The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin.

This paper reviews important aspects of infection of pigs with avian influenza viruses. Wild waterfowl are the main reservoir for influenza A viruses; other species, such as pigs, can be infected, but most avian strains are imperfectly adapted to replication and transmission in such new hosts. However, some avian-to-porcine host jumps have resulted in the emergence of stable swine influenza virus lineages, with major consequences for both animal and human health. Different categories of factors are involved in these cross-species adaptations, both epidemiological (relating to host-host interactions) and virological (relating to host-virus interactions). An understanding of the adaptation of avian influenza viruses to pigs has benefited from a number of recent studies, but more research is warranted to fully appreciate the key molecular and epidemiological factors involved in this intriguing viral host jump.

Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells-critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells-important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements.

Influenza A virus in swine (IAV-S) circulating in the United States of America are phylogenetically and antigenically distinct. A human H3 hemagglutinin (HA) was introduced in the IAV-S gene pool in the late 1990s, sustained continued circulation, and evolved into five monophyletic genetic clades after 2009, H3 IVA-E. Across these phylogenetic clades, distinct antigenic clusters were identified, with three clusters (cyan, red and green) among the most frequently detected antigenic phenotypes. Although it was demonstrated that antigenic diversity of H3N2 IAV-S was associated with changes at a few amino acid positions in the head of the HA, the implications of this diversity on vaccine efficacy was not tested. Using antigenically representative H3N2 viruses, we compared whole inactivated virus (WIV) and live attenuated influenza vaccine (LAIV) vaccines for protection against challenge with antigenically distinct H3N2 viruses in pigs. WIV provided partial protection against antigenically distinct viruses, but did not prevent virus replication in the upper respiratory tract. In contrast, LAIV provided complete protection from disease and virus was not detected after challenge with antigenically distinct viruses.IMPORTANCE Due to the rapid evolution of the influenza A virus, vaccines require continuous strain updates. Additionally, the platform used to deliver the vaccine can have an impact on the breadth of protection. Currently, there are various vaccine platforms available to prevent influenza A virus infection in swine, and we experimentally tested two: adjuvanted-whole inactivated virus and live attenuated virus. When challenged with an antigenically distinct virus, adjuvanted-whole inactivated virus provided partial protection while live attenuated virus provided effective protection. Additional strategies are required to broaden the protective properties of inactivated virus vaccines given the dynamic antigenic landscape of co-circulating strains in North America, whereas live attenuated vaccines may require less frequent strain updates based on demonstrated cross-protection. Enhancing vaccine efficacy to control influenza infections in swine will help reduce the impact it has on swine production and reduce the risk of swine-to-human transmission.

Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.

Influenza A virus (IAV) causes a disease burden in the swine industry in the US and is a challenge to prevent due to substantial genetic and antigenic diversity of IAV that circulate in pig populations. Whole inactivated virus (WIV) vaccines formulated with oil-in-water (OW) adjuvant are commonly used in swine. However, WIV-OW are associated with vaccine-associated enhanced respiratory disease (VAERD) when the hemagglutinin and neuraminidase of the vaccine strain are mismatched with the challenge virus. Here, we assessed if different types of adjuvant in WIV vaccine formulations impacted VAERD outcome. WIV vaccines with a swine δ1-H1N2 were formulated with different commercial adjuvants: OW1, OW2, nano-emulsion squalene-based (NE) and gel polymer (GP). Pigs were vaccinated twice by the intramuscular route, 3 weeks apart, then challenged with an H1N1pdm09 three weeks post-boost and necropsied at 5 days post infection. All WIV vaccines elicited antibodies detected using the hemagglutination inhibition (HI) assay against the homologous vaccine virus, but not against the heterologous challenge virus; in contrast, all vaccinated groups had cross-reactive IgG antibody and IFN-γ responses against H1N1pdm09, with a higher magnitude observed in OW groups. Both OW groups demonstrated robust homologous HI titers and cross-reactivity against heterologous H1 viruses in the same genetic lineage. However, both OW groups had severe immunopathology consistent with VAERD after challenge when compared to NE, GP, and non-vaccinated challenge controls. None of the WIV formulations protected pigs from heterologous virus replication in the lungs or nasal cavity. Thus, although the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar H1, none tested here increased the breadth of protection against the antigenically-distinct challenge virus, and some impacted immunopathology after challenge.

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.