Antigenic comparison of Canadian and US isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to the nucleocapsid protein.

Fifteen Canadian field isolates of porcine reproductive and respiratory syndrome (PRRS) virus from Quebec and Ontario were compared with 5 US PRRS virus (PRRSV) isolates and with the European Lelystad isolate using monoclonal antibodies (MAbs) SDOW17, EP147, and VO17 directed to the 15-kDa nucleocapsid protein of PRRSV. All Canadian and US isolates tested by indirect immunofluorescence were recognized by the 3 MAbs, and individual titers of MAbs towards Canadian and US PRRSV isolates were similar as well. In contrast, the Lelystad virus isolate reacted only with the SDOW17 MAb and showed no reactivity with either EP147 or VO17. The reactivity pattern with these MAbs suggests that the Canadian isolates of PRRSV tested are antigenically similar to US isolates of PRRSV, and that these North American isolates share highly conserved epitopes on the 15-kDa nucleocapsid protein that clearly differentiate them from the European Lelystad virus isolate.     

Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein.

The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV.     

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.     

Isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome.

Severe clinical signs of swine infertility and respiratory syndrome (SIRS) of unknown cause were observed in several Minnesota swine farms between November 1990 and March 1991. Forty-five lung samples of weak pigs were collected from 13 swine farms, and virus isolation was attempted using swine alveolar macrophage (SAM) cultures. A cytopathic virus was isolated from 19 lung samples collected from 6 different farms. Four pregnant sows were infected intranasally with a tissue suspension from which virus was isolated, and 4 6-week-old pigs and 2 contact pigs were infected intranasally with 1 of the isolates. The 4 sows farrowed 12 stillborn and 32 normal pigs. Virus was recovered from 10 of 19 pigs examined. Infected 6-week-old pigs were clinically normal except for slightly elevated rectal temperatures and mild respiratory signs. No or mild interstitial pneumonic lesions were observed in inoculated pigs, but the lesion was obvious in the 2 contact pigs. Seroconversion was observed in sows and pigs as measured by indirect fluorescent antibody (IFA). Serologic identification of the isolates was carried out by IFA using reference serum prepared from an experimentally infected sow. A cytoplasmic fluorescence was observed on the SAM monolayers infected with each of the 19 different isolates. Fluorescence was also observed when the monolayers were tested with SIRS virus ATCC VR-2332-infected sow sera. Replication of the isolates was not affected in the medium containing 5-iodo-2'-deoxyuridine but was inhibited by treatment with ether. The isolates were relatively stable at 56 C and did not agglutinate with various erythrocytes tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus.

A monoclonal antibody (2C12) against the 19 kDa membrane (M) protein of a Canadian isolate of porcine reproductive and respiratory syndrome (PRRS) virus was produced. By indirect immunofluorescence (IIF) cytoplasmic fluorescence was observed in infected cells, but the pattern of fluorescence was generally different and intensity was weaker than that observed using the nucleocapsid protein-directed monoclonal antibody SDOW17. When tested by IIF towards a total of 26 PRRS virus isolates from Canada, 122 isolates from the US and 13 isolates from Europe the 2C12 MAb reacted with all the North American isolates tested including the VR-2332 isolate and the vaccine (RespPRRS) isolate. However no reactivity was observed towards the European isolates tested including the Lelystad virus. This reactivity pattern suggests that the epitope recognized by this MAb on the M protein of PRRS virus appears highly conserved among North American isolates but absent or weakly expressed on European isolates of PRRS virus.     

