Interspecies Transmission of Swine Influenza A Viruses and Human Seasonal Vaccine-Mediated Protection Investigated in Ferret Model.

We investigated the infection dynamics of 2 influenza A(H1N1) virus isolates from the swine 1A.3.3.2 (pandemic 2009) and 1C (Eurasian, avian-like) lineages. The 1C-lineage virus, A/Pavia/65/2016, although phylogenetically related to swine-origin viruses, was isolated from a human clinical case. This strain infected ferrets, a human influenza model species, and could be transmitted by direct contact and, less efficiently, by airborne exposure. Infecting ferrets and pigs (the natural host) resulted in mild or inapparent clinical signs comparable to those observed with 1A.3.3.2-lineage swine-origin viruses. Both H1N1 viruses could infect pigs and were transmitted to cohoused ferrets. Ferrets vaccinated with a human 2016-17 seasonal influenza vaccine were protected against infection with the antigenically matched 1A pandemic 2009 virus but not against the swine-lineage 1C virus. Our results reaffirm the need for continuous influenza A virus surveillance in pigs and identification of candidate human vaccine viruses.

Emerging Influenza D virus infection in European livestock as determined in serology studies: Are we underestimating its spread over the continent?

Influenza D virus (IDV) is a novel orthomyxovirus that was first isolated in 2011 in the United States from a swine exhibiting influenza-like disease. To date, its detection is extended to all continents and in a broad host range: IDV is circulating in cattle, swine, feral swine, camelids, small ruminants and horses. Evidence also suggests a possible species jump to humans, underlining the issue of zoonotic potential. In Europe, serological investigations in cattle have partially allowed the understanding of the virus diffusion in different countries such as Italy, France, Luxembourg and Ireland. The infection is widespread in cattle but limited in other investigated species, consolidating the assumption of cattle as IDV primary host. We hypothesize that commercial livestock trade could play a role in the observed differences in IDV seroprevalence among these areas. Indeed, the overall level of exposure in cattle and swine in destination countries (e.g. Italy) is higher than in origin countries (e.g. France), leading to the hypothesis of a viral shedding following the transportation of young cattle abroad and thus contributing to larger diffusion at countries of destination. IDV large geographic circulation in cattle from Northern to more Southern European countries also supports the hypothesis of a viral spread through livestock trade. This review summarizes available data on IDV seroprevalence in Europe collected so far and integrates unpublished data from IDV European surveillance framework of the last decade. In addition, the possible role of livestock trade and biosecurity measures in this pathogen's spread is discussed.

Detection of a New Genetic Cluster of Influenza D Virus in Italian Cattle.

Influenza D virus (IDV) has been increasingly reported all over the world. Cattle are considered the major viral reservoir. Based on the hemagglutinin-esterase (HEF) gene, three main genetic and antigenic clusters have been identified: D/OK distributed worldwide, D/660 detected only in the USA and D/Japan in Japan. Up to 2017, all the Italian IDV isolates belonged to the D/OK genetic cluster. From January 2018 to May 2019, we performed virological surveillance for IDV from respiratory outbreaks in 725 bovine farms in Northern Italy by RT-PCR. Seventy-four farms were positive for IDV. A full or partial genome sequence was obtained from 29 samples. Unexpectedly, a phylogenetic analysis of the HEF gene showed the presence of 12 strains belonging to the D/660 cluster, previously unreported in Europe. The earliest D/660 strain was collected in March 2018 from cattle imported from France. Moreover, we detected one viral strain with a reassortant genetic pattern (PB2, PB1, P42, HEF and NP segments in the D/660 cluster, whilst P3 and NS segments in the D/OK cluster). These results confirm the circulation of IDV in the Italian cattle population and highlight the need to monitor the development of the spreading of this influenza virus in order to get more information about the epidemiology and the ecology of IDV viruses.

Antimicrobial capabilities of non-spermicidal concentrations of tea tree (Melaleuca alternifolia) and rosemary (Rosmarinus officinalis) essential oils on the liquid phase of refrigerated swine seminal doses.

Antimicrobial resistance is increasing within the porcine industry with consequential high impact on human health, leading to a need for new antimicrobials. Lately, the scientific community has turned its interest towards natural compounds, and different essential oils have been tested on spermatozoa for preliminary assessment of toxicity before considering them as good substitutes for standard antibiotics. The aim of the present work was to investigate the potential antimicrobial effect of Melaleuca alternifolia and Rosmarinus officinalis essential oils, already evaluated for toxicity, on swine artificial insemination doses deprived of spermatozoa and stored at 16 °C for 5 days. This was accomplished by setting up an in vitro model with a standardized quantity of E. coli. Essential oils, previously chemo-characterized by means of gas chromatography, were tested at 0.2 and 0.4 mg/ml. Analyses, performed at 24 and 120 h, included optical density evaluation, bacterial DNA quantification by qPCR, and colony count. The results demonstrate that both Melaleuca alternifolia and Rosmarinus officinalis essential oils, at a concentration of 0.4 mg/ml, are capable of delivering similar effects to ampicillin, used as control, on the experimental samples. At the lower concentration, M. alternifolia essential oil seemed more effective when compared to R. officinalis. Overall, these findings strengthen the hypothesis of the potential use of phyto-complexes as antimicrobial agents for reproductive biotechnologies.

