Attenuated temperature-sensitive respiratory syncytial virus mutants generated by cold adaptation.

Two strains of respiratory syncytial virus (RSV), RSV 2B and RSV 3A (representing subgroup B and A virus respectively) were cold-adapted by passaging in Vero cells for up to 42 weeks at successively lower temperatures down to 20 degrees C. Successful cold adaptation of the virus population was dependent on the amount of time the cultures were maintained at the various low temperatures, as well as on the strain of virus used. Temperature-sensitive (TS) mutants appeared in the cold passaged virus populations; however, the majority of the virus variants remained predominantly non-TS. Four RSV 2B and three RSV 3A TS mutants were selected for further characterization. These seven TS mutants retained their fusion phenotype and two major neutralizing antibody epitopes, and displayed varying levels of temperature sensitivity. Six of the seven mutants had a cold-adapted (CA) phenotype. All of the RSV 2B mutants were highly attenuated in cotton rats and two of the mutants elicited relatively high levels of neutralizing antibody and were able to protect rats against virus challenge. The RSV 3A TS mutants grew well in the nose but poorly in the cotton rat lungs, as did the parental 3A virus. All 3A mutants elicited high titers of neutralizing antibody and provided complete protection against virus challenge. These mutants showed varying levels of temperature sensitivity in vitro and attenuation in vivo and represent potential vaccine candidates.

Vaccines for neonatal viral infections: towards a live respiratory syncytial virus vaccine:a study in risk.

There is risk attached to the development of respiratory syncytial virus vaccines, live attenuated or otherwise, but without the acceptance of this risk by manufacturers, health providers and the public, the conclusion of successful Phase III trials may lie in the distant future.

Establishment and characterization of St Louis encephalitis virus persistent infections in Aedes and Culex mosquito cell lines.

Persistent infections with St Louis encephalitis (SLE) virus were established in three mosquito cell lines (Aedes albopictus, A. dorsalis and Culex tarsalis) and were maintained for over 2 years. All three persistently infected cell cultures shared two features: (i) no overt cytopathic effect and (ii) a relatively high proportion of cells infected (41 to 85%). The Aedes persistently infected cultures were resistant to superinfection with the homologous virus but not heterologous viruses. Two significant differences were observed between the Aedes and C. tarsalis persistently infected cell cultures: (i) viral titres in the A. albopictus and A. dorsalis cell cultures decreased slowly over time (the decrease was particularly marked in the A. albopictus cell cultures), whereas titres in the C. tarsalis cell cultures remained relatively constant and (ii) the addition of anti-SLE virus antibody led to decreased virus production in the C. tarsalis cell cultures (one of two cultures was cured of infection), whereas antibody had no effect on the persistently infected Aedes cell cultures. These results suggest that there may be significant differences in the regulation of viral replication and the maintenance of flavivirus persistent infections in mosquito cell lines of different origins.

Phenotypes of St Louis encephalitis virus mutants produced in persistently infected mosquito cell cultures.

Viral mutants that appeared during long-term persistent infections of mosquito cell cultures (Aedes albopictus, A. dorsalis and Culex tarsalis) with St Louis encephalitis virus were characterized. Evidence was obtained for the presence of temperature-sensitive mutants in the A. dorsalis and C. tarsalis persistently infected cultures, and small plaque mutants were predominant in all cultures except one of two cell cultures of C. tarsalis. Virus from persistently infected A. albopictus cell cultures was growth-restricted in Vero and C. tarsalis cells. One of two persistently infected A. dorsalis cell cultures also produced viral mutants that were growth-restricted in C. tarsalis cells. Further, Western blots of persistently infected A. albopictus cell extracts showed an overproduction of capsid (C) and envelope (E) structural proteins and reduced production of an Mr 27K protein (p27) which was immunologically related to the E protein. In contrast, the production of E and C proteins in persistently infected C. tarsalis cultures was consistent with the amount of infectious virus present, whereas p27 was relatively overproduced. These observations suggest that the host cell has an important influence on both the types and relative quantities of viral mutants that accumulate during long-term persistent infections.

Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures.

Cell-to-cell fusion of Aedes albopictus (mosquito) cells infected with dengue and St Louis encephalitis (SLE) flaviviruses was induced by exposure to low pH. The parameters of this low pH-induced fusion were examined. Syncytium formation was maximal in cultures 36 to 48 h post-infection and occurred when cultures were maintained at the acid pH for 15 min at 35 degrees C. The optimal pH range for fusion was 5.0 to 6.5 for dengue virus-infected cells and 5.0 to 5.5 for SLE virus-infected cells. Syncytia were not observed in vertebrate cells (Vero and BHK) under these conditions despite similar virus yields. Fusion was shown to be ATP-dependent and could be prevented by the addition of either polyclonal antiviral antibodies or monoclonal antibody to the envelope glycoprotein. The lysosomotropic amine ammonium chloride inhibited the replication of SLE virus in both mosquito and vertebrate cells, consistent with the idea that low pH-induced fusion is necessary for virus entry into both types.

