A rapid method for identifying the thymidine kinase genes of avipoxviruses

The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK⁻ mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes.     

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.     

Mapping and molecular characterization of a functional thymidine kinase from Amsacta moorei entomopoxvirus.

A thymidine kinase (TK) gene from the entomopoxvirus of Amsacta moorei (AmEPV) has been identified, mapped, cloned, and sequenced. The AmEPV TK was shown to be biologically functional as cloning of the gene into a TK-derivative of the orthopoxvirus vaccinia creates a TK+ virus. The gene has been localized to a 1.5-kb EcoRI-Q DNA fragment which maps to the far left end of the viral genome. Sequence analysis reveals an open reading frame (ORF) of 182 amino acids potentially encoding a polypeptide of 21.2 kDa. Amino acid homology comparisons indicate that the gene is most closely related to the TKs of a variety of poxviruses (approximately 45%) and less so to the TKs of vertebrates (approximately 40%). The TK from African swine fever virus (ASF) showed the least homology (31.4%) to the AmEPV TK gene, suggesting that these two viruses are not closely related although ASF shares some biological features of poxviruses, and both ASF and AmEPV can replicate within arthropod hosts.     

Organization and molecular characterization of genes in the polyhedrin region of the Anagrapha falcifera multinucleocapsid NPV

Summary. A multinucleocapsid nuclear polyhedrosis virus (MNPV) isolated from the celery looper, Anagrapha falcifera, has been proposed as a new virus based on differences in virulence and DNA fragment profiles between this isolate and the Autographa californica MNPV, the MNPV type species. In the present study, we examined the relatedness of the AfMNPV and AcMNPV genomes by (1) Southern hybridization, (2) comparison of their genetic organization in the polyhedrin gene region (AcMNPV EcoRI-I fragment), and (3) comparison of the nucleotide and deduced amino acid sequences of eight viral genes in this region. Both DNAs hybridized strongly to one another in reciprocal hybridization experiments under stringent conditions, and physical mapping showed that gene order was conserved between the two viruses in the polyhedrin gene region, though the ORF 984 and ctl genes were absent from this region in the AfMNPV. Gene and deduced amino acid sequences for p78, protein tyrosine phospatase, protein kinase, lef-2, and ORFs 327, 453 and 603, showed identity between the two viruses of greater than 91%. The sequences for the gp64 gene, located on a different EcoRI fragment, were also compared and had a nucleotide sequence identity of 97%, and amino acid sequence identity of greater than 98%. The polyhedrin gene showed the least relatedness between the two viruses, with a nucleotide sequence identity of 80%, and a deduced amino acid sequence identity of 90%. Based on these results, we conclude that the AfMNPV should be considered a variant of the AcMNPV. These results also indicate that caution must be used in basing phylogenetic relationships of NPVs on analysis of a single gene, especially the polyhedrin gene, as is the current practice. Received May 31, 1996 Accepted August 8, 1996     

Construction of recombinant fowlpox viruses as vectors for poultry vaccines

Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes. The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors. To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker. This marker operates in a wide variety of cell types and obviates the need for TK⁻ cell lines for selection of TK⁻ recombinants when foreign genes have been inserted within the TK gene of FPV. The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest. Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter. A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens. These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens. Such recombinants should be a very cost-effective means of delivering vaccines to poultry.     

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.     

The fusion protein gene of phocine distemper virus: nucleotide and deduced amino acid sequences and a comparison of morbillivirus fusion proteins

Summary The nucleotide sequence of the gene encoding the fusion protein of phocine distemper virus has been determined. The mRNA is 2206 nucleotides in length and contains one major open reading frame (ORF) of 1893 nucleotides encoding a potential protein of 631 amino acid residues. However, analogy with canine distemper virus (CDV) suggests that translation of the F protein starts at the sixth AUG codon in the mRNA sequence which is located at position 461, resulting in an F₀ protein of exactly the same size (537 aa) as that of CDV. The overall homology at nucleotide level between the CDV and PDV F genes is 66%. The homology between the two F proteins of these respective viruses is 83%.     

The matrix protein gene sequence analysis reveals close relationship between peste des petits ruminants virus (PPRV) and dolphin morbillivirus.

The gene encoding the matrix protein of peste des petits ruminants virus (PPRV) has been cloned and its nucleotide sequence determined. This gene is 1466 nucleotides long and contains an open reading frame (ORF) capable of encoding a basic protein of 335 amino acid residues with a predicted molecular weight of 38,057 Da. This ORF starts at position 33-35 and ends with the codon TAA at position 1038-1040 thus leaving a long untranslated region (426 nucleotides) at the 3' end of the messenger RNA. This fragment is very G/C rich (68.5%) and in contrast to the ORF region appears to be least conserved in the M gene sequence of the morbilliviruses. A comparison of the PPRV M protein with those of other viruses in the group confirms the previously noted high degree of conservation for this protein sequence. The percent of identity within the group ranges from 76.7 to 86.9%, the highest being with the dolphin morbillivirus matrix protein.     

