Analysis of the monkeypox virus genome.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Marker rescue of the host range restriction defects of modified vaccinia virus Ankara.

The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells, and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and SPI-1 HR genes and the repair of large deletions were determined by polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA genome because the phenotypes of some recombinants approached that of the parental virus, (2) acquisition of the K1L gene correlated with the ability of recombinant viruses to propagate in RK-13 cells but did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant viruses with intermediate HRs were isolated, and (5) at least one of the new HR genes did not map within any of the major deletions because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.

Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia.

Cytotoxic T lymphocyte (CTL) responses to the human papillomavirus (HPV) type 16 E6 and E7 proteins were measured in 20 women with known HPV and cervical disease status. CTL assays were performed after stimulation with E6 or E7 fusion proteins using autologous B lymphoblastoid cells infected with vaccinia viruses expressing E6 or E7. CTL responses to E6 and E7 were detected in 6 (75%) of 8 and 5 (56%) of 9 HPV-16-positive women without cervical intraepithelial neoplasia (CIN), respectively. Responses to E6 or E7 were each detected in only 2 (29%) of 7 HPV-16-positive women with CIN. Responses to both antigens were found in 63% of women without CIN and 14% of those with CIN. CTL responses to E6 or E7 are more commonly detectable in HPV-16-positive women without CIN than in HPV-16-positive women with CIN, suggesting that CTL response may play a role in disease protection.

Compact, synthetic, vaccinia virus early/late promoter for protein expression.

Vaccinia virus, a member of the poxvirus family, is widely used as a mammalian cell expression vector. Vaccinia virus replicates in the cytoplasm and has its own transcriptional system, making it necessary to use viral promoters. Here, we describe the design, construction and use of a 40-bp synthetic, vaccinia virus promoter with largely overlapping early and late regulatory elements. Convenient plasmid transfer vectors are depicted for expression of one or two genes under control of strong early/late promoters and allowing for thymidine kinase (TK) or antibiotic selection of recombinant viruses.

Genome sequence of a human tumorigenic poxvirus: prediction of specific host response-evasion genes.

Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.

Dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene.

Vaccinia virus strains vary considerably in the amounts of extracellular enveloped virus (EEV) that they release from infected cells. The IHD-J strain produces up to 40 times more EEV than does the related WR strain and consequently generates elongated comet-shaped virus plaques instead of sharply defined round ones in susceptible monolayer cells under liquid medium. The difference in EEV formation is due to the retention of enveloped WR virions on the cell surface (R. Blasco and B. Moss, J. Virol. 66:4170-4179, 1992). By using WR and IHD-J DNA fragments for marker transfer and analyzing the progeny virus by the comet formation assay, we determined that gene A34R and at least one other gene regulate the release of cell-associated virions. Replacement of the A34R gene of WR with the corresponding gene from IHD-J increased the amount of EEV produced by 10-fold and conferred the ability to form distinctive comet-shaped plaques. Gene A34R encodes an EEV-specific glycoprotein with homology to C-type animal lectins (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). The nucleotide sequences of the A34R genes of WR and IHD-J strains differed in six positions, of which four were silent. One of the codon mutations (Lys-151-->Glu), which is located in the putative carbohydrate recognition domain, was sufficient to transfer a comet-forming phenotype to WR virus. These data indicate that the A34R-encoded glycoprotein is involved, through its lectin homology domain, in the retention of progeny virus on the surface of parental cells and raise the possibility that the protein also has a role in virus attachment to uninfected cells.

Human immunodeficiency virus type 1 envelope glycoprotein molecules containing membrane fusion-impairing mutations in the V3 region efficiently undergo soluble CD4-stimulated gp120 release.

The ability of soluble forms of CD4 to induce gp120 release from the human immunodeficiency virus type 1 envelope glycoprotein complex may reflect molecular events associated with membrane fusion. The third hypervariable (V3) region of gp120 appears to play a role in fusion independent of CD4 binding. We demonstrate herein that envelope glycoprotein molecules rendered fusion defective by mutations in conserved residues within the V3 region nevertheless undergo efficient soluble CD4-induced gp120 release.

Association of gastric hypoacidity with opportunistic enteric infections in patients with AIDS.

To determine the relation and possible significance of gastric hypoaciditity to chronic diarrhea in AIDS, patients with and without chronic (greater than 1 month) diarrhea underwent fasting gastric juice pH measurement and microbiologic study and upper and lower endoscopy with biopsy. All 8 patients with diarrhea and high gastric pH (greater than 3; mean, 6.1 +/- 1.0) had gastric bacterial overgrowth (greater than 10(4) bacteria/mL) along with opportunistic enteropathogens in the duodenum or rectosigmoid, but only 1 of 6 patients with diarrhea and gastric pH in the normal range (less than or equal to 3; mean, 1.9 +/- 0.7) had overgrowth or an opportunistic enteropathogen. By contrast, all but 1 of 9 controls (AIDS patients without diarrhea) had normal fasting gastric pH (mean, 2.9 +/- 1.5). Overall, the presence of gastric hypoacidity was associated with identification of opportunistic enteropathogens (P = .035). Thus, gastric hypoacidity is associated with quantitative bacterial overgrowth and opportunistic enteric infections and may be etiologically important in the pathophysiology of the chronic diarrhea seen in some AIDS patients.

Frame-shift mutations within the vaccinia virus A-type inclusion protein gene.

The genetic basis for the failure of vaccinia virus (strain WR) to form a full-length 150 kiloDalton (kDa) A-type inclusion protein was determined by sequencing a 4.1-kb pair segment of DNA and analyzing its transcription products. Open reading frames predicted to encode slightly overlapping 84.5- and 27.1-kDa proteins homologous to contiguous N-terminal segments of the A-type inclusion protein of cowpox virus were found. A putative deletion of two adjacent nucleotides occurring within several consecutive AG repeats and an insertion of 8 nucleotides accounted for the first and second reading frame shifts, respectively. Additional small mutations affecting reading frames were present in the C-terminal region of the gene. The vaccinia and cowpox virus mRNAs encoding the disparate size A-type inclusion proteins were similar in length, had equivalent 5' and 3' ends, and were expressed late in infection indicating the absence of mutations affecting transcriptional signals.