Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission.

Wild-caught saithe Pollachius virens were experimentally exposed to an isolate of infectious salmon anaemia virus (ISAV) of Norwegian origin. Mortality attributable to ISAV did not occur following exposure by intra-peritoneal (i.p.) injection of virus or by cohabitation with ISAV-infected Atlantic salmon Salmo salar. Despite the individual testing of 120 ISAV-exposed saithe, ISAV was not detectable using RT-PCR, the most sensitive ISAV diagnostic tool demonstrated to date. Furthermore, saithe exposed to ISAV-infected salmon were not capable of transmitting virus when transferred to tanks containing naïve salmon. Thus saithe appear to be resistant to this Norwegian isolate of ISAV and incapable of supporting its replication. Saithe which co-exist with salmon in and around aqua-culture facilities are considered unlikely to have a significant impact on the epizootiology of ISAV.

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus.

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.

Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy.

In order to explore the occurrence of antigenic drift in swine influenza A(H1N1) viruses and the match between epidemic and vaccine strains, 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and reference strains from earlier outbreaks were examined using serological and molecular methods. In contrast to swine H3N2 viruses, no significant antigenic drift was observed in swine H1N1 viruses isolated from the late 1980s up to 1996 inclusive. However, a marked antigenic and genetic heterogeneity in haemagglutination inhibition tests and nucleotide sequence analyses was detected among the 26 recent swine H1N1 virus strains. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 virus variants at the various farms of the study and points to a marked difference between the epidemiologies of human and swine influenza viruses. The observed heterogeneity may hamper the control of swine influenza by vaccination and indicates that the efficacy of current swine influenza vaccines requires re-evaluation and that the antigenic reactivity of swine influenza viruses should be monitored on a regular basis.

Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla.

Herpesvirus of eel Herpesvirus anguillae (HVA) was isolated repeatedly from farmed eel of an outwardly healthy stock, but virus isolation was much greater in an experimental group of fish that were injected with dexamethasone. The results suggest that HVA can establish a latent infection in eel. Previous exposure of these eels to HVA virus was shown by detection of HVA-specific antibodies. These eels did not show clinical signs after a secondary infection with HVA. Tracing of seropositive eel stocks, which had previous contact with HVA, and of HVA carrier fish can be useful to control disease outbreaks due to HVA infection.

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection.

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.

Survey of infectious agents involved in acute respiratory disease in finishing pigs.

Outbreaks of respiratory disease constitute a major health problem in herds of finishing pigs and their aetiology often remains unclear. In this study, 16 outbreaks of respiratory disease with acute clinical signs in finishing pigs were investigated to determine which infectious agents were involved. From each herd four diseased and two clinically healthy pigs were examined pathologically and for the presence of viruses, bacteria and mycoplasmas. In addition, paired blood samples from 10 groupmates of the diseased pigs were tested for antibodies against commonly known causal agents of respiratory disease. A clear diagnosis was possible in 12 of the 16 outbreaks. Seven were due to an infection with influenza virus and five were due to an infection with Actinobacillus pleuropneumoniae. A combination of influenza virus and A pleuropneumoniae may have caused one other outbreak, but no clear cause could be established for the other three outbreaks.

Antigenic drift in swine influenza H3 haemagglutinins with implications for vaccination policy.

In order to explore the occurrence of antigenic drift in swine influenza A(H3N2) virus, we examined virus strains from outbreaks of respiratory disease among finishing pigs in the Netherlands in 1996 and 1997 and from earlier outbreaks. In contrast to swine H3N2 strains from the 1980s, the recent isolates did not show significant cross-reactivity with human influenza A(H3N2) viruses from 1972-1975 in haemagglutination inhibition tests. These new strains form a separate branch in the phylogenetic trec of the HA1 parts of HA. We conclude that recently there has been considerable antigenic drift within the swine H3N2 viruses in the Netherlands and Belgium and recommend replacement of the A/Port Chalmers/1/73 (H3N2) strain in the current vaccine by a more recent swine H3N2 isolate.

Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonal antibodies.

The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein of Lelystad virus and tested them in competition assays with other N-specific mAbs described previously (Drew et al., 1995; Nelson et al., 1993; van Nieuwstadt et al., 1996). Four different competition groups of mAbs were identified. Pepscan analysis with solid-phase dodecapeptides was used to identify specific antigenic regions in the N protein that were bound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequences consisted of amino acids 2-12 (site A), the mAbs of the second group reacted with peptides whose core sequences consisted of amino acids 25-30 (site B), and the mAb of the third group reacted with peptides whose core sequences consisted of amino acids 40-46 (site C). However, the fourth group of mAbs binding to an antigenic region, provisionally designated as domain D, reacted very weakly or did not react at all with solid-phase dodecapeptides. To further characterize the structure of the epitopes in domain D, we produced chimeric constructs composed of the N protein sequences of Lelystad virus and another arterivirus lactate dehydrogenase-elevating virus, which was used because its N protein has similarity in amino acid sequence and hydropathicity profile but does not react with our mAbs. When the mAbs specific to domain D were tested for binding to the chimeric N proteins expressed by Semliki Forest virus, we found that the regions between amino acids 51-67 and amino acids 80-90 are involved in the formation or are part of the epitopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, whereas the epitopes mapped to domain D are more conformation dependent or discontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are conserved in European and North American isolates; and site D contains epitopes that are either conserved or not conserved in European and North American isolates. The antigenic regions identified here might be important for the development of diagnostic test for PRRSV in particular tests that discriminate between different antigenic types of PRRSV.

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.

