Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Humans with OKT4-epitope deficiency have a single nucleotide base change in the CD4 gene, resulting in substitution of TRP240 for ARG240.

The OKT4 epitope of the CD4 cell-surface protein has been shown to be polymorphic in white, black, and Japanese populations. The variable phenotypic expression is due to an alteration of the OKT4 epitope, since those persons lacking reactivity with OKT4 monoclonal antibody (mAb) are reactive with OKT4A-F mAb as well as other mAb specific for CD4. To determine the nature of this polymorphism at the gene level, we sequenced polymerase chain reaction-amplified genomic DNA containing the CD4-V3 and -V4 exons from American black subjects who are OKT4-normal, OKT4-negative heterozygous, or OKT4-negative homozygous. Comparison of the sequences revealed that the two CD4 exons are identical except for a cytosine-to-thymidine transition occurring at nucleotide position 868. This alters the first codon position of mino acid 240 and results in a tryptophan residue replacing an arginine residue. The change was also found in white and Japanese persons who are OKT4-negative.

Venezuelan equine encephalomyelitis vaccine (strain TC-83): a field study.

In 1971, more than 370 horses in south Texas were studied with respect to their clinical, virologic, and neutralizing antibody responses to vaccination with Venezuelan equine encephalomyelitis (VEE) strain TC-83. This study confirms reported findings that the vaccine used in the 1971 epizootic in the lower Rio Grande Valley of Texas was safe and efficacious. Vaccinal virus viremia titers were generally below the postulated infection threshold of epizootic vectors. In general, reactions to the vaccine were minimal and transient, with no observed abortions or deaths attributable to use of the vaccine. Eleven months after vaccination, VEE antibody titers were demonstrable in most horses that had VEE antibodies within 30 days after vaccination. Presence of western equine encephalomyelitis antibody titers of greater than or equal to 1:50 at time of VEE vaccination appears to modify or to interfere with VEE antibody production.

Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA.

Two overlapping cDNA clones corresponding to the 5' region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an ambisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5' terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3' termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression.

Possible evidence for interference with Venezuelan equine encephalitis virus vaccination of equines by pre-existing antibody to Eastern or Western Equine encephalitis virus, or both.

During 1971, an epizootic of Venezuelan equine encephalitis (VEE) reached the United States. Laboratory tests were performed on a large number of sick, healthy, unvaccinated, and vaccinated horses. Neutralization (N) tests in cell cultures revealed that 153 of 193 (79.3%) equines outside the state of Texas and 175 of 204 (85.8%) within Texas (82.6% overall) had detectable N antibody to VEE virus a week or more after vaccination. Twenty-six of 40 (65%) non-Texas equines and 18 of 29 (62%) Texas equines which had no detectable antibody against VEE virus a week or more after vaccination had N antibody against Eastern equine encephalitis (EEE) or Western equine encephalitis (WEE) virus or both, whereas only 50 of 153 (32.7%) non-Texas equines and 82 of 175 (46.9%) Texas equines with demonstrable N antibody against VEE also had N antibody against EEE and/or WEE virus. In vaccinated equines, significant negative correlations were found between the occurrence of antibody to VEE and antibody to EEE and/or WEE virus. These findings support the hypothesis that pre-existing antibody to EEE and/or WEE virus may modify or interfere with infection by VEE virus. The epizoologic significance of this possibility is discussed briefly.

Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy.

Ultrastructural studies of glutaraldehyde-fixed viruses of the Bunyaviridae were performed by negative-stain electron microscopy. The surface structure of viruses of each genus was compared with that of the other genera and with Hantaan virus, the prototype of a proposed new genus of Bunyaviridae. Viruses of each genus had a surface structure distinct for that genus. In addition, Hantaan virus had a surface structure composed of a grid-like pattern of morphologic subunits not previously described for animal viruses. Careful morphologic studies of suspected Bunyaviridae may be used in considering preliminary generic assignment. This study also supports the assignment of Hantaan-related viruses to a separate generic status within the Bunyaviridae.

Down-regulation of interleukin-10 expression and production is associated with spontaneous proliferation by lymphocytes from human T lymphotropic virus type II-infected persons.

Cytokines from peripheral blood mononuclear cells (PBMC) from human T lymphotropic virus (HTLV)-II-infected persons were studied to delineate the mechanism(s) of spontaneous lymphocyte proliferation (SLP). Culturing HTLV-II-infected PBMC that spontaneously proliferate (SLP+) resulted in greater mRNA expression and production of interferon-gamma, interleukin (IL)-4, and IL-5, with a concomitant decrease in IL-10, than was seen with nonproliferating (SLP ) and normal PBMC. While IL-2 mRNA expression was higher, production was lower in SLP+ PBMC than in SLP and normal PBMC, implying that the proliferating cells are utilizing IL-2. Neutralization of IL-2 resulted in partial inhibition, suggesting that other cytokines also affect SLP. Addition of recombinant IL-10 inhibited the proliferation of SLP+ PBMC. Further, blocking costimulatory signals with monoclonal antibodies against CD80/CD86 resulted in increased IL-10 production with concomitant inhibition of SLP. The mechanism(s) underlying HTLV-II-associated SLP in vitro involve increased utilization of IL-2 and down-regulation of IL-10.

