非洲猪瘟病毒多样性

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起猪的一种急性、高度致死性传染病.该病在全世界多个地方流行,导致该病流行的原因之一是缺乏有效的预防及治疗药物、疫苗等.尽管ASFV基因总的突变率相对其基因组来说较低,但是,与其它病毒相比,其基因突变总数则相当巨大.研究发现ASFV的多个基因具有高突变率的特性,表现为基因多样性,此外,由于该病毒为核质大DNA病毒,编码大量蛋白,其抗原也表现为多样性.本文总结了 ASFV基因多样性和抗原多样性,分析其发生原理并综述了最新研究成果,以期为研究ASFV病毒遗传演化、开发疫苗及指导疫情防控提供思路.     

Effects of Some Disinfectants on African Swine Fever Virus

Ten commercially available disinfectants were tested at high pH in 2% sodium hydroxide and low pH in 2% acetic acid as inactivants for African swine fever (ASF) in a protein-rich blood-spleen homogenate. As assayed in leukocyte cultures, sodium hydroxide and acetic acid, sodium meta silicate and Roccal did not inactivate ASF virus in 1 hr at 22 to 25 C. Some viricidal activity as assayed in leukocyte cultures was found with Weladol, Triton X-100 Amphyl, pHisoHex, sodium dodecyl sulfate, LpH, Environ, Environ D, and One-Stroke Environ. Of these, the last four appeared to be most promising. When assayed in pigs, only One-Stroke Environ (1/E) was viricidal. Concentrations of 1.0, 0.75, and 0.5 were effective, but, at 0.25%, virus was not inactivated. The minimal time to inactivate ASF virus by 1% 1/E is 60 min. A room contaminated with ASF virus was made safe for pigs after 1 hr by spraying with 1% 1/E. The most active component of 1/E is o-phenylphenol. Although another component of 1/E, i.e., o-benzyl-p-chlorophenol, also has some activity, the mixture of the active components of 1/E is most effective against ASF virus. One of the soluble antigens associated with ASF virus is destroyed by 1/E.     

