Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States

Swine influenza viruses (SIV) of the hemagglutinin subtype 1 (H1) isolated from the United States (U.S.) have not been well-characterized in the natural host. An increase in the rate of mutation and reassortment has occurred in SIV isolates from the U.S. since 1998, including viruses belonging to the H1 subtype. Two independent animal studies were done to evaluate and compare the pathogenesis of 10 SIV isolates dating from 1930 to currently circulating isolates. In addition, the hemagglutinin and neuraminidase genes of each isolate were sequenced for genetic comparison, and serological cross-reactivity was evaluated using all sera and virus combinations in hemagglutination inhibition and serum neutralization assays. Statistically significant differences in percentage of pneumonia and virus titers in the lung were detected between isolates, with modern isolates tending to produce more severe disease, have more virus shedding and higher viral titers. However, nasal shedding and virus titers in the lung were not always correlated with one another or lung lesions. Serologically, the classic historical H1N1 viruses tended to have better cross-reaction between historical sera and antigens, with moderate to good cross-reactivity with modern viral antigens. However, the modern sera were less reactive with historical viruses. Modern viruses tended to have less consistent cross-reactivity within the modern group. Overall, H1 isolates collected over the last 75 years from the U.S. pig population exhibit considerable variability in pathogenicity. There appears to be an increase in genetic and antigenic diversity coincident with the emergence of the swine triple reassortant H3N2 in 1998.     

Comparison of three serological assays to determine the cross-reactivity of antibodies from eight genetically diverse U.S. Swine influenza viruses.

Swine influenza virus is an economically important pathogen to the U.S. swine industry. New influenza subtypes and isolates within subtypes with different genetic and antigenic makeup have recently emerged in U.S. swineherds. As a result of the emergence of these new viruses, diagnosticians' ability to accurately diagnose influenza infection in pigs and develop appropriate vaccine strategies has become increasingly difficult. The current study compares the ability of subtype-specific commercial enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HI), and serum neutralization (SN) assays to detect antibodies elicited by multiple isolates within different subtypes of influenza virus. Pigs were infected with genetically and antigenically different isolates of the 3 major circulating subtypes within populations of swine (H1N1, H1N2, and H3N2). Serum was collected when all pigs within a group collectively reached HI reciprocal titers >or=160 against that group's homologous challenge virus. The antibody cross-reactivity of the sera between isolates was determined using ELISA, HI, and SN assays. In addition, the correlation between the 3 assays was determined. The assays differed in their ability to detect antibodies produced by the viruses used in the study. The results provide important information to diagnostic laboratories, veterinarians, and swine producers on the ability of 3 common serological assays used in identifying infection with influenza in pigs.     

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.

This study reports the antigenic relatedness of isolates of Lelystad virus collected in the Netherlands, Germany, and the United States. The binding of antibodies directed against these isolates was tested in a set of field sera collected during outbreaks of porcine epidemic abortion and respiratory syndrome in Europe and outbreaks of swine infertility and respiratory syndrome (SIRS) in North America. Two sets of sera from pigs experimentally infected with Lelystad virus or SIRS virus were also tested. Although all 7 isolates reacted with anti-Lelystad virus sera, antigenic variation was considerable. The 4 European isolates resembled each other closely, but differed from the American isolates, and the 3 American isolates differed antigenically from each other. To reliably diagnose Lelystad virus infection, a common antigen must first be identified.     

Serological comparison and antigenic relationships of seven serotypes of infectious bronchitis virus using the hemagglutination-inhibition test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven isolates of infectious bronchitis virus (IBV) were examined using the hemagglutination-inhibition (HI) test. Because there was a discontinuity of antigenic relationships and a high degree of cross-reactivity among serotypes of IBV in cross-hemagglutination-inhibition tests, the range of antigenic spectrum used to group the serotypes with the HI test should be wider than the limits suggested by the plaque-reduction test. The HI test may provide valuable information in monitoring the immune status of a flock following vaccination when the area has a history of infectious bronchitis infection. It may also be used as a rapid diagnostic test if a flock is experiencing an outbreak of a disease caused by emergence of a new type of IBV. Interpretation of HI titers in evaluating immune status of chickens following infection with IBV depends on further cross-challenge and cross-protection studies of various types of IBV.     

