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.     

猪瘟病毒的重组分析

以21株猪瘟病毒的编码序列作为研究对象,通过对基因树的比较和四重体分析,研究猪瘟病毒毒株间可能的重组关系。分析结果表明,ALD(D49532)与GPE-(D49533)问可能有一重组片段E0、E1和E2,而持续感染毒株CSFV39(AF407339)中的NS5A-NS5B可能来源于Shimen强毒株(AF092448),这说明不同猪瘟病毒极有可能在自然环境或减毒疫苗的应用过程中由于混合感染而发生遗传物质的交换,从而适应宿主环境的改变,并产生特殊的表型。     

Classical Swine Fever Virus vs. Classical Swine Fever Virus: The Superinfection Exclusion Phenomenon in Experimentally Infected Wild Boar

Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01 strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated with the virulent strain Margarita (secondary virus). For exclusive detection of the Margarita strain, a specific qRT-PCR assay was designed, which proved not to have cross-reactivity with the Cat01 strain. The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the Margarita virus strain, while this virus was able to infect naive PBMCs efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally, a sequence analysis could not detect CSFV Margarita RNA in the samples tested from Group B. Our results suggested that the SIE phenomenon might be involved in the evolution and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of IFN-α, which might be associated with the lack of innate immune mechanisms.     

Lymphocyte Apoptosis during Classical Swine Fever: Implication of Activation-Induced Cell Death

Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential—a particular early marker for apoptosis—were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G₁ cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.Leukopenia can result in immunosuppression and is a hallmark of certain virus infections, such as classical swine fever (CSF), bovine viral diarrhea, and dengue fever, all caused by virus members of the family Flaviviridae (for reviews, see references 27, 54, and 55).Classical swine fever virus (CSFV), a member of the genus Pestivirus, is a small enveloped RNA virus causing an economically important and fatal disease of pigs. The virus is known to have a particular affinity for cells of the immune system, which seems to relate to the detrimental effects on the immune and hematopoietic systems (9, 41, 52, 54, 55). The target cells for CSFV in the peripheral blood appear to be mainly monocytes, although in later stages of the disease infection of lymphocytes (50, 55) as well as granulocytic cells (50) has been noted. All leukocyte populations can be depleted during CSF, but B lymphocytes are particularly sensitive (52). Despite this current knowledge, the immunopathological mechanisms and the role played by the virus infection of leukocytes with respect to the disease pathology in general, and leukocyte death in particular, have not been elucidated. Generally, leukopenia could be a result of cell death, suppression of hematopoiesis, or change in the distribution of leukocytes within different compartments of the immune system. Leukocyte death can be caused by necrosis or apoptosis. The latter, a suicide-like and genetically programmed form of cellular death, is involved in physiological as well as pathological cell death, induced by either a lack or presence of particular stimuli (10). Late stages of apoptosis are characterized by typical morphological criteria and degradation of DNA (10). It has recently been reported that other characteristic cellular features, particularly a reduction in mitochondrial transmembrane potential (ΔΨ_m), precede these morphological and nuclear changes (26, 57). Due to the important role played by apoptosis in the regulation of leukocyte numbers (2, 3, 10, 25, 29), as well as the observation that virus infections can be associated with apoptosis (1, 40, 48), an important step in understanding the pathogenesis of virus-induced leukopenia would be to determine the role played by apoptosis in the depletion of lymphocytes.In this study, we (i) analyzed the relationship between the kinetics of leukocyte depletion in the peripheral blood and the virus infection therein and (ii) determined the implication of apoptosis. A significant reduction of lymphocyte numbers in the blood of CSFV-infected pigs was noted as early as 1 day postinfection (p.i.), before viremia or virus-infected leukocytes were apparent. An increase in lymphocytes programmed to die by apoptosis was also observed early p.i., implying an important role for apoptosis in the destruction of leukocytes during this disease. We present data suggesting a mechanistic role for Fas-mediated activation-induced cell death (AICD).     