[Respiratory symptoms in meat pigs: is there a role for porcine respiratory corona virus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) in the etiology?]. / Een geval van ademhalingsproblemen bij vleesvarkens: een rol voor porcine respiratory corona virus (PRCV) en porcine reproductive and respiratory syndrome virus (PRRSV) in de etiologie?

On a pig farm with about 2000 pigs, respiratory problems regularly developed in fattening pigs 3 to 4 weeks after the start of the fattening period. A postmortem examination was carried out on four pigs during the acute phase of their illness. The animals showed signs of viral pneumonia. In two animals porcine respiratory virus (PRCV) was discovered and in the other two animals porcine reproductive and respiratory syndrome virus (PRRSV). The possible role of these two viruses in the aetiology of the health problems on this farm is discussed in the context of the results of a longitudinal serological study.

Molecular characterization of Lelystad virus.

Lelystad virus (LV), the prototype of porcine reproductive respiratory syndrome virus, is a small enveloped virus, containing a positive strand RNA genome of 15 kb. LV is tentatively classified in the family Arteriviridae, which consists of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization and replication strategy as coronaviruses, but the size of the genome is much smaller (12-15 kb) and they have different morphological and physicochemical properties. The genome of LV contains eight open reading frames (ORFs) that encode the replicase genes (ORFs 1a and 1b), envelope proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7). Genomic comparison of European and North American isolates has shown that the structural proteins encoded by ORFs 2 to 7 vary widely. The amino acid sequences of ORFs 2 to 7 of North American strains share only 55 to 79% identical amino acids with those of European strains. Using polyvalent porcine anti-LV serum, gene-specific anti-peptide sera and monoclonal antibodies, we have identified six structural proteins of LV and their corresponding genes. These are: the 15 kDa unglycosylated nucleocapsid protein (N) encoded by ORF7, an 18 kDa unglycosylated integral membrane protein M encoded by ORF6, a 25 kDa N-glycosylated protein encoded by ORF5, a 31-35 kDa N-glycosylated protein encoded by ORF4, a 45-50 kDa N-glycosylated protein encoded by ORF3 and a 29-30 kDa N-glycosylated protein encoded by ORF2. A nomenclature for these structural proteins is proposed.

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.

Porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs.

A clinical and virological study of an outbreak of porcine epidemic diarrhoea in a combined breeding and finishing pig herd used ELISA techniques to detect porcine epidemic diarrhoea virus in faeces and antibodies in blood. No seropositive pigs were found at the start of the outbreak. The first signs of the disease were observed in fattening pigs and the infection spread rapidly to pregnant sows, farrowing sows and their suckling pigs, gilts and weaners housed in separate barns. Depression and diarrhoea in the fattening pigs and pregnant sows were the clearest clinical signs. An endemic form of the disease developed which would not normally have been recognised as epidemic diarrhoea because no typical signs were apparent. Eleven groups of seronegative replacement gilts, which were brought in monthly, became infected with the virus and most of the groups developed a profuse diarrhoea lasting a few days. The infections and diarrhoea persisted in six- to 10-week-old pigs in separate barns. The suckling pigs and young weaners appeared to be spared from the infections.

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELISA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus.

A blocking ELISA was developed to detect antibodies directed against porcine epidemic diarrhea virus (PEDV). The PEDV antigen was first incubated with dilutions of test sera. Any antigen that was not blocked by antibodies in the serum was assayed in a double-antibody sandwich ELISA, using 2 monoclonal antibodies directed against different antigenic sites on PEDV as capture and detecting antibodies, respectively. The blocking ELISA was compared with a fixed-cell ELISA that used monolayers of Vero cells infected with PEDV prototype strain CV777 as a solid phase and a conjugate of an IgG-specific monoclonal antibody for antibody detection. Pigs were inoculated with PEDV strain CV777 or 1 of 2 field isolates, and antibody responses were measured by use of the 2 tests. Antibodies were detected by the blocking ELISA as early as postinoculation day 7 and, by the fixed-cell ELISA, as early as postinoculation day 14. From day 14 on, antibody titers for both tests correlated highly. Titers for the fixed-cell ELISA were 5.4 times higher than those for the blocking ELISA. The latter technique is easier to perform and discriminates well between infected and noninfected pigs, which makes this test useful for routine diagnosis and serologic surveys of porcine epidemic diarrhea.

Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus.

Previously, an epitope recognized by a set of neutralizing monoclonal antibodies directed against the S protein of transmissible gastroenteritis has been identified. This neutralization epitope can be simulated by a single peptide combining residues 380 to 387 and 1176 to 1184 of the S protein; this combination peptide (SFFSYGEI-QLAKDKVNE) was more antigenic than it single constituents. Here we describe the immunogenicity of this combination peptide, in comparison with monomer and tandem peptides of both constituents, and with a cyclic peptide consisting of residues 373 to 398. All antisera, raised in rabbits, bound to the peptide used as immunogen. Only sera that recognized the residues 380 to 387 bound to whole virus. Three of the four antisera with the highest binding titers to whole virus also had neutralization activity. Analysis of the fine-specificity of the antisera with PEPSCAN peptides indicated that the spectrum of antibodies induced by the 380 to 387 sequence depended on the presentation of this sequence in a peptide to the immune system. The nonbinding and nonneutralizing anti-(380 to 387)-sera appeared to contain a limited spectrum of antipeptide antibodies. Furthermore, the lack of neutralization of the antiserum against the combination peptide could be explained by the immunodominance in rabbits of the 1176 to 1184 sequence over the 380 to 387 sequence. These findings demonstrate a few fundamental problems of simulating discontinuous epitopes by single synthetic peptides.

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-beta-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by synthesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine.

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus.

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.