Characterization of vaccine-associated enhanced respiratory disease (VAERD) in swine administered an inactivated δ-cluster influenza vaccine and challenged with pandemic A/H1N1 virus

Influenza A viruses are an important cause of respiratory disease in swine worldwide. Contemporary influenza viruses endemic in North American swine are triple reassortants with multiple subtypes displaying marked genetic and antigenic diversity. Inactivated influenza vaccines are used in swine to protect against infection and clinical disease; however, they often lack the ability to cross-protect against heterologous viruses. In addition, inactivated vaccines may enhance clinical signs and pneumonia following challenge with divergent emerging viruses that do not demonstrate serological cross-neutralizing activity. Herein we describe enhanced disease in pigs administered an inactivated H1N2 delta-cluster vaccine followed by challenge with pandemic A/H1N1 virus known as vaccine associated enhanced respiratory disease (VAERD). Pigs in the VAERD-affected group demonstrated more severe clinical signs that included coughing, respiratory distress, anorexia and lethargy in addition to elevated body temperatures compared to naive-challenged pigs. Microscopic and macroscopic pneumonia and lung lesion scores were significantly elevated in the VAERD group with increased concentrations of pulmonary pro-inflammatory cytokines. Lung lesions at 1 and 2 days post inoculation (dpi) consisted of severe necrotizing bronchitis and bronchiolitis with interlobular and alveolar edema, hemorrhage and suppurative alveolitis. Marked peribronchiolar lymphocytic cuffing and interstitial pneumonia were consistent pathological features in the lung of VAERD-affected pigs at 5 dpi. Trachea lesions in vaccinated/challenged pigs consisted of suppurative and lymphocytic tracheitis and epithelial necrosis compared to mild lesions demonstrated in non-vaccinated/challenged pigs. The immune response to the inactivated vaccine consisted of hemagglutination inhibition (HI) and serum neutralizing (SN) antibodies to homologous virus that did not cross-react with the challenge virus. In contrast, whole virus, anti-pH1N1 IgG antibodies were detected in the serum and lung of VAERD-affected pigs. However, a cross-reactive mucosal IgA response was not detected in the lung in pigs primed with the inactivated vaccine. VAERD-affected pigs demonstrated a post-challenge primary immune response consisting of anti-pH1N1 HI, SN and ELISA antibodies to the challenge virus. The results of these studies establish a consistent swine model of VAERD using an inactivated influenza vaccine followed by challenge with heterologous virus. The coincidental increase in inactivated influenza vaccine use and the evolving antigenic diversity of influenza A viruses in swine creates a realistic potential for vaccine/challenge mismatch

Characterization of porcine circovirus type 2a and 2b infection and lesions in gnotobiotic pigs

Porcine circovirus (PCV) is a member of the genus Circovirus of the Circoviridae family. This family consists of a diverse group of animal viruses that possess a small, closed circular, single-stranded DNA genome that replicates through double-stranded intermediates. Two genotypes of PCV have been identified. PCV type 1 (PCV1) is non-pathogenic, while PCV type 2 (PCV2) has been implicated as the etiological agent of a collection of disease syndromes labeled porcine circovirus associated disease (PCVAD) which include postweaning multisystemic wasting syndrome (PMWS) in swine.;In late 2005, a postweaning, high mortality syndrome spread rapidly through fattening barns in swine dense areas of the United States. Diagnostic investigations consistently isolated porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed the infectious agent was PCV2b. Prior to late 2004, only PCV2a, but not PCV2b, had been reported in North America. In the first manuscript in this thesis, we demonstrate an acute high mortality disease in germ-free pigs using infectious viruses generated from DNA clones constructed from PCV2a and PCV2b isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12 hours of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in this study were comparable to those reported in experiments with germ-free pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus. The animals in this study were confirmed to be free of detectable porcine parvovirus, bovine viral diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine hepatitis E virus, and aerobic and anaerobic bacteria. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any recognized infectious co-factor in the gnotobiotic model.;Porcine circovirus associated disease is a disease complex affecting swine around the world. Although PCV2 is believed to negatively affect the host\u27s immune system, the mechanism by which PCV2 induces disease is not completely understood. The second manuscript in this thesis describes a series of PCV2 experiments using the gnotobiotic pig model in which a correlation was noted between abnormal leukograms and clinical disease in PCV2-infected pigs. The leukogram was characterized by a lymphopenia beginning within 14 days post inoculation (dpi) followed by an absolute neutrophilia approximately one week later. No significant changes in the circulating monocyte, basophil, and eosinophil cell populations were detected. The combination of an absolute neutrophilia and lymphopenia produced a neutrophil/lymphocyte ratio that was predictive of clinical disease and was inversely correlated with the presence of neutralizing antibodies

Porcine Parainfluenza Virus Type 1 (PPIV-1) in U.S. Swine: Summary of Veterinary Diagnostic Laboratory Data

Porcine Parainfluenza-1 (PPIV-1) is a Paramyxovirus inthe genus Respirovirus. To evaluatethe frequency of detection of PPIV-1 on farms in the United States, theIowa State University Veterinary Diagnostic Laboratory (ISU VDL) conducted an analysis of clinical samples submitted from Spring toFall 2016.Thirty-four percent of diagnostic samples tested both prospectively (70/204) and retrospectively (472/1385) were positive by polymerase chain reaction (PCR), which suggests PPIV-1 is widespread

Implementation of PRRSV status classification system in swine breeding herds from a large integrated group in Spain

Background: Porcine Reproductive and Respiratory Syndrome (PRRS) is an endemic swine disease causing significant productive and economic losses. Knowledge of PRRS epidemiology is crucial to develop control strategies against this disease. In that regard, classifying farms according to PRRS virus (PRRSV) shedding and exposure, and understanding key drivers of change in status over time, provides great applied knowledge for developing disease control programs. In most European countries, PRRSV monitoring is performed most frequently at the individual farm level although criteria selected for monitoring varies among different regions and farms. The aim of this study was to implement a systematic monitoring program for PRRSV in Spanish sow farms. Breeding herds were classified according to a standardized PRRSV infection status using sampling programs and terminology currently adopted in the United States (US), which allowed an evaluation of PRRSV epidemiology in a large integrated Spanish group during a one-year study period (February 2017–March 2018). Results: Fifteen farms achieved a stable PRRSV status after the first 4 consecutive samplings and 20 farms were classified as unstable. One of the farms maintained a stable status throughout the duration of the whole monitoring period. Among the 20 farms classified as unstable at the beginning of the monitoring protocol, 9 farms (45%) never reached the stable status and 11 farms (55%) reached stable status afterwards during the monitoring study period. From PRRSV PCR positive pools, there were 47 different PRRSV nucleotide sequences from 24 different farms. More than one PRRSV sequence was obtained from 15 farms. In the farms with more than one sequence detected, we observed recirculation of the same PRRSV field strain in 7 farms and introduction of a different PRRSV strain in 5 farms and both events in 3 farms. Conclusions: Systematic monitoring for PRRSV in breeding herds established a basis of knowledge of PRRSV epidemiology at the farm level and provided key data to classify farms according to PRRSV exposure and shedding status. These data allow further evaluation of the impact of the PRRSV farm status on production and economic performance in breeding herds and additional investigation of factors related to PRRSV epidemiology

Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type

The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines

Immunogenicity and Protective Efficacy of a Recombinant Pichinde Viral-Vectored Vaccine Expressing Influenza Virus Hemagglutinin Antigen in Pigs

Influenza A virus of swine (IAV-S) is an economically important swine pathogen. The IAV-S hemagglutinin (HA) surface protein is the main target for vaccine development. In this study, we evaluated the feasibility of using the recombinant tri-segmented Pichinde virus (rPICV) as a viral vector to deliver HA antigen to protect pigs against IAV-S challenge. Four groups of weaned pigs (T01–T04) were included in the study. T01 was injected with PBS to serve as a non-vaccinated control. T02 was inoculated with rPICV expressing green fluorescence protein (rPICV-GFP). T03 was vaccinated with rPICV expressing the HA antigen of the IAV-S H3N2 strain (rPICV-H3). T04 was vaccinated with the recombinant HA protein antigen of the same H3N2 strain. Pigs were vaccinated twice at day 0 and day 21 and challenged at day 43 by intra-tracheal inoculation with the homologous H3N2 IAV-S strain. After vaccination, all pigs in T03 and T04 groups were seroconverted and exhibited high titers of plasma neutralizing antibodies. After challenge, high levels of IAV-S RNA were detected in the nasal swabs and bronchioalveolar lavage fluid of pigs in T01 and T02 but not in the T03 and T04 groups. Similarly, lung lesions were observed in T01 and T02, but not in the T03 and T04 groups. No significant difference in terms of protection was observed between the T03 and T04 group. Collectively, our results demonstrate that the rPICV-H3 vectored vaccine elicited protective immunity against IAV-S challenge. This study shows that rPICV is a promising viral vector for the development of vaccines against IAV-S

Quantifying the Persistence of Vaccine-Related T Cell Epitopes in Circulating Swine Influenza A Strains from 2013-2017

When swine flu vaccines and circulating influenza A virus (IAV) strains are poorly matched, vaccine-induced antibodies may not protect from infection. Highly conserved T cell epitopes may, however, have a disease-mitigating effect. The degree of T cell epitope conservation among circulating strains and vaccine strains can vary, which may also explain differences in vaccine efficacy. Here, we evaluate a previously developed conserved T cell epitope-based vaccine and determine the persistence of T cell epitope conservation over time. We used a pair-wise homology score to define the conservation between the vaccine’s swine leukocyte antigen (SLA) class I and II-restricted epitopes and T cell epitopes found in 1272 swine IAV strains sequenced between 2013 and 2017. Twenty-four of the 48 total T cell epitopes included in the epitope-based vaccine were highly conserved and found in >1000 circulating swine IAV strains over the 5-year period. In contrast, commercial swine IAV vaccines developed in 2013 exhibited a declining conservation with the circulating IAV strains over the same 5-year period. Conserved T cell epitope vaccines may be a useful adjunct for commercial swine flu vaccines and to improve protection against influenza when antibodies are not cross-reactive

Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n = 8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n = 9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n = 9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n = 9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n = 9), pigs infected with VR-2385 (n = 9), and pigs infected with NC16845b (n = 9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens