvirus) genome. Flanking the two SPV genes were ORFs whose counterparts in other poxvirus genera are located at the same relative positions. SPV appeared to be most closely related to capripoxvirus, based on the organization of the four genes and on the percentage of identical amino acid residues of the respective encoded proteins.     

Nucleotide sequence analysis of the ORF0 region of Potato leafroll virus isolates from Tunisia

Potato leafroll virus (PLRV) isolates from potato plants from different regions of Tunisia were investigated for the ORF0 variable region of genomic RNA using PCR, nucleotide sequencing and sequence analysis. Based on the ORF0 variable region nucleotide sequence, individual Tunisian isolates were more homologous as a group compared to the isolates originating from other parts of the world. There was no correlation between bioclimatic origin of the isolates and their ORF0 sequence. Alignment of the deduced amino acid sequences showed that the P0 protein was not much conserved. Unexpectedly, Tunisian isolates were found to be most homologous to Peruvian ones both at nucleotide and amino acid level. A phylogenetic tree, based on the P0 amino acid sequence, showed that all the PLRV isolates were located in two major clusters regardless of their geographic origin. In the second cluster, three sub-clusters could be distinguished. These results provide valuable information for molecular characterization of the PLRV isolates occurring in Tunisia.     

Isolation and molecular characterization of the swinepox virus thymidine kinase gene.

Swinepox virus (SPV), the only member of the Suipoxvirus genus, shows little antigenic relatedness or DNA homology to members of the other poxvirus genera. A SPV thymidine kinase (TK) gene was detected and mapped to the left end of the HindIII G fragment using degenerate oligonucleotide probes. Cloning and sequencing of a 1.8-kb HindIII-BamHI fragment containing the SPV TK gene revealed an open reading frame (ORF) of 181 amino acids yielding a predicted polypeptide of Mr 20.6 kDa with significant homology to both poxvirus and vertebrate thymidine kinases. Comparison with other TK protein sequences showed that the SPV thymidine kinase was closely related to the TK genes of avipoxviruses (52.0%) and vertebrates (57.1-59.7%). The TK gene from African swine fever virus (ASF) showed little homology (30.5%) to the SPV TK gene suggesting that these two viruses are not closely related though they share many biochemical features and infect a single, common mammalian host (swine). The SPV TK gene, like that of other poxviruses, is transcribed early, and when cloned into a TK- strain of vaccinia converted the virus to a TK+ phenotype. BUdRR mutants of SPV contained frameshift, deletion, and missense mutations in the TK ORF.     

The Myxoma Virus EcoRI-O Fragment Encodes the DNA Binding Core Protein and the Major Envelope Protein of Extracellular Poxvirus

The nucleotide sequences of the myxoma virus gene homologs encoding the DNA binding core protein (MF17) and the major envelope protein of the extracellular poxvirus particle (MF13) have been localized to the myxoma virus 4 kB EcoRI-O fragment. The EcoRI-O fragment is located approximately 22 kb from the left end of the 163 kb DNA genome and encodes homologs of the F12L, F13L, F15L, F16L, F17R and E1L genes of the Copenhagen strain of vaccinia virus. The inferred amino acid sequences of the myxoma virus EcoRI-O encoded products have been compared to the protein databases to identify related proteins. The myxoma virus open reading frames MF12, MF15, MF16, MF17 and ME1 encode homologs of poxvirus specific proteins while the MF13 envelope protein also shares amino acid similarity with other poxvirus and cellular proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogenetic analyses indicate little variation among reticuloendotheliosis viruses infecting avian species, including the endangered Attwater's prairie chicken

Reticuloendotheliosis virus infection, which typically causes systemic lymphomas and high mortality in the endangered Attwater's prairie chicken, has been described as a major obstacle in repopulation efforts of captive breeding facilities in Texas. Although antigenic relationships among reticuloendotheliosis virus (REV) strains have been previously determined, phylogenetic relationships have not been reported. The pol and env of REV proviral DNA from prairie chickens (PC-R92 and PC-2404), from poxvirus lesions in domestic chickens, the prototype poultry derived REV-A and chick syncytial virus (CSV), and duck derived spleen necrosis virus (SNV) were PCR amplified and sequenced. The 5032bp, that included the pol and most of env genes, of the PC-R92 and REV-A were 98% identical, and nucleotide sequence identities of smaller regions within the pol and env from REV strains examined ranged from 95 to 99% and 93 to 99%, respectively. The putative amino acid sequences were 97-99% identical in the polymerase and 90-98% in the envelope. Phylogenetic analyses of the nucleotide and amino acid sequences indicated the closest relationship among the recent fowl pox-associated chicken isolates, the prairie chicken isolates and the prototype CSV while only the SNV appeared to be distinctly divergent. While the origin of the naturally occurring viruses is not known, the avian poxvirus may be a critical component of transmission of these ubiquitous oncogenic viruses.     

The complete DNA sequence of myxoma virus

Myxomatosis in European rabbits is a severely debilitating disease characterized by profound systemic cellular immunosuppression and a high rate of mortality. The causative agent, myxoma virus, is a member of the poxvirus family and prototype of the Leporipoxvirus genus. As a major step toward defining the genetic strategies by which the virus circumvents host antiviral responses, the genomic DNA sequence of myxoma virus, strain Lausanne, was determined. A total of 171 open reading frames were assigned to cover the 161.8-kb genome, including two copies each of the 12 genes that map within the 11.5-kb terminal inverted repeats. Database searches revealed a central core of approximately 120 kb that encodes more than 100 genes that exhibit close relationships to the conserved genes of members of other poxvirus genera. Open reading frames with predicted signal sequences, localization motifs, or homology to known proteins with immunomodulatory or host-range functions were examined more extensively for predicted features such as hydrophobic regions, nucleic acid binding domains, ankyrin repeats, serpin signatures, lectin domains. and structural cysteine spacings. As a result, several novel, potentially immunomodulatory proteins have been identified, including a family with multiple ankyrin-repeat domains, an OX-2 like member of the neural cell adhesion molecule family, a third myxoma serpin, a putative chemokine receptor fragment, two natural killer receptor-like species, and a variety of species with domains closely related to diverse host immune regulatory proteins. Coupled with the genomic sequencing of the related leporipoxvirus Shope fibroma virus, this work affirms the existence of a conserved complement of poxvirus-specific core genes and expands the growing repertoire of virus genes that confer the unique capacity of each poxvirus family member to counter the immune responses of the infected host.     

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.