Coupled PCR-restriction enzyme analysis for rapid identification of structural gene relationships among strains of eastern equine encephalitis virus.

We have used restriction endonuclease digestion analysis of polymerase chain reaction (PCR)-amplified gene regions to rapidly examine individual structural gene relationships among field isolates of eastern equine encephalitis (EEE) virus. The E1+ (E1 gene plus 292 nucleotides 3' of the coding region), E2, and C gene regions from North American (NA) variety viruses and the E1 and C gene regions of South American (SA) variety viruses were successfully amplified by RT-PCR using a single primer set for each locus. The products were then digested with a panel of restriction endonucleases and the resulting DNA fragments electrophoretically compared. Our findings revealed marked similarity among the E1+ and the E2 gene restriction patterns, respectively, of most NA strains. In contrast, the restriction patterns exhibited by the E1+ gene of SA strains differed substantially from those of NA strains and also appeared more heterogeneous. The digestion patterns of the C gene were generally similar for all strains of the virus examined. These results thus demonstrate that EEE viral E1+ and C structural gene sequences can be amplified from an assortment of both NA and SA varieties of the virus by RT-PCR using a single primer set per locus, and that both varietal and individual isolate distinctions can be identified by comparison of subsequent restriction digestion patterns. This technique should prove useful as an epidemiological tool for rapid identification of EEE isolates from clinical and field specimens, and as a rapid screen for alterations within structural gene regions.

Enhancement of antigen presentation of influenza virus hemagglutinin by the natural human anti-Gal antibody.

Immunogenicity of inactivated virus or subviral vaccines may be enhanced by complexing with an IgG antibody. Such antibody would increase the uptake, processing and presentation of the vaccine's antigens by antigen presenting cells (APC), via the adhesion of the antibody-vaccine complex to Fc-receptors on macrophages and other APC. A natural antibody in humans, which may be generally exploited for this purpose, is the natural anti-Gal antibody. This antibody is ubiquitously produced as 1% of circulating IgG in humans and Old World primates, and it interacts specifically with the carbohydrate epitope Gal alpha 1-3 Gal beta 1-4 GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is synthesized in large amounts in cells of nonprimate mammals and New World monkeys by the glycosylation enzyme alpha 1,3 galactosyltransferase. Here we describe in vitro studies on the ability of anti-Gal to bind to alpha-galactosyl epitopes on influenza virus propagated in mammalian cells, and to enhance presentation by APC of viral hemagglutinin antigenic determinants to specific helper T cell clones. The various approaches for achieving alpha-galactosyl epitope expression on virion and subviral vaccines are discussed.

Differential reactivity of immune sera from human vaccinees with field strains of eastern equine encephalitis virus.

Eastern equine encephalitis (EEE) virus is a mosquito-borne alphavirus that can produce a severe and often fatal acute encephalitis in humans, with significant neurologic sequelae in survivors. Due to the serious nature of the disease, an investigational inactivated EEE vaccine (PE-6) is available to individuals at risk for infection. Both serologic and recent molecular analyses of EEE viruses have demonstrated marked differences between the two antigenic varieties of EEE virus, designated North American (NA) and South American (SA). In view of these findings, we have examined the reactivity of sera from three individuals immunized with the EEE vaccine, derived from an NA isolate, with field strains of EEE virus. Anti-EEE serum antibodies from vaccinees reacted strongly in Western blot assays with both of the envelope (E1 and E2) glycoproteins of each NA strain examined, while reactivities with the glycoproteins of SA strains were substantially weaker and variable and dependent upon both the immune response of the vaccinee and the virus isolate assayed. Most striking was the modest to virtual lack of reactivity with the E2 protein of SA strains. Antigenic differences among the glycoproteins of EEE viruses were not as pronounced in immunoprecipitation analysis. Most significantly, although human immune sera displayed high neutralizing titers against each of the NA isolates examined, only negligible neutralizing titers were obtained against SA isolates. These data suggest that immunized individuals would mount an effective antibody response against infection with NA strains of EEE virus, but that further investigation is clearly warranted to fully assess the protective capability of the vaccine against infection with SA strains.

Structural protein relationships among eastern equine encephalitis viruses.