Antigenic variation and genotype of isolates of porcine reproductive and respiratory syndrome virus in Korea

A panel of three anti-glycoprotein 5 (gp5) protein monoclonal antibodies (mAbs) (15, 28 and 246) and three anti-nucleocapsid (N) protein mAbs (SDOW17, VO17 and EP147) was used to investigate, by an indirect fluorescent antibody test, the antigenic variations of 50 Korean isolates of porcine reproductive and respiratory syndrome virus (PRRSV), and compare them with a us ATCC vR2332-derived attenuated vaccine strain and the reference European Lelystad strain of PRRSV. A multiplex PCR assay for the differentiation of European and North American genotypes of PRRSV was used to determine the genotype of the 50 Korean isolates. Forty-six (92 per cent) of the 50 Korean isolates shared the epitopes recognised by the anti-N protein mAb SDOW17. No reactivity to the anti-gp5 and anti-N protein mAbs was observed with the other four isolates. Six distinct patterns could be identified on the basis of their reactivities with the anti-PRRSV mAbs. All 50 isolates were identified as North American genotypes by the differential PCR.     

Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates.     

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.     

Isolation of a Lelystad virus-like agent from British pigs and scanning electron microscopy of infected macrophages.

Six, one-week-old gnotobiotic piglets were inoculated with tissues or sera collected from field cases of porcine reproductive and respiratory syndrome. The piglets showed little or no illness, and two that were necropsied at 8 and 9 days post infection appeared grossly normal. However, a Lelystad virus-like agent was isolated from most of the inoculated pigs using porcine alveolar macrophage cultures. Seroconversion to the Lelystad virus was observed and some animals developed microscopically detectable interstitial pneumonias. Scanning electron microscopy was used to study the in vitro cytopathic effect of the Lelystad virus on porcine alveolar macrophages.     

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Performance of ELISA antigens prepared from 8 isolates of porcine reproductive and respiratory syndrome virus with homologous and heterologous antisera.

Porcine reproductive and respiratory syndrome virus (PRRSV) ELISA antigens of high quality were produced using 8 different isolates of PRRSV: the European Lelystad virus (LV), the U.S. MN-1b, 89-46448, 93-44927, and 93-24025B, and the Canadian LHVA-93-3, PA-8 and GH-6 virus isolates. The performance of each of these 8 antigens and a commercial PRRSV antibody test kit (Idexx's HerdChek) were measured against antisera raised in 5 groups of 6 piglets inoculated with either LV, MN-1b, 89-46448, 93-44927, or 93-24025B. Among the 8 isolates, the 89-46448 isolate produced the broadest spectrum of antigen and resulted in earlier detection of antibodies to various North American PRRSV isolates, followed by MN-1b as the 2nd best ELISA antigen for the detection of North American PRRSV antibodies. The GH-6 and PA-8 viral antigens exhibited restricted detection of PRRSV antibodies. The LV and 89-46448 combined antigens produced the best performance for the detection of antibodies against both European and North American antigenic types of PRRSV. Using 173 panel samples collected at 11 to 60 d after intranasal inoculation with 1 of the 5 PRRSV isolates, the sensitivities of the indirect ELISA used were 73.4%, 98.3%, 90.8%, 98.3%, 83.2%, 93.1%, 77.1%, 64.2%, 98.8% and 95.9% for LV, MN-1b, LHVA-93-3, 89-46448, 93-44927, 93-24025B, PA-8, GH-6 antigens, 89-46448-LV combined antigens and Idexx's PRRSV antibody test kit, respectively. All 8 antigens gave negative results with preinfection porcine sera (n = 30); high background or nonspecific reactions were not observed with the antigens.     

Highly cited article published in the Veterinary Quarterly in 1991

In early 1991, the Dutch pig industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the aetiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hysonoviae, M. hyopneumoniae, and Acheloplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterized as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly affected by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

猪繁殖与呼吸综合征病毒的分离与鉴定

从广东地区发病猪场的病料中,分离到1株致Marc-145细胞病变的病毒ShB6。扩增猪繁殖与呼吸综合征病毒(PRRSV)主要结构蛋白基因ORF2-ORF7并进行序列分析,结果表明,该分离株与国内PRRSV分离株HB-1(sh)/2002的同源性为96.9%;与ATCC VR-2332株的同源性为91%;而与Lelystad株的同源性仅为59.8%;用美洲型PRRSV单抗进行免疫组化染色,结果显示在细胞病变处呈现明显的阳性着色(为棕黄色)。综合可见,所分离的病毒为美洲型PRRSV。     

