Séverine Hervé

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.

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.

Study of influenza A virus in wild boars living in a major duck wintering site

Wild birds, which are reservoirs of influenza viruses, are believed to be the original source of new influenza viruses-including highly pathogenic ones-that can be transmitted to domestic animals as well as humans and represent a potential epizootic and/or pandemic threat. Despite increasing knowledge on influenza A virus dynamics in wild birds, the viral circulation in wild boars remains largely unknown. This is of particular interest since pigs can be infected with both human and avian viruses; upon co-infection, they can act as a mixing vessel through reassortment, a mechanism that resulted in the emergence of the pandemic H1N1 virus in 2009. The Camargue (Southern France) appears as an ideal study area to investigate inter-species transmission of influenza A viruses from wild birds and possibly humans to wild boars. Indeed, the important local wild boar population shares wetland use with humans and the largest concentration of wintering ducks in France, that are both susceptible to infection by influenza A viruses. Additionally, wild boars occasionally prey on ducks. We conducted a virological and serological survey on wild boars in the Camargue (Southern France) between September 2009 and November 2010. No influenza A virus was detected in the collected nasal swabs (n=315) and no influenza specific antibodies were observed in the serological samples (n=20). As the study was mainly focused on viral excretion, which is limited in time, we cannot exclude that low or occasional influenza A virus circulation took place during the study period. Although, wild boars did not seem to be a key element in the dynamics of influenza A virus circulation in the Camargue, wild boar influenza A virus infections should be more widely studied to determine if the pattern observed here represents the normal situation or an exceptional one.

Influenza A(H1N1)pdm09 Virus in Pigs, Réunion Island

During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended.

Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis

BackgroundSwine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important.ResultsSeveral reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146–147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the β-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts.ConclusionThe RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146–147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.

Maternally-derived antibodies do not inhibit swine influenza virus replication in piglets but decrease excreted virus infectivity and impair post-infectious immune responses

Maternally-derived antibodies (MDA) reduce piglet susceptibility to swine influenza A virus, but interfere with post-infectious immune responses, raising questions about protection after waning of passive immunity. We therefore analysed the impact of different levels of residual MDA on virus excretion and immune responses in piglets born to vaccinated sows (MDA+) and infected with H1N1 at 5, 7 or 11 weeks of age, in comparison to piglets born to unvaccinated sows (MDA-). Subsequent protection against a second homologous infection occurring 4 weeks after the primo-infection was also investigated. MDA- pigs showed clinical signs, shed the virus, and developed specific immune responses despite some age-dependent differences: 7-week-old pigs were less affected clinically, showed a 2-day delayed excretion peak and excreted less virus than younger pigs. In MDA+ animals, clinical signs increased together with the decrease of MDA levels related to the age at infection-time. Virus shedding was not prevented and genome quantification profiles were similar to those obtained in MDA- piglets. However, viral particles excreted by 5-week-old MDA+ piglets appeared to be less infectious than those shed by MDA- piglets at the same age. Humoral response was affected by MDA as illustrated by the absence of HI and neutralizing response regardless the infection age, but anti-NP/M responses were less affected. Proliferative T cell responses were slightly delayed by high MDA levels. Nevertheless, MDA+ animals were all protected from a second infection, like MDA- piglets. In conclusion, responses of pigs to H1N1 were affected by both the physiological development of animals at infection and the MDA level.

Serological survey of influenza A viruses in domestic and wild Suidae in Corsica (France), a Mediterranean island environment

Corsica is a mountainous French island in the north-western Mediterranean Sea. It is a rural area, where pig farming is a major economic activity. Although no acute respiratory outbreaks due to swine influenza A viruses (swIAVs) have ever been reported in this free-ranging pig breeding system, influenza A viruses (IAVs) could be circulating within this pig population. A serological study was conducted as a first approach to domestic pigs and wild boars. Serum samples from 543 pigs raised on 91 different farms were collected during the 2013-2014 slaughtering season, and 279 sera from wild boars were obtained over four hunting seasons (between 2009 and 2014). They were first analysed by ELISA and then IAV positive and doubtful sera were subjected to haemagglutination inhibition tests using antigens representative of the four major enzootic swIAV lineages in Europe, i.e. avian-like swine H1N1 (H1avN1), pandemic-like swine H1N1 (H1N1pdm), H1N2 and H3N2. According to the ELISA results, 26.4% (CI95%: 17.7-36.7%) of herds had at least one positive animal (positive or doubtful by ELISA) and 12.4% (CI95%: 7.8-19.8%) of the pigs tested positive. Using the test characteristics (sensitivity and specificity), the true seroprevalence among Corsican pigs was estimated to be 16.4% (95% CI: 9.9-26.3). Antibodies directed against two different viral lineages were identified: H1N1pdm (in 66.2% and 45.8% of the IAV positive pigs and farms respectively) and H1avN1 (15.0% and 20.8% respectively). Evidence of exposure to viruses from two distinct lineages were detected on a single farm but in two different animals. Among the wild boars, 1.4% (CI95%: 0.4-3.6%) tested positive by ELISA and antibodies against the same two viruses were detected. Altogether, these results suggest that swIAVs from at least two different lineages are circulating among Corsican pigs, i.e. the H1N1pdm virus, probably introduced during the 2009 pandemic, and the H1avN1 virus, which is the most frequent swIAV in Europe. The low frequency of positive results observed in the Corsican wild boars hunted suggests that they would not play a major role in IAV dispersion dynamics on the island.

Erratum to: Maternally-derived antibodies do not prevent transmission of swine influenza A virus between pigs

A transmission experiment involving 5-week-old specific-pathogen-free (SPF) piglets, with (MDA+) or without maternally-derived antibodies (MDA−), was carried out to evaluate the impact of passive immunity on the transmission of a swine influenza A virus (swIAV). In each group (MDA+/MDA−), 2 seeders were placed with 4 piglets in direct contact and 5 in indirect contact (3 replicates per group). Serological kinetics (ELISA) and individual viral shedding (RTPCR) were monitored for 28 days after infection. MDA waning was estimated using a nonlinear mixed-effects model and survival analysis. Differential transmission rates were estimated depending on the piglets’ initial serological status and contact structure (direct contact with pen-mates or indirect airborne contact). The time to MDA waning was 71.3 [52.8–92.1] days on average. The airborne transmission rate was 1.41 [0.64–2.63] per day. The compared shedding pattern between groups showed that MDA+ piglets had mainly a reduced susceptibility to infection compared to MDA− piglets. The resulting reproduction number estimated in MDA+ piglets (5.8 [1.4–18.9]), although 3 times lower than in MDA− piglets (14.8 [6.4–27.1]), was significantly higher than 1. Such an efficient and extended spread of swIAV at the population scale in the presence of MDAs could contribute to swIAV persistence on farms, given the fact that the period when transmission is expected to be impacted by the presence of MDAs can last up to 10 weeks.