Search

African swine fever (ASF) causes fatal disease in pigs and is an escalating threat to the global swine industry. ASF has re-emerged from Africa as a transcontinental epidemic spreading through the Caucasus into Europe, Russia, China, numerous Asian countries, and the Caribbean. ASF virus (ASFV) is a U.S. select agent requiring handling in high-containment biosafety level 3 (BSL-3) laboratories for pathogen work. Formalin-fixation eliminates infectivity and preserves the genome, providing noninfectious specimens for BSL-2 work. Recovery of DNA from formalin-fixed, paraffin-embedded tissue (FFPET) is challenging and cumbersome. A reliable and easy-to-perform method for DNA recovery from FFPET would facilitate surveillance. To meet this objective, we developed a high-throughput protocol for the recovery of ASFV DNA from FFPET. Deparaffinization, tissue lysis, and reversal of cross-linking were performed in a single tube, followed by DNA purification via automated magnetic bead extraction. Quantitative PCR (qPCR) detection was used to determine the copy number of the B646L gene that encodes for the ASFV p72 protein in tissues (5 pigs, 4 tissues) from pigs with lesions consistent with acute ASF. Copy numbers obtained from FFPET were within one log of copy numbers obtained from fresh tissue, thus enabling ASF qPCR surveillance from formalin-inactivated and preserved tissues at BSL-2 at diagnostic sensitivity similar to fresh tissues tested at BSL-3.

Introduction The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig’s body. Material and Methods A total of 25 in vivo avIAV passages of the A/duck/Bavaria/77 strain were performed by inoculation of 50 piglets, and after predetermined numbers of passages 20 uninoculated piglets were exposed to the virus through contact with inoculated animals. Clinical signs of swine influenza were assessed daily. Nasal swabs and lung tissue were used to detect IAV RNA by real-time RT-PCR and isolates from selected passages were sequenced. Results Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet. Conclusion Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passagesA total of 25Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet.Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes periodic outbreaks of abortion in ruminant species and hemorrhagic disease in humans in sub-Saharan Africa. These outbreaks have a significant impact on veterinary and public health. Its introduction to the Arabian Peninsula in 2003 raised concerns of further spread of this transboundary pathogen to non-endemic areas. These concerns are supported by the presence of competent vectors in many non-endemic countries. There is no licensed RVF vaccine available for humans and only a conditionally licensed veterinary vaccine available in the United States. Currently employed modified live attenuated virus vaccines in endemic countries lack the ability for differentiating infected from vaccinated animals (DIVA). Previously, the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins, derived from the 1977 human RVFV isolate ZH548, was demonstrated in sheep. In the current study, cattle were vaccinated subcutaneously with the Gn only, or Gn and Gc combined, with either one or two doses of the vaccine and then subjected to heterologous virus challenge with the virulent Kenya-128B-15 RVFV strain, isolated from Aedes mosquitoes in 2006. The elicited immune responses by some vaccine formulations (one or two vaccinations) conferred complete protection from RVF within 35 days after the first vaccination. Vaccines given 35 days prior to RVFV challenge prevented viremia, fever and RVFV-associated histopathological lesions. This study indicates that a recombinant RVFV glycoprotein-based subunit vaccine platform is able to prevent and control RVFV infections in target animals.

The recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

The 2 predominant circulating subtypes of influenza A virus in the dog population, equine-origin H3N8 and avian-origin H3N2, constitute a potential zoonotic risk. We determined the prevalence of influenza A antibodies in 496 dogs in Poland and found 2.21% of sera positive by commercial ELISA. Hemagglutination inhibition (HI) assays indicated 7.25% of sera positive using equine H3N8, swine H3N2, and pandemic H1N1 antigens, with the most frequently detected immune response being to H3N2. Considering interspecies transfer, reassortment ability, and close contact between dogs and humans, infections of dogs with influenza A virus should be monitored.

