The spread of drug-resistant influenza viruses type A to close contacts in families, schools, and nursing homes has been well documented. To investigate whether drug-resistant influenza viruses circulate in the general population, 2017 isolates collected in 43 countries and territories during a 4-year period were tested for drug susceptibility in a bioassay. Drug resistance was confirmed by detection of specific mutations on the M2 gene that have been shown to confer resistance to amantadine or rimantadine. Sixteen viruses (0.8%) were found to be drug-resistant. Only 2 of these resistant viruses were isolated from individuals who received amantadine or rimantadine treatment at the time the specimens were collected. For 12 individuals use of amantadine or rimantadine could be excluded, and from the remaining 2 patients information about medication was unavailable. These results indicate that the circulation of drug-resistant influenza viruses is a rare event, but surveillance for drug resistance should be continued.

Affinity-purified type A influenza virus nucleocapsid protein expressed by a recombinant baculovirus vector was used in in vitro RNA transcription reactions to create RNP complexes containing a synthetic influenza A virus NS gene. When used in transfection assays, the baculovirus-expressed NP was shown to be biologically active allowing the efficient isolation of transfectant viruses containing the artificially-introduced NS gene. The results demonstrate that NP is the only virion protein necessary in the reconstituted RNP complexes used for transfection thus eliminating the need for purified RNP complexes containing active polymerase.

Four influenza type B viruses isolated in Russia during periods of relatively low (1987-8) or high (1990-1) influenza B activity were characterized antigenically using a microneutralization assay. These isolates were antigenically similar to contemporary reference strains from either of two separate lineages represented by B/Victoria/2/87 and B/Yamagata/16/88. The evolutionary relationships of the variable portion of the haemagglutinin (HA1) genes of these viruses were determined by comparison with influenza B HA1 sequences previously obtained. The Isolate B/USSR/2/87, collected during the 1987-8 influenza season, was found to be closely related to viruses on the B/Victoria/2/87 lineage that circulated during the 1988-9 influenza season in the United States. Sequence analysis of the isolates from the 1990-1 influenza season demonstrated co-circulation of viruses from both the B/Victoria/2/87 and B/Yamagata/16/88 lineages in Russia, confirming the antigenic analysis.

The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991, B/Hong Kong/22/89- or B/Yamagata/16/88-like viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA1 domains of representative viruses confirmed the continued existence of two main lineages among recent strains of influenza B virus and identified unique amino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.

The generation of defective-interfering (DI) particles by rubella virus during serial undiluted passage and persistent infection in Vero cells was studied. A series of 24 serial undiluted passages was initiated with plaque-purified virus. The virus titer remained relatively constant through the first nine passages, after which it declined, reaching a low level of 20-fold less than the originating stock by passage 15. In subsequent passages, the titer cycled. Intracellular DI RNAs were first detectable at passage 4, at which time DI RNAs of 7500 and 1400 nucleotides in length were observable. Thus, the rate of which DI RNAs were generated by rubella virus during serial undiluted passage was similar to the rate of DI generation by other enveloped RNA viruses during serial undiluted passage. The longer rubella DI RNA was present in all passages subsequent to passage 4, while the 1400-nucleotide DI RNA was replaced by a DI RNA of 800 nucleotides in length by passage 15. Subsequent to passage 7, the relative amount of genomic RNA declined dramatically and the DI RNAs became the predominant intracellular virus-specific RNA species. Negative-polarity RNA species corresponding to the 7500- and 800-nucleotide DI RNA species were indentified. The 7500- and 1400-nucleotide DI RNA species were encapsidated into virus particles while the presence of the 800-nucleotide DI RNA species in virus particles could not be detected. Interestingly, the rubella virus subgenomic RNA was present in virus particles in preparations containing DI RNAs. A persistent infection was initiated by subculturing the surviving cells from a high multiplicity of infection with plaque-purified virus. Intracellular DI RNAs were first detectable at Day 19 after initiation of persistence and became significant by Day 26. The amount of genomic RNA began to decrease at Day 47 and was undetectable after Day 68. Through Day 54, there were several DI RNA species present, but at later times, one of these species became predominant. Thus, DI particles were generated during persistent infection, but their presence was not necessary for initiation of persistence.

Virus specific macromolecular synthesis was studied in Vero cells infected with plaque-purified rubella virus under one-step multiplication conditions. Under these conditions, the rate of virus production was found to increase rapidly until 24 hr postinfection after which time the rate of virus production rose more slowly, reaching a peak level at 48 hr postinfection. This peak rate of virus production was maintained through 72 hr postinfection. A majority of the cells remained alive through 96 hr postinfection, although a 20 to 30% decrease in the number of living cells occurred between 24 and 48 hr postinfection, the time period at which cytopathic effect was first observed. The virus structural proteins were first detected intracellularly at 16 hr postinfection. The rate of synthesis of these proteins was already maximal at 16 hr postinfection and remained constant through 48 hr postinfection. By immunofluorescence, cells expressing virus proteins were first observed at 12 hr postinfection. At 24 hr postinfection, 35 to 50% of the cells in the infected culture were exhibiting immunofluorescence, at 36 hr postinfection, 65 to 90% of the cells were exhibiting immunofluorescence, and at 48 hr postinfection, all of the cells were exhibiting immunofluorescence. The virus genomic and subgenomic RNA species were first detectable by 12 hr postinfection. The rate of synthesis of both of these species peaked at 26 hr postinfection. Rubella virus infection was found to have no effect on total cell RNA synthesis. However, a modest inhibition of total cell protein synthesis which reached 40% by 48 hr postinfection was observed. When Northern analysis of RNA extracted from infected cells was performed, a negative-polarity, virus-specific RNA probe hybridized only to the virus genomic and subgenomic RNA species. A positive-polarity, virus-specific RNA probe hybridized predominantly to a negative-polarity RNA of genome length indicating that both the genomic and subgenomic RNAs are synthesized from a genome-length negative-polarity template. Defective interfering (DI) RNAs were not detected in infected cells through 96 hr postinfection or in cells onto which virus released through 96 hr postinfection was passaged. Thus, the generation of DI particles by rubella virus appears to play no role in the slow, noncytopathic replication of this virus or in the ability of rubella virus-infected cells to survive for extended periods of time.

The sequence of the 1600 3' terminal nucleotides of the RNA of rubella virus was determined from cDNA synthesized from both virion and intracellular RNA using reverse transcriptase and an oligodeoxythymidine primer and cloned into a bacterial plasmid vector. This sequence contained the complete coding sequence for virion envelope protein E1 and a 57 nucleotide nontranslated region between the stop codon for E1 and the poly A tract. The predicted size for E1 was 481 amino acids and within this sequence were three potential N-linked glycosylation sites and a putative trans-membrane domain near the carboxy terminus. Immediately preceding the E1 coding region was a putative signal sequence. No homology was found at either the amino acid or nucleotide level between the region of the rubella virus genome sequenced and corresponding regions of the genomes of the alphaviruses, the other genus of the family Togaviridae for which sequence information has been obtained.