Serological Survey of Aujeszky's Disease in Wild Boar from Southeastern France.

Aujeszky's disease virus (ADV), also known as pseudorabies virus, causes an important neurological infection with a major economic and health impact on animal husbandry. Here, we serologically screened muscle fluid from wild boar (Sus scrofa) for the presence of anti-ADV antibodies. Animals were caught during two hunting seasons (2019−2020 and 2021−2022) from three areas in southeastern France known to be endemic with wild boar populations. A total of 30.33% of the 399 tested animals scored positive for anti-glycoprotein B antibodies directed against ADV using a commercial competitive ELISA test. A significant effect (p-value < 0.0001) of the geographical location and animal age on ADV seroprevalence was observed. The results of this study confirmed the importance of wild boar in the epidemiology of ADV in southeastern France.

Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (swIAV) are major pathogens of the porcine respiratory disease complex, but little is known on their interaction in super-infected pigs. In this study, we investigated clinical, virological and immunological outcomes of successive infections with PRRSV-1 and H1N2 swIAV. Twenty-four specific pathogen-free piglets were distributed into four groups and inoculated either with PRRSV at study day (SD) 0, or with swIAV at SD8, or with PRRSV and swIAV one week apart at SD0 and SD8, respectively, or mock-inoculated. In PRRSV/swIAV group, the clinical signs usually observed after swIAV infection were attenuated while higher levels of anti-swIAV antibodies were measured in lungs. Concurrently, PRRSV multiplication in lungs was significantly affected by swIAV infection, whereas the cell-mediated immune response specific to PRRSV was detected earlier in blood, as compared to PRRSV group. Moreover, levels of interferon (IFN)-α measured from SD9 in the blood of super-infected pigs were lower than those measured in the swIAV group, but higher than in the PRRSV group at the same time. Correlation analyses suggested an important role of IFN-α in the two-way interference highlighted between both viral infections.

Concomitant Swine Influenza A Virus Infection Alters PRRSV1 MLV Viremia in Piglets but Does Not Interfere with Vaccine Protection in Experimental Conditions.

Modified-live vaccines (MLVs) against porcine reproductive and respiratory syndrome viruses (PRRSVs) are usually administrated to piglets at weaning when swine influenza A virus (swIAV) infections frequently occur. SwIAV infection induces a strong interferon alpha (IFNa) response and IFNa was shown to abrogate PRRSV2 MLV replication and an inherent immune response. In this study, we evaluated the impacts of swIAV infection on the replication of a PRRSV1 MLV (MLV1), post-vaccine immune responses and post-challenge vaccine efficacy at both the systemic and pulmonary levels. Piglets were either swIAV inoculated and MLV1 vaccinated 6 h apart or singly vaccinated or mock inoculated and mock vaccinated. Four weeks after vaccination, the piglets were challenged with a PRRSV1 field strain. The results showed that swIAV infection delayed MLV1 viremia by six days and post-vaccine seroconversion by four days. After the PRRSV1 challenge, the swIAV enhanced the PRRSV1-specific cell-mediated immunity (CMI) but the PRRSV1 field strain viremia was not better controlled. High IFNa levels that were detected early after swIAV infection could have been responsible for both the inhibition of MLV1 replication and CMI enhancement. Thus, whereas swIAV infection had a negative impact on humoral responses post-vaccination, it did not interfere with the protective effectiveness of the PRRSV MLV1 in our experimental conditions.

Genetic and Antigenic Evolution of European Swine Influenza A Viruses of HA-1C (Avian-Like) and HA-1B (Human-Like) Lineages in France from 2000 to 2018.

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1av) and -1B (H1hu), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1avNy and 105 H1huNy strains were submitted to hemagglutination inhibition tests. H1avN1 (66.5%) and H1huN2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named "Δ146-147" harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the "Eurasian avian-like lineage", with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1av/H1hu strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.

Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus.

The surveillance of swine influenza A viruses in France revealed the emergence of an antigenic variant following deletions and mutations that are fixed in the HA-encoding gene of the European human-like reassortant swine H1N2 lineage. In this study, we compared the outcomes of the parental (H1huN2) and variant (H1huN2Δ146-147) virus infections in experimentally-inoculated piglets. Moreover, we assessed and compared the protection that was conferred by an inactivated vaccine currently licensed in Europe. Three groups of five unvaccinated or vaccinated piglets were inoculated with H1huN2 or H1huN2Δ146-147 or mock-inoculated, respectively. In unvaccinated piglets, the variant strain induced greater clinical signs than the parental virus, in relation to a higher inflammatory response that involves TNF-α production and a huge afflux of granulocytes into the lung. However, both infections led to similar levels of virus excretion and adaptive (humoral and cellular) immune responses in blood. The vaccinated animals were clinically protected from both infectious challenges and did not exhibit any inflammatory responses, regardless the inoculated virus. However, whereas vaccination prevented virus shedding in H1huN2-infected animals, it did not completely inhibit the multiplication of the variant strain, since live virus particles were detected in nasal secretions that were taken from H1huN2Δ146-147-inoculated vaccinated piglets. This difference in the level of vaccine protection was probably related to the poorer ability of the post-vaccine antibodies to neutralize the variant virus than the parental virus, even though post-vaccine cellular immunity appeared to be equally effective against both viruses. These results suggest that vaccine antigens would potentially need to be updated if this variant becomes established in Europe.

