Susceptibility of mink to certain viral animal diseases foreign to the United States.

Mink (Mustela vison) were inoculated with viruses: African horse sickness (AHS), African swine fever (ASF), bovine herpes virus II (BHV2), foot-and-mouth disease (FMD), goat pox (GP), hog cholera (HC), peste des petits ruminants (PPR), rinderpest (RP), swine vesicular disease (SVD), vesicular exanthema of swine (VES) and vesicular stomatitis (VS). Their susceptibility was measured by development of clinical signs, virus isolation and detection of precipitin and/or virus neutralizing antibodies. SVD virus produced a lesion in one mink. Virus was isolated from mink inoculated with SVD, FMD and BHV2. Neutralizing and/or precipitin antibodies were detected in mink inoculated with ASF, FMD, GP, RP, SVD and VS viruses. Mink were not susceptible to AHS, HC, PPR and VES viruses.     

Hairpin loop structure of African swine fever virus DNA.

The ends of African swine fever virus genome are formed by a 37 nucleotide-long hairpin loop composed, almost entirely, of incompletely paired A and T residues. The loops at each DNA end were present in two equimolar forms that, when compared in opposite polarities, were inverted and complementary (flip-flop), as in the case of poxvirus DNA. The hairpin loops of African swine fever and vaccinia virus DNAs had no homology, but both DNAs had a 16 nucleotide-long sequence, close to the hairpin loops, with an homology of about 80%. An analysis of African swine fever virus replicating DNA showed head-to-head and tail-to-tail linked molecules that may be replicative intermediates.     

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.     

Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture.

An analysis of the binding properties of a collection of monoclonal antibodies to African swine fever virus particles showed that virus field isolates passaged in porcine macrophages changed antigenically more than a strain of a cell-adapted virus passaged in Vero cells. From seven clones isolated from the spleen of a field-infected pig, we found four clones that had the same antigenic properties, one clone that had large changes in proteins p150 and p27 and small changes in proteins p37 and p14, and two clones that had minor changes in proteins p150 and p27, respectively. An analysis of the binding properties of the monoclonal antibodies to 23 field isolates from Africa, Europe, and America showed that the African isolates differed among themselves more than the European and the American isolates; in this study we found changes in 8 of the 10 virus proteins tested. The most variable proteins in the African isolates were p150, p27, p14, and p12. In contrast to the African isolates, protein p12 from the non-African viruses did not change. The clustering of the field virus isolates in six antigenic homology groups indicated the existence of a complex variety of African swine fever virus serotypes.     

Viruses in boar semen: detection and clinical as well as epidemiological consequences regarding disease transmission by artificial insemination

Many viruses have been reported to be present in boar semen, particularly during the viremic phase of the diseases. Some of them, such foot-and-mouth disease virus, porcine reproductive and respiratory syndrome virus, swine vesicular disease virus, porcine parvovirus, picornaviruses, adenoviruses, enteroviruses, Japanese encephalitis virus, pseudorabies virus, African swine fever virus and reoviruses are of particular importance and accurate monitoring prior to and during the presence of boars in AI stations is essential. Various methods may be used to detect these viruses in the animals, or even directly in batches of semen. Cell culture, ELISA and PCR are the most accurate and widely used. Because of the high risk of dissemination of disease via AI, the absolute goal is to provide pathogen-free semen and this is feasible with the adequate measures that are discussed briefly in this paper.     

Phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of African swine fever virus

We have modeled in vitro infection of African swine fever virus (ASFV) in primary unstimulated cells of the porcine bone marrow and have studied the phenotypical changes in the population of porcine lymphoid cells by cytophotometry. Monocytes and large-sized lymphocytes completely vanished in 72 h of infection which is result of high sensitivity of those cells to ASFV. We describe DNA synthesis in monocytes at 24 h post infection. Cytophotometry of the uninfected cells revealed the few number of atypical lymphocytes and lymphoblasts after 72 h of cultivation; whereas in viral infected cultures, atypical cells appeared in large quantity (about 14%) with 24 h. Most of atypical lymphocytes and lymphoblasts had altered nucleus, and only a small number of atypical cells had additional nucleus. The cytophotometry of main and additional nuclei showed that DNA content didn't exceed diploid standard which indicates that the additional nuclei were consequence of fragmentation of nuclei in lymphocytes.     