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.     

The coat protein genes of squash mosaic virus: cloning,sequence analysis,and expression in tobacco protoplasts

Summary Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3 end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.     

Structure of the gene encoding the M1 protein of sonchus yellow net virus

The gene encoding the M1 protein of sonchus yellow net virus (SYNV), a plant rhabdovirus, has been sequenced and identified by Western blot analysis of SYNV proteins using antibodies directed against a fusion protein derived from a portion of the sequenced gene. The M1 gene is positioned between nucleotides 4039 and 5109 relative to the 3' end of the viral RNA and is the fourth gene from the 3' end of the genome. The 1071-nucleotide (nt) M1 gene lies between a putative nonstructural gene of unknown function and the gene encoding the glycoprotein and is bordered on either side by the same GG intergenic dinucleotide that separates other genes in the SYNV genome. The M1 mRNA (scRNA 6) consists of a 71-nt untranslated region at the 5' terminus followed by an 858-nt open reading frame (ORF) capable of encoding a protein with a calculated molecular weight of 31,779. The amino acid sequence deduced from this ORF is not highly homologous to those of other rhabdovirus matrix proteins, but has some localized regions of similarity. The UGA codon that terminates the M1 ORF is followed by a 3' untranslated region of 142 nt. The viral RNA (minus-sense) sequence corresponding to the extreme 3' end of the mRNA contains a 9-nt tract (3'-AUUGUUUUU-5') that is identical to the sequences at the termini of other SYNV genes.     

Analysis of nucleotide sequence variations in herpes simplex virus types 1 and 2, and varicella-zoster virus.

To analyze the difference in the degree of divergence between genes from identical herpesvirus species, we examined the nucleotide sequence of genes from the herpes simplex virus type 1 (HSV-1) strains VR-3 and 17 encoding thymidine kinase (TK), deoxyribonuclease (DNase), protein kinase (PK; UL13) and virion-associated host shutoff (vhs) protein (UL41). The frequency of nucleotide substitutions per 1 kb in TK gene was 2.5 to 4.3 times higher than those in the other three genes. To prove that the polymorphism of HSV-1 TK gene is common characteristic of herpesvirus TK genes, we compared the diversity of TK genes among eight HSV-1, six herpes simplex virus type 2 (HSV-2) and seven varicella-zoster virus (VZV) strains. The average frequency of nucleotide substitutions per 1 kb in the TK gene of HSV-1 strains was 4-fold higher than that in the TK gene of HSV-2 strains. The VZV TK gene was highly conserved and only two nucleotide changes were evident in VZV strains. However, the rate of nonsynonymous substitutions in total nucleotide substitutions was similar among the TK genes of the three viruses. This result indicated that the mutational rates differed, but there were no significant differences in selective pressure. We conclude that HSV-1 TK gene is highly diverged and analysis of variations in the gene is a useful approach for understanding the molecular evolution of HSV-1 in a short period.     

Identification of a Gene Cluster within the Genome of Chilo Iridescent Virus Encoding Enzymes Involved in Viral DNA Replication and Processing

The nucleotide sequence of the genome of Chilo iridescent virus (CIV) between the genome coordinates 0.974 and 0.101 comprising 27,079 bp was determined. Computer-assisted analysis of the DNA sequence of this particular region of the CIV genome revealed the presence of 42 potential open reading frames (ORFs) with coding capacities for polypeptides ranging from 50 to 1,273 amino acid residues. The analysis of the amino acid sequences deduced from the individual ORFs resulted in the identification of 10 potential viral genes that show significant homology to functionally characterized proteins of other species. A cluster of five viral genes that encode enzymes involved in the viral DNA replication was identified including the DNA topoisomerase II (A039L, 1,132 amino acids (aa)), the DNA polymerase (ORF A031L, 1,273 aa), a helicase (ORF A027L, 530 aa), a nucleoside triphosphatase I (ORF A025L, 1,171 aa), and an exonuclease II (ORF A019L, 624 aa), all ORFs possessing the same genomic orientation. The DNA polymerase of CIV showed the highest homology (24.8% identity) to the DNA polymerase of lymphocystis disease virus lymphocystis disease virus 1 (LCDV-1), a member of the family Iridoviridae, indicating the close relatedness of the two viruses. In addition, four putative gene products were found to be significantly homologous to previously identified hypothetical proteins of CIV.