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published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Influenza A virus infections in pigs is a disease of concern to the swine industry and to the ecology and epidemiology of influenza viruses in humans. Pigs have been proposed as a “mixing vessel” for generation of pandemics via reassortment between avian and mammalian viruses. The H1N1pdm 2009 virus probably emerged from swine into humans though reassortment between the recent North American triple reassortant H1N2 swine viruses and Eurasian avian-like swine viruses. Swine influenza viruses of H1N1, H1N2 and H3N2 subtypes have been regularly detected in pigs in most parts of the world. Nevertheless, ecological and virological data on swine influenza is not available in Sri Lanka, and indeed, little documented data is available in the South Asian continent. The swine population in Sri Lanka is about 80,000, and live pigs are not regularly imported to the country. Swine husbandry is largely confined to four neighboring administrative districts in the country. Systematic virological and serological surveillance carried in swine abattoirs in Sri Lanka during 2009-2013 detected H1N1pdm 2009 like virus in local herds. Infection in pigs followed each of the H1N1pdm 2009 outbreaks in humans; October 2009 – January 2010, October 2010 – February 2011 and November 2012 – March 2013, respectively. Genetic, phylogenetic, and epidemiologic analysis of the human, and swine influenza viruses indicated spillover events of H1N1pdm 2009 from humans into pigs, with self-limited transmission and extinction within pig herds. The data also indicated that although H1N1pdm 2009 was able to spill over from humans to swine, it is not ideally adapted to establish sustained transmission among swine in the absence of further reassortment with other swine influenza virus lineages. Theses finding might reflect characteristics of swine husbandry in Sri Lanka, which has a low density pig population and remains isolated from global swine influenza viruses because of the absence of regular cross-border and cross-continental movements of swine. In contrast to some other parts of the world, we failed to isolate established lineages of swine influenza viruses, viz. Classical, North American triple reassortant and European Avian lineages. Sero prevalence to these endemic swine viruses was largely absent in local swine herds. In vitro replicative kinetic study indicated that H1N1pdm 2009 viruses isolated from swine have undergone some adaptation to swine led to decreased fitness for replication in human cells. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