Biochemical characterizations of two temperature-sensitive and attenuated strains of respiratory syncytial virus subgroup B.

Cold-adapted, temperature-sensitive (ts), attenuated strains of respiratory syncytial virus have been developed from a B subgroup clinical isolate for potential use as vaccine candidates. The replication of two B subgroup ts mutant viruses (2B33F and 2B20L) at the permissive and nonpermissive temperatures have been compared with that of the parental 2B virus to establish differences that may account for their ts and/or attenuated phenotypes. We have shown that the ts restriction at 39 degrees C in the replication of the two mutant viruses in tissue culture occurs at a step after virus adsorption but before or including initiation of virus-specific mRNA transcription. At the permissive temperature of 32 degrees C a 12- to 24-h delay in the accumulation of mRNA for both mutant viruses in comparison to that of the parental 2B virus was exhibited. This effect was mirrored by equivalent delays in viral protein synthesis and production of infectious virus. By 36 h postinfection both mutants had produced levels of viral mRNA, protein, and infectious virus that were similar to those of the parent virus at 32 degrees C. ts+ revertant viruses derived from both mutants have also reverted in their viral mRNA, protein, and infectious virus production kinetics at 32 degrees C to rates more like those exhibited by the parental 2B virus. This suggests a positive correlation between the ts step in the replication of the mutant viruses and the initial delay in mRNA production that occurs at the permissive temperature.

Acidotropic amines inhibit proteolytic processing of flavivirus prM protein.

Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weight, glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM occurs in the acidic post-Golgi vesicles.

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer.

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NS1 (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NS1 is consistent with the idea that the high-molecular-weight form of NS1 is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NS1 is a general feature of flavivirus infection. It is formed between 20 and 40 min after NS1 is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NS1 are also found as the high-molecular-weight form.

Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant.

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription-PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

Identification of mutations contributing to the reduced virulence of a modified strain of respiratory syncytial virus.

The nucleotide sequences of the genome of the RSS-2 wild type strain of respiratory syncytial (RS) virus, which is known to induce upper respiratory tract infection in adults, and that of the attenuated ts1C candidate vaccine derived from it by three cycles of mutagenesis and selection of temperature-sensitive (ts) mutants, have been determined. Comparison of the sequences has located the genetic changes which contribute to the reduced pathogenicity in adults of the candidate vaccine. Thirty-seven nucleotide changes distinguish the wild type and ts1C, 13 of which confer amino acid substitutions; no mutations are present in extragenic regions. Partial nucleotide sequencing of the genomes of the first stage ts mutant (ts1A) and the second stage ts mutant (ts1B), which were intermediates in the derivation of the third stage mutant ts1C, established that five mutations resulting in amino acid substitutions had been induced in the first cycle of mutagenesis, one in the second cycle, and seven in the third cycle. The unique mutation differentiating ts1B from ts1A substitutes an alanine for a threonine at residue 736 in the polymerase (L) protein. The occurrence of a mutation in ts1C inducing substitution of a phenylalanine for a serine residue at an adjacent site (731) suggests that mutations in this region of the polymerase can have significant attenuating effects. The data suggest also that a mutation in the F gene may contribute to the attenuated phenotype.

Evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy.

A live-attenuated, intranasal respiratory syncytial virus (RSV) candidate vaccine, cpts-248/404, was tested in phase 1 trials in 114 children, including 37 1-2-month-old infants-a target age for RSV vaccines. The cpts-248/404 vaccine was infectious at 104 and 105 plaque-forming units in RSV-naive children and was broadly immunogenic in children >6 months old. Serum and nasal antibody responses in 1-2 month olds were restricted to IgA, had a dominant response to RSV G protein, and had no increase in neutralizing activity. Nevertheless, there was restricted virus shedding on challenge with a second vaccine dose and preliminary evidence for protection from symptomatic disease on natural reexposure. The cpts-248/404 vaccine candidate did not cause fever or lower respiratory tract illness. In the youngest infants, however, cpts-248/404 was unacceptable because of upper respiratory tract congestion associated with peak virus recovery. A live attenuated RSV vaccine for the youngest infant will use cpts-248/404 modified by additional attenuating mutations.