African Swine Fever Virus Is Wrapped by the Endoplasmic Reticulum

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249–278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.African swine fever (ASF) virus is a large icosahedral enveloped DNA virus that causes a lethal hemorrhagic disease in domestic pigs. The virus is endemic in areas of southern Europe and in Africa where it causes major problems for the development of pig industries. At present there are no vaccines, and the disease is controlled through the slaughter of infected animals. The economic importance of ASF virus has made the virus the focus of much research since it was first described in 1921 (32). ASF virus is unique among animal viruses, and its classification has been controversial. ASF virus shares the striking icosahedral symmetry of iridoviruses (5, 8, 13, 34), while the presence of inverted terminal repeats and covalently linked ends in the 170-kDa genome suggests similarities with poxviruses (16). The ASF virus genome encoding at least 150 proteins has been sequenced (17, 51), and the amino acid sequences of at least 11 structural proteins are known. p73 is the major structural protein (14, 28) and has sequence similarities to the capsid protein of iridoviruses (39). The ordered proteolysis of pp220 produces p150, p37/p34 and p14 (40), which together comprise 25% of the viral proteins (3). These proteins localize to the interior of the virion (3). Three proteins, J13L/p54, I1L/p17, and p22, with membrane-spanning domains localize to the viral envelope (10, 37, 41, 43). Three other structural proteins, p14.5 encoded by E120R (30), p10 encoded by K78R (35), and p5AR encoded by A104R (7), have DNA-binding properties (51) and may be involved in DNA packaging. The virus has been the subject of several detailed electron microscopy studies (2–4, 8, 9, 11, 13, 34, 47). Electron micrographs of sections taken through ASF virus assembly sites reveal fully assembled virions as 200-nm hexagons and an ordered series of assembly intermediates with one to six sides of a hexagon. Close inspection of intracellular virions identifies multiple concentric layers of differing electron densities. According to recent models, the layers represent a central electron-dense nucleocapsid core, surrounded by an inner core shell, an inner envelope, and an outer capsid layer (3). The mechanism of formation of the inner envelope of ASF virus has not been resolved.Most viruses gain a single membrane envelope by budding into intracellular membrane compartments or from the plasma membrane, as reviewed in reference 21. When viruses bud into an intracellular compartment, the domains of the membrane proteins that are initially located in the lumen of membrane compartments are exposed on the outside of the virion after release from the cell (Fig. ​(Fig.1a).1a). A second mechanism of envelopment, described recently for poxviruses and herpesviruses (18, 20, 24, 38, 42, 46, 50), is more complex and involves the wrapping of virions by membrane cisternae derived from specific membrane compartments. Wrapping provides two membrane envelopes in one step and leaves the virion free in the cytoplasm. When compared with budding, wrapping reverses the orientation of membrane proteins within the virus such that the domains of membrane proteins located in the lumen of the wrapping organelle are confined to the interior of the virus after release from the cell, whereas cytoplasmic tails are exposed on the outside of the virus (Fig. ​(Fig.1b).1b). Given these important consequences for understanding the mechanism of assembly of the virus and for determining the final orientation of membrane proteins in virions, we have set out to determine whether ASF virus acquires its membranes by the conventional budding mechanism or whether the virus is wrapped by intracellular membrane compartments before release from the cell. Open in a separate windowFIG. 1Schematic comparison of budding and wrapping mechanisms of virus envelopment. (a) Budding. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins (|, membrane glycoproteins). Interactions between viral proteins lead to membrane curvature, and the virion gains a single membrane by budding into the lumen of the membrane compartment. When the virion is released from the cell, oligosaccharides () are exposed on the surface of the virus, and the cytoplasmic tail of the membrane glycoprotein is buried within the virion. (b) Wrapping. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins. The nucleoprotein complex is then wrapped by the membrane cisternae, and the virus gains two membranes. The particle remains in the cytosol. When the virion is released from the cell by cell lysis, oligosaccharides () are buried within the two membranes of the virion while the cytoplasmic tail of the membrane glycoprotein is exposed on the surface of the virus.In this study we have taken advantage of thin cryoelectron microscopic sections to enhance the definition of viral membranes. The micrographs show two membranes within mature intracellular virions and all structural intermediates. They also show assembly intermediates in continuity with cellular membrane compartments. Consistent with our earlier study showing that p73 was enveloped by the endoplasmic reticulum (ER) (15), immunogold labelling experiments show resident proteins of the ER within membranes found at assembly sites, in virus assembly intermediates, and in mature virions. Importantly, we have identified a protein (pXP124L) encoded by ASF virus that translocates completely into the lumen of the ER and is incorporated as a structural protein of the virus. The presence of two membranes within intracellular virions and structural intermediates and the packaging of a structural protein from the lumen of the ER into the virus, strongly suggest that ASF virus is wrapped by the ER.     

Role of Wild Suids in the Epidemiology of African Swine Fever

There is presently no vaccine to combat African swine fever (ASF), a viral hemorrhagic fever of domestic pigs that causes up to 100% morbidity and mortality in naive, commercial pig populations. In its endemic setting, ASF virus cycles between asymptomatic warthogs and soft ticks, with persistence in exotic locations being ascribed to the almost global distribution of susceptible soft tick and suid hosts. An understanding of the role played by diverse hosts in the epidemiology of this multi-host disease is crucial for effective disease control. Unlike the intensively studied Ornithodoros tick vector, the role of many wild suids remains obscure, despite growing recognition for suid-exclusive virus cycling, without the agency of the argasid tick, at some localities. Because the four wild suid genera, Phacochoerus, Potamochoerus, Hylochoerus, and Sus differ from each other in taxonomy, distribution, ecology, reservoir host potential, virus shedding, ASF symptomology, and domestic-pig contact potential, their role in disease epidemiology is also varied. This first consolidated summary of ASF epidemiology in relation to wild suids summarizes current knowledge and identifies information gaps and future research priorities crucial for formulating effective disease control strategies.     

Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na⁺/H⁺ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.Many animal viruses have evolved to exploit endocytosis to gain entry into host cells after initial attachment of virions to specific cell surface receptors. To date, a number of different routes of endocytosis used by viruses have been characterized, including clathrin-mediated endocytosis, uptake via caveolae/lipid rafts, macropinocytosis, phagocytosis, and other routes that are currently poorly understood.In recent years, viruses have also been used as tools to study cellular endocytosis and membrane trafficking at the molecular level, with there being special interest in the regulation of the diverse routes (31), since examples of viruses using each route can be found (reviewed in references 26, 31, and 38). The clathrin-mediated endocytic route has been the most extensively studied at the molecular level, and it has been shown to be used by diverse mammalian enveloped viruses, such as vesicular stomatitis virus (42), Semliki Forest virus (19), and West Nile virus (11), to infect cells. Influenza virus and HIV-1 also can use this pathway as an alternative route of entry (12, 39). Clathrin is assembled on the inside face of the plasma membrane to form a characteristic coated pit (CCP). During this process, clathrin also interacts with a number of essential molecules, including Eps15, adapter protein AP2, and dynamin GTPase (9). Additionally, clathrin-mediated endocytosis also provides endocytic vesicles as an acidified environment for those viruses that require a low-pH step during the first stages of infection to initiate capsid destabilization and genome uncoating. On the other hand, the lipid raft/caveola-based route is generally used by those acid-independent viruses. Recently, macropinocytosis is generating growing interest, since it has been demonstrated to be induced by some viruses from diverse families, such as vaccinia virus and adenovirus serotype 3 (5, 29), to gain entry into cells.In this study, we have focused on the entry of African swine fever virus (ASFV), a large enveloped DNA virus with a genomic composition similar to that of poxviruses, although the virion structure and morphology resemble those of iridoviruses. At present, it is the sole member of the newly created family Asfarviridae (16, 43). It is the etiologic agent responsible for a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in affected countries because of the high rate of mortality associated with this illness and the lack of an effective vaccine.Early studies of the entry of BA71V, a Vero cell-adapted ASFV strain, into host cells showed that this internalization of virus particles is a temperature-, energy-, and low-pH-dependent process, since it does not occur at 4°C or in the presence of inhibitors of cellular respiration or lysosomotropic agents (2, 44). All these features are consistent with a receptor-mediated endocytosis mechanism of entry. However, there are still numerous questions to be answered. One of them is the nature of the cellular receptor(s) that mediates ASFV entry, which remains largely unknown, although a correlation between cell susceptibility to infection and expression of porcine CD163 on the surface of swine monocytes/macrophages has been reported (36). In regard to the viral components involved in this initial step, the p12 and p54 proteins were shown to play a role during attachment to the cell surface and p30 during internalization, as inferred from previous studies with neutralizing antibodies against p30 and p54 (17) and blockage of infection after saturation of virus binding sites with recombinant p12 (6). Early electron microscopy (EM) studies (2, 45) revealed that attachment of ASFV virions to the cell surface often occurs in coated pits; however, their later presence inside coated vesicles is not fully clear. After attachment, virions are detected inside endosomes, where fusion with the viral membrane takes place. The ASFV cycle continues with the transport of viral cores via retrograde transport along microtubules to reach a perinuclear area, known as the viral factory, where replication occurs (4).In recent times, knowledge about different endocytic pathways and their regulatory molecules has notably increased, and the development of molecular tools to study these processes is becoming increasingly precise (38). In the present study, we examined the ASFV infection using a variety of chemical inhibitors and dominant negative molecules to disrupt different endocytic pathways. Our results confirmed a major role for dynamin-dependent and clathrin-mediated endocytosis during the first stages of ASFV infection, with no significant differences in the behavior of the two ASFV strains and the two cell lines analyzed.     

