Progenitor Virus Research Articles

Emergence of Subtype Zaire Ebola Virus in Gabon

Gabon has recently been struck three times by Ebola hemorrhagic fever. The first isolate originating from the 1994 outbreak has been subjected to molecular characterization of its GP and VP24 genes. Sequence analysis demonstrates that the agent, Gabon-94 virus, belongs to subtype Zaire of Ebola virus. The isolate is closely related to the Kikwit-95 isolate, and both viruses seem to have evolved from a progenitor virus different from that of the Zaire-76 isolates. The relatively close relationship of all subtype Zaire viruses isolated at different geographical locations and up to 20 years apart suggests an extreme conservation in the yet unknown natural reservoir of Ebola viruses. The level of genetic variability in the human host might be different as indicated by the comparison of isolates from a single outbreak (Mayinga-76 and Eckron-76), but needs further investigation on clinical material of patients by PCR since both isolates have different levels of passages in tissue culture.

Analysis of live, oral poliovirus vaccine monopools for human immunodeficiency virus type 1 and simian immunodeficiency virus.

Although there is no evidence for transmission of mammalian retroviruses to humans via vaccine immunization, the allegations of contamination of oral poliovirus vaccines with human immunodeficiency virus (HIV) type 1 or a hypothetical progenitor virus from monkeys has created controversy and dispute regarding the origin of AIDS in humans. Twelve monovalent lots of live, attenuated oral poliovirus vaccine types 1, 2, and 3, which were released for use by a North American manufacturer between 1976-1989, were tested for the presence of HIV-1 and simian immunodeficiency virus (SIV). HIV/SIV were not detected in these monovalent poliovirus vaccine lots with the reverse transcriptase assay, a general detection assay, and highly sensitive and specific polymerase chain reaction assays.

Nucleotide Sequence Analysis of the Respiratory Syncytial Virus Subgroup A Cold-Passaged (cp) Temperature Sensitive (ts)cpts-248/404 Live Attenuated Virus Vaccine Candidate

The complete nucleotide sequence of the RSVcpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passagedcp-RSV, andcpts-248 virus, which constitute the series of progenitor viruses. RSVcpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36°) than itscpts-248 parent virus (shut-off temperature 38°) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited bycp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for thetsand attenuation phenotypes of its chemically mutagenized derivatives,cpts-248 andcpts-248/404. Compared with itscpts-248 parent, thecpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, thecpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of thecpts-248/404 in tissue culture. Biologically cloned derivatives of RSVcpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of thecpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans.

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

Restriction of Fusion Protein mRNA as a Mechanism of Measles Virus Persistence

Vero cells persistently infected with the hamster neurotropic strain (HNT-PI) of measles virus are deficient in the release of extracellular virus and syncytia formation, suggesting that mutations occur within the viral envelope proteins. Nucleotide sequence comparisons indicated that the coding regions of the matrix (M) and fusion (F) genes of HNT-PI were relatively conserved compared with those of their lytic progenitor virus Philadelphia 26 (Ph26), whereas the hemagglutinin (H) gene differed by 4.2% at the amino acid level. Northern blot analyses demonstrated the predominance of bicistronic M/F transcripts in HNT-PI at a 5:1 ratio over F monocistronic mRNA. Accordingly, no F protein could be detected in the HNT-PI cell line, although both the M and H proteins were produced in amounts comparable to those of Ph26. When the Semliki Forest virus replicon was used, coexpression of the HNT-PI F and Ph26 H genes resulted in the formation of multinucleated syncytia in transfected Veto cell cultures. Since the HNT-PI F protein was fusogenic, the restriction of its monocistronic mRNA is postulated to be a contributing factor in the reduction of cell fusion and ultimately in the maintenance of the persistent infection.

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.