Different cross-reactivity of human and rodent sera to Tula virus and Puumala virus

Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans.     

Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs.

A new antigenic variant of swine influenza virus was isolated from the lungs of pigs experiencing respiratory problems in 7 different swine herds in Quebec. Pigs of different ages were affected, and the main clinical signs were fever, dyspnea, and abdominal respiration. Coughing was not a constant finding of the syndrome. At necropsy, macroscopic lesions included the overall appearance of pale animals, general lymphadenopathy, hepatic congestion, and consolidation of the lungs. Histopathologic findings were mainly proliferative pneumonia with a significant macrophage invasion, necrotic inflammatory cells in the alveoli and the airways, a marked proliferation of type II pneumocytes, and thickening of the alveolar septae. Fluorescent antibody examination of lungs of sick piglets did not demonstrate porcine parvovirus, transmissible gastroenteritis virus, or encephalomyocarditis virus. However, evidence of the presence of an influenza type A infection was demonstrated by indirect immunofluorescence (IIF) staining using monoclonal antibody directed to nucleocapsid protein (NP) of human type A influenza virus. The virus was isolated either by intra-allantoic inoculation of specific-pathogen-free embryonating hens' eggs or propagation in canine kidney (MDCK) cells in the presence of trypsin. By hemagglutination inhibition tests, no cross-reactivity was demonstrated with human influenza H1N1, H2N2, and H3N2 strains, and infected MDCK cells did not react by IIF with monoclonal antibodies to NP protein of type B influenza virus. The hemagglutination activity of plaque-purified isolates was only partly inhibited by hyperimmune serum produced to subtypes A/Wisconsin/76/H1N1 and A/New Jersey/76/H1N1 of swine influenza virus. Gnotobiotic piglets that were infected intranasally with egg-adapted isolates of this new antigenic variant of swine influenza virus developed the very same type of lesions observed in field cases.     

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.     

Immunological cross-reactivity of pathogenic Nocardia and related organisms.

Aerobic actinomycetes associated with various clinical conditions in man and animals were studied for their antigenic relationships along with related organisms. Culture filtrate antigens obtained from 16 species were tested by antigen-antibody crossed-immunoelectrophoresis and by agar gel double diffusion against rabbit antibodies to Nocardia asteroides, N. brasiliensis and N. caviae. Weak cross-reactivity was detected among the three, Nocardia species and Micropolyspora brevicatena, but no cross-reactivity was noted with antigens from any of the other species studied.     

Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

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.     

Avian paramyxovirus serotype 1 isolates from the spinal cord of parrots display a very low virulence

The spinal cord of 32 psittacines suffering from proventricular dilatation disease (PDD) was investigated. In six cases, a virus was isolated which upon electron microscopic examination revealed morphological details typical of members of the Paramyxoviridae. All isolates were subsequently characterized as avian paramyxovirus serotype 1 (APMV-1) by type-specific polyclonal antisera. According to their reactivity with APMV-1 specific monoclonal antibodies, the six isolates shared epitopes within the haemagglutinin-neuraminidase spike protein, distinct from pigeon-type paramyxoviruses and the LaSota vaccine strain. This grouping was further corroborated by properties of the haemagglutinin: all isolates showed a very thermosensitive haemagglutination activity and were rapid eluters. Virulence of the APMV-1 isolates in 1-day-old specific pathogen free (spf) chicken was very low, with intracerebral pathogenicity indices between 0 and 0.1. In embryonated spf chicken eggs, psittacine isolates replicated to high titres (10(8.6)-10(10.7) EID50/ml). However, they exhibited a reduced lethality over an observation time of 7 days (10(6.1)-10(8.3) ELD50/ml). In a haemagglutination inhibition test with parrot sera from birds with no history of APMV-1 vaccination, sera reacted preferentially with two isolates compared with APMV-1 vaccine strains LaSota and B1. The other four isolates exhibited a differentiated reaction pattern with the parrot sera, indicating an antigenic inhomogeneity. This is the first report of isolating very low virulent APMV-1 from neuronal tissue of parrots and implications for a possible role in slow progressing disease will be discussed.     