Characterization of Essential Domains and Plasticity of the Classical Swine Fever Virus Core Protein

Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins E^rns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala₂₅₅ at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.Several important pathogens of cloven-hoofed animals, such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), comprise the genus Pestivirus. The latter, together with the genera Flavivirus and Hepacivirus, belong to the family Flaviviridae. Pestiviruses possess a single-stranded, positive-sense RNA genome of at least 12.3 kb, coding for one polyprotein. It is processed into 12 mature viral proteins by cellular and viral proteases.Pestiviral virions contain four structural proteins, the small, basic Core protein and three envelope glycoproteins, E^rns, E1, and E2. The nucleocapsid consists of Core protein and the viral RNA genome (13, 38, 42). While Flaviviridae typically encode Core protein as the first product of the polyprotein, pestiviruses encode the unique N-terminal protease N^pro at the analogous position (41). N^pro facilitates the degradation of interferon regulatory factor 3 (IRF3) (1) and generates the N terminus of Core by autoproteolytic action. If cleavage is blocked, no generation of infectious particles can be observed and the N^pro-Core protein accumulates in the cytoplasm (39). However, nonviral proteins can be expressed between N^pro and Core if an additional protease cleavage site (2A protease of foot-and-mouth disease virus) is integrated at the Core N terminus (8). The C terminus of Core is created through an intramembrane cleavage by signal peptide peptidase (12). The same proteolytic mechanism is employed in the biosynthesis of Core protein of hepatitis C virus (HCV) (29) but not by members of the genus Flavivirus. Here, the C terminus of the Capsid protein is generated by the viral NS3 protease (4).Recent studies on the structure of the pestiviral Core protein describe it as an intrinsically disordered protein on the basis of far-UV circular dichroism and intrinsic fluorescence spectroscopy analysis (15, 32). Its disordered nature is highlighted in analogy to the Core N terminus of other members of the Flaviviridae, which is often found to be responsible for RNA binding (3, 6, 7, 9, 25). Neither a C-terminal ordered domain, apart from 15 amino acids at the C terminus, nor assembly to an alphahelical, dimeric structure, as described for flaviviruses and HCV, has been reported for the pestiviral Core protein (3, 15, 32). Its interaction with nucleic acids is of low affinity and low specificity, and no specific RNA packaging signals have been identified (32). This unspecific interaction with RNA was further supported by the functional replacement of the RNA binding domain of Sindbis virus Capsid protein by BVDV Core (32). Recently, RNA chaperone activity of BVDV Core protein has been reported, which is responsible for changes in RNA structure without the need of chemical energy provided as ATP (15). RNA chaperone activity relies on a disordered protein stretch that is insensitive to heat.A large internal deletion in Core protein was lethal for recombinant BVDV. However, infectious virus could be recovered by providing Core along with other structural proteins in trans (37). Thus, the importance of pestiviral Core protein for the generation of infectious virus particles is known, but no reports exist on the functional organization of this protein.This study analyzes the domain structure of the pestiviral Core protein by mapping regions important for virus assembly. Truncations and deletions within the Core protein, as well as a dramatic increase of molecular mass, reveal a plasticity that does not fit the strict symmetric requirements that are to be expected for icosahedral nucleocapsids.     

Inferring the Dynamics of Diversification: A Coalescent Approach

Recent analyses of the fossil record and molecular phylogenies suggest that there are fundamental limits to biodiversity, possibly arising from constraints in the availability of space, resources, or ecological niches. Under this hypothesis, speciation rates decay over time and biodiversity eventually saturates, with new species emerging only when others are driven to extinction. This view of macro-evolution contradicts an alternative hypothesis that biodiversity is unbounded, with species ever accumulating as they find new niches to occupy. These contrasting theories of biodiversity dynamics yield fundamentally different explanations for the disparity in species richness across taxa and regions. Here, we test whether speciation rates have decayed or remained constant over time, and whether biodiversity is saturated or still expanding. We first derive a general likelihood expression for internode distances in a phylogeny, based on the well-known coalescent process from population genetics. This expression accounts for either time-constant or time-variable rates, time-constant or time-variable diversity, and completely or incompletely sampled phylogenies. We then compare the performance of different diversification scenarios in explaining a set of 289 phylogenies representing amphibians, arthropods, birds, mammals, mollusks, and flowering plants. Our results indicate that speciation rates typically decay over time, but that diversity is still expanding at present. The evidence for expanding-diversity models suggests that an upper limit to biodiversity has not yet been reached, or that no such limit exists.     

Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.     

猪瘟病毒Erns在杆状病毒系统中的表达和纯化

采用Bac-to-Bac表达系统构建重组杆状病毒rAcV-MBP-Erns,感染Sf9昆虫细胞,经免疫荧光及Western blot分析证实MBP-Erns融合蛋白在Sf9细胞中高效表达。表达的MBP-Erns以可溶和包涵体两种形式存在。在Sf9细胞中规模化增殖重组病毒,经Amylose Resin亲和层析纯化获得高纯度MBP-Erns,制备的MBP-Erns具有良好免疫原性,这些工作为研究该蛋白的生物学功能和免疫原性奠定基础。     

New Routes to Phylogeography: A Bayesian Structured Coalescent Approximation

Phylogeographic methods aim to infer migration trends and the history of sampled lineages from genetic data. Applications of phylogeography are broad, and in the context of pathogens include the reconstruction of transmission histories and the origin and emergence of outbreaks. Phylogeographic inference based on bottom-up population genetics models is computationally expensive, and as a result faster alternatives based on the evolution of discrete traits have become popular. In this paper, we show that inference of migration rates and root locations based on discrete trait models is extremely unreliable and sensitive to biased sampling. To address this problem, we introduce BASTA (BAyesian STructured coalescent Approximation), a new approach implemented in BEAST2 that combines the accuracy of methods based on the structured coalescent with the computational efficiency required to handle more than just few populations. We illustrate the potentially severe implications of poor model choice for phylogeographic analyses by investigating the zoonotic transmission of Ebola virus. Whereas the structured coalescent analysis correctly infers that successive human Ebola outbreaks have been seeded by a large unsampled non-human reservoir population, the discrete trait analysis implausibly concludes that undetected human-to-human transmission has allowed the virus to persist over the past four decades. As genomics takes on an increasingly prominent role informing the control and prevention of infectious diseases, it will be vital that phylogeographic inference provides robust insights into transmission history.     

Bayesian Inference of Reticulate Phylogenies under the Multispecies Network Coalescent

The multispecies coalescent (MSC) is a statistical framework that models how gene genealogies grow within the branches of a species tree. The field of computational phylogenetics has witnessed an explosion in the development of methods for species tree inference under MSC, owing mainly to the accumulating evidence of incomplete lineage sorting in phylogenomic analyses. However, the evolutionary history of a set of genomes, or species, could be reticulate due to the occurrence of evolutionary processes such as hybridization or horizontal gene transfer. We report on a novel method for Bayesian inference of genome and species phylogenies under the multispecies network coalescent (MSNC). This framework models gene evolution within the branches of a phylogenetic network, thus incorporating reticulate evolutionary processes, such as hybridization, in addition to incomplete lineage sorting. As phylogenetic networks with different numbers of reticulation events correspond to points of different dimensions in the space of models, we devise a reversible-jump Markov chain Monte Carlo (RJMCMC) technique for sampling the posterior distribution of phylogenetic networks under MSNC. We implemented the methods in the publicly available, open-source software package PhyloNet and studied their performance on simulated and biological data. The work extends the reach of Bayesian inference to phylogenetic networks and enables new evolutionary analyses that account for reticulation.     