We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates. In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA- and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA- and SA-serotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NA- and SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.

Differential host-dependent expression of alpha-galactosyl epitopes on viral glycoproteins: a study of eastern equine encephalitis virus as a model.

The carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl) is abundantly expressed on cells of non-primate mammals, prosimians and New World monkeys, where it is synthesized by the enzyme alpha 1,3-galactosyltransferase (alpha 1,3GT). Old World monkeys, apes and humans lack alpha 1,3GT and hence do not synthesize alpha-galactosyl epitopes. Instead, these species produce a natural antibody, anti-Gal, which interacts specifically with alpha-galactosyl epitopes and which constitutes up to 1% of circulating immunoglobulins in humans. We have used eastern equine encephalitis (EEE) virus as a model to examine the differential expression of alpha-galactosyl epitopes on the glycoproteins of virus propagated in cells that either produce or lack alpha 1,3GT. As predicted, virus propagated in Vero cells (derived from the African green monkey, an Old World monkey) did not express alpha-galactosyl epitopes. In contrast, virus propagated in mouse 3T3 cells (EEE3T3) expressed approximately 80 alpha-galactosyl epitopes per virion on both the E1 and the E2 envelope glycoproteins. Thus, expression of the alpha-galactosyl epitope on virions paralleled that on host cells. The binding of anti-Gal antibody to these epitopes on EEE3T3 virions partially neutralized virus infectivity, raising the possibility that anti-Gal production in hosts may influence the initial infectious stage of viruses expressing alpha-galactosyl epitopes.

Diversity within natural populations of eastern equine encephalomyelitis virus.

We evaluated genetic and phenotypic diversity within natural populations of the alphavirus, Eastern equine encephalomyelitis (EEE) virus. RNA fingerprinting revealed that most populations within infected hosts (unpassaged isolates) contained a consensus genotype along with minority genotypes differing in one to three T1-resistant oligonucleotides. Mutation frequencies appeared to be similar to those reported for other RNA viruses, suggesting that the slow rate of EEE virus evolution is not limited by fidelity of genome replication. Within a given year, genetic diversity was generally greater among geographically distant isolates than among those from the same transmission focus, suggesting that dispersal among EEE viruses in North America is not complete annually. Two of three bird isolates from Maryland and New York contained relatively distantly related genotypes, differing in 15-19 oligonucleotides. A 1985 mosquito isolate from Maryland contained stable, small plaque variants which comprised the majority of that population. These small plaque variants differed by up to eight T1-resistant oligonucleotides when compared with their large plaque counterparts. Temperature sensitive virus was not detected in six unpassaged mosquito isolates from Maryland and New York.

Detection of eastern equine encephalomyelitis virus deposition in Culiseta melanura following ingestion of radiolabeled virus in blood meals.

We examined deposition of eastern equine encephalomyelitis virus (EEEV) in the alimentary tract of its enzootic mosquito vector, Culiseta melanura, to detect potential sites of initial infection. Artificial viremias were created by injecting purified [3H]uridine-labeled EEEV intravenously into one-day-old chicks. Mosquitoes were allowed to engorge, incubated 1-2 hr, fixed, and whole abdomens and thoraces of 25 specimens embedded. Tagmata were sectioned, mounted on slides and coated with autoradiographic emulsion. Following exposures of 1-4 months, slides were developed and examined microscopically. In the majority of mosquitoes, label (virus) was detected only within the midgut; most virus was concentrated in a band of expressed serum adjacent to the abdominal midgut epithelium. Small amounts of virus were also deposited within folds in the cardial thoracic midgut of 96% of mosquitoes. Viral penetration into epithelial cells was detected throughout the abdominal midgut in all mosquitoes, and in the thoracic midgut of 20%. A small number (4/25) of mosquitoes examined showed signs of leaky abdominal midguts, with virus detected in the abdominal hemocoel. Concentration of EEEV in expressed serum adjacent to the abdominal midgut epithelium may enhance initial midgut infection. Leaky abdominal midguts exhibited by some mosquitoes may also facilitate rapid systemic infections of Cs. melanura. Deposition of virus in thoracic alimentary tissues suggests the possibility of early EEEV infection of the anterior midgut.

