A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.     

Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease

We have previously shown that replication of foot-and-mouth disease virus (FMDV) is highly sensitive to alpha/beta interferon (IFN-alpha/beta). In the present study, we constructed recombinant, replication-defective human adenovirus type 5 vectors containing either porcine IFN-alpha or IFN-beta (Ad5-pIFNalpha or Ad5-pIFNbeta). We demonstrated that cells infected with these viruses express high levels of biologically active IFN. Swine inoculated with 10(9) PFU of a control Ad5 virus lacking the IFN gene and challenged 24 h later with FMDV developed typical signs of foot-and-mouth disease (FMD), including fever, vesicular lesions, and viremia. In contrast, swine inoculated with 10(9) PFU of Ad5-pIFNalpha were completely protected when challenged 24 h later with FMDV. These animals showed no clinical signs of FMD and no viremia and did not develop antibodies against viral nonstructural proteins, suggesting that complete protection from infection was achieved.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

African Swine Fever Virus MGF-110-9L-deficient Mutant Has Attenuated Virulence in Pigs

African swine fever virus(ASFV) is the etiological agent of African swine fever(ASF), an often lethal disease in domestic and wild pigs. ASF represents a major threat to the swine industry worldwide. Currently, no commercial vaccine is available because of the complexity of ASFV or biosecurity concerns. Live attenuated viruses that are naturally isolated or genetically manipulated have demonstrated reliable protection against homologous ASFV strain challenge. In the present study, a mutant ASFV strain with the deletion of ASFV MGF-110-9 L(ASFV-D9 L) was generated from a highly virulent ASFV CN/GS/2018 parental strain, a genotype II ASFV. Relative to the parental ASFV isolate, deletion of the MGF-110-9 L gene significantly decreased the ability of ASFV-D9 L to replicate in vitro in primary swine macrophage cell cultures. The majority of animals inoculated intramuscularly with a low dose of ASFV-D9 L(10 HAD50) remained clinically normal during the 21-day observational period. Three of five ASFV-D9 L-infected animals displayed low viremia titers and low virus shedding and developed a strong virus-specific antibody response, indicating partial attenuation of the ASFV-D9 L strain in pigs. The findings imply the potential usefulness of the ASFV-D9 L strain for further development of ASF control measures.     

A Serological Survey of Ruminant Livestock in Kazakhstan During Post-Soviet Transitions in Farming and Disease Control

The results of a serological survey of livestock in Kazakhstan, carried out in 1997–1998, are reported. Serum samples from 958 animals (cattle, sheep and goats) were tested for antibodies to foot and mouth disease (FMD), bluetongue (BT), epizootic haemorrhagic disease (EHD), rinderpest (RP) and peste des petits ruminants (PPR) viruses, and to Brucella spp. We also investigated the vaccination status of livestock and related this to changes in veterinary provision since independence in 1991. For the 2 diseases under official surveillance (FMD and brucellosis) our results were similar to official data, although we found significantly higher brucellosis levels in 2 districts and widespread ignorance about FMD vaccination status. The seroprevalence for BT virus was 23%, and seropositive animals were widespread suggesting endemicity, despite the disease not having being previously reported. We found a few seropositives for EHDV and PPRV, which may suggest that these diseases are also present in Kazakhstan. An hierarchical model showed that seroprevalence to FMD and BT viruses were clustered at the farm/village level, rather than at a larger spatial scale. This was unexpected for FMD, which is subject to vaccination policies which vary at the raion (county) level.     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

Experimental Infection of Ornithodoros erraticus sensu stricto with Two Portuguese African Swine Fever Virus Strains. Study of Factors Involved in the Dynamics of Infection in Ticks

