Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.     

Blood pH in the umbilical artery at birth: an analysis of data from patients delivered in Hesse between 1986 and 1989

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus

Three groups of 10 pigs were vaccinated with an American serotype porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and three groups of 10 pigs were vaccinated with a European serotype PRRSV vaccine. A control group of 12 pigs was left unvaccinated. Four weeks after vaccination the PRRSV-specific antibody titres were determined and each group was challenged with either a Spanish, German or Dutch PRRSV wild-type strain. The serological responses four weeks after vaccination confirmed that the two vaccines were of different serotypes. Vaccination with the American serotype vaccine hardly reduced the level of viraemia after challenge with the European PRRSV wild-type strains, and only after challenge with the Spanish PRRSV strain was a moderate, statistically significant reduction in viraemia observed. In contrast, after vaccination with the European serotype vaccine, viraemia was completely suppressed after challenge with the German PRRSV isolate and almost completely suppressed after challenge with the Spanish and Dutch PRRSV isolates.     

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.     

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds

Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2-99.5% to the vaccine virus RespPRRS and 99.0-99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus.     

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M(r)) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP3) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP3 protein was present in amounts equivalent to those of the N, M, and GP5 proteins in the infected cells, whereas no GP3 could be detected in purified virions. During the first 30 min of chase, the GP3 undergoes a gradual downward shift of its apparent M(r), thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural.     

Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine

In 1979, an H1N1 avian influenza virus crossed the species barrier, establishing a new lineage in European swine. Because there is no direct or serologic evidence of previous H1N1 strains in these pigs, these isolates provide a model for studying early evolution of influenza viruses. The evolutionary rates of both the coding and noncoding changes of the H1N1 swine strains are higher than those of human and classic swine influenza A viruses. In addition, early H1N1 swine isolates show a marked plaque heterogeneity that consistently appears after a few passages. The presence of a mutator mutation was postulated (C. Scholtissek, S. Ludwig, and W. M. Fitch, Arch. Virol. 131:237-250, 1993) to account for these observations and the successful establishment of an avian H1N1 strain in swine. To address this question, we calculated the mutation rates of A/Mallard/New York/6750/78 (H2N2) and A/Swine/Germany/2/81 (H1N1) by using the frequency of amantadine-resistant mutants. To account for the inherent variability of estimated mutation rates, we used a probabilistic model for the statistical analysis. The resulting estimated mutation rates of the two strains were not significantly different. Therefore, an increased mutation rate due to the presence of a mutator mutation is unlikely to have led to the successful introduction of avian H1N1 viruses in European swine.     

Studies of porcine reproductive and respiratory syndrome (PRRS) virus infection in avian species

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently recognized virus of swine. As a newly emerging virus, much of the basic information regarding PRRSV is in the process of discovery. We report three experiments with PRRSV in birds, and a fourth experiment to evaluate the infectivity and transmissibility of avian-derived PRRSV in swine. Experiment 1 compared the susceptibility of Muscovy ducks, Mallard ducks, guinea fowl, and chickens to PRRSV. Birds were exposed to PRRSV (ATCC VR-2402) in drinking water and virus isolation was attempted from feces collected from cages. Based on the duration of fecal shedding of the virus, this experiment showed that Mallard ducks were particularly susceptible to PRRSV. Experiment 2 was done in mallards to corroborate and augment the observations of experiment 1. Virus was isolated from pooled mallard feces up to 25 days post exposure (PE) and from the intestinal contents of 8 of 20 birds euthanized on day 38 PE. No gross or microscopic lesions were observed in ducks collected between 0 and 15 days PE. Experiment 3 evaluated the infectivity and transmissibility of mallard-derived PRRSV in mallards. A cage of mallards orally exposed to PRRSV shed the virus in feces. Exposure of a second cage of mallards to feces from the first cage resulted in fecal shedding of PRRSV by birds in cage two. In turn, exposure to feces from the second cage led to fecal shedding by mallards in a third cage. Experiment 4 assessed the infectivity and transmissibility of mallard-derived virus in swine. Pigs intranasally exposed to PRRSV isolaed from mallard feces in experiment 2 became viremic, seroconverted by ELISA, and transmitted the virus to sentinel swine. Collectively, these studies show that the possibility exists for avian species to be involved in the epidemiology of PRRSV. This is the first report of PRRSV infection in a species other than swine.     

Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus

Monoclonal antibodies (MAbs) against porcine reproductive and respiratory syndrome virus (PRRSV) were prepared and characterized. Four MAbs were developed from the mice immunized with the recombinant GP4 protein expressed in insect cells, and six MAbs were derived from the immunization with recombinant GP5 protein. All of the MAbs showed strong perinuclear fluorescence in PRRSV VR2385 infected cells by immunofluorescence staining. Among the MAbs to GP5 protein, one showed strong reactivity in ELISA and recognized a 26 kDa band of PRRSV in a western blot assay, while another showed neutralizing activity against the VR2385 isolate. Out of the four MAbs to GP4 protein, one showed mild reactivity in ELISA with detergent extracted antigen, but had no reactivity in a western-blot assay. The failure of MAb binding to detergent extracted antigen in ELISA or in western-blot analysis indicated that the MAbs were against conformationally dependent epitopes. Reactivity patterns of the MAbs with PRRSV field isolates tested by fixed-cell ELISA showed that there are antigenic variations in PRRSV GP4 and GP5 proteins. Development of these MAbs will benefit further studies on PRRSV structural proteins as well as in understanding their roles in PRRSV pathogenesis.     

Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview

PRRS disease was first recognised in the USA in 1987 and in Europe in 1990 and since then the disease has spread widely throughout many pig-producing countries. After a severe epidemic phase, the infection has become endemic. The prevalence of infection is generally high in infected countries. However, in areas with a low density of pigs, infection may spread slowly and if infected animal movements are not significant, farm-to-farm spread can be controlled and prevalence of infection maintained at a low level. The PRRS virus (PRRSV) was completely unknown before 1986, and the question of its origin remains unanswered. The exact epidemiologic relationship between American and European strains of PRRSV is difficult to establish because different isolates appear to belong to two distinct sub-populations which are only distantly antigenically related. In the environment, virus survival is optimal when temperature is cold and when ultra-violet light exposure is low (little sunshine). These conditions are easily attained in winter and that may explain why virus spread increases during this period. Pigs of any age (including wild boars) are the only animals known to be naturally infected with PRRSV. Relatively close contact between pigs is the primary factor in virus transmission. Aerial transmission is a second mechanism of spread, particularly in winter and particularly over distances of less than 3 km. A third route of transmission is via semen. The role of fomites is not clearly documented, however since the virus is excreted in faeces and urine, slurry should be considered as a potential source of contamination. Within herds, the virus spreads rapidly with up to 85 to 95% of pigs in a herd becoming sero-positive within two to three months. Thereafter, virus activity persists for extended periods (several month to years). Nevertheless, some authors have reported spontaneous elimination of PRRSV from infected farms. For the future, there remain questions concerning the possible evolution of the disease (in terms of its sanitary and economic impacts), and the possible influence of vaccines on the epidemiological features of PRRS.     

Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis

The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B. afzelii or B. garinii should be used solely, or in addition to B. burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.     

Functionality and cell anchorage dependence of the African swine fever virus gene A179L, a viral bcl-2 homolog, in insect cells

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.     

Direct isolation and identification of recombinant pseudorabies virus strains from tissues of experimentally co-infected swine

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wild-type or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.     

Tackling the "dropped nucleus"

The ability of male rats to accumulate menaquinone-4 (MK-4) in tissues when fed a vitamin K-deficient diet supplemented with intraperitoneal phylloquinone (K) as the sole source of vitamin K for 14 d was assessed. In both conventionally housed controls and gnotobiotic rats, supplementation with the equivalent of 1500 microg vitamin K/kg diet increased (P < 0.001) tissue MK-4 concentrations above those of controls fed a vitamin K-deficient diet. MK-4 concentrations were approximately 5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary glands of gnotobiotic rats. MK-4 concentrations in conventionally housed rats were higher than in gnotobiotic rats in heart (P < 0.01), brain (P < 0.01) and kidney (P < 0.05) but lower in salivary gland (P < 0.05). Cultures of a kidney-derived cell line (293) converted K to the expoxide of MK-4 in a manner that was dependent on both time of incubation and concentration of vitamin K in the media. A liver-derived cell line (H-35) was less active in carrying out this conversion. These data offer conclusive proof that the tissue-specific formation of MK-4 from K is a metabolic transformation that does not require bacterial transformation to menadione as an intermediate in the process.     

Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate

The complete sequence of a North American tobacco rattle virus (TRV) isolate, 'Oregon yellow' (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 to TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than--SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses. ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses is also discussed.     

Moredun Bordetella Medium, an improved selective medium for isolation of Bordetella parapertussis

Bordetella parapertussis, previously thought to be an obligate human respiratory tract pathogen, has been isolated from sheep. Attempts to assess the prevalence of B. parapertussis in conventionally reared sheep by nasal swabbing proved futile with existing selective media because of extensive overgrowth with Mucor spp. and other nasal commensals. Moredun Bordetella Medium (MBM), which contains cycloheximide and spectinomycin at final concentrations of 0.5 mg/ml and 100 mu g/ml, respectively, was developed as an improved selective medium to isolate B. parapertussis from the nasal cavities of conventionally reared sheep. The selective ability of MBM was evaluated with 200 nasal swabs from conventionally reared sheep, and B. parapertussis was recovered from 31.5% of the samples. MBM facilitated the simple and effective isolation of B. parapertussis from ovine nasal swabs and, in successfully excluding overgrowth with other contaminants, proved superior to other test formulations evaluated and to existing conventional media.