野猪在非洲猪瘟流行中的重要作用

非洲猪瘟因其高死亡率、没有疫苗防疫、影响国际贸易而备受广泛关注。1921年首次确认非洲猪瘟疫情以来,先后在非洲、欧洲、美洲等多个国家和地区发病造成重大经济损失。野猪作为该病传播的重要生物媒介在俄罗斯等多个国家非洲猪瘟疫情散播中发挥了重要作用。充分了解全球非洲猪瘟疫情状况和野猪在俄罗斯非洲猪瘟疫情中的影响,分析我国野猪分布、疫病监测和管理现状,将为我国非洲猪瘟外来疫情防控策略制定提供参考。     

African Swine Fever Virus Protein E199L Promotes Cell Autophagy through the Interaction of PYCR2

African swine fever virus(ASFV), as a member of the large DNA viruses, may regulate autophagy and apoptosis by inhibiting programmed cell death. However, the function of ASFV proteins has not been fully elucidated, especially the role of autophagy in ASFV infection. One of three Pyrroline-5-carboxylate reductases(PYCR), is primarily involved in conversion of glutamate to proline. Previous studies have shown that depletion of PYCR2 was related to the induction of autophagy. In the present study, we found for the first time that ASFV E199 L protein induced a complete autophagy process in Vero and HEK-293 T cells. Through co-immunoprecipitation coupled with mass spectrometry(CoIP-MS)analysis, we firstly identified that E199 L interact with PYCR2 in vitro. Importantly, our work provides evidence that E199 L down-regulated the expression of PYCR2, resulting in autophagy activation. Overall, our results demonstrate that ASFV E199 L protein induces complete autophagy through interaction with PYCR2 and down-regulate the expression level of PYCR2, which provide a valuable reference for the role of autophagy during ASFV infection and contribute to the functional clues of PYCR2.     

非洲猪瘟病毒多基因家族研究进展

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的一种急性、热性、高度接触性、致死性动物传染病.由于ASFV基因组庞大,变异能力强,免疫逃逸机制复杂,至今无有效药物和疫苗.近年来,对多基因家族的研究取得了很大的进展,可变区多基因家族拷贝数变化是导致ASFV变异的主要原因,且陆续发现多基因家族在决定细胞宿主范围、影响病毒毒力强弱、抑制Ⅰ型干扰素信号通路、抑制干扰素抗病毒效应和促进病毒蜱源感染中具有重要作用.本文对当前的ASFV多基因家族研究进展进行总结,阐述其在基因变异和病毒感染中的作用,以期为非洲猪瘟免疫逃逸机制的探索和疫苗的研发提供理论依据.     

非洲猪瘟病毒的分子生物学研究进展

非洲猪瘟是一类动物传染病,致死率高达100%,在我国虽未发现该病,但一旦发生会给畜牧养殖业带来巨大经济损失。文中概述了非洲猪瘟病毒的分类、形态、基因组特征、主要结构蛋白,以及分子生物学诊断技术的研究进展。为进一步研究该病毒的复制机理、毒力、致病机理及疫苗的开发提供参考依据。     

Regulation of Host Translational Machinery by African Swine Fever Virus

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host''s defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.     

Apoptosis of thymocytes in experimental African Swine Fever virus infection

This paper report on the lesions occurred in the thymus in experimental acute African swine fever (ASF). Twenty-one pigs were inoculated with the highly virulent ASF virus (ASFV) isolate Spain-70. Animals were slaughtered from 1 to 7 days post infection (dpi). Three animals with similar features were used as controls. Thymus samples were fixed in 10% buffered formalin solution for histological and immunohistochemical study and in 2.5% glutaraldehyde for ultrastructural examination. For immunohistochemical study, the avidin-biotin-peroxidase complex (ABC) technique was used to demonstrate viral protein 73 and porcine myeloid-histiocyte antigen SWC3 using specific monoclonal antibodies. Cell apoptosis was evaluated by the TUNEL assay. Blood samples were taken daily from all pigs and were used for leukocyte counts. The results of this study show a severe thymocyte apoptosis not related to the direct action of ASFV on these cells, but probably to a quantitative increase in macrophages in the thymus and their activation. A decrease in the percentage of blood lymphocytes was observed at the same time No significant vascular changes were observed in the study. With these results we suggest that ASFV infection of the thymus does not seem to play a critical role in the acute disease. Although severe apoptosis was observed, animals died because of the severe lesions found in the other organs.     

