Long-Term Serological Investigations of Influenza A Virus in Free-Living Wild Boars (Sus scrofa) from Northern Italy (2007-2014).

Influenza A viruses (IAV) have been repeatedly demonstrated to circulate in wild suid populations. In this study, serum samples were collected from 2618 free-ranging wild boars in a protected area of Northern Italy between 2007 and 2014, and firstly screened by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against IAV. The ELISA-positive samples were further tested by hemagglutination inhibition (HI) assays performed using antigen strains representative of the four major swine IAV (sIAV) lineages circulating in Italy: avian-like swine H1N1, pandemic-like swine H1N1, human-like swine H1N2 and human-like swine H3N2. An overall seroprevalence of 5.5% (145/2618) was detected by ELISA, with 56.7% (80/141) of screened sera tests positive by HI assay. Antibodies against H1N1 subtypes were the most prevalent beginning in 2009-with the highest detection in the first quarter of the year-until 2013, although at a low level. In addition, antibodies to H3N2 subtype were found during six years (2007, 2009, 2010, 2011, 2012 and 2014) whereas H1N2 antibodies were detected in 2012 only. Of the HI-positive samples, 30% showed reactivity to both H1N1 and H3N2 subtypes. These results provide additional insight into the circulation dynamics of IAV in wild suid populations, suggesting the occurrence of sIAV spillover events from pigs to wild boars.

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.

Genetic Variability among Swine Influenza Viruses in Italy: Data Analysis of the Period 2017-2020.

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs' evolution could generate strains with zoonotic potential.

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.

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.

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses.

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, p<0.01) compared to the MDCK cells and embryonated chicken eggs for the isolation of H1N1 and H1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p<0.01).

[Further research on the kinetics of inactivation of HAV and enteroviruses with peracetic acid. Note II]. / Ulteriori indagini comparative sulle cinetiche di inattivazione dell'Hav e di enterovirus con acido peracetico. Nota II.

Further research was performed on the disinfectant efficacy of peracetic acid on viruses. Hepatitis A virus (HM-175 strain), Poliovirus 2 (attenuated strain) and Coxsackie virus B5 (isolated strain) were tested using peracetic acid concentrations between 160 mg/l and 1280 mg/l. Inactivation tests were carried out at +20 degrees C and at 3 different pH conditions, acid, neutral and basic. The results confirmed that HAV virus, which showed a decline with concentrations above 320 mg/l, has the highest resistance with respect to the other viruses. The other two Enteroviruses were found to be less resistant, especially in neutral and acid conditions.