Comparative sequence analysis of classical swine fever virus isolates from the epizootic in The Netherlands in 1997-1998.

Sixteen classical swine fever virus (CSFV) field isolates from outbreaks of classical swine fever from the period between February 1997 and March 1998 in the Netherlands were sequence analysed. Parts of the 5' noncoding region (5'NCR) and the E1/E2 gene were sequenced after RT-PCR. The obtained sequences were compared with isolates of recent outbreaks in Europe and those of former outbreaks in the Netherlands. Sequence alignment of the 5'NCR region (321 bp) revealed that the isolates of the Dutch outbreak of 1997-1998 were closely linked to an isolate of the CSF outbreak that started in Paderborn, Germany in 1996. A relatively large fragment of the E1/E2 gene of 850 bp, including the antigenic region of E2, which is one of the most variable regions of the CSFV genome, was sequenced to determine whether this region can be used for epidemiology within an epizootic. Epidemiological tracing of transmission of virus was followed, starting from the first isolate and a line of five generations of viruses was analysed. Besides this, new isolates which could not be epidemiologically linked to preceding ones were also characterised. Differences between the isolates of the Dutch outbreak were minor both for the linked as well as for the non-linked isolates, indicating that all isolates have a common origin. Furthermore, our data show for the first time the genetic stability of CSFV even in the highly variable antigenic region of the E2 gene during a major epidemic lasting more than 1 year.     

Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs

The clinical diagnosis of classical swine fever (CSF) still caused problems to the veterinarians during the last decade. The primary CSF outbreak was often detected too late and, meanwhile, the virus had spread. Consequently, the recent classical swine fever virus isolates (CSFV) were suspected to be of low virulence. The purpose of the study was to quantify the virulence of four recent CSFV by evaluating the clinical and pathological signs caused by different CSFV. Pigs of the same breed and age group were inoculated intranasally with CSFV from recent epidemics in European Union (EU) member states. The CSFV used are registered in the data base of the EU Reference Laboratory for CSF and belong to different genotypes: 2.1, 2.2 and 2.3 respectively. Clinical signs of CSF were evaluated by using a score system suggested previously (Mittelholzer et al., 2000: Vet. Microbiol. 74, 293). For the evaluation of pathological lesions, a new pathological score was introduced. The four CSFV tested here were classified as moderately virulent in general, although one CSFV may cause different clinical courses, ranging from highly virulent to avirulent. This indicates the importance of additional factors in the host animal for virulence. Differences in the clinical and pathological signs between these four recent CSFV were rather minor, emphasizing that the genetic typing of CSFV is absolutely essential. Differences towards former CSFV (e.g. reference virus strain Alfort 187) were more pronounced, especially regarding the onset and duration of the disease, the occurrence of skin haemorrhages and pathological lesions of kidney, subcutis and serosae. It is concluded that clinical diagnosis of CSF is rather difficult in pigs up to 14 days post-CSFV infection using these four CSFV, emphasizing the need for careful differential diagnosis and the laboratory investigation for CSF at an early stage.     

Molecular epidemiology of classical swine fever in the Russian Federation

The ability to discriminate between various classical swine fever virus (CSFV) strains and isolates is a prerequisite for following the spread of the virus after an outbreak. To determine the relatedness between Russian CSFV isolates from different geographical regions, three fragments of the viral genome (5' NTR, the variable region of the E2 gene and a fragment of the NS5B gene) were sequenced and used for genetic typing. Thirty-one field isolates were obtained from CSF outbreaks which occurred between 1994 and 1999. In addition, three attenuated strains were included in the study, namely the LK and CS vaccine strains, and the moderately virulent 238H isolate. The vaccine strains have been used in Russia for more than 30 years. Our results showed that all field isolates are in subgroup 1.1 together with Alfort 187 and with the highly virulent strain Shimen. In contrast, the CS and LK vaccine strains belong to subgroup 1.2. While there is no evidence for the reversion of the two vaccine strains to wild type, it is feasible that the highly virulent Shimen strain, which has been used as a challenge strain for many years, contributed to field strain generation. The Russian field isolates from the 1990s can be distinguished from the CSF virus isolates which occurred in the EU Member States in the same decade, as here all outbreaks were caused by CSF viruses belonging to subgroup 2.     

