The detection of bovine viral diarrhoea virus in bulk milk samples by the use of a single-tube RT-PCR.

A single step, single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) test was developed to detect the presence of bovine viral diarrhoea virus (BVDV) in somatic cells from bulk milk samples. The test was configured using commercial kit-form RNA extraction and RT-PCR procedures. The test was validated by examining bulk milk samples from approximately 80 herds with a history of BVDV and comparing results with those obtained from samples from a similar-sized control group. The test proved highly specific, giving a positive result in 20.5% of herds with a history of BVDV, with no control herds positive. Its sensitivity was likewise high, detecting, at its maximum, one persistently infected (PI) cow in a herd of 162 lactating animals. In 19 herds where follow-up blood tests were performed, the RT-PCR gave a positive result in all ten herds where at least one lactating PI animals was present. In control involving the detection of PI cattle, the test provides a rapid and inexpensive alternative to individual animal testing for those cows in milk at the time of sampling.

Variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the UK.

Previous studies using monoclonal antibodies (mAbs) have revealed antigenic variation among UK isolates of porcine reproductive and respiratory syndrome viruses (PRRSV) and the use of in vitro translation products has shown that this variation lies in the protein encoded by open reading frame (ORF) 3. This protein has been shown to be present in purified virion preparations, suggesting that it is a structural protein. The original objective was to investigate the degree of variation of ORF3 among a number of UK isolates of different mAb reactivity and diverse chronology by sequencing and to correlate this with the mAb reactivity, in an attempt to define conserved and variable antigenic sites. A number of PRRSV isolates, from different outbreaks in the UK between 1991 and 1994, were propagated in pig alveolar macrophages and RNA extracted. The ORF3 and ORF7 regions of the individual viruses were amplified by the polymerase chain reaction (PCR) and their sequences were determined using internal sense and antisense primers. A number of differences among the sequences were noted within specific regions of the ORF3, with a hypervariable area detected at the carboxyterminal end, in the area of overlap with ORF4. With the most divergent isolate, 9.5% of the 84 translated amino acids encoded by the area of overlap were different from Lelystad isolate, translating the sequence in both reading frames. In view of consistent changes elsewhere in the ORF that suggest a common ancestry among the isolates studied, we conclude that this region may be subject to rapid change in comparison to other regions studied, and therefore may be an area subjected to immunoselective pressure.

Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species.

A pestivirus was transmitted by contact from a persistently infected (P.I.) bullock to pregnant sheep. This resulted in the birth of P.I. lambs, one of which in turn was able to transmit virus by contact to pregnant cattle. Two of these animals gave birth to P.I. calves, from one of which the virus was again transmitted by contact with pregnant sheep, leading to another generation of P.I. lambs. The expression of one or more epitopes on the E2 glycoprotein of the viruses isolated from this series of alternate cattle-sheep transmissions appeared to depend on the host species. Thus, several monoclonal antibodies which bound strongly to, and neutralised, viruses isolated from the bovine hosts, failed to bind or neutralise in the case of sheep isolates. The viral consensus sequences of the E2 gene as well as parts of the 5' untranslated region and of the Npro and capsid genes were compared between the different isolates. This revealed a high degree of genetic stability. However, a single codon change at amino acid position 9 of the E2 gene correlated with and was able to cause the loss of particular epitopes.