African swine fever (ASF) is a frequently devastating hemorrhagic disease of domestic pigs and wild boar and Ornithodoros erraticus sensu stricto argasid ticks are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe. Recently this disease emerged in Eastern Europe and Russian Federation, showing a huge potential for a rapid spread between countries. There is some risk of re-emergence of ASF in the countries where these ticks exist, that can contribute for the persistence of infection and compromise control measures. In this study we aimed to identify factors that determine the probability of infection and its dynamics in the tick vector Ornithodoros erraticus sensu stricto, with two Portuguese strains of ASFV. Our results suggest that these ticks have a high likelihood of excreting the two haemadsorbing ASF viruses of different host origins and that, in field surveys, the analysis of adults and 5^th nymphal stage can provide the best chance of detecting virus infection. The results also indicate that infection of pigs with highly virulent ASF viruses will promote higher rates of infection and a higher likelihood for virus excretion by ticks. Nevertheless, there is also a risk, although lower, that ticks can become infected on pigs that have overcome the acute phase of infection, which was simulated in our study by membrane feeding ticks with low titres of virus. We believe these results can be valuable in designing and interpreting the results of ASF control programmes, and future work can also be undertaken as our dataset is released under open access, to perform studies in risk assessment for ASFV persistence in a region where O. erraticus sensu stricto ticks are present.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Efficacy of vaporized hydrogen peroxide against exotic animal viruses.

The efficacy of vapor-phase hydrogen peroxide in a pass-through box for the decontamination of equipment and inanimate materials potentially contaminated with exotic animal viruses was evaluated. Tests were conducted with a variety of viral agents, which included representatives of several virus families (Orthomyxoviridae, Reoviridae, Flaviviridae, Paramyxoviridae, Herpesviridae, Picornaviridae, Caliciviridae, and Rhabdoviridae) from both avian and mammalian species, with particular emphasis on animal viruses exotic to Canada. The effects of the gas on a variety of laboratory equipment were also studied. Virus suspensions in cell culture media, egg fluid, or blood were dried onto glass and stainless steel. Virus viability was assessed after exposure to vaporphase hydrogen peroxide for 30 min. For all viruses tested and under all conditions (except one), the decontamination process reduced the virus titer to 0 embryo-lethal doses for the avian viruses (avian influenza and Newcastle disease viruses) or less than 10 tissue culture infective doses for the mammalian viruses (African swine fever, bluetongue, hog cholera, pseudorabies, swine vesicular disease, vesicular exanthema, and vesicular stomatitis viruses). The laboratory equipment exposed to the gas appeared to suffer no adverse effects. Vaporphase hydrogen peroxide decontamination can be recommended as a safe and efficacious way of removing potentially virus-contaminated objects from biocontainment level III laboratories in which exotic animal disease virus agents are handled.     

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.     

Serologic surveillance for vesicular stomatitis virus on Ossabaw Island, Georgia

Seventeen species of mammals and seven species of birds from Ossabaw Island, Georgia, were tested for vesicular stomatitis (VS) neutralizing antibodies. Seropositive results were restricted to mammals with six of 17 species testing seropositive for VS (New Jersey type) neutralizing antibodies. Seropositive species included: raccoons (Procyon lotor), white-tailed deer (Odocoileus virginianus), feral swine (Sus scrofa), cattle (Bos taurus), horses (Equus caballus), and donkeys (Equus asinus). All tests for VS (Indiana type) were negative.     

非洲猪瘟防控及疫苗研发：挑战与对策

非洲猪瘟是由非洲猪瘟病毒引起的一种接触传染性、广泛出血性猪烈性传染病,最急性和急性感染死亡率高达100%。自2018年8月我国发生首起非洲猪瘟疫情后,3个多月内,已有18个省份累计暴发69起,给我国养猪业造成了沉重打击。从目前非洲猪瘟全球流行态势及世界各国防控经验来看,我国非洲猪瘟防控和根除面临的形势不容乐观,亟需安全有效的疫苗用于该病的防控。文中结合当前非洲猪瘟病原学最新研究成果,系统总结了非洲猪瘟防控策略、疫苗研究进展及其面临的挑战,重点分析了疫苗研发历程、存在的问题、未来发展方向以及商业化应用所面临的关键科学问题,以期为我国非洲猪瘟防控及病原和疫苗研究提供借鉴。     

Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

ABSTRACT: BACKGROUND: Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East-South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. RESULTS: The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHA topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. CONCLUSIONS: Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHA topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China.     

