Sequence Determination and Molecular Analysis of Two Strains of Bovine Parainfluenza Virus Type 3 that are Attenuated for Primates

The Kansas/15626/84 (Ka) and Shipping Fever (SF) strains of bovine parainfluenza virus type 3 (BPIV3) replicate less efficiently than human PIV3 (HPIV3) in the upper and lower respiratory tract of rhesus monkeys, and BPIV3 Ka is also highly attenuated in humans and is in clinical trials as a candidate vaccine against HPIV3. To initiate an investigation of the genetic basis of the observed attenuation phenotype of BPIV3 in primates, the complete genomic sequences of Ka and SF genomes were determined and compared to those of BPIV3 strain 910N and two HPIV3 strains, JS and Wash/47885/57. There is a high degree of identity between the five PIV3 viruses in their 55 nucleotide (nt) leader (83.6%) and 44 nt trailer (93.2%) sequences. The five viruses display amino acid sequence identity ranging from 58.6% for the phosphoprotein to 89.7% for the matrix protein. Interestingly, the majority of amino acid residues found to be variable at a given position in a five-way protein alignment are nonetheless identical within the viruses of either host species (BPIV3 or HPIV3). These host-specific residues might be products of distinct selective pressures on BPIV3 and HPIV3 during evolution in their respective hosts. These host-specific sequences likely include ones which are responsible for the host range differences, such as the efficient growth of BPIV3 in bovines compared to its restricted growth in primates. It should now be possible using the techniques of reverse genetics to import sequences from BPIV3 into HPIV3 and identify those nt or protein sequences which attenuate HPIV3 for primates. This information should be useful in understanding virus-host interactions and in the development of vaccines to protect against HPIV3-induced disease.     

Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the P/C gene. Because CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the P/C mutants, r-CF170S and r-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).     

Herpes Simplex Virus Type 1 (HSV-1) Strain HSZP Host Shutoff Gene: Nucleotide Sequence and Comparison with HSV-1 Strains Differing in Early Shutoff of Host Protein Synthesis

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL4l gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accesion numbers Z72337 and Z72338. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ⅰ型单纯疱疹病毒糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

目的构建单纯疱疹病毒Ⅰ型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础.方法PCR扩增HSV1-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K-gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性.结果获得了重组的酵母表达载体pPIC9K-gD.测序结果证实为HSV1-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量为40.52 ku,pI为7.67,包含完整成熟肽分值达1.7的多个抗原决定簇.结论成功构建了HSV1-gD成熟肽基因的毕赤酵母表达载体.     

Capsid Protein-Encoding (P1) Sequence of Foot and Mouth Disease Virus Type Asia 1 Ind 63/72

Variations in the amino acid sequence of Foot-and-Mouth Disease Virus (FMDV) structural proteins are the basis for the antigenic diversity of the virus. Majority of antigenic sites for the virus neutralization are present on VP1, the major immunogenic protein. However, a few conformational epitopes are present on the structural proteins VP2 and VP3. The nucleotide sequence encoding all the four structural proteins (P1 region) of FMDV type Asia 1 Ind 63/72 was determined. The nucleotide and the deduced amino acid sequence of P1 of Asia 1 of Indian strain was compared with that of Asia 1 Israel strain. Differences were observed at 284 (14%) nucleotide positions resulting in 69 (10%) amino acid changes. The variation in the derived amino acid sequence is the highest in VP1 (14.4%) followed by VP2 (10%), VP3 (6.4%) and VP4 (3%). Deletion of two amino acids, which was observed in the case of Indian strain as well as in Israel strain indicated that these deletions are specific for type Asia 1. The P1 sequence was also compared with the corresponding region of the other serotypes O1K, A12, C1 and SAT-1. The sequence has been submitted to EMBL data bank, under accession number Y09949.     

The Foot and Mouth Disease Virus Type O Outbreak of 1992 Is Not Related to Vaccine Strain (O/R2/75)

Vaccination is the only pragmatic approach to control foot and mouth disease in India. Strict quality control measures are essential to supply potent vaccine to the field application, in addition to monitoring the performance of the vaccine in the field. During the process of monitoring, an outbreak of FMD in vaccinated animals caused by type “O” virus in Tanjavur district of Tamil Nadu and a type “O” virus from unvaccinated herd of Karnataka were studied. Field isolates and vaccine virus were sequenced and analyzed. Data indicated that the virus from the outbreak in vaccinated cattle was a variant which could escape neutralization by antibodies against vaccine virus. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris)

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8⁺ T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.     

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.