Serologic and Virologic Evidence of Influenza A Viruses in Wild Boars ( Sus scrofa) from Two Different Locations in Italy.

Swine influenza viruses (SIVs) have been repeatedly demonstrated to circulate in wild boar ( Sus scrofa) populations, whereas no evidence of exposure to avian influenza viruses (AIVs) has been described in wild boar. To better understand how different environments may influence the ecology of influenza A viruses (IAVs) in wild suid populations, we examined biologic samples of wild boars from two study areas represented by an upland (UL) and a wetland (WL) in northern and central Italy, respectively. Serum samples were collected from 388 wild boars sampled in the UL, whereas both a serum sample and a nasal swab were obtained from each of 35 wild boars sampled in the WL. Twenty of 388 (5.2%) sera from the UL were positive by enzyme-linked immunosorbent assay for the presence of antibodies against influenza A nucleoprotein and some of these samples showed antibodies by hemagglutination inhibition to SIVs of H1N1 (1/20), H1N2 (10/20), and H3N2 (1/20) antigenic subtypes. No IAV-seropositive wild boar was detected in the WL, although one of 35 animals was found to be IAV-positive by both a reverse transcriptase PCR and a real-time reverse transcriptase PCR. We hypothesize an SIV exposure for IAV-seropositive wild boars occupying the UL, whereas a possible AIV spillover from aquatic bird species-natural reservoirs of IAVs-to wild boars in the WL cannot be ruled out. Further research is needed to better understand the role played by wild boars in IAV ecology in Mediterranean habitats.

MAb-based competitive ELISA for the detection of antibodies against influenza D virus.

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb-based competitive ELISA for the detection of antibodies against IDV. Thirty-one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724-3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid-phase competitive ELISA (IDV-cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase-conjugated MAb as competitor, which had shown wide intratype cross-reactivity and positivity in HI. To evaluate the diagnostic performances of IDV-cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut-off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV-cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI -0.899 to 0.961) between HI test and IDV-cELISA. ROC analysis showed that IDV-cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993-1.000). A cut-off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1-99.9) and 98.75 (95% CI 97.1-99.6). These results proved excellent diagnostic performances of IDV-cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti-IDV antibodies in different animal species.

Influenza D in Italy: towards a better understanding of an emerging viral infection in swine.

Influenza D virus (IDV), a new member of the Orthomyxoviridae family, was first reported in 2011 in swine in Oklahoma, and consequently found in cattle across North America and Eurasia. To investigate the circulation of IDV among pigs in Italy, in the period between June 2015 and May 2016, biomolecular and virological tests were performed on 845 clinical samples collected from 448 pig farms affected by respiratory distress located in the Po Valley. Serological tests were conducted on 3698 swine sera, including archive sera collected in 2009, as well as samples collected in 2015 from the same region. Viral genome was detected in 21 (2.3%) samples from 9 herds (2%), while virus was successfully isolated from 3 samples. Genetic analysis highlighted that Italian swine IDVs are closely related to the D/swine/Oklahoma/1334/2011 cluster. Sera collected in 2015 showed a high prevalence of IDV antibody titers (11.7%), while archive sera from 2009 showed statistically significant lower positivity rates (0.6%). Our results indicate an increasing epidemiological relevance of the pathogen and the need for in-depth investigations towards understanding its pathogenesis, epidemiology and possible zoonotic potential of this emerging virus.

Expression Dynamics of Innate Immunity in Influenza Virus-Infected Swine.

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.

Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus.

The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains. A new Real-Time PCR, specific for influenza D virus (IDV), was developed and evaluated. The target sequences of primers and probe are highly conserved among IDV strains currently known. The specificity of the method was demonstrated in silico by BLAST, and in vitro with a huge panel of common swine and bovine respiratory pathogens. The analytical sensitivity of the Real-Time PCR was estimated through synthetic RNA molecules and the limit of detection was about 20 copies/µL. The assay was assessed in field and proved to be a valuable tool for the detection of IDV strains.

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.

H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion.

UNLABELLED: The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCE: Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis.

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

UNLABELLED: 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. IMPORTANCE: Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs.

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

UNLABELLED: 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.

Temporal insight into the natural generation of a new reassortant porcine influenza virus in a swine holding.