Risk of African swine fever introduction into the European Union through transport-associated routes: returning trucks and waste from international ships and planes

ABSTRACT: BACKGROUND: The uncontrolled presence of African swine fever (ASF) in Russian Federation (RF) poses a serious risk to the whole European Union (EU) pig industry. Although trade of pigs and their products is banned since the official notification in June 2007, the potential introduction of ASF virus (ASFV) may occur by other routes, which are very frequent in ASF, and more difficult to control, such as contaminated waste or infected vehicles. This study was intended to estimate the risk of ASFV introduction into the EU through three types of transport routes: returning trucks, waste from international ships and waste from international planes, which will be referred here as transport-associated routes (TAR). Since no detailed and official information was available for these routes, a semi-quantitative model based on the weighted combination of risk factors was developed to estimate the risk of ASFV introduction by TAR. Relative weights for combination of different risk factors as well as validation of the model results were obtained by an expert opinion elicitation. RESULTS: Model results indicate that the relative risk for ASFV introduction through TAR in most of the EU countries (16) is low, although some countries, specifically Poland and Lithuania, concentrate high levels of risk, the returning trucks route being the analyzed TAR that currently poses the highest risk for ASFV introduction into the EU. The spatial distribution of the risk of ASFV introduction varies importantly between the analyzed introduction routes. Results also highlight the need to increase the awareness and precautions for ASF prevention, particularly ensuring truck disinfection, to minimize the potential risk of entrance into the EU. CONCLUSIONS: This study presents the first assessment of ASF introduction into the EU through TAR. The innovative model developed here could be used in data scarce situations for estimating the relative risk associated to each EU country. This simple methodology provides a rapid and easy to interpret results on risk that may be used for a target and cost-effective allocation of resources to prevent disease introduction.     

Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV.     

非结构蛋白在非洲猪瘟病毒感染中作用

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的一种急性、出血性猪传染病,给疫情发生国家(地区)的养猪业造成重大经济损失.ASFV为双股DNA病毒,基因组含有150～167个开放阅读框(ORFs),可编码150～200种蛋白质,其中非结构蛋白有100余种.ASFV编码的酶、转录因子、调节宿主细胞功能蛋白和病毒免疫逃逸相关蛋白等作为重要的非结构蛋白,在病毒核苷酸代谢、DNA复制、修复、转录、蛋白修饰以及病毒与宿主细胞相互作用等过程中发挥重要作用,但仍有许多非结构蛋白的功能尚不明晰.因此,本文综述了 ASFV非结构蛋白在病毒感染中的作用,以期为ASFV非结构蛋白的进一步研究提供参考.     

African Swine Fever virus proteinase is essential for core maturation and infectivity

African swine fever virus (ASFV) encodes two polyprotein precursors named pp220 and pp62 that are sequentially processed during viral infection, giving rise to six major structural proteins. These reside at the core shell, a matrix domain located between the endoplasmic reticulum-derived inner envelope and the DNA-containing nucleoid. Proteolytic processing of the polyprotein precursors is catalyzed by the viral proteinase pS273R, a cysteine proteinase that shares sequence similarity with the SUMO1-processing peptidases. We describe here the construction and characterization of an ASFV recombinant, vS273Ri, that inducibly expresses the ASFV proteinase. Using vS273Ri, we show that repression of proteinase expression inhibits polyprotein processing and strongly impairs infective virus production. Electron microscopic examination of vS273Ri-infected cells showed that inhibition of proteolytic processing leads to the assembly of defective icosahedral particles containing a noncentered electron-dense nucleoid surrounded by an abnormal core shell of irregular thickness. The analysis of purified extracellular defective particles revealed that they contain the unprocessed pp220 and pp62 precursors, as well as the major DNA-binding nucleoid proteins p10 and pA104R. Altogether, these results indicate that the proteolytic processing of the polyproteins is not required for their incorporation into the assembling particles nor for the incorporation of the DNA-containing nucleoid. Instead, the ASFV proteinase is involved in a late maturational step that is essential for proper core assembly and infectivity.     

