Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: construction and characterization of mutant viruses.

The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK(2) cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK(2) cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37 degrees C and 39 degrees C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.

[Circulation of West Nile virus (Flaviviridae, Flavivirus) and some other arboviruses in the ecosystems of Volga delta, Volga-Akhtuba flood-lands and adjoining arid regions (2000-2002)]. / Osobennosti tsirkuliatsii virusa Zapadnogo Nila (Flaviviridae, Flavivirus) i nekotorykh drugikh arbovirusov v ékosistemakh del'ty Volgi, Volgo-Akhtubinskoi poimy i sopredel'nykh aridnykh landshaftakh (2000-2002 gg.).

Comprehensive virological, serological as well as genetic studies of the ecology of West Nile Virus (WNV) as well as of some other arboviruses were undertaken in different ecosystems in the territories of the Astrakhan Region and of the Kalmyk Republic. The main carriers (mosquitoes, ticks, birds and mammals) were defined as involved in the circulation of viruses within the natural and anthropogenic biocenosis. Phylogenetic examinations of isolated strains and samples, which were positive in RT-PCR, showed an absolute predominance of genotype I virus that was most closely related to American and Israeli strains. At the same time, epidemic strains had up to 6% of nucleotide differences versus the historic strains isolated in the same region 20-30 years ago. Besides, the circulation of genotype IV was discovered; it was characterized by a lower pathogenicity, which, possibly, ensures the shaping of a pronounced immune interlayer bearing no epidemic consequences. An analysis of the study results on the WNV ecology denotes the epicenter of the endemic territory located in the middle part of the Volga delta.

Molecular determinants of virulence of West Nile virus in North America.

West Nile virus (WNV) is a mosquito-borne flavivirus that until very recently had not been found in the Americas. In 1999, there was an outbreak of West Nile encephalitis in New York and surrounding areas, involving 62 human cases, including 7 fatalities. The virus has subsequently become established in the United States of America (U.S.) with 4156 human cases, including 284 deaths, in 2002. The WNV strains found in the U.S. are members of "lineage I", a genetic grouping that includes viruses from Europe, Asia and Africa. Molecular epidemiologic studies indicate that two genetic variants of WNV emerged in 2002. The major genetic variant is found in most parts of the U.S., while the minor genetic variant has been identified only on the southeast coast of Texas. Investigation of WNV in mouse and hamster models demonstrated that strains from the U.S. are highly neurovirulent and neuroinvasive in these laboratory rodents. Other strains, such as Ethiopia 76a from lineage I, are not neuroinvasive and represent important viruses which can be used to elucidate the molecular basis of virulence and attenuation of WNV. To identify putative molecular determinants of virulence and attenuation, we have undertaken comparative nucleotide sequencing of Ethiopia 76a and strains from the U.S. The results show that the two viruses differ by 5 amino acids in the envelope (E) protein, including loss of the glycosylation site. Comparison of our panel of 27 WNV strains suggests that E protein glycosylation is a major determinant of the mouse neuroinvasive phenotype.

[Analysis of new variants of West Nile fever virus]. / Analiz novykh variantov virusa likhoradki Zapadnogo Nila.

The complete nucleotide sequences for 6 strains of the West Nile fever virus were determined. For the first time the complete nucleotide sequences of the Indian isolate and Krsn190 strain, that is the most far phylogenetically from all isolates known at present time were established. The scheme for separation of virus variants into 4 groups and criteria for determination the group to which the isolate belongs are suggested.

Specific detection of chikungunya virus using a RT-PCR/nested PCR combination.

Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.

Development of new vaccines against dengue fever and Japanese encephalitis.

Mosquito-borne dengue (DEN) and Japanese encephalitis (JE) viruses are the leading causes of arthropod-transmitted viral disease in humans. A licensed tetravalent vaccine that provides effective, long-term immunity against all four serotypes of DEN virus is needed, but is currently unavailable. Improvements to currently available JE vaccines are also needed. Past and recent strategies for the development of new DEN and JE vaccines include inactivated and live attenuated viruses, engineered viruses and chimeric viruses derived from infectious cDNA clones of DEN or JE virus, recombinant poxviruses, recombinant baculoviruses, protein expression in Escherichia coli, and naked DNA vaccines. This report summarizes some of the recent developments in DEN and JE vaccinology, particularly vaccine strategies that involve live attenuated viruses, engineered viruses derived from infectious cDNA clones, and naked DNA vaccines.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses.