The influenza A virus (IAV) subtypes H1N1, H3N2 and H1N2 are the most prevalent subtypes in swine in Italy. Reassortant influenza A viruses subtypes in swine appeared in European pig population. In particular reassortant viruses carrying genome segment from the pandemic H1N1 (H1N1pdm) are reported in many European countries, including Italy. In a 1000 sows farrow-to feeder farm, in Northern Italy, we isolated 10 IAVs from recurrent episodes of respiratory disease in 45-70 days-old pigs from September 2012 to June 2013. The antigenic and genetic characterization of the swine IAV isolates showed the contemporary circulation of H1N1 avian-like and H1N1pdm strains in the first outbreak. The analysis of the viruses isolated subsequently showed the circulation of H1N1pdm IAV and then the establishment of a new previously undescribed H1N1 reassortant strain with a pandemic derived hemagglutinin gene and all the other seven segments of swine H1N1 avian-like lineage.

Is there a relation between genetic or social groups of mallard ducks and the circulation of low pathogenic avian influenza viruses?

We investigated the circulation dynamics of low pathogenic avian influenza viruses (LPAIVs) in the mallard (Anas platyrhynchos) reservoir in Italy. In particular, we evaluated the temporal distribution of virologic findings by combining virus isolation data with a new population genetic-based study approach. Thus, during 11 consecutive sampling periods (wintering periods between 1993/94 and 2003/04), categorised into 40 sampling sub-periods, cloacal swab samples were collected from 996 wild and 16 captive-reared mallards, to be screened by RT-PCR before attempting influenza A virus isolation in embryonated eggs. Forty-eight LPAIVs were isolated from wild mallards and antigenically characterised by haemagglutination-inhibition and neuraminidase-inhibition assays. When considering LPAIV antigenic subtypes in which more than one mallard tested virus isolation positive (H1N1, n. 22; H2N3, n. 2; H5N3, n. 2; H6N5, n. 3; H6N8, n. 2; H7N3, n. 3; H11N6, n. 5), at least two birds infected with a specific HN subtype clustered within one same sampling sub-period. In the context of the novel population genetic approach, total DNA was extracted from a subset of 16 captive-reared and 65 wild ducks (2000/01 and 2001/02 sampling periods) to assess genetic diversity by amplified fragment length polymorphisms (AFLP) markers. Analyses of AFLP results showed that captive-reared mallards clustered together, whereas two main independent clusters characterised the distribution pattern of most wild mallards. Within this subset of samples, nearly identical H7N3 LPAIV strains were isolated from two wild mallards belonging to the same genetic cluster. Blood sera were also collected from the above subset of mallards and examined for antibodies to the homologous H7N3 virus strain. Four out of six wild mallards testing H7N3-seropositive by haemagglutination-inhibition assay (2001/02 period) belonged to the genetic cluster including H7N3 virus shedding ducks. Overall, our data raise the possibility of an enhanced transmission and circulation of LPAIVs in genetic or social groups of wild mallards, gathered in flocks possibly related by parentage and/or geographic origin.

Epidemiological survey of swine influenza A virus in the wild boar population of two Italian provinces.

OBJECTIVES: An epidemiological survey was carried out in order to obtain a better understanding of the role of wild boars in the epidemiology of the influenza virus. DESIGN: The samples were submitted to Real-Time PCR testing for gene M of the swine influenza virus (SIV), and virus isolation was performed from the positive PCR samples. Genome sequence analysis was performed on the isolates. Additionally, 1,977 boar sera samples were analyzed using ELISA and hemoagglutination inhibition. SETTING: Over recent years, the wild boar population has greatly increased in Italy, including in areas of high-density industrial pig farming, where the influenza virus is widespread. From July to December 2012, wild boar lung samples were collected in the Parma and Piacenza area, in the Emilia Romagna region. SAMPLE: 354 wild boar lung samples were collected. MAIN OUTCOME MEASURES: Wild-boar influenza A virus infection should be studied more broadly in order to obtain a better understanding of the epidemiological role played by this species. RESULTS: Three SIV strains were isolated out of 12 samples that resulted positive using PCR analysis and they were identified as avian-like SIV subtype H1N1. Phylogenetic analysis of the sequences obtained from isolate A/wild boar/291320/2012 showed that it clustered with recent Italian avian-like H1N1 SIVs isolated from domestic pigs. Sixty-eight sera samples showed a positive titer to the isolate A/wild boar/291320/2012. CONCLUSIONS: This study suggests that SIV actively circulates in the wild boar population in the investigated. area.

Full-Genome Sequence of a Reassortant H1N1 Swine Influenza Virus Isolated from Pigs in Italy.

In this study, the full-genome sequence of a novel reassortant H1N1 swine influenza virus (SIV) is reported. The isolate has a hemagglutinin (HA) gene of the pandemic H1N1 influenza virus, but it carries the seven genome segments of the avian-origin H1N1 SIV currently circulating in European pig farms.