Variation in dengue type 2 viruses isolated in Bangkok during 1980.

Dengue type-2 viruses isolated in metropolitan Bangkok during 1980 (Bangkok/80) were characterized by oligonucleotide fingerprinting, restriction enzyme (RE) mapping and antigenic analysis using monoclonal antibody probes. Of 10 isolates analysed by oligonucleotide fingerprinting, nine were very closely related, showing 72.5% to 91.4% oligonucleotide homology. One isolate (D80-141) produced a distinctly different fingerprint (55.7% to 58.0% homology) and was less related to other Bangkok/80 dengue-2 virus isolates than to a 1964 Bangkok isolate (16681). RE mapping conducted on complementary dsDNA prepared from three Bangkok/80 isolates, strain 16681 and the prototype New Guinea C strain confirmed that D80-141 was genetically distinct. On antigenic analysis, only one of 22 monoclonal antibody probes produced against representative 1980 Bangkok dengue-2 isolates, D80-100 and D80-141, was able to distinguish between these virus strains. Monoclonal antibody 47-10/10, prepared using D80-100 virus and directed at the NS1 non-structural glycoprotein, had a significantly lower (100-fold) solid phase radioimmune assay endpoint titre for D80-141 antigen than for D80-100 antigen. By the indirect immunofluorescence assay, 47-10/10 had lower antibody endpoint titres against D80-141, the NGC strain and 13 (12%) of 110 Bangkok/80 isolates than to a control antibody preparation. These results suggest that strain D80-141 represents a second minor topotype of dengue-2 which was circulating concurrently with the major endemic topotype in Bangkok in early 1980.

Development of an attenuated strain of chikungunya virus for use in vaccine production.

An attenuated chikungunya (CHIK) virus clone was developed for production of a live vaccine for human use. CHIK strain 15561 was subjected to 18 plaque-to-plaque passages in MRC-5 cultures before CHIK 181/clone 25 was selected as vaccine seed based on homogeneous small plaque size, suckling mouse avirulence, reduced monkey viraemia and genetic stability. Oligonucleotide mapping demonstrated differences between parent and clone. Vaccine (pilot-lot production) elicited neutralizing antibody and protected mice and rhesus monkeys against challenge. After challenge, viraemias were absent in vaccinated monkeys. Vaccine was then produced and tested in accordance with governmental regulatory requirements of human use.

Identification of an antigenic and genetic variant of dengue-4 virus from the Caribbean.

Twenty-one dengue (DEN) viruses isolated from the Caribbean (Dominica and Jamaica) during the 1981-1982 epidemic year were distinct serological and genetic variants of DEN-4 virus. These isolates were clearly identified as DEN-4 viruses using type-specific monoclonal antibodies in indirect immunofluorescence assays. However, they either were not neutralized, or were neutralized poorly using hyperimmune mouse ascitic fluids (HMAF) or rhesus monkey serum directed against the H-241 prototype strain of DEN-4 virus isolated in the Philippines in 1956. HMAF prepared against a representative Caribbean isolate, however, neutralized with similar effectiveness the homologous virus, the H-241 prototype strain, and virus strains isolated from the Pacific and Southeast Asian areas from 1973 to 1984. The Caribbean isolate exhibited no more than 30% and 16% oligonucleotide spot homology with the H-241 and Bangkok viruses, respectively, by RNA fingerprint analysis, while demonstrating 82% and 89% homology with the Gilbert and Niue Island isolates, respectively. The isolation of dengue viruses which are serologically and genetically distinct from the prototype virus emphasizes the need for continued dengue virus surveillance. The recognition of unique dengue isolates should allow the selection of reference strains and vaccine candidate strains which will induce antibodies that are equally effective in neutralizing viruses from all geographic areas.