Genome affinities and epitope profiles of whitefly‐transmitted geminiviruses from the Americas

SummaryThe relationships among fifteen isolates of whitefly‐transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple‐antibody‐sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA‐A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV‐T). Conversely, the probe for ACMV DNA‐A did not detect any of the American viruses, and that for TYLCV‐T DNA‐A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA‐B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA‐B probe and both chino del tomate virus (CdTV)‐DNA and SLCV‐DNA. However, a probe for DNA‐B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV‐PR DNA, and a probe for DNA‐B of CdTV from Mexico detected several American viruses.Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and‐ACMV MAbs reacted with all, and one anti‐ACMV MAb and two anti‐ICMV MAbs reacted with nearly all the American viruses, one anti‐ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV‐CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA‐A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA‐B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA‐B diverging to a greater extent than DNA‐A and its particle‐protein gene.

Genetic evolution and tropism of transmissible gastroenteritis coronaviruses

Transmissible gastroenteritis virus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S protein of six PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have S proteins with a size similar to the enteric viruses. NEB72 and TOY56 viruses have in the S protein 2 and 15 specific amino acid differences with the enteric viruses. Four of the residues changed (aa 219 of NEB72 isolate and as 92, 94, and 218 of TOY56) are located within the deletion present in the PRCVs and may be involved in the receptor binding site (RBS) conferring enteric tropism to TGEVs. A second RBS used by the virus to infect ST cells might be located in a conserved area between sites A and D of the S glycoprotein, since monoclonal antibodies specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor virus which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BR170, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the fixation of mutations with time, that is, the existence of a well-defined molecular clock. A mutation fixation rate of 7 ± 2 × 10 −4 nucleotide substitutions per site and per year was calculated for TGEV-related viruses. This rate falls in the range reported for other RNA viruses. Point mutations and probably recombination events have occurred during TGEV evolution.

Genetic evidence for variant selection in the course of dilute passaging of mumps vaccine virus

Mumps vaccine viruses, Leningrad-3 (L-3) strain, harvested at the 8th (8P) and 38th (38P) passage levels, were compared by nucleotide sequencing of the fusion (F) and the phosphoprotein (P) genes, and for replication efficiency in cell culture. Sequencing revealed only one clear base substitution throughout the entire F gene, and no substitutions in the variable 183-nucleotide-long region of the P gene. However, the 8P virus, unlike the 38P variant, contained multiple "ambiguous" nucleotide regions, i.e., additional bases positioned at the level of the principal ones. The 38P variant replicated faster and appeared more homogeneous by its plaque character compared to the 8P virus. The results indicate that the 8P progenitor virus consisted of more than one viral variant and that one of these was selected on repeated passage due to its higher replication efficiency.

Human Chromosome-Dependent and -Independent Pathways for HIV-2 Trans-Activation

Human immunodeficiency virus (HIV types 1 and 2) replication is controlled by the interaction of viral-encoded regulatory proteins and host cellular proteins with the viral long terminal repeat (LTR). The presence of HIV-1 and HIV-2 trans-activator proteins, tat1 and tat2, respectively, greatly increases viral gene expression from their homologous LTRs. It is unclear if the cellular factors that support tat1-directed trans-activation of the HIV-1 LTR are the same for tat2 trans-activation of the HIV-2 LTR. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV-1 and HIV-2 tat-directed transactivation. DNA transfection experiments showed that the presence of human chromosome 12 in human-hamster hybrid clones was necessary for high-level tat-directed trans-activation of the HIV-1 and -2 LTR. Cross-trans-activation of the HIV-2 LTR by tat1 was found to be chromosome 12 independent. In addition, chromosome 12 did not support trans-activation of another human retrovirus (human T-cell leukemia virus type I). Our results suggest that HIV-1 and -2 have evolved to employ a cellular pathway(s) encoded on human chromosome 12 for supporting homologous tat-directed trans-activation. Trans-activation of the HIV-2 LTR by tat1 in chromosome 12-minus cells suggests that multiple cellular pathways can be recruited to trans-activate the HIV-2 LTR and that these pathways may have been important in an HIV-like progenitor virus.