Influenza in swine in Belgium (1969–1986) Epizootiologic aspects

From 1984 until 1986, influenza isolates were obtained from 59 outbreaks of respiratory tract disease in Belgium. In 21 of the outbreaks, H₃N₂-influenza virus isolates, related to the human A/Port Chalmers/1/73 strain were obtained. All other isolates were H₁N₁-influenza virus strains. The prevalence of variants of the human H₃N₂-influenza virus in the Belgium swine population was determined by examining sow sera which had been collected between 1969 and 1984. The results of this serological study showed that, although a Port Chalmers-like strain was associated with outbreaks of respiratory tract disease in swine only since 1984, such strain was already present in the swine population in 1974, when a low percentage of sow sera (7%) reacted with A/Port Chalmers/1/73. Between 1975 and 1984, antibody against this strain were present in 28–61% of the sera. Furthermore, 3–6% of the sera collected between 1971 and 1980 reacted with the A/Hong Kong/1/68 strain. There were no indications that more recent human H₃N₂-strains (A/Texas/1/77, A/Bangkok/1/79 and A/Belgium/2/81) circulated in the Belgian swine population.     

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.     

Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines

In this study, the efficacy of two attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines was assessed. The virological protection in the lungs of vaccinated pigs upon challenge was studied. Also, challenged pigs were exposed to lipopolysaccharide (LPS) to evaluate clinical protection. Six-week-old pigs were immunized intramuscularly with commercial vaccines based on either an attenuated American or an attenuated European virus strain. Non-immunized pigs and pigs intramuscularly inoculated with the virulent Lelystad strain were included as controls. Six weeks after immunization, pigs were challenged either intratracheally or intranasally with the Lelystad strain, and 3 and 6 days later intratracheally exposed to Escherichia coli LPS. After LPS administration, pigs were monitored for clinical signs. At 4 and 7 days after challenge, pigs were euthanized to determine virus quantities in broncho-alveolar lavage (BAL) fluids and in lungs. Challenge virus was recovered from three out of eight pigs that had been primo-inoculated with the Lelystad strain with titers ranging between 0.3 and 3.1 log(10). Fifteen out of sixteen pigs vaccinated with the attenuated American strain were positive for challenge virus and their mean virus titers were similar to those of non-immunized challenge controls. Eleven out of 16 pigs vaccinated with the attenuated European strain were positive for challenge virus and their mean virus titers were 2.0-2.5 log(10) lower than those of non-immunized challenge controls. Thus, the virological protection in the lungs of vaccinated pigs upon challenge was incomplete, but was more pronounced in the homologous situation. Clinical signs upon LPS exposure in both vaccinated groups were not reproducible in two experiments.     

Genetic analysis of ORF5 of recent Korean porcine reproductive and respiratory syndrome viruses (PRRSVs) in viremic sera collected from MLV-vaccinating or non-vaccinating farms

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.     

Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.     

Differentiation of vaccine strains and Georgia field isolates of infectious laryngotracheitis virus by their restriction endonuclease fragment patterns

Seven restriction endonucleases (REs) were used to cleave the DNA from seven vaccine strains of infectious laryngotracheitis (ILT) virus and from six Georgia field isolates of ILT virus. After electrophoresis of the resulting RE fragments, the patterns were compared in order to differentiate strains of ILT virus. The six chicken-embryo-origin (CEO) vaccines were identical with each RE, but the tissue-culture-origin (TCO) vaccine strain differed from the CEO vaccines using five of the REs. Four of the six field isolates were identical by each RE, but two field isolates differed from each other and from the four identical field isolates on the basis of patterns produced by some but not all of the REs. The four identical field isolates could not be differentiated from the CEO vaccine strains by any RE, but the other two field isolates were not identical to either strain of vaccine virus. This work demonstrates that differentiable strains of ILT virus exist in the United States and that viruses other than vaccine viruses are involved in field outbreaks of ILT.