Epizootic hemorrhagic disease virus (EHDV) is an arthropod-transmitted RNA virus and the causative agent of epizootic hemorrhagic disease (EHD) in wild and domestic ruminants. In North America, white-tailed deer (WTD) experience the highest EHD-related morbidity and mortality, although clinical disease is reported in cattle during severe epizootics. No commercially licensed EHDV vaccine is available in North America. The objective of this study was to develop and evaluate a subunit vaccine candidate to control EHD in WTD. Recombinant VP2 (rVP2) outer capsid proteins of EHDV serotypes 2 (EHDV-2) and 6 (EHDV-6) were produced in a baculovirus-expression system. Mice and cattle vaccinated with EHDV-2 or EHDV-6 rVP2 produced homologous virus-neutralizing antibodies. In an immunogenicity/efficacy study, captive-bred WTD received 2 doses of EHDV-2 rVP2 or sham vaccine, then were challenged with wild-type EHDV-2 at 30 d post vaccination. None of the rVP2-vaccinated deer developed clinical disease, no viral RNA was detected in their blood or tissues (liver, lung, spleen, kidney), and no EHDV-induced lesions were observed. Sham-vaccinated deer developed clinical disease with viremia and typical EHD vascular lesions. Here, we demonstrate a rVP2 subunit vaccine that can provide protective immunity from EHDV infection and which may serve as an effective tool in preventing clinical EHD and reducing virus transmission.

Rift Valley fever virus, a zoonotic arbovirus, poses major health threats to livestock and humans if introduced into the United States. White-tailed deer, which are abundant throughout the country, might be sentinel animals for arboviruses. We determined the susceptibility of these deer to this virus and provide evidence for a potentially major epidemiologic role.

African swine fever virus (ASFV) causes high morbidity and mortality in swine (Sus scrofa), for which there is no commercially available vaccine. Recent outbreaks of the virus in Trans-Caucasus countries, Eastern Europe, Belgium and China highlight the urgent need to develop effective vaccines against ASFV. Previously, we evaluated the immunogenicity of a vaccination strategy designed to test various combinations of ASFV antigens encoded by DNA plasmids and recombinant proteins with the aim to activate both humoral and cellular immunity. Based on our previous results, the objective of this study was to test the combined DNA-protein vaccine strategy using a cocktail of the most immunogenic antigens against virulent ASFV challenge. Pigs were vaccinated three times with a cocktail that included ASFV plasmid DNA (CD2v, p72, p32, +/−p17) and recombinant proteins (p15, p35, p54, +/−p17). Three weeks after the third immunization, all pigs were challenged with the virulent ASFV Armenia 2007 strain. The results showed that vaccinated pigs were not protected from ASFV infection or disease. Compared to the non-vaccinated controls, earlier onset of clinical signs, viremia, and death were observed for the vaccinated animals following virulent ASFV challenge. ASFV induced pathology was also enhanced in the vaccinated pigs. Furthermore, while the vaccinated pigs developed antigen-specific antibodies, immunized pig sera at the time of challenge lacked the capacity to neutralize virus, and instead was observed to enhance ASFV infection in vitro. The results of this work points to a putative immune enhancement mechanism involved in ASFV pathogenesis that warrants further investigation. This pilot study provides insight for the selection of appropriate combinations of ASFV antigens for the development of a rationally-designed, safe, and efficacious vaccine for ASF., This work was funded by grants from the U.S. Department of Homeland Security under Grant Award Number DHS-2010-ST-061-AG0001 for the Center of Excellence for Emerging and Zoonotic Animal Disease (CEEZAD) and the State of Kansas National Bio and Agro-Defense Facility (NBAF) transition fund, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

The aim of the study was to monitor genetic diversity and antigenic changes in the genome of influenza A(H1N1)pdm09 viral isolates detected during the post-pandemic period in Poland. Clinical specimens obtained from three suspected cases of influenza were analysed by sequencing. Among the differences identified in amino acids sequences, nine substitutions were located within the antigenic HA1 sites and in five residues forming receptor-binding pocket. The HA(D222G) mutation was shown in the isolate Swine/Poland/134312/12 obtained from a mild case of the disease. It must be emphasized that, in general, clinically mild cases are caused by the viruses in which that specific mutation, i.e. haemagglutinin (D222G), does not occur.

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, H1 hu N2 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.