Identification of an antigenic and genetic variant of dengue-4 virus from the Caribbean

Twenty-one dengue (DEN) viruses isolated from the Caribbean (Dominica and Jamaica) during the 1981-1982 epidemic year were distinct serological and genetic variants of DEN-4 virus. These isolates were clearly identified as DEN-4 viruses using type-specific monoclonal antibodies in indirect immunofluorescence assays. However, they either were not neutralized, or were neutralized poorly using hyperimmune mouse ascitic fluids (HMAF) or rhesus monkey serum directed against the H-241 prototype strain of DEN-4 virus isolated in the Philippines in 1956. HMAF prepared against a representative Caribbean isolate, however, neutralized with similar effectiveness the homologoous virus, the H-241 prototype strain, and virus strains isolated from the Pacific and Southeast Asian areas from 1973 to 1984. The Caribbean isolate exhibited no more than 30 percent and 16 percent oligonucleotide spot homology with the H-241 and Bankok viruses, respectively, by RNA fingerprint analysis, while demonstrating 82 percent and 89 percent homology with the Gilbert and Niue Island isolates, respectively. The isolation of dengue viruses which are serologically and genetically distinct from the prototype virus emphasizes the need for continued dengue virus surveillance. The recognition of unique dengue isolates should allow the selection of reference strains and vaccine candidate strains which will induce antibodies that are equally effective in neutralizing virus from all geographic areas. (AU)

Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization.

A newly modified semimicro plaque reduction neutralization test (PRNT) in BHK cells was compared with a standard PRNT in bottles with LLC-MK2 monolayers and with an LLC-MK2 PRNT adapted to semimicro methods. The BHK semimicro PRNT compared favorably in terms of sensitivity in detecting dengue antibody (96%), specificity at a screening dilution (95%), and ability to detect seroconversion to dengue viruses of three serotypes (93%). Disagreements between the BHK test and the LLC-MK2 tests were attributed to greater sensitivity of the BHK test in detecting dengue type 2 (DEN-2) antibody in acute-phase sera and to apparent low-level DEN-1/DEN-3 cross-reactions in some sera in all three tests. The BHK PRNT was easier, faster, and more economical than either of the LLC-MK2 tests. Many of the benefits of the BHK PRNT derive from the fact that cells are infected while still in suspension, at the time of cell splitting, hence the term "BHK suspension test."

RNA fingerprinting as a method for distinguishing dengue 1 virus strains.

Virion RNAs of 12 geographically distinct dengue type 1 (DEN-1) virus isolates were clearly unique by RNA fingerprinting. Isolates from the same geographic area were very similar but differed from those of other areas, allowing us to establish three geographical groupings based upon percent shared oligonucleotides. Three Caribbean strains were virtually identical (85-91% homologous oligonucleotides) whereas Pacific/S.E. Asian strains exhibited considerably less homology to one another (44-49%). The Pacific/S.E. Asian strains exhibited little relationship (20-30%) to the Caribbean and African strains. A Sri Lankan isolate displayed a relatively high degree of homology to Nigerian isolates (60-66% homologous oligonucleotides), suggesting that the Sri Lanka DEN-1 infection originated from Africa. A 1978 Nigerian DEN-1 isolate and the 1969 Sri Lankan strain each exhibited greater than 50% homology with a 1977 Jamaican strain. The similarities observed between the African/Sri Lankan and Jamaican strains suggest that the DEN-1 virus which caused the 1977 Jamaican epidemic may have originated from Africa or Sri Lanka. The RNA fingerprint is a unique characteristic of DEN-1 strains from a particular geographic region, suggesting this technique as a useful tool for dengue epidemiological investigations.

In vivo transcription and protein synthesis capabilities of bunyaviruses: wild-type snowshoe hare virus and its temperature-sensitive group I, group II, and group I/II mutants.

The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280:213-215, 1975; M. Bouloy and C. Hannoun, Virology 69:258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16:1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 mug/ml), alpha-amanitin (25 mug/ml), or rifampin (100 mug/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40 degrees C) for virus replication gave values comparable to those obtained at permissive temperatures (33 degrees C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33 degrees C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40 degrees C than those of the SSH group I ts mutants or wild-type SSH virus at 40 degrees C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40 degrees C were significantly higher than their primary transcription capabilities (as measured at 33 degrees C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using (32)P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33 degrees C (primary and secondary transcription) or 40 degrees C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33 degrees C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40 degrees C, low levels of N and G1 polypeptides were evident.