A serological survey of selected pathogens in wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) in northern Turkey

During the hunting season in March 2012, a total of 93 blood samples were collected from wild boars (Sus scrofa) shot in the area of northern Turkey (Samsun and Gumushane provinces). These blood samples were examined by enzyme immunoassay (ELISA) for the presence of antibodies to classical swine fever virus (CSFV), Aujeszky’s disease virus (ADV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), swine influenza virus (SIV), porcine parvovirus (PPV), swine vesicular disease virus (SVDV), hepatitis E virus (HEV), African swine fever virus (ASFV), porcine rotavirus (PRV), transmissible gastroenteritis virus (TGEV) and bovine viral diarrhoea virus (BVDV). Out of 93 serum samples examined, 65 (69.9 %) were positive for PRV, 22 (23.7 %) were positive for ADV, 5 (5.4 %) were positive for BVDV, 4 (4.3 %) were positive for PPV and 2 (2.2 %) were positive for PRRSV. All sera were negative for ASFV, SVDV, HEV, SIV, PRCV, TGEV and CSFV. The results, recorded for the first time in Turkey, supported the hypothesis that wild boar act as a potential reservoir of selected viruses and thus have a role in the epidemiology of these diseases.     

Residual viruses in pork products.

Partly cooked canned hams and dried pepperoni and salami sausages were prepared from the carcasses of pigs infected with African swine fever virus and pigs infected with hog cholera virus. Virus was not recovered from the partly cooked canned hams; however, virus was recovered in the hams before heating in both instances. Both African swine fever virus and hog cholera virus were recovered from the dried salami and pepperoni sausages, but not after the required curing period.     

Residual viruses in pork products

Partly cooked canned hams and dried pepperoni and salami sausages were prepared from the carcasses of pigs infected with African swine fever virus and pigs infected with hog cholera virus. Virus was not recovered from the partly cooked canned hams; however, virus was recovered in the hams before heating in both instances. Both African swine fever virus and hog cholera virus were recovered from the dried salami and pepperoni sausages, but not after the required curing period.     

Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.     

An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1.

An open reading frame, LMW5-HL, in the African swine fever virus genome displays a high degree of similarity to the proto-oncogene bcl-2 and, to a lesser degree, the Epstein-Barr virus gene BHRF1. A highly conserved central region is found in all three proteins. LMW5-HL encodes a protein of 18 kDa that is present in infected porcine macrophages at both early and late times postinfection. The similarity of LMW5-HL to bcl-2 and BHRF1 suggests a role for it in cell maintenance during productive or persistent viral infection.     

Embryo transfer as a means of controlling the transmission of viral infections: V. the in vitro exposure of zona pellucida-intact porcine embryos to African swine fever virus

African swine fever virus (ASFV) was detected on or in zona pellucida-intact porcine embryos that had been exposed to 10^6.6 hemadsorption dose 50%/ml (HAdD₅₀/ml) of ASFV for 18 hours, washed and then cultured. Ninety-five percent of the embryos retained infectious virus after washing. Treating the embryos with papain, EDTA or ficin had no effect on the retained virus, whereas treating them with trypsin or pronase reduced the number of embryos carrying detectable virus (30% instead of 95%) and lowered the amount of virus on the embryos. It has not yet been determined whether ASFV enters the embryonic cells but the evidence suggests that most of the virus, and possibly all of it, is bound to the zona pellucida.     

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

African swine fever(ASF) is an infectious disease caused by African swine fever virus(ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification(RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13 a(previously known as C2 c2) through quantitative real-time PCR(qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Requirement of cell nucleus for African swine fever virus replication in Vero cells.

The role of the cell nucleus in the development of African swine fever virus in Vero cells has been studied. No viral growth could be detected in enucleated cells under conditions that allow normal development of Sindbis virus. Furthermore, African swine fever virus DNA synthesis was inhibited more than 95% after infection of enucleated Vero cells as compared with normal cells.     

Establishment of a Vero cell line persistently infected with African swine fever virus.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.     

A serosurvey for Brucella suis, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus in feral swine (Sus scrofa) of eastern North Carolina

As feral swine (Sus scrofa) populations expand their range and the opportunity for feral swine hunting increases, there is increased potential for disease transmission that may impact humans, domestic swine, and wildlife. From September 2007 to March 2010, in 13 North Carolina, USA, counties and at Howell Woods Environmental Learning Center, we conducted a serosurvey of feral swine for Brucella suis, pseudorabies virus (PRV), and classical swine fever virus (CSFV); the samples obtained at Howell Woods also were tested for porcine circovirus type 2 (PCV-2). Feral swine serum was collected from trapped and hunter-harvested swine. For the first time since 2004 when screening began, we detected B. suis antibodies in 9% (9/98) of feral swine at Howell Woods and <1% (1/415) in the North Carolina counties. Also, at Howell Woods, we detected PCV-2 antibodies in 59% (53/90) of feral swine. We did not detect antibodies to PRV (n=512) or CSFV (n=307) at Howell Woods or the 13 North Carolina counties, respectively. The detection of feral swine with antibodies to B. suis for the first time in North Carolina warrants increased surveillance of the feral swine population to evaluate speed of disease spread and to establish the potential risk to commercial swine and humans.     