The kinetics of C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA), and pig major acute protein (Pig-MAP) response in pigs co-infected with H3N2 swine influenza virus (SwH3N2) and Bordetella bronchiseptica (Bbr) was studied, with assessment of potential correlations between the concentration of acute phase proteins (APPs) in serum samples, lung lesions, and the clinical course of the disease in co-infected pigs. The standard bacteriological methods for detection of Bbr and PCR technique for identification of Bbr and SwH3N2 were used. The serum concentrations of APPs were measured using ELISA. The concentration of CRP, SAA, and Pig-MAP was significantly higher from 2 to 4 or 5 dpi. The concentration of Hp was elevated until the end of the study. Significant correlations were found between the serum concentration of SAA and Pig-MAP and clinical score, and between the concentration of SAA and lung score. Apart from their potential as biological markers for co-infections, SAA and Pig-MAP levels have additive value since they are related to the severity of infection. The results indicate that measurement of APP (i.e SAA) may prove valuable in assessing the severity of respiratory infection in pigs, and may be of supportive value in the clinical evaluation of animals and in the selection of more appropriate treatment.

Background The influenza A virus is highly variable, which, to some degree, is caused by the reassortment of viral genetic material. This process plays a major role in the generation of novel influenza virus strains that can emerge in a new host population. Due to the susceptibility of pigs to infections with avian, swine and human influenza viruses, they are considered intermediate hosts for the adaptation of the avian influenza virus to humans. In order to test the reassortment process in pigs, they were co-infected with H3N2 A/swine/Gent/172/2008 (Gent/08) and H1N1 A/duck/Italy/1447/2005 (Italy/05) and co-housed with a group of naïve piglets. Results The Gent/08 strains dominated over Italy/05, but reassortment occurred. The reassortant strains of the H1N1 subtype (12.5%) with one gene (NP or M) of swine-origin were identified in the nasal discharge of the contact-exposed piglets. Conclusions These results demonstrate that despite their low efficiency, genotypically and phenotypically different influenza A viruses can undergo genetic exchange during co-infection of pigs. Electronic supplementary material The online version of this article (doi:10.1186/s12917-017-1137-x) contains supplementary material, which is available to authorized users.

The outbreaks of pandemic H1N1 influenza A virus (pdm-like H1N1 2009), detected for the first time in farrow-to-finish farms in Poland, were described. The nasal swabs and lung tissue collected from diseased/dead animals were tested using molecular techniques (RRT-PCR, MRT-PCR, RT-PCR, SSG-PCR, sequencing) and virus isolation. The amplification of the genetic material extracted from the tested samples confirmed the presence of the M1 gene sequence of type A influenza virus. Using MRT-PCRs no products characteristic for HA and NA of any swine influenza virus subtypes were obtained. Using SSGPCR, products specific for pandemic HA and NA gene fragments were detected. Six new pdm-like H1N1 2009 strains were isolated and characterised. Phylogenetic analysis of the HA and NA genes revealed that they belong to one lineage with the pandemic strain A/California/04/2009 and other human strains, including human strains isolated in Poland in 2011.

The paper describes an outbreak of swine influenza, which was the first H1N2 outbreak confirmed in pig farm in Poland. The outbreak occurred in October 2011 in the farrows to finish farm, first in the fattening units, and subsequently in the reproduction and weaning sectors. Samples of the lungs taken from dead gilts were tested by the use of real time and multiplex PCR, sequencing, and virus isolation methods. The amplification of the genetic material extracted from the lungs confirmed the presence of the M1 gene sequence of type A influenza virus. Using multiplex PCR, the bands of 241 bp, typical for HA1 human-like type, and of 791 bp, typical for NA2, were demonstrated. The new isolates of swine influenza virus, named A/swine/Poland/15817/2011, were obtained in embryonated SPF chicken eggs and MDCK cells. Phylogenetic analysis of the HA and NA genes of the isolate revealed that the virus is of human swine linkage closely related to European human-like viruses. On the basis of such results, it can be stated that A/Swine/Poland/15817/2011 is a new swine influenza virus, representing the H1N2 subtype strain, which emerged in Poland.

Phylogenetic analysis of the genes determining influenza virus subtype - haemagglutinin (HA) and neuraminidase (NA), was performed. The results showed that the Polish H1N2 isolate (A/Swine/Poland15817/2011) was reassortant of human-like swine H1N1 and human-like swine H3N2 origin. The novel isolate was presented to have a close phylogenic relationship with one of the latest European isolates of H1N2 (A/SW/Gent/102/2007 and A/SW/Hungary/13509/2007). Our evolutionary analyses also suggested that the HA and NA genes evolved in a significantly higher rate of synonymous substitutions after they were introduced from human to swine and established the European H1N2 swine lineage.