Human Immunodeficiency Viruses and the Pathogenesis of AIDS

THE ACQUIRED immunodeficiency syndrome (AIDS) is caused by a newly recognized member of the lentivirus family, the human immunodeficiency virus (HIV). 1 This retrovirus family is distinct from the other subfamilies of human retroviruses, the Spumavirinae and Oncovirinae (Table 1). In particular, lentivirus genomes are large and contain several viral genes; the viruses frequently induce cytopathic effects in infected cells, and the disease they cause has a long incubation period resulting in immunologic disorders and neurological disease. 2 Lentiviruses have been identified in several other animal species, including horses, sheep, goats, cows, cats, and monkeys. 1,2 This finding suggests that a progenitor virus entered the animal kingdom in the distant past and various strains evolved over time within each species. This could be the reason that the primate and human lentiviruses (the simian immunodeficiency virus and HIV) resemble each other. 3 Two subtypes of HIV, HIV-1 and HIV-2, have been

AIDS in Africa: Emerging trends

This paper reviews the literature on AIDS in Africa. By 29 February, 1988, 9760 cases of AIDS had been reported in Africa. This review addresses the currently accepted belief in the African origin of AIDS, the distribution (cluster) of AIDS in Africa, identified modes of transmission, and changing sexual and drug use behaviors in Africa. There is no conclusive evidence to show that the AIDS virus originated in Africa. Even if the progenitor virus had a habitat in central Africa, certain high-risk sexual behaviors which were introduced in the 60s and 70s may have initiated the infectious process. The distribution pattern does not suggest a transcontinental spread; rather, it suggests a contigual and transatlantic spread. The primary mode of transmission is heterosexual activity. This paper raises a number of questions relating to heterosexual behaviors. Other issues addressed include drug use behavior, homosexuality, and high-risk sexual activities of Africans and foreigners in Africa. Other modes of transmission are transfusion of contaminated blood and blood products, use of nonsterile needles, and perinatal transmission. It is not likely that traditional (folk) medicine will contribute to the spread of AIDS. Traditional doctors use fresh razor blades for cuts and not hollow instruments such as needles. The impact of the political and socioeconomic climate in most of Africa during the 60s and early 70s is evaluated. Finally, we make suggestions for future direction, which include confirmatory testing of HIV Positive samples, conducting clinical epidemiology and social science-based research, and developing innovative education programs that are culturally relevant.

Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS.

In a study of genetic variation in the AIDS virus, HTLV-III/LAV, sequential virus isolates from persistently infected individuals were examined by Southern blot genomic analysis, molecular cloning, and nucleotide sequencing. Four to six virus isolates were obtained from each of three individuals over a 1-year or 2-year period. Changes were detected throughout the viral genomes and consisted of isolated and clustered nucleotide point mutations as well as short deletions or insertions. Results from genomic restriction mapping and nucleotide sequence comparisons indicated that viruses isolated sequentially had evolved in parallel from a common progenitor virus. The rate of evolution of HTLV-III/LAV was estimated to be at least 10(-3) nucleotide substitutions per site per year for the env gene and 10(-4) for the gag gene, values a millionfold greater than for most DNA genomes. Despite this relatively rapid rate of sequence divergence, virus isolates from any one patient were all much more related to each other than to viruses from other individuals. In view of the substantial heterogeneity among most independent HTLV-III/LAV isolates, the repeated isolation from a given individual of only highly related viruses raises the possibility that some type of interference mechanism may prevent simultaneous infection by more than one major genotypic form of the virus.