Determination of the antigenic relationships among six serotypes of infectious bronchitis virus using the enzyme-linked immunosorbent assay and serum-neutralization test

The indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect antibody to infectious bronchitis (IB) virus in chickens. A cross-reactivity study was performed to determine the antigenic relationships among six strains: Massachusetts, Connecticut, Beaudette, and three Ontario field isolates. For comparison, the cross-reactivity study was performed in eggs using the serum-neutralization (SN) test for IB. The ELISA proved to be more broadly cross-reactive than the IB-SN test. This suggests that the ELISA for IB will be useful as a rapid diagnostic test for serum profiling in unvaccinated flocks, regardless of the IB serotype involved.     

H3N2亚型猪流感病毒中国分离株的克隆纯化及生物学特性

以有限稀释克隆法对29株H3N2亚型猪流感病毒(SIV)不同地区分离株进行纯化，并对其生物学特性进行了研究。结果，8株对鸡呈现中等致病力，21株对鸡呈现低致病力。不同地区SIV分离株(第5代)的EID50差异较大，以安徽分离株最高，为10^-10.77／0．2mL，其他毒株在10^-5.5～10^-10.56／0．2mL之间。黑龙江省分离株和浙江省分离株的LD50高达10^-2.84／0．1mL，其他分离株在10^-1.17～10^-2.56／0．1mL之间。经鸡胚分离传代后，SIV分离株均能凝集0．7％人“O”型血、绵羊、兔、豚鼠、小鼠、大鼠及鸡的红细胞，其红细胞凝集谱的差异主要表现在对马、牛、驴、猪红细胞的凝集特性上。大部分SIV分离株为热不稳定型，部分毒株表现为中等热稳定型和热稳定型。从其抗原特性看，大部分SIV分离株表现为亲和相，对AIV参考毒株DKUK63和SIV参考毒株SWTN77表现出较高的HI滴度；部分分离株经鸡胚传代后，出现了相别的变异。     

Genetic and antigenic changes in porcine rubulavirus

Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains.     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum

Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity.     

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.     

Single-dilution indirect solid-phase radioimmunoassay for the detection of anti-pseudorabies immunoglobulin G in swine sera

A single-dilution indirect solid-phase radioimmunoassay (IRIA) was developed for the detection of low levels of anti-pseudorabies immunoglobulin G in swine sera. The assay derived increased sensitivity from the use of a second amplifying antibody. The IRIA was examined for its stoichiometry, amplification by secondary antibody, advantage of a single-dilution assay vs an end-point titration, sensitivity, and specificity. The assay had a near linear dose-response relationship with positive sera (serum-neutralization titer less than or equal to 1:16) and lacked a dose response with negative sera. With addition of the secondary antibody, the IRIA was enhanced 8.5-fold in net specific binding, and the end-point titer was amplified 32-fold. The single-dilution assay was proved to be a feasible test, compared with end-point titration. Anti-pseudorabies virus titers were at least 128-fold higher by IRIA than those by serum-neutralization test. Evidence indicated that there may be minimal or no cross-reactivity of IRIA antigen with anti-infectious bovine rhinotracheitis sera. The single-dilution IRIA was a rapid and sensitive test for anti-pseudorabies virus immunoglobulin G in swine sera.     

Serologic response of vaccinated cattle to strains of Moraxella bovis isolated during epizootics of keratoconjunctivitis.

In studies to determine whether there were antigenic differences between strains (isolates) of Moraxella bovis, the sera from vaccinated calves were tested with isolates of M bovis while the calves were experiencing epizootics of infectious bovine keratoconjunctivitis (IBK). Before the epizootics of IBK, the calves were intramuscularly vaccinated with a formalin-killed autogenous M bovis bacterin. During the epizootics, the eyes were examined by cultural technique, and isolates which were obtained were categorized by catalase activity, source (diseased or nondiseased eyes), and reactivity with the various sera. The serum reactivity of the isolates was compared with that of the vaccinal strain. The vaccinal strain and 8 of the 1 5 selected isolates obtained during the 1974 epizootic were catalase negative. Seven of the 15 isolates from the 1974 epizootic and all of the selected isolates from the 1975 epizootic were catalase positive. A significantly higher (P less than 0.01) percentage of calf sera were serologically reactive with the vaccinal strain and other catalase-negative isolates (45.0%) than with catalase-positive isolates (34.8%). The results, although not definitive, suggest that there may be antigenic differences among strains of M bovis. These differences should be considered when cattle are vaccinated against IBK under natural conditions of exposure.