猪瘟病毒结构蛋白Erns在E.Coli中的高效表达及纯化

猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swinefever virus,CSFV)引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一.CSFV基因组为单股正链RNA分子,长约12.3kb,仅编码一个开放性阅读框.位于5'端的囊膜糖蛋白Erns、E1和E2构成了CSFV的外壳,其中Erns和E2参与病毒感染细胞的过程,并能诱导宿主产生保护性免疫应答[1].目前研究的CSFV基因工程疫苗主要以E2蛋白作为抗原,并通过检测Erns的抗体来区分E2标记疫苗免疫猪和野毒感染猪,有利于剔除猪群中潜在的传染源,达到最终消灭猪瘟的目的.氨基酸序列比较发现,CSFV的Erns氨基酸序列中有地衣类与植物核苷酸酶家族的特征序列,属于胞外RNase家族,具有RNase活性,Erns可降解病毒和细胞的RNA,在研究CSFV的致病机制方面具有重要意义[2].本研究利用RT-nPCR技术,克隆到了Erns基因,并利用大肠杆菌表达系统高效表达了Erns蛋白,纯化后的蛋白具有良好的生物学活性,为进一步建立Erns抗体的检测方法和探讨Erns蛋白在CSFV致病过程中的作用奠定了基础.     

Prediction of Recognition Sites for Genomic Replication of Classical Swine Fever Virus with Information Analysis

In order to explore the mechanism for the genomic replication of classical swine fever virus (CSFV), so as to make a basis for investigating its pathogenicity, an introduction of the information theory is presented in connection with the statistical mechanics, whence small-sample statistics appears naturally as a consequence of the Bayesian approach. Furthermore, a selection rule for identifying the pattern of a recognition site for an RNA-binding protein is proposed by means of the maximum entropy principle. Based on those, the information contents of 3"-untranslated regions (3"UTRs) of genomes of 20 CSFV strains and 5"-untranslated regions (5"UTRs) of genomes of 58 CSFV strains are analyzed with a computational algorithm in a reduction mode, and the 3"UTR sites of 20 strains and 5"UTR sites of 58 strains containing important motifs are extracted from the unaligned RNA sequences of unequal lengths. These sites, which have the patterns of sequence and structure similar to the putative cis elements related to the regulation of genomic replication, would be identified as the potential recognition sites in 3"UTRs and 5"UTRs for CSFV replicase responsible for classical swine fever virus genomic replication, and to some extent, this identification is supported by experimental evidence. Finally, information analysis allows a presumption to be made about the CSFV RNA replication initiation mechanism.     

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447_Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447_Δc. Upon infection of the natural host, Vp447_Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.     

猪瘟病毒结构蛋白E^ms在E．coli中的高效表达及纯化

猪瘟（Classical swine fever,CSF）是由猪瘟病毒（Classical swine fever virus,CSFV）引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一。CSFV基因组为单股正链RNA分子,长约12．3kb,仅编码一个开放性阅读框。位于5’端的囊膜糖蛋白E^ms、E1和E2构成了CSFV的外壳,     

猪瘟病毒石门株NS2—3基因片段的序列测定及比较

根据猪瘟病毒Ｃ株的序列,以计算机辅助设计,化学合成１对引物（ＰＦ５６４８／ＰＲ６６０４）,应用ＲＴＰＣＲ技术从感染猪血中成功地扩增了我国猪瘟病毒强毒石门株ＮＳ２３基因片段,大小为９５７ｂｐ,位于ＮＳ３基因的中部ＮＴＰａｓｅ和Ｈｅｌｉｃａｓｅ活性区。克隆后测序,结果表明该段基因产物具有解旋酶超家族全部七个特征性保守序列,包括共同的ＮＴＰ结合基序Ａ位点（ＧＸＧＫＴ／Ｓ）和Ｂ位点（３ｈｙ,２ｘ）Ｄ。序列同源性比较表明,石门株与日本的ＡＬＤ和ＧＰＥ－株同源性最高,与其它３株猪瘟病毒（Ｃ株、Ｂｒｅｓｃｉａ株和Ａｌｆｏｒｔ株）的同源性也很高,并与２株牛病毒性腹泻病毒（ＢＶＤＶ）（ＮＡＤＬ株和ＳＤ１株）也有较高的同源性,尤其是由核苷酸序列推导的氨基酸序列,同源性均大于９０％,是瘟病毒属基因组中最保守的区段,这与该基因产物在病毒复制及聚蛋白前体加工过程中所具有的重要功能是一致的