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3.

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants.

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex.

The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.

Genetic evidence for the origins of Venezuelan equine encephalitis virus subtype IAB outbreaks.

Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.

Resistance to alpha/beta interferons correlates with the epizootic and virulence potential of Venezuelan equine encephalitis viruses and is determined by the 5' noncoding region and glycoproteins.

We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.

The alphavirus 3'-nontranslated region: size heterogeneity and arrangement of repeated sequence elements.

The 3'-nontranslated region (NTR) of representative strains of all known alphavirus species was amplified by reverse transcription-polymerase chain reaction. For 23 of them, the 3'-NTR sequence was determined. Together with previously published data, this allowed an analysis of the 3'-NTR of the viruses in the genus Alphavirus. The length of the 3'-NTRs varied from 77 nt for Pixuna virus to 609 nt for Bebaru virus. The 19-nt conserved sequence element directly adjacent to the poly(A) tract was found in all viruses, supporting the hypothesis that this region is a cis-acting sequence element during viral replication and essential for virus growth in vitro. Within the 3'-NTR of all alphaviruses, repeated sequence elements of various numbers and lengths were found. Their composition was very consistent in both the Venezuelan equine encephalitis (VEE) and the Sindbis-like viruses, although their number was constant only within the latter group. For the VEE viruses, our data suggested that insertion events rather than deletions from an ancestor with a long 3'-NTR created the various number of repeated sequence elements. Among the remaining viruses, both the number and the composition of repeated sequence elements varied remarkedly.

Nucleotide sequences of the 26S mRNAs of the viruses defining the Venezuelan equine encephalitis antigenic complex.

Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.

Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53.

We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.

Genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction.

A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the genus-specific detection of alphaviruses. Based on the available published sequences, degenerate primers were designed to ensure hybridization to a conserved region within the nonstructural protein 1 gene of all alphavirus species. The expected 434-basepair (bp) cDNA fragment was amplified from all 27 alphavirus species by using RNA extracted from 200 microl of infected cell culture supernatant. In addition, eight strains of Venezuelan equine encephalitis (VEE) virus and 10 strains of Sindbis virus were amplified. The viral origin of the amplicons was confirmed by restriction enzyme analysis and comparison with the expected cleavage pattern based on published sequence data. The PCR products of alphavirus species with thus far unknown nucleotide composition were sequenced. About 120 nucleotides downstream of the forward primer, a region showing sufficient homology for the design of another forward primer was found and used in a semi-nested PCR. The expected 310-bp semi-nested fragment was demonstrated for all viruses investigated. The sensitivity of the RT-PCR was about 1,200 plaque-forming units (PFU) for VEE virus reference strain Trinidad donkey. The detection limit after the semi-nested PCR was 1.2 PFU. The sensitivity was not hampered by the presence of human serum, thus making this test suitable for an application in viremic individuals. Chikungunya virus RNA was amplified from infected mouse brain tissue by the described RT-PCR assay. Our data suggest that the semi-nested RT-PCR may be applied as a highly sensitive alternative to virus isolation in the rapid screening and diagnosis of alphavirus infections, including post-mortem diagnosis. Phylogenetic analysis of the amplicon sequence data identified six genotypes within the Alphavirus genus.

Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas.

Murine T-helper cell immune response to recombinant vaccinia-Venezuelan equine encephalitis virus.

The T-helper (Th) cell immune response following immunization of C3H (H-2k) mice with a recombinant vaccinia (VAC) virus (TC-5A) expressing the structural proteins (capsid, E1 and E2) of the attenuated vaccine strain (TC-83) of Venezuelan equine encephalitis (VEE) virus was compared with the immune response induced in mice after immunization with TC-83 virus. TC-5A virus elicited Th cells that strongly recognized both VAC and TC-83 viruses in in vitro lymphoblastogenesis tests. Th-cell activation was associated with elevated levels of interleukin-2. TC-5A virus induced long-term humoral immunity; VEE virus-binding and neutralizing antibodies were detected in mouse sera collected from mice 16 months after a single immunization.

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.