Variation in resistance to the porcine reproductive and respiratory syndrome virus (PRRSV) in Pietrain and Miniature pigs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the economically most important diseases of swine. Viraemia and the prolonged persistence of the virus are among the most critical factors. Virus replication and severity of disease vary with virus isolates, and there is rising evidence for a genetic component of the host susceptibility. Dissecting the genetic basis of resistance/susceptibility to PRRS virus (PRRSV) might lead to improved knowledge on the molecular mechanisms of PRRS and the establishment of genetic markers for future disease control. The aim of this study was to establish a porcine model with emphasized genetic differences in PRRSV susceptibility. Seven 'Wiesenauer Miniature' pigs (MI), a local German breed and eight commercial Pietrain (PI) pigs were challenged with 10(5) TCID(50) of an attenuated PRRSV strain (Ingelvac PRRSV MLV). Clinical status, viraemia and seroconversion of the pigs were compared. No clinical signs were observed during the experiment. Viraemia peaked on day 6 p.i., with 100% of viraemic pigs in PI and on day 12 p.i with 87% of viraemic MI. Viraemia lasted for up to 35 days in MI and for at least 72 days in PI. This surprising result was confirmed by a second study with another four MI. MI and PI showed maximum virus titres of 10(2.5) TCID(50)/ml of serum and 10(4.5) TCID(50)/ml, respectively, indicating a virus replication in MI of approximately 3.3% that of PI over the complete period. MI were more efficient in antibody production. With such pronounced breed differences, the model is of high relevance for the genetic dissection of PRRS pathogenesis and susceptibility.

High risk of false positive results in a widely used diagnostic test for detection of the porcine reproductive and respiratory syndrome virus (PRRSV).

During 2003 and 2004, increasing numbers of positive PRRSV RT-PCR results were reported from herds negative for PRRSV infection. Interestingly, three herds represent nucleus herds with no animal contacts from outside and without clinical symptoms of PRRS until now. Since these positive results that were obtained using a PCR protocol adapted to routine laboratory conditions could not be reproduced with other PRRSV specific RT-PCRs, controlled negative and positive samples were used to examine this phenomenon. A RT-PCR assay for detection and differential diagnosis of the European and North American genotypes of the porcine reproductive and respiratory syndrome virus (PRRSV) according to the method previously published by Oleksiewicz et al. [Oleksiewicz, M.B., Botner, A., Madsen, K.G., Storgaard, T., 1998. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet. Microbiol. 64, 7-22] was investigated in parallel to another recently published method [Pesch, S., 2003. Etablierung einer Nachweismethode für die zwei Genotypen von dem porcine reproductive and respiratory syndrome virus (PRRSV) und ein Beitrag zu seiner molekularen Epidemiologie. Thesis, Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig]. A panel of 228 clinical samples sent in for PRRSV routine diagnostics served as test panel. It was found that both methods have similar analytical sensitivity. However, the primers published by Oleksiewicz were shown to yield a very high proportion of false positive results under routine diagnostic laboratory conditions, i.e. they resulted in RT-PCR products with non-PRRSV sequences, that were indistinguishable from truly positive reagents in standard gel electrophoresis settings. The reason for and possible implications of this finding as well as the risk of modifying published methods without control are discussed.

New insights into the genetic diversity of European porcine reproductive and respiratory syndrome virus (PRRSV).

The complete ORF5 sequences of 66 porcine reproductive and respiratory syndrome (PRRS) field virus strains (1991-2001) and three European modified live vaccine strains were determined, as well as ORFs 6 and 7 of 19 selected strains. The variability of the deduced ORF5 amino acid sequences was analysed using statistical process control (SPC), allowing for the objective assessment of variable and conserved regions. Four variable and four conserved regions as well as five hypervariable amino acid positions were defined. The effects of genetic variability on possible structural and functional properties were discussed with emphasis on immunogenic features. Phylogenetic analysis and pairwise comparison of the nucleotide sequences revealed that the genetic distances between the strains has greatly increased over time. The data do not support an evolutionary influence of the geographical location or the time of sample collection, nor of PRRSV vaccination on strain development. In contrast to other authors who tended to concentrate on the samples from either a common geographic origin or a short sampling period, we could not confirm geographically separate PRRSV clusters nor did we find evidence of positive selective pressure as measured by the ratio of synonymous to non-synonymous substitutions in ORF5, 6 or 7. Immunological implications and vaccination strategies are discussed.

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands.

Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.

Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94.

