Newcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960's and in Hungary in the early 1980's. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980's.

A 75% region of the F gene (between nucleotides 334 and 1682) of Newcastle disease virus (NDV) RNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). PCR products were cleaved by three restriction endonucleases and the positions of thirty cleavage sites were mapped in more than 200 NDV strains. Restrictions site analysis established six major groups of NDV isolates and unique fingerprints of vaccine strains. Group I comprised lentogenic strains isolated mainly from waterfowl with some from chickens. "Old" (prior to 1960s) North American isolates of varying virulence including lentogenic and mesogenic vaccine strains belonged to group II. Group III included two early isolates from the Far East. Early European strains (Herts 33 and Italien) of the first panzootic (starting in the late 1920s) and their descendants with some modifications were placed into group IV. NDV strains isolated during the second panzootic of chickens (starting in the early 1960s) were classified into two groups. Group V included strains originating in imported psittacines and in epizootics of chickens in the early 1970s. Group V1 comprised strains from the Middle East in the late 1960s and later isolates from Asia and Europe. Pigeon paramyxovirus-1 strains that were responsible for the third panzootic formed a distinct subgroup in group V1. Our grouping of NDV strains has confirmed group differences established by monoclonal antibodies. It is concluded that restriction site analysis of F gene PCR amplicons is a relatively fast, simple and reliable method for the differentiation and identification of NDV strains.

No abstract

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme Scal, which specifically cleaved products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (890%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.

The in vivo distribution of bovine herpesvirus type 4 (BHV-4) was examined by testing nasal and conjunctival exudates, peripheral blood leukocytes, and various organs of experimentally infected calves. For virus detection, a nested PCR assay, virus isolation, and immunohistochemistry were applied. The nervous system and the muscles were free of viral DNA. Liver and intestinal lymph nodes contained low amounts of virus (less than two copies per 1 g of cellular DNA). Intestinal, tonsil, thymus, and kidney tissues contained more viral DNA copies (5 to 50 copies per 1 g of cellular DNA). The highest amounts of BHV-4 DNA (50 to 500 copies per 1 g of cellular DNA) were found in the spleen, lungs, trachea, and nasal epithelium. Amplification of DNA from blood lymphocytes through postinoculation (p.i.) day 48 proved that the virus started to replicate in these cells immediately after inoculation of the calves and that intensive virus growth took place during the 7 to 8 weeks of the infection. The number of virus-infected lymphocytes reached the maximum on p.i. days 22 to 26 and slowly declined thereafter. Virus-infected cells were found only in the spleen on p.i. day 48 by immunohistochemistry. Western blotting (immunoblotting) detected signs of an immune response against 9 of the 29 BHV-4 proteins.

No abstract

No abstract

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the previously established subgroups A and AB. A third pattern was produced by five Scandinavian strains and a fourth was observed from a single Dutch isolate. The genetic diversity of 27 strains of BRSV was investigated by comparative nucleotide sequence analysis of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were ob-