Antibodies to selected viral and bacterial pathogens in European wild boars from southcentral Spain

Serum samples from 78 European wild boars (Sus scrofa) harvested during the 1999-2000 hunting season were tested for antibodies to Brucella spp., classical swine fever virus, Erysipelothrix rhusiopathiae, Haemophilus parasuis, Leptospira interrogans serovar pomona, Mycoplasma hyopneumoniae, pseudorabies virus (PRV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus, Salmonella serogroups B, C, and D, Streptococcus suis, and swine influenza virus (SIV) serotypes H1N1 and H3N2. Samples were collected from Sierra Morena and Montes de Toledo in southcentral Spain. Antibodies were detected to PRV (36%), L. interrogans serovar pomona (12%), PPV (10%), E. rhusiopathiae (5%), SIV serotype H1N1 (4%), Salmonella serogroup B (4%), and Salmonella serogroup C (3%). Our results suggest that more research is needed to describe the epidemiology of infectious diseases of Spanish wild boars.     

Antibodies to selected pathogens in wild boar (Sus scrofa) from Catalonia (NE Spain)

From 2004 to 2007, blood samples from 273 healthy wild boars (Sus scrofa), culled during the hunting season, were obtained in three areas of Catalonia (NE Spain): Pyrenees, Sant Llorenç del Munt i l’Obac Natural Park (SLM), and Ports de Tortosa i Beseit National Hunting Reserve (PTB). We investigated the presence of antibodies against classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine vesicular disease virus (PVDV), porcine respiratory and reproductive syndrome virus (PRRSV), Aujeszky’s disease virus (ADV), porcine influenza A virus (PIV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Salmonella spp., and Toxoplasma gondii. Four wild boars were suspicious for CSFV, but the infection was discarded with a virus neutralization test, and infection with a border disease virus was confirmed. Negative results were obtained against ASFV and PVDV. Antibodies were detected against PRRSV (3%), ADV (0.8%), PIV (6.4%), PCV2 (64.6%), PPV (54.7%), M. hyopneumoniae (26.6%), E. rhusiopathiae (5.3%), Salmonella spp. (11.3%), and T. gondii (43.5%). In SLM, we detected a higher seroprevalence for PIV and M. hyopneumoniae and a lower seroprevalence for E. rhusiopathiae than in the other two areas. In PTB, seroprevalence was higher for PPV, Salmonella spp., and PCV2. Adult wild boar displayed higher seroprevalence for PPV, PIV, and M. hyopneumoniae, whereas presence of antibodies for Salmonella spp. was higher in juveniles compared with adults and piglets.

     

Introduction of African Swine Fever into the European Union through Illegal Importation of Pork and Pork Products

Transboundary animal diseases can have very severe socio-economic impacts when introduced into new regions. The history of disease incursions into the European Union suggests that initial outbreaks were often initiated by illegal importation of meat and derived products. The European Union would benefit from decision-support tools to evaluate the risk of disease introduction caused by illegal imports in order to inform its surveillance strategy. However, due to the difficulty in quantifying illegal movements of animal products, very few studies of this type have been conducted. Using African swine fever as an example, this work presents a novel risk assessment framework for disease introduction into the European Union through illegal importation of meat and products. It uses a semi-quantitative approach based on factors that likely influence the likelihood of release of contaminated smuggled meat and products, and subsequent exposure of the susceptible population. The results suggest that the European Union is at non-negligible risk of African swine fever introduction through illegal importation of pork and products. On a relative risk scale with six categories from negligible to very high, five European Union countries were estimated at high (France, Germany, Italy and United Kingdom) or moderate (Spain) risk of African swine fever release, five countries were at high risk of exposure if African swine fever were released (France, Italy, Poland, Romania and Spain) and ten countries had a moderate exposure risk (Austria, Bulgaria, Germany, Greece, Hungary, Latvia, Lithuania, Portugal, Sweden and United Kingdom). The approach presented here and results obtained for African swine fever provide a basis for the enhancement of risk-based surveillance systems and disease prevention programmes in the European Union.