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A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

Numerous viruses of the Flaviviridae family, including dengue, yellow fever, Japanese encephalitis, and West Nile, cause significant disease in humans and animals. The structure and function of the molecular components of the flavivirus envelope are therefore of significant interest. To our knowledge, a membrane (M) protein mutation which affects the pH at which flavivirus particles are inactivated in vitro has never been reported. Here we show that substitution of proline for glutamine at residue M5 (MQ5P) of a Japanese encephalitis-yellow fever chimera (ChimeriVax-JE) increases its acid sensitivity in vitro by 0.3 pH units (i.e., increases the pH at which virus titer is reduced by 50% from 6.08 to 6.38). In addition, growth kinetics of this mutant virus are accelerated in Vero cells, while neurovirulence and neuroinvasiveness measured in a mouse model are unaffected. A possible interpretation of these observations is that M can modulate the envelope (E) protein function during cell infection.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

Rubella virus (RUB), a small plus-strand RNA virus, is a significant human pathogen. The RA27/3 vaccine strain of RUB is one of the most successful live attenuated vaccines developed. In this article, we report the construction of an RA27/3 infectious clone, a complete cDNA copy of the RA27/3 genome that can be transcribed in vitro to generate infectious RNA molecules. Virus generated from such in vitro transcripts was phenotypically similar to RA27/3 virus. To investigate the attenuation of the RA27/3 strain, a series of chimeras was made by the insertion of different fragments of the RA27/3 genome into an infectious clone based on the Therien wild-type strain of RUB. Analysis of the resulting chimeric viruses revealed that the pattern of RA27/3 attenuation in cell culture is complex: attenuating elements in the RA27/3 genome were found in the 5' untranslated region (UTR), a region of the nonstructural proteins containing the protease motif and the capsid gene. Within the 5' UTR, the attenuation determinant was mapped to nt 7. Surprisingly, these analyses also revealed a potentiating mutation at nt 164 of the RA27/3 genome. Although this determinant was within the coding sequences of the nonstructural proteins, the encoded amino acid had no effect on cell culture phenotype and thus the determinant may operate at the level of RNA structure. In addition to investigation of the mechanisms of RA27/3 attenuation, the availability of the RA27/3 infectious clone offers the opportunity for strict genetic control over RUB vaccine manufacturing, for development of novel DNA-based vaccines against RUB, and for development of recombinant RUB vaccines that also target other diseases., (Copyright 2000 Academic Press.)

The replication of rubella virus (RUB) in Vero cells, an adherent cell line, results in apoptotic death of infected cells as detected by chromatin fragmentation assays. In infected cultures, virtually all of the cells that had become detached (a hallmark feature of RUB-induced cytopathology) were apoptotic; they were predominantly dead as shown by propidium iodide and trypan blue exclusion tests. In contrast, the majority of the cells in the infected monolayers that remained adherent were alive and contained intact chromatin. Thus simple counting of detached cells in the medium is a convenient way of measuring the extent of RUB-induced apoptosis. RUB-induced cytopathology was inhibited by z-VAD-fmk, an inhibitor of caspases that are involved in the execution stages of apoptosis, confirming the induction of apoptosis by RUB. The lack of apoptotic adherent cells (maximally 1% at any time point through 6 days postinfection) indicates that the induction of apoptosis is asynchronous since cells become uniformly virus antigen-positive by day 2 postinfection. To elucidate whether this asynchronicity and the ability of RUB to persistently infect Vero cells were due to a suppression of apoptosis, we examined whether RUB can suppress chemically induced apoptosis. Staurosporine (ST) was found to be an efficient inducer of apoptosis in Vero cells. ST treatment of RUB-infected and RUB persistently infected cells resulted in a much higher proportion of detached cells, higher even than in Vero cells treated with ST alone. This indicates that RUB does not suppress ST-induced apoptosis and, rather, that ST and RUB acted cumulatively in inducing apoptosis, possibly indicating that they use different induction pathways., (Copyright 1998 Academic Press.)

A series of double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding the immunogenic proteins of Japanese encephalitis virus (JEV) [prM-E, prM-E-NS1, NS1-NS2A, 80%E (encodes the amino-terminal 80% part of E), and NS1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent JEV. The cassettes were introduced into both 5' [second subgenomic promoter of the vector precedes the SIN structural open reading frame (SP-ORF)] and 3' (the promoter follows the SP-ORF) dsSIN vectors. The longest cassette (prM-E-NS1) was 3.2 kb in length, which is remarkable for such a small vector virus as SIN (SIN genome is roughly 11.8 kb in length). The level of expression of JEV proteins appeared similar for both 5' and 3' recombinants. In general, the stability of the recombinants obtained was found to be low (expression was lost following one to five passages at low multiplicity of infection, depending on the recombinant). However, 5' recombinants containing longer cassettes (prM-E-NS1, prM-E, NS1-NS2A) were more stable than the corresponding 3' recombinants. Intraperitoneal inoculation of mice with 10(7) PFU of dsSIN-JEV recombinants induced antibodies against JEV proteins and low titers of JEV-neutralizing antibodies were produced by mice inoculated with recombinants expressing 80%E, prM-E, and prM-E-NS1. A single immunization of mice with the dsSIN-prM-E or dsSIN-prM-E-NS1 recombinants provided 40-65% protection against peripheral lethal challenge with 10(4) LD50 of neurovirulent JEV. The results demonstrate that genetically engineered togaviruses can be successfully used as vaccine vectors.

Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants was observed. This suppression was more pronounced late in infection (< 5% of total product was secreted during an 8-hr chase) than early in infection (15% secretion during a 6-hr chase). In comparison, a vaccinia virus-prM-E recombinant (vP829) described previously (E. Konishi et al. (1991) Virology 185, 401-410) was shown to secrete 35-50% of total product during a 6- to 8-hr chase both early and late in infection. In contrast, secretion of prM-E from dsSIN-prM-E-infected mosquito (C6/36) cells was found to be efficient (> 50% during an 8-hr chase). The prM-E secreted from both mammalian and mosquito cells was in the form of subviral particles as determined by velocity gradient centrifugation, sensitivity to nonionic detergent, and analysis of processing of N-linked glycans. The truncated E protein expressed by the dsSIN recombinants was secreted efficiently from both mammalian and mosquito cells. Coinfection experiments with the dsSIN-JEV recombinants + wild-type vaccinia virus and vP829 + SIN demonstrated that the reduced level of secretion of subviral particles exhibited by the dsSIN-JEV recombinants was due to an inhibitory effect of the dsSIN vectors. Furthermore, this inhibitory effect was accounted for by the SIN nonstructural proteins since SIN replicons that express prM-E cassette in place of the SIN structural protein open reading frame exhibited a low level of subviral particle secretion. No self-propagating infectious particles were produced in cells transfected with SIN replicons that encode the JEV prM-E cassette. The suppression of subviral particle secretion was apparently correlated with the inhibition of cell protein synthesis which is mediated in SIN-infected vertebrate cells by expression of the SIN nonstructural proteins.