非洲猪瘟在俄罗斯的流行与研究现状

非洲猪瘟(African swine fever,ASF)由非洲猪瘟病毒(ASFV)引起,是家猪和野猪的一种高度接触性、致死性传染病,可表现为最急性、急性、亚急性和慢性四种形式。猪感染后以发热、高病毒血症和出血性病变为特征。有的毒株可引起高发病率和高死亡率。自2007年ASF传入格鲁吉亚以来,该病在高加索地区(包括俄罗斯)逐步蔓延,造成多地大量家猪和野猪病死,经济损失惨重。2017年3月,ASF突然在远东地区伊尔库茨克州出现,疫点距中国北方最大陆路口岸满洲里仅约1 000 km,使得传入中国的风险空前提高。为此,本文对该病10年间在俄罗斯的流行状况和研究情况进行总结,以期为我国对该病的防控提供参考。     

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.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

Swine vesicular disease viruses isolated from healthy pigs in non-epizootic period. I. Isolation and identification

A total of 524 fecal samples were collected from healthy swine of 36 hog farms scattered all over Japan from which had been detected neutralizing antibody against swine vesicular disease (SVD) virus. A virus was isolated from 21 of them. It was neutralized by antiserum against SVD virus. Of the 21 samples, 19 were derived from six farms, in areas which remained to be free from SVD in the past years and two from a farm where SVD broke out 18 months before. The cross-neutralization test was carried out with seven strains of SVD virus isolated from healthy pigs (SVDV-H) and five strains of SVD virus isolated from diseased pigs (SVDV-D). There was, however, no significant difference in antigenicity between the two groups. Some strains of SVDV-H were antigenically close to the Faulkner strain of Coxsackie B5 virus, and others to the freshly isolated strain of this virus. Neutralizing antibody of low titer against SVD virus was detected from pigs kept in areas free from SVD. It was presumed to have been produced in these pigs involved in silent infection with this virus.     

非洲猪瘟病毒的免疫逃逸策略

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病。目前无商品化的ASF疫苗,一旦发病,仅能依靠快速扑杀进行防控,严重威胁我国养猪及相关行业的健康发展。ASF疫苗研发面临的主要困难是对ASFV的毒力相关基因、致病及其免疫逃逸机制知之甚少。本文对ASFV的免疫逃逸研究进行了总结,探讨了ASFV免疫逃逸基因及其编码蛋白的功能,以便加深对ASFV及其免疫逃逸策略的认知,为致病机制研究和疫苗研发提供借鉴。     

Ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies

Using the vesicular stomatitis virus (VSV) pseudotype system, we studied the functional properties of the Ebola virus glycoprotein (GP). Amino acid substitutions at the GP cleavage site, which reduce glycoprotein cleavability and viral infectivity in some viruses, did not appreciably change the infectivity of VSV pseudotyped with GP. Likewise, removal of two acylated cysteine residues in the transmembrane region of GP showed no discernible effects on infectivity. Although most filoviruses are believed to target endothelial cells and hepatocytes preferentially, the GP-carrying VSV showed greater affinity for epithelial cells than for either of these cell types, indicating that Ebola virus GP does not necessarily have strong tropism toward endothelial cells and hepatocytes. Finally, when it was used to screen for neutralizing antibodies against Ebola virus GP, the VSV pseudotype system allowed us to detect strain-specific neutralizing activity that was inhibited by secretory GP (SGP). This finding provides evidence of shared neutralizing epitopes on GP and SGP molecules and indicates the potential of SGP to serve as a decoy for neutralizing antibodies.     

Infectivity-enhancing antibodies to Ebola virus glycoprotein

Ebola virus causes severe hemorrhagic fever in primates, resulting in mortality rates of up to 100%, yet there are no satisfactory biologic explanations for this extreme virulence. Here we show that antisera produced by DNA immunization with a plasmid encoding the surface glycoprotein (GP) of the Zaire strain of Ebola virus enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP. Substantially weaker enhancement was observed with antiserum to the GP of the Reston strain, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. We also generated an anti-Zaire GP monoclonal antibody that enhanced viral infectivity and another that neutralized it, indicating the presence of distinct epitopes for these properties. Our findings suggest that antibody-dependent enhancement of infectivity may account for the extreme virulence of the virus. They also raise issues about the development of Ebola virus vaccines and the use of passive prophylaxis or therapy with Ebola virus GP antibodies.