Viruses from the recent epidemic of swine vesicular disease (SVD) in Europe have been isolated and characterized by antigenic and genetic methods to examine the likely epidemiological origins of the disease. Antigenic analysis was performed on 77 SVD viruses (SVDV) isolated in Europe between 1966 and 1994 using two panels of monoclonal antibodies (MAb) in a trapping ELISA. Genetic analysis of 33 of the SVD viruses by reverse transcription-polymerase chain-reaction (RT-PCR) amplification and nucleotide sequencing of the ID (VP1) coding region was also performed. Comparison of the nucleotide sequences with each other and with three other previously published SVDV sequences revealed four distinct groups which correlated exactly with the results of the pattern of reactivity with MAbs. The first group consisted solely of the earliest SVD virus isolated (ITL/1/66) while the second group comprised viruses present in Europe and Japan between 1972 and 1981. The third group consisted of viruses isolated from outbreaks of SVD in Italy between December 1988 and June 1992. Viruses isolated between 1987 and 1994 from Romania, the Netherlands, Italy and Spain formed a fourth group. The genetic and antigenic similarity of the most recent virus isolates from Western Europe to a virus isolated in Romania 5 years previously suggests that the possible origin of the recent epidemic of swine vesicular disease in Western Europe was in Eastern Europe.

Differential diagnosis and genetic analysis of the antigenically related swine vesicular disease virus and coxsackie viruses.

Monoclonal antibodies directed against an isolate of swine vesicular disease virus (SVDV), characterized by virus neutralization tests and competition assays, were used to compare SVDV isolates and isolates of the antigenically related Coxsackie viruses by ELISA. SVDV-specific reaction patterns and one specific for Coxsackie viruses were observed. This provided a method for distinguishing between these enteroviruses. In addition, RT-PCRs were undertaken with Coxsackie virus and SVDV genomes. Different product patterns were obtained which correlated with the genetic differences revealed by nucleotide sequence determination. RT-PCR distinguished between SVDV and Coxsackie viruses by pattern differences. Further SVDV-specific PCRs were carried out with clinical samples. Viral genomes were detected with a sensitivity equivalent to that of virus isolation in cell culture. Sequencing of the Coxsackie virus-derived 2A-coding PCR products resulted in a not previously described sequence of a B5 isolate and in SVDV-specific sequence of two Coxsackie virus A16 isolates. The differences of the isolates by ELISA and PCR reactivity, as well as the nucleotide sequence differences are consistent with the quasispecies concept of RNA viruses.

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.

Rabbit hemorrhagic disease (RHD): characterization of the causative calicivirus.

Rabbit hemorrhagic disease (RHD) which was first recognized in China in 1984 spread via Eastern Europe to many countries of Western Europe and other parts of the world. The analysis of the virus outlined in this review comprises: 1) physico-chemical properties, 2) electron microscopy including immunoelectron microscopy, 3) demonstration of capsid protein, 4) in vivo neutralization with monoclonal antibodies (mabs), 5) infectivity of purified RNA, and 6) characterization of the viral genome. Also included are clinical, pathological and epidemiological findings, different diagnostic methods as well as disease control measures. Finally, similarities between RHD and the European brown hare syndrome (EBHS) are pointed out. The latter disease is caused by a calicivirus different from RHDV.

Identification of the viral haemorrhagic disease virus of rabbits as a calicivirus.

Liver tissue from rabbits which had died of viral haemorrhagic disease (VHD) was used to identify the causative agent of the disease. After extraction of liver homogenates and density gradient ultracentrifugation, viral particles with buoyant densities between 1.32 to 1.38 g/ml and estimated sedimentation coefficients between 100 S and 175 S were obtained. The isolated virions were determined to have a diameter of 40 nm. The particles morphologically resembled those of the Caliciviridae family, had positive reactions both in ELISA and haemagglutination assays, and were infective for rabbits. By immunoblotting, a major structural protein with a molecular weight of 60 kDa was identified. RNA of high purity and of approximately 8 kb was isolated from virions. Labelled cDNA of virion RNA detected two RNAs of 8 kb and 2 kb in Northern blots of liver RNA from animals infected with the VHD virus (VHDV). Finally, isolated virion RNA injected directly into the liver or rabbits produced a disease with clinical symptoms and pathological findings typical of VHD. A calicivirus originally designated "rabbit haemorrhagic disease virus (RHDV)" was thus identified as the causative agent of VHD. The agent causing the European brown hare syndrome (EBHS) is also a calicivirus (EBHSV). VHDV and EBHSV are different members of the Caliciviridae family; their relationship can be clearly demonstrated by using different approaches of investigation.

Identification and characterization of the virus causing rabbit hemorrhagic disease.

Liver tissue from animals that died of rabbit hemorrhagic disease (RHD) was used to identify the causative agent. After extraction of liver homogenates and sucrose density gradient ultracentrifugation, distinct bands were obtained. The respective gradient fractions reacted positively in an enzyme-linked immunosorbent assay as well as in hemagglutination assays and were infective for rabbits. These fractions contained virions which had a diameter of 40 nm and resembled morphologically those of the family Caliciviridae. By immunoblotting, a major structural protein with a molecular weight of 60,000 was identified. Highly pure RNA of about 8 kilobases was isolated from virions. Labeled cDNA synthesized from virion RNA detected two RNAs of 8 and 2 kilobases in Northern (RNA) blots of liver RNA from animals infected with RHD virus. Finally, isolated virion RNA injected into the liver of rabbits produced a disease with clinical symptoms and pathological findings typical of RHD. We conclude that a calicivirus represents the causative agent of RHD.