- Volume 75, Issue 3, 1994

In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80 %; of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the crossreactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.

The virulent strain SFV4 of Semliki Forest virus (SFV), produced from the infectious clone pSP6-SFV4, is lethal after intranasal (i.n.) infection of adult mice and for pregnant mice after intraperitoneal (i.p.) infection. In contrast, the A7 strain of SFV is avirulent when given i.n. to adult mice, but induces fetal death in pregnant mice after i.p. infection. The nucleotide and deduced amino acid sequences of part of the core and all of the envelope region of A7-SFV were determined and compared to those of SFV4. A7 differed from SFV4 at 80 nucleotides (nt) in the coding sequence, 15 of which were associated with amino acid differences and seven of which (two in the E2 protein and five in El) were nonconservative. The 3′ non-coding sequence of A7 was longer (415 nt) than that of SFV4 (263 nt) and a divergent sequence of 181 nt was present adjacent to the end of the El coding region. The effects on virulence of two mutations in the E2 gene of SFV4, resulting in the non-conservative amino acid substitutions present in A7, were analysed. One mutation (mut 8729 a/c) resulted in only slight attenuation, whereas the other (mut 8902 a/g) resulted in avirulence for pregnant mice. However, mut 8902 a/g was lethal for the majority of developing fetuses after i.p. infection of the mother.

Antigenic differences between three of six equine influenza virus (H3N8) MDCK cell- and egg-derived pairs have been demonstrated using monoclonal and polyclonal antibodies. Sequencing of the haemagglutinin (HA) genes revealed amino acid changes in four of the six virus pairs. These data contrast with those for human isolates of influenza virus in that it was predominantly tissue culture-isolated equine virus and not egg-derived virus which displayed heterogeneity. Some of the molecular changes involved are located within the vicinity of the cell receptor-binding site (positions 156, 158 and 222) whereas others are in the vicinity of the HA1-HA2 cleavage site (positions 18 and 32 of HA1 and position 12 of HA2). Our results indicate that the host cell can play a part in selecting antigenic variants of equine influenza virus and suggest that the egg, and not cell culture as is the case for human isolates, is the preferred host for vaccine and antigenic studies.

Part of genomic segment A of five recent European ‘very virulent’ (W) isolates of infectious bursal disease virus, encoding the major, conformational, protective epitope, have been reverse-transcribed, amplified and cloned. Sequence analysis of 582 bases of VP2 showed that the four U.K. isolates are closely related to each other, differing by no more than two bases, but are distinct from the ‘classical’ type 1 strains (differing by at least 29 bases, or four amino acids, from any other strain and by 15 bases, or three amino acids, from the consensus sequence of all other strains). The U.K. isolates differ from an earlier W isolate from the Netherlands by no more than five bases or two amino acids. The U.K. isolates differ by only one or two bases, with no amino acid changes, from the recently published sequence of a Japanese W isolate.

The sequence of the 3′-terminal region of the genome of Québec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by analysis of four cDNA clones. The 3′-terminal 530 nucleotides (nt) encompassed a large open reading frame with a coding capacity of 123 amino acids (M r 13649). The predicted protein was extremely basic and hence was considered to correspond to the nucleocapsid (N) protein gene. When compared to the homologous sequences of two reference Netherlands strains (Lelystad and isolate 10) of PRRSV, the IAF-exp91 N protein was found to be five amino acids shorter and displayed a high degree of divergence. Overall, IAF-exp91 strain showed identities of 63 %; and 59%; with both reference European strains at the nucleotide and amino acid level, respectively. Two amino acid stretches, STAPM and SQGAS, present respectively at the N- and C-terminal regions of the N protein of European strains, were missing in the IAF-exp91 N protein sequence. The 3 -terminal non-coding region (151 nt) of the IAF-exp91 strain was 22 nt longer than that of the European strains. The aligned nucleotide sequence of this non-coding region exhibited an overall identity of 59 %; with that of the European strains. The Québec reference strain of PRRSV appeared to be related more closely to equine arteritis virus and lactate dehydrogenase-elevating virus than are the two European strains of the virus. Preliminary data obtained by reverse transcription-PCR experiments, using specific or common oligonucleotide primers, suggested that this approach could be useful for distinguishing between PRRSV strains from different geographic origins.

To analyse the intratypic genome variability of coxsackievirus B1, 17 coxsackievirus B1 isolates were collected over a period of 10 years. Nucleotide sequences of the 2A coding region of the various coxsackievirus B1 isolates and known sequences of other enteroviruses were compared. The maximum diversity observed within the entire group of coxsackievirus B1 isolates was 25 %;. Comparison of deduced amino acid sequences revealed a maximum diversity of 5%;. Phylogenetic analysis demonstrates a close genetic relationship between these clinical isolates and the other different coxsackievirus B serotypes. Further analysis of nucleotide and amino acid sequences of the 2A region of known enteroviruses demonstrated that the genus enterovirus can be subdivided into a coxsackievirus B-like group, including the coxsackie B viruses, coxsackievirus A9, echovirus 11 and swine vesicular disease virus, and a poliovirus-like group including the polioviruses 1 to 3, and the coxsackieviruses A21 and A24. Enterovirus type 70 and bovine enterovirus represent distinct groups. The 2A coding region of coxsackievirus B1 strains cannot be distinguished from that of other members of the coxsackievirus B-like group. Although conservation of the overall amino acid sequence was almost limited to residues essential for proposed enzymatic activity, the predicted secondary structures were well conserved within the genus enterovirus.

The genome of broad bean mottle bromovirus (BBMV) contains three positive-sense ssRNA segments, each capped with m7GpppA. Full-length transcribable cDNA clones for four strains of BBMY were constructed by employing reverse transcriptase-PCR (RT-PCR) and a high fidelity Vent DNA polymerase. The transcribed BBMY RNAs contained a 5′ non-viral G residue and, although delayed, produced symptoms similar to those observed in plants infected with authentic virion RNAs. The transcripts replicated inefficiently in protoplasts. In contrast, transcript-derived progeny BBMV RNAs had the repaired termini, were as infectious as the authentic BBMV RNAs and replicated to high levels in protoplasts. In vitro translation of synthetic RNAs confirmed the previously proposed gene expression strategy for BBMV. Sequencing of virion RNAs from the Bawden strain revealed two forms of BBMV RNA 3 components, the longer form containing 21 5′ extra nucleotides derived by the duplication of two short 5′ leader regions. The relative concentration of the two RNA 3 forms was found to be host-dependent, with the longer form prevailing in broad bean and Nicotiana clevelandii infections and the shorter form in bean infections.