Morphological identification of the agent of Korean haemorrhagic fever (Hantaan virus)as a member of the Bunyaviridae.

Korean haemorrhagic fever (KHF) (Hantaan virus), a rodent-borne viral illness, is an important cause of human disease throughout much of Asia and Eastern Europe. The agent responsible for KHF has not yet been conclusively identified. Plaque-purified KHF virus was concentrated and then banded in a potassium tartrate gradient. Material from the 1.17-1.19 g/ml band was examined by electron microscopy and particles with a morphology identical to that of the family Bunyaviridae were found. The particles were aggregated by KHF serum but not by saline solution or non-immune serum. Identification of KHF virus as a member of the family Bunyaviridae implies a potential for spread by arthropod vectors.

Labile serum factor and its effect on arbovirus neutralization.

Enhancement of neutralization of Sindbis, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, and St. Louis encephalitis viruses by labile serum factor (LSF) in human serum and plasma was demonstrated. Human serum and plasma could be diluted 1:8 and 1:16 and still retain some LSF activity. Satisfactory storage temperatures for retention of LSF activity were -20 or -56 C. Repeated freeze-thaw cycles of serum did not alter LSF activity, but the activity was completely eliminated by heating at 56 C for 5 min. LSF of human serum equally enhanced neutralization by Sindbis immune mouse and rabbit sera; these results suggest a lack of species specificity. Rehydrated lyophilized gunea pig complement did not restore LSF activity to heated human plasma. Serum components responsible for LSF activity were not dialyzable. Discovery of fresh serum without LSF activity established the need to pretest all sera used as LSF sources.

Physiological and immunologic disturbances associated with shock in a primate model of Lassa fever.

The degree of cell and organ damage in clinical and histological studies of patients dying of Lassa fever has been insufficient to explain the catastrophic shock characteristic of the fatal illness. To explore this issue further, we conducted a study of the evolution of shock in three Lassa virus-infected rhesus monkeys. By the sixth day after infection, a marked, progressive reduction of in vitro platelet aggregation occurred despite normal numbers of circulating platelets and a normal platelet survival time and was accompanied by loss of prostacyclin production by postmortem endothelium. Both of these functions recovered rapidly in a surviving animal. There was no evidence of disseminated intravascular coagulation, nor were clotting factors significantly abnormal. We observed association of viral antigen with neutrophils and progressive neutrophilia. Viremia was not reduced by a brisk antibody response in our animals, and there was a general depression of response to mitogens in mixed lymphocyte stimulation assays. Our findings suggest that shock in Lassa fever is due to biochemical dysfunctions of platelets and endothelial cells and results from loss of intravascular plasma volume, effusions, and hemorrhage.

Comparison of three methods used to isolate dengue virus type 2.

During the 1969 dengue epidemic in Puerto Rico, human sera and Aedes aegypti mosquitoes were collected for virus isolation and identification. Three methods of isolation were used and compared. In the first method, we inoculated newborn mice by the intracranial route, noted any signs of illness, and serially passed specimens in mice until virus was isolated. In the second method, we inoculated tube cultures of LLC-MK(2) cells, noted any cytopathic effect (CPE), and assayed fluids for virus by plaque formation in LLC-MK(2) cell monolayers. The third method was different from the second only in that the original specimens were first inoculated into fluid cultures of Singh's A. albopictus cells. No significant CPE was seen in LLC-MK(2) cultures; however, distinct syncytial CPE was observed in A. albopictus cells. About the same number of virus isolates were made in each isolation system. Virus isolates from both sera and mosquitoes were identified as dengue type 2 by a plaque-reduction neutralization test in LLC-MK(2) cells. The utility of the three methods, individually or in combination, is discussed and related to diagnostic and epidemic situations.

Serological and virological evidence of a Hantaan virus-related enzootic in the United States.

The first isolation of a Hantaan-related virus from a feral rat in the United States was made from a Rattus norvegicus caught in New Orleans. The strain, designated Tchoupitoulas virus, is antigenically related to, but distinct from, the prototype strain 76-118 of Hantaan virus and is the first Hantaan-like virus isolated from the pancreas of a naturally infected animal. Serosurveys of wild rodents from urban and rural areas in the United States indicated that Hantaan-related viruses infected urban rats in coastal and inland cities and infected five species of New World rodents in the western United States (Peromyscus maniculatus, Peromyscus difficilis, Peromyscus californicus, Neotoma mexicana, and Neotoma cinerea). Serosurveys disclosed no evidence of Hantaan-virus infection in rats in large-scale breeding colonies.

Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.