A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

African swine fever(ASF) is a devastating infectious disease in swine that is severely threatening the global pig industry. An efficacious vaccine is urgently required. Here, we used the Chinese ASFV HLJ/18 as a backbone and generated a series of genedeleted viruses. The virulence, immunogenicity, safety, and protective efficacy evaluation in specific-pathogen-free pigs,commercial pigs, and pregnant sows indicated that one virus, namely HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, cannot convert to the virulent strain, and provides complete protection of pigs against lethal ASFV challenge.Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV, and as such is expected to play an important role in controlling the spread of ASFV.     

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.     

The persistence of African swine fever virus in field-infected Ornithodoros erraticus during the ASF endemic period in Portugal

African swine fever (ASF) is an important disease of pigs and outbreaks of ASF have occurred in Europe on multiple occasions. To explore the period for which the European soft tick species Ornithodoros erraticus (Acari: Argasidae) is able to act as a reservoir of African swine fever virus (ASFV) after infected hosts are removed, we collected specimens from farms in the provinces of Alentejo and Algarve in Portugal during the endemic period and tested them subsequently using cell culture and experimental infection. We show that ticks from previously infected farms may contain infectious virus for at least five years and three months after the removal of infectious hosts. Furthermore, in two cases infectious virus was successfully isolated from ticks on restocked farms that had not yet suffered a re-emergence of disease. Experimental transmission to pigs was demonstrated in batches tested up to 380 days after an outbreak. These results clarify the epidemiological role of O. erraticus ticks in the persistence of ASFV in the field, provide additional evidence to support its role in the re-emergence of a sporadic outbreak of ASF in Portugal in 1999 and suggest that the current quarantine legislation and restocking advice when these ticks are present on the pig farm premises is appropriate.     

非洲猪瘟病毒的生物学特性与疫苗研制的难点

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,是全球养猪业的"头号杀手",强毒株引发的超急性和急性感染死率高达100%。2018年8月ASF首次传入我国,截止2019年6月6日,已有32个省份累计暴发137起疫情,给我国社会、经济构成巨大威胁。ASF疫苗的研制始于20世纪60年代,但均以失败而告终,其主要原因是对ASFV生物学特性缺乏深入的研究。有效控制当前ASF疫情扩散、研制安全有效的疫苗将是我国面临的巨大挑战。本文对ASFV形态与基本结构、传播途径、致病机制、基因组及编码蛋白、入侵机制、免疫逃逸等生物学特性进行了概述,并分析了当前疫苗研制面临的难点,以期为我国有效控制ASF疫情及病原研究提供参考。     

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.     

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。     

Recovery and assay of African swine fever and swine vesicular disease viruses from pig slurry

Assaying samples for infectious virus is more difficult when the sample is toxic to cells used in the assay, e.g. with samples of infected pig slurry. Various techniques were compared for the recovery of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV) in pig slurry. Extraction with Freon led to 80-100% recovery of SVDV added to pig slurry. The assay sensitivity enabled undiluted, centrifuged sample to be put directly onto monolayers of IB-RS2 cells, allowing a minimum detection level of 100.7 pfu ml-1. ASFV was difficult to recover intact, and the best technique allowed a recovery of 60% with a minimum detectable level of 101.8 HAD50 ml-1, due to toxicity to the cells at low sample dilutions. Extraction with the addition of an equal volume of ox serum to inoculated slurry was best at recovering ASFV. Poor recoveries with the other techniques may have been due to the inactivation of the virus while in the slurry rather than as a result of the inability of the method to extract ASFV.     

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。     

The medical and veterinary role of Ornithodoros erraticus complex ticks (Acari: Ixodida) on the Iberian Peninsula

Argasid ticks of the Ornithodoros erraticus complex are associated with traditional pig‐farming practices on the Iberian Peninsula and are also found elsewhere in North Africa, West Africa, and western Asia. The ticks associated with pig farming on the Iberian Peninsula are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe, and their ecology makes them an extremely effective reservoir of both ASFV and the Borrelia species which cause tick‐borne relapsing fever (TBRF) in humans. The recent reappearance of ASFV in the European Union, coupled with evidence that Portuguese tick populations continue to harbor Borrelia despite a lack of confirmed human infections, suggest that these populations merit closer attention. In Portugal, a series of surveys over the last twenty‐five years indicates that the number of farm sites with tick infestations has declined and suggest that populations are sensitive to changes in farm management, particularly the use of modern pig housing. Various technologies have been suggested for the control of farm‐associated Ornithodoros ticks and related species but, in our opinion, farm management changes are still the most effective strategy for population control. Furthermore, we suggest that this species could probably be eradicated from Iberian pig farms.     

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.     

Regulation of antiviral immune response by African swine fever virus (ASFV)

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality approaching 100% in domestic pigs. ASF is an endemic in countries in sub-Saharan Africa. Now, it has been spreading to many countries, especially in Asia and Europe. Due to the fact that there is no commercial vaccine available for ASF to provide sustainable prevention, the disease has spread rapidly worldwide and caused great economic losses in swine industry. The knowledge gap of ASF virus (ASFV) pathogenesis and immune evasion is the main factor to limit the development of safe and effective ASF vaccines. Here, we will summarize the molecular mechanisms of how ASFV interferes with the host innate and adaptive immune responses. An in-depth understanding of ASFV immune evasion strategies will provide us with rational design of ASF vaccines.     

African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N_1–107). Interestingly, unlike the effect of GSDMD-N_1–279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N_1–107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.     

The African Swine Fever Virus Thymidine Kinase Gene Is Required for Efficient Replication in Swine Macrophages and for Virulence in Swine

African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter–β-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK⁻ ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK⁺ revertant virus. Swine surviving TK⁻ ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

非洲猪瘟病毒的非必需基因：真的可有可无吗？

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种出血性、致死性的猪烈性传染病。ASF在全球广泛传播,给养猪业造成重大的经济损失。ASFV基因组庞大,可编码150多种蛋白,一些非必需基因编码的蛋白与调控病毒毒力、复制和免疫逃逸等相关。通过删除ASFV毒力相关的非必需基因所构建的减毒株是当前比较有前景的疫苗,然而其安全性有待提高。系统地鉴定ASFV非必需基因及其功能,不仅有助于ASF基因缺失疫苗的研发,也有益于ASFV致病机制研究。本文对目前已鉴定的ASFV非必需基因及其功能研究进行了总结分析,着重讨论了影响ASFV毒力、调控病毒复制、参与免疫逃逸的非必需基因及其编码蛋白的功能,旨在加深对ASFV病原学的认识,为新的ASFV非必需基因的鉴定和功能研究提供参考。     

非洲猪瘟病毒编码蛋白功能研究进展

非洲猪瘟(African swine fever,ASF)是非洲猪瘟病毒(African swine fever virus,ASFV)感染家猪或野猪引起的一种急性、出血性、高度接触性传染病,其特征是病程短、高热和出血性病变,急性感染死亡率高达100%,严重威胁全球养猪业但目前尚未开发出有效的疫苗和治疗方法。ASFV是非洲猪瘟病毒科非洲猪瘟病毒属的唯一成员,为大型双链DNA病毒,主要在巨噬细胞胞质中复制,其基因组约170?193 kb,含有150?167个开放阅读框,编码150?200种蛋白质。目前已知功能的病毒编码蛋白约有50个,大部分为病毒的结构蛋白,仍有一半以上的ASFV编码蛋白功能尚不清楚。除结构蛋白以外,病毒含有完整的酶和与病毒转录有关的因子,编码调节宿主细胞功能及与病毒免疫逃逸相关的蛋白等。本文综述了ASFV的结构蛋白、非结构蛋白以及参与免疫逃逸等相关蛋白功能的研究进展,以期为ASFV病毒蛋白研究及疫苗研发提供相关借鉴。     

Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.     

Purification and characterisation of a P-selectin-binding molecule from the salivary glands of Ornithodoros moubata that induces protective anti-tick immune responses in pigs

Ornithodoros moubata is an argasid tick that lives in Africa in wild and synanthropic habitats. It feeds on warthogs, domestic swine and humans, and is able to transmit severe diseases such as human relapsing fever and African swine fever. The elimination of O. moubata from the synanthropic surroundings should improve control of the diseases it transmits. Previous attempts to develop a vaccine against O. moubata showed that a salivary gland extract (SGE) induced a protective response that inhibited the feeding of the ticks by up to 60%. Our aim in the present work was to isolate and characterise the salivary antigens responsible for the protective immune response induced with the SGE. The work reported here describes the finding and partial characterisation of a tick salivary glycoprotein of 44 kDa (Om44) that binds host P-selectin, presumably preventing the adhesion of leucocytes and platelets to vessel walls, thus allowing the ticks to complete their feeding. Preliminary analysis indicated that Om44 is not a homologue of the mammalian PSGL-1s and lacks sialyl-Lewis^X, Lewis^X and Lewis^Y determinants but carries heparin, which is the P-selectin-binding motif. Om44 is not recognised by the pig immune system after natural contact with O. moubata, but it can be neutralised by specific vaccine-induced antibodies, resulting in feeding inhibitions of around 50% in adults and nymphs-4, 25% in nymphs-3, 5% in nymphs-2 and 0% in nymphs-1 when they are fed on Om44-vaccinated pigs. In addition, the fecundity of females was inhibited by up to 43.8%. Om44 provides new prospects for the development of new anti-tick vaccines.