Polymorphisms in the haemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine.

Interactions between the viral surface glycoprotein haemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in HA genes of two pandemic 2009 H1N1 (H1N1pdm09) isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at positions 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published H1N1pdm09 consensus sequence, the P186 genotype was more readily detected in primary infected and contact-naïve pigs when inoculated with a heterogeneous mixed stock of Ca/09. Using reverse genetics, we engineered Ca/09 and Mx/09 genomes by introducing Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either the Ca/09 or Mx/09 backbone resulted in higher and prolonged viral shedding in naïve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occurred within the receptor-binding pocket and the Sb antigenic site, they did not affect serological cross-reactivity. Relative increases of P186 in publicly available sequences from swine H1N1pdm09 viruses supported the experimental data, indicating this amino acid substitution conferred an advantage in swine.

Contemporary epidemiology of North American lineage triple reassortant influenza A viruses in pigs.

The 2009 pandemic H1N1 infection in humans has been one of the greatest concerns for public health in recent years. However, influenza in pigs is a zoonotic viral disease well-known to virologists for almost one century with the classical H1N1 subtype the only responsible agent for swine influenza in the United States for many decades. Swine influenza was first recognized clinically in pigs in the Midwestern U.S. in 1918 and since that time it has remained important to the swine industry throughout the world. Since 1988, however, the epidemiology of swine influenza changed dramatically. A number of emerging subtypes and genotypes have become established in the U.S. swine population. The ability of multiple influenza virus lineages to infect pigs is associated with the emergence of reassortant viruses with new genomic arrangements, and the introduction of the 2009 pandemic H1N1 from humans to swine represents a well-known example. The recent epidemiological data regarding the current state of influenza A virus subtypes circulating in the Canadian and American swine population is discussed in this review.

Developing One Health surveillance systems.

The health of humans, domestic and wild animals, plants, and the environment are inter-dependent. Global anthropogenic change is a key driver of disease emergence and spread and leads to biodiversity loss and ecosystem function degradation, which are themselves drivers of disease emergence. Pathogen spill-over events and subsequent disease outbreaks, including pandemics, in humans, animals and plants may arise when factors driving disease emergence and spread converge. One Health is an integrated approach that aims to sustainably balance and optimize human, animal and ecosystem health. Conventional disease surveillance has been siloed by sectors, with separate systems addressing the health of humans, domestic animals, cultivated plants, wildlife and the environment. One Health surveillance should include integrated surveillance for known and unknown pathogens, but combined with this more traditional disease-based surveillance, it also must include surveillance of drivers of disease emergence to improve prevention and mitigation of spill-over events. Here, we outline such an approach, including the characteristics and components required to overcome barriers and to optimize an integrated One Health surveillance system.

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses.

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.

Detection of anti-influenza A nucleoprotein antibodies in pigs using a commercial influenza epitope-blocking enzyme-linked immunosorbent assay developed for avian species.

Influenza virus causes acute respiratory disease in pigs and is of concern for its potential public health significance. Many subtypes of influenza virus have been isolated from pigs, and the virus continues to evolve in swine populations. Current antibody assays have limited antigenic recognition, and accurate, broad-spectrum, high through-put screening tests are needed to detect infections in swine herds and to aid in the implementation of control measures. In the current study, a commercial blocking enzyme-linked immunosorbent assay (ELISA) developed for the detection of Influenza A virus nucleoprotein antibodies in avian species was evaluated for the detection of anti-influenza serum antibodies in swine. Serum samples used to evaluate the test were archived samples from influenza research conducted at the U.S. Department of Agriculture-Agricultural Research Service-National Animal Disease Center and included samples from influenza-inoculated pigs (H1N1, H1N2, H2N3, and H3N2), contact-infected pigs, vaccinated pigs, and negative controls. Based on samples of known status (n = 453), a receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity at 96.6% (95% confidence interval [CI]: 92.3, 98.9) and 99.3% (95% CI: 97.6, 99.9), respectively. By using the cutoff established in the ROC analysis, the assay was evaluated in pigs infected with 2 isolates of the 2009 pandemic H1N1 virus. Overall, the assay showed excellent diagnostic performance against the range of influenza subtypes investigated and could serve as a useful screening assay for swine.

Serological surveillance and factors associated with influenza A virus in backyard pigs in Southern Brazil.

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP-ELISA, subtype-specific serology based on hemagglutination inhibition (HI) assay of the ELISA-positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%-7.33%) and 2.3% (1.34%-3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as "age," "sex," "number of suckling pigs" and "neighbours raising pigs" that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human-to-pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.

A TaqMan-based real-time PCR for detection and quantification of porcine parvovirus 4.

Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was detected recently in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time PCR (qPCR) targeting a conserved region of the ORF3 gene of PPV4 was developed. The qPCR detection limit was 9.5 × 10(1) DNA copies/µL. There was no cross-reaction with porcine parvovirus, torque teno virus 1, torque teno virus 2, porcine circovirus type 1, porcine circovirus type 2, or with pseudorabies virus. Two hundred and seventy-two samples, including serum, semen, lungs, feces, ovarian follicular fluids, ovaries and uterus, were evaluated by qPCR and PPV4 was detected in 36 samples (13.2%). When compared with a conventional PCR (cPCR), the qPCR assay was 10 times more sensitive and the detection of PPV4 DNA in field samples was increased 2.5 times. Partial sequencing of PPV4 ORF3 gene, obtained from two pooled samples of uterus and ovaries, revealed a high nucleotide identity (98-99%) with a reference PPV4 sequence. The qPCR can be used as a fast and accurate assay for the detection and quantification of PPV4 in field samples and for epidemiological studies in swine herds.

Distribution of antibodies against influenza virus in pigs from farrow-to-finish farms in Minas Gerais state, Brazil.

BACKGROUND: Swine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds. OBJECTIVES: The objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil. METHODS: Thirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2-3 weeks), nursery (4-7 weeks), grower pigs (8-14 weeks), and finishing pigs (15-16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain. RESULTS: The percentages of seropositive animals for H1N1pdm09 and H3N2 were 26.23% and 1.57%, respectively, and the percentages of seropositive herds for both viruses were 96.6% and 13.2%, respectively. CONCLUSIONS: The serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.

Serological and molecular evidence of hepadnavirus infection in swine.

INTRODUCTION AND OBJECTIVE: Recently, investigations in a swine herd identified evidence of the existence of a novel member of the Hepadnavirus family endemic in swine. The aim of this study was to investigate the serological and molecular markers of Hepadnavirus circulation in Brazilian domestic swine and wild boar herds, and to evaluate the identity with HBV and other Hepadnaviruses reported previously. MATERIALS AND METHODS: For the study, 376 swine were screened for hepatitis B virus serological markers. Analyses were performed in serum samples using commercial enzyme-linked immunosorbent assay (ELISA) kits (DiaSorin®) for anti-HBc, HBsAg and anti-HBs. Reactive and undetermined swine serum samples were selected to perform DNA viral extraction (QIAamp DNA Mini Kit, Qiagen®), partial genome amplification and genome sequencing. RESULTS: From 376 swine samples analysed, 28 (7.45%) were reactive to anti-HBc, 3 (0.80%) to HBsAg and 6 (1.6%) to anti-HBs. Besides, more 17 (4.52%) swine samples analyzed were classified in the grey zone of the EIA test to anti-HBc and 2 (0.53%) to HBsAg. From 49 samples molecularly analyzed after serological trial, 4 samples showed a positive result for the qualitative PCR for Hepadnavirus. Phylogenetic reconstruction using partial genome sequencing (360 bp) of 3 samples showed similarity with HBV with 90.8-96.3% of identity. CONCLUSIONS: Serological and molecular data showed evidence of the circulation of a virus similar to hepatitis B virus in swine.

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus.

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.

Serological evidence of swine influenza in Brazil.

The aim of this work was to detect serum antibodies specific to influenza viruses in swine in Brazil. Serum samples of 355 pigs from 17 herds in Minas Gerais state were tested by hemagglutination inhibition (HI) for antibodies against H1N1 swine (SIV) and human influenza viruses, and H3N2 SIV. HI revealed that 158 animals (44·5%) and 11 herds (64·7%) were positive for H1N1 SIV, 36 animals (10·1%) and four herds (23·5%) were positive for H3N2 SIV, and 136 animals (38·3%) and 10 herds (58·8%) were positive for H1N1 human. This study indicates that swine influenza is disseminated throughout Minas Gerais state, Brazil.

Natural co-infection of torque teno virus and porcine circovirus 2 in the reproductive apparatus of swine.

This work aimed to detect and study natural co-infection of Circoviridae torque teno virus (TTV) and porcine circovirus 2 (PCV2) in the swine reproductive apparatus. Semen and organs from 17 boars were tested by nested and real-time PCR. PCV2 was amplified from semen (47%), lymph nodes (84.6%) and testicles (35.3%). TTV2 was amplified from 16/17 testis and 13/13 lymph nodes. TTV1 DNA was detected in fewer testicle samples (2/17), which were also TTV2 positive. Analyzed ovaries, follicular fluid and uteri of 83 culled sows showed TTV2, TTV1 and PCV2 from 49.3%, 30.1% and 6.0% of the sows, respectively. Sperm analysis indicated insignificant differences between PCV2 and TTVs positive and negative boars. The most frequent pathologic lesion in sows was endometritis (28.9%), but this was unassociated with PCV2 or TTVs detection. These findings question the importance of PCV2 and TTV2 natural co-infection in the pathology of porcine reproductive failures.

Comparison of Human-Like H1 (δ-Cluster) Influenza A Viruses in the Swine Host.

Influenza A viruses cause acute respiratory disease in swine. Viruses with H1 hemagglutinin genes from the human seasonal lineage (δ-cluster) have been isolated from North American swine since 2003. The objective of this work was to study the pathogenesis and transmission of δ-cluster H1 influenza viruses in swine, comparing three isolates from different phylogenetic subclusters, geographic locations, and years of isolation. Two isolates from the δ2 subcluster, A/sw/MN/07002083/07 H1N1 (MN07) and A/sw/IL/00685/05 H1N1 (IL05), and A/sw/TX/01976/08 H1N2 (TX08) from the δ1 sub-cluster were evaluated. All isolates caused disease and were transmitted to contact pigs. Respiratory disease was apparent in pigs infected with MN07 and IL05 viruses; however, clinical signs and lung lesions were reduced in severity as compared to TX08. On day 5 following infection MN07-infected pigs had lower virus titers than the TX08 pigs, suggesting that although this H1N1 was successfully transmitted, it may not replicate as efficiently in the upper or lower respiratory tract. MN07 and IL05 H1N1 induced higher serum antibody titers than TX08. Greater serological cross-reactivity was observed for viruses from the same HA phylogenetic sub-cluster; however, antigenic differences between the sub-clusters may have implications for disease control strategies for pigs.

Efficacy of inactivated swine influenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs.

The gene constellation of the 2009 pandemic A/H1N1 virus is a unique combination from swine influenza A viruses (SIV) of North American and Eurasian lineages, but prior to April 2009 had never before been identified in swine or other species. Although its hemagglutinin gene is related to North American H1 SIV, it is unknown if vaccines currently used in U.S. swine would cross-protect against infection with the pandemic A/H1N1. The objective of this study was to evaluate the efficacy of inactivated vaccines prepared with North American swine influenza viruses as well as an experimental homologous A/H1N1 vaccine to prevent infection and disease from 2009 pandemic A/H1N1. All vaccines tested provided partial protection ranging from reduction of pneumonia lesions to significant reduction in virus replication in the lung and nose. The multivalent vaccines demonstrated partial protection; however, none was able to prevent all nasal shedding or clinical disease. An experimental homologous 2009 A/H1N1 monovalent vaccine provided optimal protection with no virus detected from nose or lung at any time point in addition to amelioration of clinical disease. Based on cross-protection demonstrated with the vaccines evaluated in this study, the U.S. swine herd likely has significant immunity to the 2009 A/H1N1 from prior vaccination or natural exposure. However, consideration should be given for development of monovalent homologous vaccines to best protect the swine population thus limiting shedding and the potential transmission of 2009 A/H1N1 from pigs to people.

Experimental inoculation of pigs with pandemic H1N1 2009 virus and HI cross-reactivity with contemporary swine influenza virus antisera.

BACKGROUND: A novel A/H1N1 was identified in the human population in North America in April 2009. The gene constellation of the virus was a combination from swine influenza A viruses (SIV) of North American and Eurasian lineages that had never before been identified in swine or other species. OBJECTIVES: The objectives were to (i) evaluate the clinical response of swine following experimental inoculation with pandemic H1N1 2009; (ii) assess serologic cross-reactivity between H1N1 2009 and contemporary SIV antisera; and (iii) develop a molecular assay to differentiate North American-lineage SIV from H1N1 2009. METHODS: Experiment 1: Weaned pigs were experimentally infected with A/California/04/2009 (H1N1). Experiment 2: The cross-reactivity of a panel of US SIV H1N1 or H1N2 antisera with three isolates of pandemic A/H1N1 was evaluated. Experiment 3: A polymerase chain reaction (PCR)-based diagnostic test was developed and validated on samples from experimentally infected pigs. RESULTS AND CONCLUSIONS: In experiment 1, all inoculated pigs demonstrated clinical signs and lesions similar to those induced by endemic SIV. Viable virus and antigen were only detected in the respiratory tract. In experiment 2, serologic cross-reactivity was limited against H1N1 2009 isolates, notably among virus antisera from the same HA phylogenetic cluster. The limited cross-reactivity suggests North American pigs may not be fully protected against H1N1 2009 from previous exposure or vaccination and novel tests are needed to rapidly diagnose the introduction of H1N1 2009. In experiment 3, an RT-PCR test that discriminates between H1N1 2009 and endemic North American SIV was developed and validated on clinical samples.

Molecular epidemiology of Brazilian pseudorabies viral isolates.

Pseudorabies is a disease caused by pseudorabies virus (PRV) and is responsible for considerable economic losses in the swine industry. The objective of this work was to use molecular epidemiology as a tool to facilitate the study of PRV outbreaks in Brazil. The standard PRV strain Shope, the vaccine strain Bartha and isolates from the south and the southeast regions of Brazil, were amplified for gE and gC partial genes by PCR. Results indicated that Brazilian PRV isolates are grouped in two clusters, A and B, except for one isolate that grouped with Bartha and Shope. Most Brazilian PRV isolates belonged to cluster B and diverged from virus isolated from other countries.

Absence of 2009 pandemic H1N1 influenza A virus in fresh pork.

The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.