Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool.We thank Jaime Maldonado and HIPRA (Spain) for the A/Swine/Spain/ 54008/2004 (H3N2) strain, Edward J. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) IAV strain, T. M. Chambers and the University of Kentucky for the A/Equine/OH/1/03 (H3N8) IAV strain, and Hon Ip and the U.S. Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbor Seal/New Hampshire/179629/2011 (H3N8) IAV strains. We thank Sergio López, David Solanes, Francisco X. Abad, Jordi Alberola, Jaume Martorell, and Eduard J. Cunilleras for help in providing different samples and during the experimental infections, as well as the personnel in Cat3 laboratories and the animal house. We thank Adolfo García-Sastre for providing materials and for support as the principal investigator of the NIAID-funded Center for Research in Influenza Pathogenesis (HHSN266200700010C). The research leading to these results received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project), and projects AGL2010-22200-C02-01 and AGL2007-60274 of the Spanish Ministry of Science and Innovation

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs

BACKGROUND: A diversifying pool of mammalian‐adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. OBJECTIVES: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost‐effective large‐scale analysis. METHODS: New SIV haemagglutinin (HA) and neuraminidase (NA) subtype‐ and lineage‐specific multiplex real‐time RT‐PCRs (RT‐qPCR) have been developed and validated with reference virus isolates and clinical samples. RESULTS: A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M‐gene‐specific influenza A virus RT‐qPCR. In a second step, positive samples are examined by tetraplex HA‐ and triplex NA‐specific RT‐qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian‐derived), “hu” (European human‐derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT‐qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT‐PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT‐qPCR subtyping. CONCLUSIONS: These new multiplex RT‐qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe

Expression dynamics of innate immunity in influenza virus-infected swine

We would like to thank Dr. Jaime Maldonado and HIPRA, Spain for the A/swine/Spain/54008/2004 (H3N2) influenza virus; Dr. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) influenza virus; Dr. Chambers and University of Kentucky for the A/Equine/OH/1/03 (H3N8) influenza virus; and Dr. Hon Ip and the US Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbour Seal/New Hampshire/179629/2011 (H3N8) influenza viruses. The authors thank Sergio López, David Solanes and Francisco X. Abad for their help during the experimental infections as well as the personnel in Cat3 laboratories and animal house. The authors also wish to thank Dr. I. L. Archetti (IZSLER, Brescia, Italy) for the invaluable help in measuring some clinical immunology parameters, Dr. L. Fraile (UdL, Spain) for assistance in statistical analysis, Dr. J. Domínguez (INIA, Spain) for porcine antibodies, Dr. M. Gennari and Dr. M. Giunta (S.S. Genova, IZSPLV, Italy) for assistance in real-time PCR analyses. The skillful technical assistance of Mrs. C. Mantovani (IZSLER, Brescia, Italy) is also gratefully acknowledged. The research leading to these results has received funding from: the European Community's Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228393 (NADIR project), and from the project AGL2010-22200-C02-01 of Spanish Ministry of Science and Innovation.Geological Survey National Wildlife Health Center/[u'duck/Maine/44411-532/2008', u'H3N8']The current circulating swine influenza virus (IV) subtypes in Europe (H1N1, H1N2, and H3N2) are associated with clinical outbreaks of disease. However, we showed that pigs could be susceptible to other IV strains that are able to cross the species barrier. In this work, we extended our investigations into whether different IV strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. For this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a H3N2 Swine IV and four different H3N8 IV strains circulating in different animal species. Pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. In the present study, all groups but mock exhibited antibody responses to IV nucleoprotein protein. Pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. Interestingly, pigs infected with avian and seal H3N8 strains also showed moderate lesions and viral replication, whereas equine and canine IVs did not cause overt pathological signs, and replication was barely detectable. Swine IV infection induced interferon (IFN)-alpha and interleukin-6 responses in bronchoalveolar fluids (BALF) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. However, IFN-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of IFN-alpha genes. Remarkably, the Equine strain gave rise to a Serum Amyloid A response in BALF despite little if any replication. Each virus strain could be associated with expression of cytokine genes and/or proteins after infection. These responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system

Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013

The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like (“avian-like”) genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level.IMPORTANCE The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like (“avian-like”) lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine

European Surveillance Network for Influenza in Pigs : Surveillance Programs, Diagnostic Tools and Swine Influenza Virus Subtypes Identified in 14 European Countries from 2010 to 2013

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections

Detection of Verocytotoxin-producing Escherichia coli serogroups O157 and O26 in the cecal content and lymphatic tissue of cattle at slaughter in Italy

Verocytotoxin-producing Escherichia coli (VTEC) has emerged as a foodborne pathogen that can cause severe and potentially fatal illnesses, such as hemorrhagic colitis or the hemolytic uremic syndrome. In this study, 182 cattle at slaughter (119 dairy cows and 63 feedlot cattle) were randomly selected and tested for the presence of VTEC serogroups O26, O103, O111, O145, and O157 in their cecal content and lymphatic tissue (tonsils or mesenteric lymph nodes). A total of 364 samples were evaluated with an immunomagnetic separation technique followed by slide agglutination. Presumptive VTEC O26, O103, O111, O145, and O157 isolates were tested by Vero cell assay for verocytotoxin production and by multiplex PCR assay for the detection of vtx1, vtx2, eae, and E-hlyA genes. VTEC O157 was detected in 6 (3.3%) of 182 animals, and VTEC O26 was detected in 1 (0.5%) of 182 animals. No VTEC O103, VTEC O111, or VTEC O145 isolates were found in cattle feces, but one VTEC O91:H vtx2 , eae, E-hlyA strain nonspecifically cross-reacted with the VTEC O103 type. The prevalence of VTEC O157 in the lymphatic tissue of cattle was 1.1% in both tonsils (1 of 93 samples) and mesenteric lymph nodes (1 of 89 samples). Lymphatic tissue contamination was observed only in VTEC O157 intestinal carriers; two (33.3%) of six fecal carriers were simultaneously VTEC O157 lymphatic carriers. This finding suggests that VTEC O157 contamination of meat does not necessarily come from feces or the environment. No other VTEC serogroups were detected in the lymphatic tissue of slaughtered cattle