Coinfections and their molecular consequences in the porcine respiratory tract.

Understudied, coinfections are more frequent in pig farms than single infections. In pigs, the term "Porcine Respiratory Disease Complex" (PRDC) is often used to describe coinfections involving viruses such as swine Influenza A Virus (swIAV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine CircoVirus type 2 (PCV2) as well as bacteria like Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Bordetella bronchiseptica. The clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. In this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. Coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. We discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health.

Genetic Diversity among Pseudorabies Viruses Isolated from Dogs in France from 2006 to 2018.

Pseudorabies (PR), also known as Aujeszky's disease, is an economically important disease for the pig industry. It has been eradicated in domestic pigs in many European countries, including France, but its causative agent-Suid Herpesvirus 1-is still circulating in wild boars. The risk of endemic PR in wild fauna lies in reintroducing the virus among domestic pigs and transmitting it to other mammals, especially hunting dogs for which the disease is rapidly fatal. As such infections are regularly reported in France, this study genetically characterized canine PR virus strains in the country to obtain information on their diversity and evolution. Partial sequencing of the glycoprotein C-encoding gene from 55 virus strains isolated from dogs between 2006 and 2018 showed that 14 strains belonged to genotype I-clade A and another 38 to genotype I-clade B, two clades usually reported in Western Europe. More surprisingly, three strains were found to belong to genotype II, suggesting an Asian origin. Genotype I-clade A strains exhibited the highest diversity as five geographically segregated genogroups were identified.

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.

Involvement of swine influenza A viruses in the porcine respiratory disease complex.

Respiratory diseases constitute a major problem in pig farms, often resulting from multiple infections with different respiratory pathogens. This phenomenon is referred to as porcine respiratory disease complex (PRDC). Swine influenza viruses of type A (swIAV) affect half of French farms and are often isolated from PRDC cases. The flu severity is recognized to be strongly influenced by the presence of other respiratory pathogens but interactions between microorganisms are still poorly understood. Development of experimental models of co-infections is required to better understand mechanisms underlying flu exacerbation that is a prerequisite for improving measures for disease control. This review summarizes current knowledge on flu disease in pig and swIAV's involvement in PRDC, as well as results of in vivo co-infections studies involving swIAVs. The mechanisms responsible for the flu exacerbation in case of co-infection with Mycoplasma hyopneumoniae (Mhp) are more particularly discussed, the swIAV/Mhp experimental model having been the most studied to date. In this case, it appears that the severe flu is due to additive inflammatory responses rather than due to a synergistic effect between both pathogens.

Mycoplasma hyopneumoniae does not affect the interferon-related anti-viral response but predisposes the pig to a higher level of inflammation following swine influenza virus infection.

In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.

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 (RT-PCR) 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.

Effect of feed restriction on performance and postprandial nutrient metabolism in pigs co-infected with Mycoplasma hyopneumoniae and swine influenza virus.

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.

Validation of a commercial real-time PCR kit for specific and sensitive detection of Pseudorabies.

Pseudorabies virus is the causative agent of Aujeszky's disease, one of the OIE listed diseases that mainly affects swine, but also can affect other animal species, and which can lead to heavy economic losses in pig industry. This study was designed to evaluate the performance of the ADIAVET(®) PRV REALTIME kit, a new commercial real time PCR kit for Pseudorabies virus genome detection developed by the French manufacturer Adiagène. It can be used on pig biological samples such as nasal swab supernatant, tonsil, brain or lung samples, or on samples from other susceptible animals, such as domestic carnivores. This ready-to-use duplex PCR assay contains an external positive control, appropriate for assessing DNA extraction efficiency and the presence of PCR inhibitors. The analytical specificity and sensitivity, intra- and inter-assay repeatability and diagnostic characteristics of the kit were determined and compared with virus isolation, which is the gold standard. Based on these results, the ADIAVET(®) PRV REALTIME kit received full validation for diagnostic purposes.

Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs.

Swine influenza, apart from its importance in animal health, may also be of public health significance. Although the first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure, this virus was shown to be related genetically to swine influenza viruses (SIV) and easily transmissible to pigs. In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences on the risk of human infection by increasing the reservoir of this virus and the risk of possible emergence of new reassortant viruses with increased virulence for pigs and/or humans. Sensitive tools to monitor and detect rapidly such an infection are therefore mandatory. In this study, five commercial real-time RT-PCR assays developed by manufacturers LSI and Adiagène were assessed and validated, (i) for rapid detection of influenza A viruses, including pH1N1/09, in pig and (ii) for the differentiation of pH1N1/09 in that species. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene. All five kits are ready-to-use, one-step duplex RT-PCR and contain an internal positive control (IPC), appropriate for porcine biological samples, for assessing RNA extraction efficiency and the presence of PCR inhibitors. They have been used successfully by veterinary laboratories and shown to be powerful tools for the diagnosis and epidemiological surveillance of influenza virus infections in pigs.

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells.

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication.