[Comparative analysis of the morphogenesis of the Getah virus in cell cultures of vertebrates and mosquitoes]. / Sravnitel'nyi analiz morfogeneza virusa Geta v kletkakh kul'tur pozvonochnykh zhivotnykh i komarov.

Reproduction of Getah virus (the genus Alphavirus) in piglet kidney (PK) and A. aegypti mosquito cell cultures was studied morphologically. Inoculation of PK cells resulted in the development of a lytic infection. Virus morphogenesis in these cells showed the features known for other alphaviruses. In mosquito cell cultures persistent infection developed which was not accompanied by any marked changes in morphology or proliferative activity of the culture. All the morphological and cytochemical data indicate that a certain role in the establishment and maintenance of virus persistence in A. aegypti cell culture may be played by exo- and endophagocytic processes owing to which a peculiar "self-purification" of the cells from the virus and its components occurs. Incubation of the persistently infected culture of mosquito cells at a suboptimal temperature resulted in a marked reduction in the number of newly formed virions which sharply increased with the shift up to the optimal temperature. The experimental results give some insights into certain aspects of alphavirus ecology.

Inhibition of transcription by immunoglobulins directed against the ribonucleoprotein of homotypic and heterotypic vesicular stomatitis viruses.

Specific antisera were raised by immunization of rabbits with purified nucleocapsids containing only RNA and N protein (ribonucleoprotein, RNP) obtained from vesicular stomatitis (VS) virions of the Indiana (VSInd) and the New Jersey (VSNJ) serotypes. The specificity of anti-RNPInd serum was demonstrated by selective precipitation of homotypic RNPInd devoid of L and NS proteins; anti-RNPInd serum also selectively precipitated soluble N protein present in cytoplasm of infected cells, but co-precipitated a limited amount of contaminating soluble NS protein. Immunoglobulins prepared from each homotypic antiserum markedly inhibited in vitro transcription of VSInd and VSNJ virions. Anti-RNPInd and anti-RNPNJ immunoglobulins also exhibited cross-reactivity by inhibiting transcription of heterotypic virions, but only to a much lesser degree than in the homotypic reaction. Anti-RNPInd immunoglobulin did not inhibit transcription of the antigenically unrelated Chandipura rhabdovirus, but anti-RNPNJ immunoglobulin did to a very limited extent. The transcription inhibitory activity of anti-RNPInd immunoglobulin was not dependent on RNP immunoprecipitation activity, which could be diluted out well before loss of antitranscriptase activity. Anti-RNPind immunoglobulin appeared to exert its effect on transcription by blocking elongation rather than initiation or reinitiation of RNA transcripts.

Non-specific inhibitory fraction for hemagglutination of some togaviruses obtained from Swiss albino mouse brain.

A fraction from Swiss albino mouse brain homogenate obtained by DEAE cellulose chromatography showed a non-specific hemagglutination-inhibitory property for some Togaviruses. Evidence are presented indicating that the inhibitory property is related to a protein-rich fraction with sialic acid as an active group.

Uukuniemi virus contains an RNA polymerase.

An RNA-dependent RNA polymerase activity has been found associated with Uukuniemi virions. The enzyme activity is expressed only after disrupting the virions with the nonionic detergent Triton X-100 and is absolutely dependent on Mn2+, whereas Mg2+ is not required, a finding that distinguishes this polymerase from those of other enveloped minus-strand RNA viruses. Within the range pH 7.2 to 8.5 no distinct optimum was found. The optimum temperature was between 37 and 40 C. The reaction was not inhibited by actinomycin D, rifampin, or DNase, whereas RNase was completely inhibitory. The partially RNase-resistant product consisted of rather small-sized RNA, which contained sequences complementary to Uukuniemi virus RNA as shown by hybridization to the template L, M, and S RNA species of Uukuniemi virus.

[RNA polymerase activity associated with a bunya virus (Lumbo)]. / Activité RNA polymérasique à un bunyavirus (Lumbo)

RNA dependent RNA polymerase has been demonstrated in purified Lumbo virus (Bunyavirus) which contains a single stranded segmented RNA. Divalent cations (Mn++ and Mg++) are required for optimal in vitro activity. Reaction products can be specifically annealed with the viral genome.

Reconstitution of infectivity and transcriptase activity of homologous and heterologous viruses: vesicular stomatitis (Indiana serotype), Chandipura, vesicular stomatitis (New Jersey serotype), and Cocal viruses.

RNA TRANSCRIPTASE ACTIVITIES HAVE BEEN RECONSTITUTED FROM FRACTIONATED COMPONENTS OF FOUR RHABDOVIRUSES: vesicular stomatitis virus (VSV) (Indiana serotype), Cocal, VSV (New Jersey), and Chandipura viruses. Heterologous reconstitution of transcription has been observed between template and enzyme fractions of VSV Indiana and Cocal viruses but not to any significant extent between components of the other two viruses with VSV Indiana template or enzyme. Homologous reconstitution of infectivity has been obtained for each virus from their respective parts as well as heterologous reconstitution between Cocal and VSV Indiana components.

RNA transcription by the virion polymerases of five rhabdoviruses.

The presence of a virion-associated RNA-dependent RNA polymerase in five serologically distinct rhabdovirus isolates (vesicular stomatitis virus [VSV] Indiana, VSV New Jersey, Cocal, Chandipura, and Piry viruses) has been demonstrated. The enzyme for each virus has been shown to be a transcriptase capable of synthesizing in vitro RNA which is complementary to the viral genome. By sequence analyses it has been shown that, for each rhabdovirus, transcription is multiply initiated with specific 5' nucleotide sequences. The results indicate that, for all five viruses, the initiation of transcription involves similar sequences (e.g., pppApCpGp..., pppGpCp..., and possibly one or two other sequences), suggesting relatedness and some genome conservation among these viruses.

Association of viral ribonucleic acid with cellular membranes in chick embryo cells infected with Sindbis virus.

Membranes from cells infected with Sindbis virus had associated with them viral ribonucleic acid (RNA) polymerase and about 60 to 70% of the viral RNA labeled when short pulses were used. This RNA contained most of the replicative intermediate and replicative form of viral RNA found in the infected cells. The use of "Mg(2+) sarkosyl crystals" permitted the isolation of membrane-bound nucleic acids and allowed the demonstration that Sindbis virus RNA was synthesized on a membrane-viral RNA complex. Viral RNA from the infecting virions first became associated with the membranes during the latent period and, subsequently, slowly detached. The attachment of the viral RNA to the membranes did not require active viral RNA polymerase, since RNA from ts6, an RNA(-) temperature-sensitive mutant of Sindbis virus, associated with cellular membranes at a nonpermissive temperature. However, the subsequent detachment of the RNA from the membranes was restricted in the absence of viral RNA synthesis. The results indicate that association of viral RNA with cellular membranes may represent an early step occurring during the replication of Sindbis virus RNA.

Sindbis virus-induced viral ribonucleic acid polymerase.

A cytoplasmic structure containing the viral ribonucleic acid (RNA) polymerase has been isolated by sucrose density centrifugation from cells infected with Sindbis virus. Uninfected cells did not contain any such structure. Preliminary experiments indicated that the structure may be associated with membranes. This structure incorporated (3)H-guanosine triphosphate in vitro in the absence of added template. The RNA synthesized in vitro by the enzyme consisted of single-stranded 40S RNA, the ribonuclease-resistant replicative form, and possibly the replicative intermediate form of viral RNA. The products formed in vitro by the enzyme are identical in sedimentation rates to those formed in the infected cells in vivo.