The aim of the study was to estimate the current epidemiological situation concerning swine influenza (SI) in Poland. The study was based on an annual passive survey of 11,770 fatteners’ sera from 584 herds, taken at slaughterhouses within the last 30 months (from January 2010 till June 2012), as well as, an active monitoring conducted in 2011 and 2012, in 25 farms, using 388 sera taken from life pigs of different age/technological groups. The analysis of simultaneous circulation of different swine influenza virus (SIV) subtypes was taken into a deep consideration. The wide spread of SIV in Poland, including the occurrence of multiple SIV infections was demonstrated. In 2010 and 2011, the domination of H1N1 subtype and the most frequently co-circulation of H1N1 and H1N2 viruses was evidenced, while in the first 6 months of 2012, the co-circulation of H1N1 and H3N2 viruses was detected more often. Based on the obtained results, it can be stated that the epidemiological situation concerning SI in Poland is dynamic and similar to that observed in other European regions with high pigs’ density; however, the prevalence of antibodies and the occurrence of mixed SIV infections is lower than in Western Europe.

Influenza A viruses (IAVs) are zoonotic agents, capable of crossing the species barriers. Nowadays, they still constitute a great challenge worldwide. The natural reservoir of all influenza A viruses are wild aquatic birds, despite the fact they have been isolated from a number of avian and mammalian species, including humans. Even when influenza A viruses are able to get into another than waterfowl population, they are often unable to efficiently adapt and transmit between individuals. Only in rare cases, these viruses are capable of establishing a new lineage. To succeed a complete adaptation and further transmission between species, influenza A virus must overcome a species barrier, including adaptation to the receptors of a new host, which would allow the virus-cell binding, virus replication and, then, animal-to-animal transmission. For many years, pigs were thought to be intermediate host for adaptation of avian influenza viruses to humans, because of their susceptibility to infection with both, avian and human influenza viruses, which supported hypothesis of pigs as a 'mixing vessel'. In this review, the molecular factors necessary for interspecies transmission are described, with special emphasis on adaptation of avian influenza viruses to the pig population. In addition, this review gives the information about swine influenza viruses circulating around the world with special emphasis on Polish strains.

The genetic material of influenza A virus consists of eight negative-sense RNA segments. Under suitable conditions, the segmented structure of the viral genome allows an exchange of the individual gene segments between different strains, causing formation of new reassorted viruses. For reassortment to occur, co-infection with two or more influenza virus strains is necessary. The reassortment is an important evolutionary mechanism which can result in antigenic shifts that modify host range, pathology, and transmission of the influenza A viruses. In this process, the influenza virus strain with epidemic and/or pandemic potential can be created. Cases of this kind were in 1957 (Asian flu), 1968 (Hong Kong flu) and recently in 2009 (Mexico). Viruses containing genes of avian, swine, and/or human origin are widespread around the world, for example the triple reassortant H1N1 virus causing the 2009 influenza pandemic in 2009 that has become a seasonal virus. The aim of the study is to present the mechanism of reassortment and the results of experimental co-infection with different influenza viruses.

A total of 209 Bordetella bronchiseptica (Bbr) strains isolated from pigs were examined. Phenotypic study included: biochemical characterization (motility, catalase, oxidase, urease activity, nitrate reduction and growth on MacConkey agar) and antimicrobial susceptibility (disc diffusion method). Genotypic studies based on detection of three genes encoded virulence factors, such as: flagella (fla), dermonecrotoxin (dnt), and exogenous ferric siderophore receptor (bfrZ), using PCR.Most of the Bbr strains tested had a homogeneous biochemical profile. 97.6% of them provided suitable results in biochemical tests. All Bbr isolates tested showed high resistance to penicillin (100%), linco-spectin (100%) and ceftiofur (97.9%). Over 57% and 43% of Bbr strains were resistant to ampicillin and amoxicillin, respectively. All Bbr isolates showed high sensitivity to most chemotherapeutics used such as enrofloxacin (97.9%), tetracycline (97.9%), oxytetracycline (97.9%), amoxicillin with clavulonic acid (95.8%), florfenicol (90.4%), and gentamicine (77.6%). Over of 94% of Bbr strains were moderately susceptible to norfloxacine.Molecular analysis confirmed that almost all evaluated Bbr strains (94.7%) possessed the fla gene. A lower percentage of isolates had the dnt gene (72.7%) and the lowest percentage of strains (51.7%), had the bfrZ gene.