African Swine Fever Virus Infection in the Argasid Host,Ornithodoros porcinus porcinus

The pathogenesis of African swine fever virus (ASFV) infection in Ornithodoros porcinus porcinus was examined in nymphal ticks infected with the ASFV isolate Chiredzi/83/1. At times postinfection (p.i.) ranging from 6 h to 290 days, ticks or dissected tick tissues were titrated for virus and examined ultrastructurally for evidence of virus replication. The ASFV infection rate in ticks was 100% in these experiments, and virus infection was not associated with a significant increase in tick mortality. Initial ASFV replication occurred in phagocytic digestive cells of the midgut epithelium. Subsequent infection and replication of ASFV in undifferentiated midgut cells was observed at 15 days p.i. Generalization of virus infection from midgut to other tick tissues required 2 to 3 weeks and most likely involved virus movement across the basal lamina of the midgut into the hemocoel. Secondary sites of virus replication included hemocytes (type I and II), connective tissue, coxal gland, salivary gland, and reproductive tissue. Virus replication was not observed in the nervous tissue of the synganglion, Malpighian tubules, and muscle. Persistent infection, characterized by active virus replication, was observed for all involved tick tissues. After 91 days p.i., viral titers in salivary gland and reproductive tissue were consistently the highest detected. Successful tick-to-pig transmission of ASFV at 48 days p.i. correlated with high viral titers in salivary and coxal gland tissue and their secretions. A similar pattern of virus infection and persistence in O. porcinus porcinus was observed for three additional ASFV tick isolates in their associated ticks.African swine fever (ASF) is a highly lethal disease of domestic pigs for which animal slaughter and area quarantine are the only methods of disease control. African swine fever virus (ASFV), the causative agent of ASF, is a large double-stranded DNA virus which is the only member of an unnamed family of viruses. ASFV is the only known DNA arbovirus (4, 6, 12). The natural arthropod host for ASFV is Ornithodoros porcinus porcinus (Walton) ticks (40). Some confusion exists in earlier reports since ticks that should be classified as O. porcinus porcinus are often referred to as either O. moubata porcinus or simply O. moubata (59).ASFV can infect hosts through either a sylvatic cycle or a domestic cycle. In the sylvatic cycle, ASFV infects warthogs (Phacochoerus aethiopicus) and bushpigs (Potamochoerus spp.) as well as ticks of the genus Ornithodoros (7–10, 36, 55). In sub-Saharan Africa, warthogs occupy burrows which are frequently infested with large numbers of O. porcinus porcinus ticks (38, 45, 57, 58), and a correlation, though not absolute, has been established between ASFV infection of warthogs and the presence of O. porcinus porcinus ticks in burrows (57). In ASFV-enzootic areas, adult warthogs are typically nonviremic, although most are seropositive (28, 41, 46, 53, 58), and virus can usually be isolated only from lymph nodes (28, 41). Young warthogs, which are confined to the burrow for the first months of life, are most likely to be infected through feeding of infected O. porcinus porcinus ticks. Infection in young warthogs is subclinical, with viremic titers ranging from 2 to 3 log₁₀ 50% hemadsorption dose (HAD₅₀)/ml (56, 57), a level sufficient to infect a low percentage of naive ticks (42, 58, 30). The sylvatic ASFV cycle is further maintained by transovarial (43) and venereal (44) transmission in ticks. In burrows containing ASFV-infected ticks, infection rates are typically low (<2%), with the highest rate occurring in adult females (40, 45, 57, 65). The mechanism of ASFV transmission from the sylvatic cycle in Africa to the domestic cycle is most likely through feeding of infected ticks on pigs (41, 58), since direct contact between infected warthogs and domestic pigs has failed to result in transmission (36, 10, 28, 58), except in a single case (8). The virus may be transmitted between domestic pigs by either direct or indirect contact (33).Various characteristics of ASFV infection have been studied in a number of Ornithodoros spp. ticks. The first association of ASFV with a tick was made by Sanchez-Botija (50), who reported isolation of ASFV from O. erraticus, a tick native to the Iberian peninsula and later considered important to maintenance of ASFV in an enzootic cycle in that region (51). In the first experimental infection, striking differences were found in the percentage of O. moubata porcinus ticks infected by two different ASFV isolates, a low infectious dose for ticks (ranging from of 0.9 to 4 log₁₀ HAD₅₀) was demonstrated, and transmission out to 469 days postinfection (p.i.) was successful with single ticks (42). Experimental ASFV infection and transmission to pigs has been demonstrated for O. savignyi, a tick found in Africa (34), O. coriaceus (23, 25) and O. turicata (25), ticks indigenous to the United States, and O. puertoricensis (25, 14), a tick indigenous to the Caribbean. A 40% mortality rate was found in infected O. coriaceus (25) and O. puertoricensis ticks (15). O. marocanus, which was formerly referred to as O. erraticus, transmitted ASFV out to 588 days p.i., although 73% mortality was reported for infected ticks (16, 17). A number of reports have not found significant virus-induced mortality in O. moubata porcinus ticks (22, 40–44). In contrast, mortality rates were 35% higher in infected O. moubata porcinus females in the only study to examine mortality during the gonotrophic cycle (26).Specific aspects of ASFV infection in the natural host remain poorly understood. Greig (22) experimentally infected O. moubata porcinus ticks with pathogenic ASFV isolates and used virus titration and immunofluorescence of dissected tissues to determine that the midgut was the initial site of viral replication and the site of longest persistence. Several other tissues were also found to have detectable levels of virus, although the midgut was the only tissue which was consistently positive. The presence of ASFV has been demonstrated in hemocytes of infected O. coriaceus ticks by electron microscopy and immunofluorescence studies, but the presence or nature of virus replication was not addressed (13).Here we describe the pathogenesis and persistence of ASFV infection in O. porcinus porcinus ticks. Our data indicate that initial ASFV replication occurs in phagocytic digestive cells of the midgut epithelium, with secondary replication occurring in undifferentiated midgut cells at later times p.i. Generalization of virus infection from the midgut to other tick tissues required 2 to 3 weeks. Secondary sites of virus replication include hemocytes (type I and II), coxal gland, salivary gland, connective tissue, and reproductive tissue. Successful tick-to-pig transmission correlated with relatively high viral titers in salivary and coxal glands. Persistent infection in the tick involves continuous viral replication in several tissues and is associated with minimal cytopathology.     

