猪瘟和猪繁殖与呼吸综合征混合感染的快速检测及地域流行性分析

呼吸道和繁殖障碍疾病是猪场最常见的两种疾病,严重影响猪群的健康和正常生产,已经给世界养猪业造成了很大的经济损失。而且这两种疾病的病原复杂多样,不同的猪场病因可能完全不同,从而给疾病的诊断与防治带来了极大的困难。多年的研究表明,呼吸道和繁殖障碍疾病的病原包     

猪瘟和猪繁殖与呼吸综合征混合感染的快速检测及地域流行性分析

呼吸道和繁殖障碍疾病是猪场最常见的两种疾病,严重影响猪群的健康和正常生产,已经给世界养猪业造成了很大的经济损失[1].而且这两种疾病的病原复杂多样,不同的猪场病因可能完全不同,从而给疾病的诊断与防治带来了极大的困难.     

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.     

African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.     

Inactivation of the RNase Activity of Glycoprotein Erns of Classical Swine Fever Virus Results in a Cytopathogenic Virus

Envelope glycoprotein E^rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E^rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E^rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E^rns, the mutated proteins had lost their RNase activity. The mutated proteins reacted with E^rns-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E^rns. This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E^rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E^rns gene and by neutralizing polyclonal antibodies directed against E^rns, indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E^rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.Classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus belong to the genus Pestivirus within the family Flaviviridae (10). The viruses are structurally, antigenically, and genetically closely related. BVDV and border disease virus can infect ruminants and pigs. CSFV infections are restricted to pigs (6). Pestiviruses are small, enveloped, positive-stranded RNA viruses (23). The genome of pestiviruses varies in length from 12.5 to 16.5 kb (1, 2, 7, 17, 19, 25, 26, 28, 32) and contains a single large open reading frame (ORF) (1, 7, 8, 17, 26). The ORF is translated into a polyprotein which is processed into mature proteins by viral and host cell proteases (30). The envelope of the pestivirus virion contains three glycoproteins, E^rns, E1, and E2 (35). Animals infected with pestiviruses raise antibodies against at least two viral glycoproteins, namely, E^rns and E2 (16, 34, 42). Inhibition studies with E2 and E^rns produced in insect cells showed that both envelope proteins are indispensable for viral attachment and entry of pestiviruses into susceptible cells (13). In the virion, E^rns is present as a homodimer with a molecular mass of about 100 kDa (35). E^rns lacks a membrane anchor, and association with the envelope is accomplished by an as-yet-unknown mechanism. Significant amounts of E^rns are secreted from infected cells (30). A unique feature is that E^rns, besides being an envelope protein, possesses RNase activity (12, 31). E^rns belongs to the family of extracellular RNases consisting of several fungal (e.g., RNase T₂ and Rh) and plant (e.g., S glycoproteins of Nicotiana alata) RNases (12, 31). These RNases contain two homologous regions of 8 amino acids each which are spaced by 38 (E^rns) nonhomologous amino acids and which form the RNase active site. Histidine residues in both regions appear to be essential for RNase catalysis (15).The role of this RNase activity in the replication of pestiviruses or in the pathogenesis of a pestivirus infection is an interesting issue that, as yet, has not been studied. The availability of a recently generated infectious DNA copy of CSFV strain C (24) has given us the opportunity to study the effect of defined mutations in a pestivirus genome. In this paper, we report the inactivation of the RNase activity of E^rns by mutagenesis. To characterize the mutated proteins, we produced large amounts of them in insect cells (12). By reverse genetics, we generated an RNase-negative CSFV recombinant. The effect of the inactivation of the RNase activity of E^rns on the replication of CSFV in vitro was studied.     

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。     

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.     

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.     