O vírus da influenza tipo A (VIA) é um importante agente em rebanhos suínos ao redor do mundo. Alguns subtipos do vírus podem ser transmitidos entre espécies diferentes, como aves, homem e suínos, proporcionando o aumento de mutações genômicas e de novas cepas circulantes. Os suínos são considerados hospedeiros de "mixagem", uma vez que possuem receptores para cepas de aves, de humanos e suínos. A doença clínica em suínos é caracterizada por quadro respiratório agudo e brando, com duração de 5 a 7 dias. Em rebanhos de reprodutores – como as Unidade Produtoras de Leitões (UPL) – um surto epidêmico de influenza pode levar o estabelecimento de uma infecção endêmica com duração de semanas a meses, sem produção de sinais clínicos evidentes. Protocolos de biosseguridade vêm sendo incorporados e padronizados pelas agroindústrias, visando prevenir a introdução de doenças infecciosas em rebanhos. Entretanto, existem lacunas no conhecimento dos fatores associados à biosseguridade em rebanhos de matrizes suínas brasileiras e sua relação com doenças infecciosas. Por essa razão, um estudo transversal foi realizado para estimar a soroprevalência do VIA em matrizes de UPL e explorar práticas de biosseguridade associadas à presença de anticorpos contra o vírus da influenza. Ao todo, foram amostradas 404 matrizes em 21 granjas. O diagnóstico sorológico foi realizado pelo ELISA (protocolo in house). Todas as amostras positivas pelo ELISA foram testadas usando a inibição de hemaglutinação (IH) para diagnosticar a presença de H1N1pdm2009, H1N2 e H3N2 como subtipos de vírus influenza. As informações sobre práticas de biosseguridade foram obtidas através da aplicação de um questionário. A associação entre o resultado do diagnóstico do ELISA de cada uma das matrizes amostradas e as práticas de biosseguridade da propriedade foi feitas através de um modelo de Regressão de Poisson Robusta, estimando a Razão de Prevalência (RP) como medida da associação. A prevalência estimada de anticorpos anti-VIA nas matrizes foi de 63,9% (IC 95%: 55% - 72%), sendo que todas as granjas tiveram resultados positivos. A frequência dos subtipos nas matrizes usando IH foi 51,9% para H1N1, 27,8% H1N2 e 0,6% H3N2. Coinfecções entre H1N1 e H1N2 foram observadas em 19 granjas. As práticas de biosseguridade associadas significativamente com a presença de anticorpos (p-valor <0,05) foram a "presença de tela anti-pássaros" (RP = 0,75) e "local de aclimatação para leitoas" (RP = 0,57) como fatores protetivos e "reposição externa de leitoas" (RP = 1,38) como associada a uma maior prevalência do vírus da influenza suína. Foi possível verificar que a soroprevalência do VIA nas matrizes comerciais da população estudada é alta, indicando que os animais são frequentemente expostos ao patógeno, e que algumas medidas de biosseguridade estão associadas com a ocorrência da doença, fornecendo subsídios técnicos sobre a importância dos protocolos de biosseguridade para a promoção da saúde do plantel. / Influenza A virus (IAV) is an important infectious agent in pig herds across the globe. Some subtypes of this virus can be transmitted between different species, such as birds, human and pig, increasing genomic mutations and evolving of new circulating strains. Pigs are considered "mixed vessel" for influenza A, since they have cell receptors for birds, humans and pigs strains. The clinical disease in pigs is characterized by an acute and mild respiratory disease, lasting from 5 to 7 days. In breeding herds such as sow farms, an epidemic outbreak of influenza can lead to the establishment of an endemic infection lasting weeks to months without clinical signs. Biosecurity procedures were incorporated and standardized by the agroindustry in order to prevent both the introduction and dissemination of infectious diseases. However, there are gaps in the knowledge about what biosecurity factors are associated with infectious diseases in Brazilian herds. For this reason, a cross-sectional study was carried out to estimate IAV seroprevalence in sows and assess which biosecurity practices are associated with the prevalence of influenza virus antibodies. Four hundred forty-four sows were sampled from 21 farms. Serological assays were performed using an ELISA test (in-house protocol). All ELISA positive samples were tested using the hemagglutination inhibition test (HI) to identify the presence of H1N1pdm2009, H1N2 and H3N2 subtypes. Information of biosecurity practices was obtained through the application of a questionnaire. Association between ELISA diagnostic result of each sampled sow and biosecurity practices was assessed using a robust Poisson regression and the Prevalence Ratio (PR) was used as the measure of association. The estimated prevalence of anti-IAV antibodies in sows was 63.9% (95% CI: 55% - 72%), and all farms had at least one seropositive sow s. The frequency of subtypes using HI was 51.9% for H1N1, 27.8% for H1N2 and 0.6% for H3N2. Co-infections with H1N1 and H1N2 were observed in 19 farms. Biosecurity practices such as "presence of bird-proof" (PR = 0.75), and "presence of an acclimatization unit" (PR = 0.57), protective ones, and "external replacement of gilts" (PR = 1.38), which was positively associated with the IAV prevalence, were statistically significant in the final model (p-value <0.05). It was possible to verify that IAV seroprevalence is high and some biosecurity procedures were associated with the serologic status, offering technical subsidies about the importance of the biosecurity for the herd heath.

published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / Politics and Public Administration / Master / Master of Public Administration

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is an enveloped, segmented, negative-sense RNA virus that infects avian species and mammals. Its segmented feature enables antigenic shift which can generate novel IAVs that pose a threat to animal and public health due to lack of immunity to these viruses. Pigs have been considered the “mixing vessels” of influenza A viruses to generate novel reassortant viruses that may threaten animal and public health. Therefore, it is necessary to understand the pathogenicity and transmissibility of newly emerged reassortant viruses in swine. Adding to this complexity is the newly identified bat influenza A-like viruses which have roused interest in understanding the evolutionary history and pandemic potential of bat influenza. At least 10 different genotypes of novel reassortant H3N2 IAVs with gene(s) from 2009 pandemic H1N1 [A(H1N1)pdm09] have been identified in pigs in the United States. To date, only three genotypes of these viruses have been evaluated in animal models leaving the pathogenicity and transmissibility of the other seven genotype viruses unknown. We showed that reassortant viruses with genes from A(H1N1)pdm09 are pathogenic and transmissible in pigs. Further studies showed that avian-like glycine at position 228 of the HA receptor binding site is responsible for inefficient transmission of the reassortant H3N2 IAV with five A(H1N1)pdm09 genes. Studying the recently discovered IAV-like sequences from bats has been hindered by the lack of live virus isolation or culturing. Using synthetic genomics, we successfully rescued modified bat influenza viruses that had the HA and NA coding regions replaced with two classical IAVs. Additional studies were performed with truncations on NS1 protein and substitution of a putative virulence mutation in bat influenza PB2. Virus reassortment experiments demonstrated that bat influenza has limited genetic and protein compatibility with other influenza viruses; however, it readily reassorts with another divergent bat influenza virus. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 IAVs in pigs. It also indicates that the bat influenza viruses recently identified are viable viruses that pose little pandemic threat to humans. Moreover, they provide new insights into the evolution and basic biology of influenza viruses.

La grippe chez le porc est due aux virus Influenza de type A dont plusieurs sous types sont devenus enzootiques dans cette population. Ces virus sont à l’origine d’épisodes grippaux répétés sur chaque bande successive et à âge fixe mais les déterminismes de leur persistance au sein de la population de l’élevage restaient mal connus. Un modèle stochastique de métapopulation représentant la co-circulation de deux sous-types de virus influenza de type A en élevage de type naisseur-engraisseur, alimenté par des données produites en conditions expérimentales, a permis d’identifier l’immunité maternelle, la transmission par voie aéroportée et la structuration de l’élevage (grande taille, conduite en bandes en flux tendu) comme facteurs déterminant la persistance des virus Influenza en élevage. Les mesures de maitrise évaluées à l’aide de ce modèle soulignent les limites des moyens actuels (vaccination notamment) et identifient l’export de bandes de porcelets au sevrage comme mesure permettant de rompre la répétition quasi-mécanique du processus infectieux. Des évènements de co-infections entre les deux sous-types viraux ont également été mis en évidence avec ce modèle, soulignant l’intérêt de poursuivre les investigations pour prendre en compte la génération de virus réassortants à partir de ces évènements et leur potentielle propagation puis maintien sur l’élevage, le porc étant considéré comme le vecteur de mélange des virus Influenza / Swine Influenza A Viruses (swIAVs) are known to persist in an endemic form in farrow-to-finish pig herds, leading to recurrent swine flu outbreaks in successive batches at a similar age. However, determinants for propagation and maintenance at the herd level were unknown. A stochastic metapopulation model, fed with experimental data, has been developed to represent the co-circulation of two distinct swIAVs subtypes within a typical farrow-to-finish pig herd in order (i) to understand the conditions for persistence and (ii) to evaluate the risk of reassortant viruses generation as well as the efficacy of control strategies. The passive immunity conferred to new-born piglets, the transmission by airborne route and the herd structure (herd size, intensive bath rearing systems) were found as determinants of swIAVs persistence. The currently used control strategies such as vaccination could hardly achieve swIAVs fade-out within the farrow-to-finish pig herds while the export of piglets batches at weaning evidenced the best ability to block the permanent transfer of infectious process from batch to batch. Co-infection events have been also evidenced highlighting the interest to account for reassortant viruses generation from these events and their further spread and persistence at the herd level from a veterinary and public health point of view, pigs being considered as a mixing vessel for influenza viruses

Three main research products are reported in this dissertation. This research focused on estimation of the seroprevalence rates in agricultural workers with exposure to pigs and poultry, and determination of risk factors for infection. Chapter 2, "Are swine workers in the United States at increased risk of infection with zoonotic influenza virus?", reports controlled, cross-sectional seroprevalence studies among farmers, meat processing workers, veterinarians, and control subjects. Using a hemagglutination inhibition assay against six influenza A virus isolates, all 3 exposed study groups demonstrated markedly elevated titers against the H1N1 and H1N2 swine influenza virus isolates, compared with control subjects. Chapter 3, "Infection due to 3 avian influenza subtypes in United States veterinarians", describes a controlled, cross-sectional seroprevalence study that examined veterinarians in the United States for evidence of previous avian influenza virus infection. Using a microneutralization assay against 9 influenza A virus strains, veterinarians exposed to birds demonstrated statistically significant elevated titers against the H5, H6, and H7 avian influenza virus isolates compared with control subjects. In chapter 4, "Cases of swine influenza in humans: a review of the literature", all known human cases of swine influenza are compiled and analyzed. Fifty cases of apparent zoonotic swine influenza virus infection, including 37 civilians and 13 military personnel, were identified, with a case-fatality rate of 14% (7 of 50 persons). Most civilian subjects (61%) reported exposure to swine. These studies provide strong evidence that transmission of zoonotic influenza likely occurs much more frequently than previously thought, and that individuals with occupational exposure to pigs and birds are at elevated risk for acquiring zoonotic influenza infections. Agricultural workers should be included in pandemic influenza planning, should receive information and training on how to use personal protective equipment, and should be offered human influenza vaccine to reduce the risk of creating viral reassortants. In the event of a pandemic, workers should be considered for antiviral medications and pandemic strain vaccines.

Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks. / United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) / North Dakota State Agricultural Products Utilization Committee (ND APUC) / North Dakota State Board of Agricultural Research (ND SABRE)