Development of a reverse transcription polymerase chain reaction procedure for the detection of marine caliciviruses with potential application for nucleotide sequencing.

A reverse transcription polymerase chain reaction (RT-PCR) procedure is described for the detection of marine caliciviruses including vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), bovine Tillamook virus (BCV Bos-1) and caliciviruses (CV) isolated from dolphin (Cetacean CV), gorilla (Primate CV) and rattlesnake (Reptile CV) using primers (1F and 1R) designed from the capsid-coding region of the viral genome. These primers were compared with those described by Neill, J.D. and Seal, B.S., 1995: Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea lion and vesicular exanthema of swine viruses, Mol. Cell. Probes 9, 33-38 (Hel1/Hel2), which had been designed from the 2C-like region of the calicivirus genome. Both sets proved to be extremely useful diagnostic tools for all of the known marine calicivirus serotypes with the exception of three: SMSV-8 and -12 and mink CV suggesting that these three caliciviruses may belong to a different group. Neither of the two primer sets reacted with strains of the vesicular disease viruses of foot-and-mouth disease (FMD), swine vesicular disease (SVD) or vesicular stomatitis (VS) nor with two feline caliciviruses (FCV). The 1F/1R primer set has the advantage over the Hel1/Hel2 set in that it generates a larger PCR product for nucleotide sequence investigations and so provides greater opportunity for identifying molecular differences between the viruses.

Genetic relationships between foot-and-mouth disease type Asia 1 viruses.

The sequence of 165 nucleotides at the 3' end of the 1D (VP1) gene of foot-and-mouth disease (FMD) virus was determined for 44 type Asia 1 strains isolated from throughout Asia between 1954-92. Analysis of the relationships between the virus genomes showed epidemiological links not previously evident. The possible origin of the only outbreak of FMD Asia 1 to have occurred in Europe, in Greece in 1984, was identified because the nucleotide sequence of this virus was closely-related to the sequences of those present in the Middle East between 1983-5. Variation in the region sequenced was not as great as that seen in the other FMDV serotypes and all viruses shared greater than 85% nucleotide identity. Thus all the virus isolates examined were considered to belong to a single genotype. A database of Asia 1 virus sequences has been established which will facilitate the rapid analysis of new outbreaks strains.

Field and laboratory analysis of an outbreak of foot and mouth disease in Bulgaria in 1991.

In July 1991, an outbreak of foot and mouth disease (FMD) occurred near Stefan Karadjovo village in Boliarovo (south-east Bulgaria, close to the Turkish border). The virus isolated was identified in Bulgaria as serotype O and this was subsequently confirmed by the World Reference Laboratory for Foot and Mouth Disease in Pirbright (United Kingdom). Serological studies using bovine sera and monoclonal antibody analysis were made. In addition, the sequence of approximately 170 nucleotides at the 3' end of the 1D gene was determined for the field isolate and for vaccine strains used in Bulgaria. These were compared with other sequences of type O FMD viruses from outbreaks in the Middle East. Serum samples were taken from domestic animals in the region close to the outbreak and examined for anti-FMD virus antibodies to assess the extent (if any) of spread of the virus before or after the outbreak. No evidence of infection was found in these animals. The virus involved in the Bulgarian outbreak was antigenically similar to the O1 vaccine strains but probably did not originate from these strains. The virus was closely related genetically to a group of viruses isolated in the Middle East since 1987, suggesting that it may have been introduced into Bulgaria from an area in the Middle East by unidentified means.

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses.

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.