多重RT-PCR一步法技术同时检测猪瘟病毒和蓝耳病病毒方法的建立以及初步应用

猪瘟病毒和蓝耳病病毒均能导致猪繁殖障碍,对养猪生产影响很大。根据猪瘟病毒(CSFV)和猪蓝耳病(PRRS)的基因保守序列设计了2对针对这2种病毒的特异引物,并建立了多重RT-PCR方法,分别对其最佳反应条件、特异性及敏感性进行了测定,结果表明能同时扩增得到2条与试验设计相符的167bp(CSFV)和320bp(PRRS)特异性条带,同时具有较好的特异性;敏感性检测结果表明,临床阳性的样品提取的核酸稀释1000倍后仍能检测出CSFV和PRRSV。本方法的建立对于这2种病毒病的早期快速检测具有十分重要的意义。     

Highly Sensitive,Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.     

Development of an Accurate and Sensitive Diagnostic System Based on Conventional PCR for Detection of African Swine Fever Virus in Food Waste

African swine fever virus (ASFV), a highly contagious virus, can cause diseases with high mortality rates in pigs, making it a pathogen of social and economic significance. ASFV has been reported to show potential long-term survival in living livestock, such as pigs, but also in leftover cooking meat and undercooked pork meat. Hence, it is possible that there could be direct reinfection or secondary infection through feed produced from household food waste and treatment facilities. Many polymerase chain reaction (PCR)-based molecular diagnostic techniques to detect ASFV in clinical swine samples have been reported. However, those with applicability for food waste samples, which contain relatively low viral copy numbers and may contain various unknown inhibitors of PCR, are still lacking. In this study, we developed a conventional PCR-based diagnostic system that can detect ASFV with high sensitivity from food waste sample types. The technique shows a 10–100 times higher limit of detection compared to that of previously reported methods based on conventional PCR and quantitative real-time PCR. It is also capable of amplifying a sequence that is approximately 751 nucleotides, which is advantageous for similarity analysis and genotyping. Moreover, a ASFV-modified positive material different from ASFV that could synthesize 1400 nucleotide amplicons was developed to identify false-positive cases and thus enhance diagnostic accuracy. The method developed herein may be applicable for future ASFV monitoring, identification, and genotyping in food waste samples.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-022-01007-y.     

Development of a Highly Sensitive Method for Detection of Clarithromycin-Resistant Helicobacter pylori from Human Feces

Gastric infection of clarithromycin (CAM)-resistant Helicobacter pylori is one of the major causes of failure to eradicate this organism. A noninvasive and useful method for the detection of CAM-resistant H. pylori from human feces by restriction fragment length polymorphism (RFLP)-nested polymerase chain reaction (PCR) targeting the mutation of the 23S rRNA gene that confers CAM-resistance in H. pylori was developed in this study. Our nested PCR method detected DNA of H. pylori in feces with high sensitivity and specificity compared with both an enzyme-linked immunoadsorbent assay (ELISA) of H. pylori in feces and the isolation of H. pylori from gastric biopsy. Furthermore, the results of mutation analysis of the H. pylori 23S rRNA gene amplified from feces completely correlated with both that of the H. pylori 23S rRNA gene amplified from the isolates of gastric biopsy and the susceptibility of H. pylori isolates to CAM. Therefore, our results show that this RFLP/nested PCR method is useful for the accurate diagnosis of CAM-resistant H. pylori infection from feces.     

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.     

猪瘟病毒感染性cDNA克隆的构建及其致病性

为了建立研究猪瘟病毒的技术平台,利用RT-PCR技术构建了猪瘟病毒中国石门株(Shimen)的全长有感染性cDNA克隆pT7SM。通过体外转录线性化的pT7SM并将转录的RNA转染至PK-15细胞中,获得了猪瘟病毒粒子。再用间接免疫荧光法测定了其生长曲线,通过电镜观察到重组病毒呈球形,具有囊膜结构。进一步把获得的猪瘟病毒粒子感染非免疫实验猪,结果子代病毒与石门株类似,对非免疫实验猪有强烈的致病性,证明该全长cDNA克隆可靠稳定,忠实地保留了石门株病毒的感染性和致病特征。由此为进一步探讨猪瘟病毒的致弱机制及病毒与机体相互作用的机制打下了良好的基础。