Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro.

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Early results suggested that the RNA polymerase "switched" from a transcriptase to a replicase during the viral life cycle in response to the expression of viral proteins. However, recently an alternative model suggesting that replication of influenza virus is regulated by stabilization of the replicative intermediates was proposed. According to this model, the virion-associated polymerase is capable of synthesizing both mRNA and cRNA. We now demonstrate that virion-derived viral ribonucleoproteins (vvRNPs) synthesize both mRNA and cRNA in vitro in the absence of non-virion-associated RNA polymerase or nucleoproteins. The authenticity of the in vitro-transcribed mRNA and cRNA was confirmed by terminal sequence analysis. The addition of non-virion-associated polymerase or NP had no effect on vvRNP activity. De novo synthesis of cRNA was found to be more sensitive than the capped primer-dependent synthesis of mRNA to the concentration of ATP, CTP, and GTP. We conclude that vvRNPs intrinsically possess both transcriptase and replicase activities and that there is no switch in the synthesis of mRNA to cRNA during the influenza virus life cycle.

Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of Lumpy skin disease virus.

The genomic sequences of 3 strains of Lumpy skin disease virus (LSDV) (Neethling type) were compared to determine molecular differences, viz. the South African vaccine strain (LW), a virulent field-strain from a recent outbreak in South Africa (LD), and the virulent Kenyan 2490 strain (LK). A comparison between the virulent field isolates indicates that in 29 of the 156 putative genes, only 38 encoded amino acid differences were found, mostly in the variable terminal regions. When the attenuated vaccine strain (LW) was compared with field isolate LD, a total of 438 amino acid substitutions were observed. These were also mainly in the terminal regions, but with notably more frameshifts leading to truncated ORFs as well as deletions and insertions. These modified ORFs encode proteins involved in the regulation of host immune responses, gene expression, DNA repair, host-range specificity and proteins with unassigned functions. We suggest that these differences could lead to restricted immuno-evasive mechanisms and virulence factors present in attenuated LSDV strains. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this assumption. The molecular design of an improved LSDV vaccine is likely to be based on the strategic manipulation of such genes.

Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR.

A triple one-step RT-PCR was developed to screen and differentiate virulent from avirulent Newcastle disease virus (NDV) isolates. Three sets of oligonucleotides were designed, each specific for amplifying NDV fusion protein gene-specific RNA from virulent, avirulent or all isolates respectively. The sensitivity of one-step RT-PCR was determined using viral RNA extracted from serially diluted NDV-infected allantoic fluid and found to be 10(-5) HA units. Application of one-step RT-PCR to various NDV samples, including wild-type virulent isolates and avirulent vaccine strains, demonstrated the potential for rapid identification (3-4 h) of NDV isolates as well as the differentiation of virulent from avirulent strains.

Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation.

A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3-4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3' poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro.

Sequence analysis of the RNA polymerase gene of African horse sickness virus.

The gene encoding the inner core protein VP1 of African horse sickness virus (AHSV) serotype 9 has been cloned, expressed in vitro and entirely sequenced, completing molecular characterization of the AHSV genome. An analysis of the sequence supporting the identity of AHSV VP1 as the putative viral RNA polymerase is presented.

Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3.

The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35% identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculovirus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.