表达荧光素酶和绿色荧光蛋白双报告基因重组非洲猪瘟病毒的构建

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染家猪和野猪引起的一种高致病性传染病,家猪感染ASFV强毒株后死亡率接近100％.为了研究ASFV的致病机制,利用绿色荧光蛋白(EGFP)作为报告基因构建重组病毒已经广泛应用,但易于定量检测且适用于高通量筛选的重组ASFV目前没有报道.本研究以ASFVHLJ/18分离株为亲本病毒,采用CRISPR/Cas9基因编辑技术和同源重组技术将表达Gaussia荧光素酶(Gluc)和EGFP的报告基因表达盒插入到ASFV的K145R基因的位置,构建可以同时表达Gluc和EGFP的重组ASFV(rASFV-Gluc-EGFP).通过PCR鉴定、Gluc和EGFP表达的检测,确定获得的重组ASFV能够感染猪肺泡巨噬细胞且表达Gluc和EGFP.对构建的重组病毒和亲本病毒进行生长曲线比较,结果表明插入的报告基因不影响病毒在猪肺泡巨噬细胞中的复制.F5、F10和F15代重组病毒基因鉴定和报告基因表达检测结果表明,重组病毒能稳定表达插入的双报告基因.本研究成功构建了表达Gluc和EGFP的重组ASFV,可以针对报告基因进行定量检测和高通量筛选,为ASFV的感染和致病机制研究奠定坚实的基础.     

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.