Genetic relatedness between A/Swine/Iowa/15/30(H1N1) and human influenza viruses

The nucleotide sequences of the M and NS1 genes of influenza virus A/Swine/Iowa/15/30 (A/SW/IW/30)(HlN1) were determined with cloned DNAs and compared with reported sequences of human and avian influenza viruses. A/SW/IW/30 virus was found to be closely similar to A/PR/8/34(HlN1) virus in the nucleotide sequences of the M and NS1 genes, the base differences between the two strains being 64 out of 1027 nucleotides in the M gene and 52 out of 740 in the NS1 gene. Based on the assumptions that these two viruses were derived from a common ancestor and that the rate of base changes per year was the same in man and in swine, it was estimated that the progenitor virus was in circulation during the period from 1915 to 1920. This estimation was compatible with the epidemiological findings suggesting that the progenitor of the swine influenza virus was the agent of the 1918 influenza pandemic. Furthermore, the M and NS1 gene sequences of A/FPV/Rostock/34(H7N6) virus were much closer to those of A/SW/IW/30 and A/PR/8/34 viruses than to A/duck/Alberta/60/76(H12N5) virus, but not as close as the A/SW/IW/30 virus was to A/PR/8/34 virus.

Role of the middle-sized bunyavirus RNA segment in mouse virulence

The middle-sized (M) RNA segment of California group viruses (Bunyaviridae) is a major determinant of virulence for mice. This has been demonstrated by intraperitoneal (ip), or intracerebral (ic), inoculation of 4-week-old mice with prototype or reassortant bunyaviruses, the latter obtained from mixed infection of BHK-21 cells with temperature-sensitive ( ts) mutants of La Crosse (LAC), snowshoe hare (SSH), and Tahyna (TAH) viruses. Reassortant viruses were not more virulent than wild-type progenitor viruses. All reassortants, and wild-type cloned viruses having the LAC, or SSH, M RNA segment, killed mice following ip inoculation; reassortant and wild-type viruses having the TAH M RNA did not kill 4-week-old mice after ip inoculation. In addition, reassortant viruses with the TAH M RNA killed mice after is inoculation more rapidly than did viruses having the LAC M RNA segment, irrespective of the parental origin of the small (S), or large (L), viral RNA species. The correlation of the M RNA segment with virulence implies that G1 and/or G2 determine major murine virulence properties, perhaps through attachment which may influence tropism. These experiments are an initial step in understanding the mechanism of virulence for mice, and eventually for man. They could also lead to the rational design of live attenuated bunyaviruses as vaccines for man and domestic animals.

Epidemic mechanisms of Type A influenza

The antigenic varieties of influenza A virus isolated from 1968 to 1976 in a surveillance of a small, rather remote population were similar to those from England and Wales as a whole, despite frequent antigenic changes during the period. Household studies in the first two H3N2 influenza A epidemics found low attack rates within households, a high proportion (70%) of affected households with only one case of influenza, similar distributions of affected households in the two epidemics by the number of cases of influenza and similar distributions of the influenza cases by the day of their onset in the household outbreak. No serial interval could be demonstrated by cumulating household outbreaks. More than one minor variant was causing influenza contemporaneously in the same villages in several seasons, and different variants were on one occasion found on successive days in bedfellows. The regular occurrence of epidemics in winter was often accompanied by the disappearance of the epidemic variants and their replacement, after a virus-free interval, by new variants. These epidemiological findings seem best interpreted on the following tentative hypothesis. Influenza A sufferers do not transmit the virus during their illness; instead it rapidly becomes latent in their tissues so that they become symptomless carrier-hosts and develop specific immunity. Next season an extraneous seasonally mediated stimulus reactivates the latent virus residues so that the carrier-host becomes briefly infectious, though symptomless. Antigenic drift occurs because particles reconstituted to be identical with the progenitor virus cannot escape the specific immunity it has provoked in the carrier host. He can shed only mutants also determined by the progenitor virus. From the assortment of mutants shed by the carrier-host, his non-immune companions select that (those) which is best fitted to survive, and it rapidly causes influenzal illness. Epidemics consist largely or entirely of such persons sick with influenza caused by reactivated virus caught from symptomless carrier-hosts.