Phylogenetic analysis of recent classical swine fever virus (CSFV) isolates from Assam, India

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5′ UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF.     

猪瘟诊断方法的研究进展

猪瘟是由猪瘟病毒引起的一种急性、发热性、接触性传染病,可引起各种年龄猪发病。随着对猪瘟病毒研究的深入,猪瘟在一定程度上得到了有效控制。但是近年来,世界各国流行的猪瘟在流行病学、临床症状和病理变化等方面出现了一些新的变化,猪瘟的防控出现了许多新的情况。我国猪瘟的发病率亦呈上升趋势,严重威胁着我国养猪业的发展,给养猪业造成了极大的经济损失。因此,建立准确的实验室诊断方法,对于预防和控制猪瘟有重要意义。本文综述了猪瘟诊断技术方面的研究进展,为猪瘟的及时诊断提供参考。     

分子生物学技术在猪瘟诊断中的应用

猪瘟是由猪瘟病毒引起的一种急性、发热性、接触性传染病,可引起各种年龄猪发病.随着对猪瘟病毒研究的深入,猪瘟在一定程度上得到有效的控制.但是近年来,世界各国流行的猪瘟在流行病学、临床症状和病理变化等方面出现了一些新的变化,猪瘟的防控出现了许多新的情况.早已宣布消灭了猪瘟的一些欧洲国家又报道有猪瘟发生.我国猪瘟的发病率亦呈上升趋势,严重威胁着我国养猪业的发展,给养猪业造成极大的经济损失.论文综述了猪瘟分子生物学诊断技术的研究进展,为猪瘟的及时诊断提供参考.     

中国猪瘟发生风险定性评估

为定性评估中国猪瘟发生风险,本研究通过收集整理国内外猪瘟研究资料,运用动物疫病风险分析方法对猪瘟病原特性、传染源、传播途径、易感动物和已采取防控措施效果5个主要风险因素进行定性分析。风险评估认为目前全国范围均存在猪瘟发生风险,由于实施了猪瘟强制免疫政策,猪群均存在一定的免疫抗体,因此猪瘟大规模流行几率很低,但点状散发流行几率较高。现阶段疫苗免疫仍是中国防控猪瘟的重要手段。     

Clinical signs and epidemiology of classical swine fever: a review of new knowledge

Although classical swine fever (CSF) has been well known for decades and epidemics still occur, clinical diagnosis continues to cause problems for veterinary practitioners. This is due to the extensive differential diagnosis, further complicated by the emergence of new diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine dermatitis and nephropathy syndrome (PDNS). In addition, acute, chronic and prenatal courses of CSF have to be distinguished. As a cause of considerable economical losses within the EU, control of CSF requires knowledge of the primary outbreaks and spread of the disease. Genetic typing of CSF virus isolates has proved to be a potent method of supporting epidemiological investigations. Phylogenetic analysis of CSF virus strains and isolates originating from different continents has allowed three genetic groups and several subgroups within these groups to be distinguished. Whereas isolates belonging to group 3 seem to occur solely in Asia, all CSF virus isolates of the 1990s isolated in the EU belonged to one of the subgroups within group 2 (2.1, 2.2, or 2.3) and were clearly distinct from former CSF reference viruses, which belong to group 1. Within the EU, different strategies are followed for the eradication of CSF in domestic pigs and in wild boar. While a strict non-vaccination policy is followed for domestic pigs, eradication of the disease in wild boar is more complex, and oral immunisation together with special hunting strategies have been applied. Recently, marker vaccines with a companion discriminatory test designed to allow differentiation between vaccinated animals and animals having recovered from field virus infection have been developed. Preliminary studies indicated that the discriminatory tests had a reduced sensitivity and specificity. Further improvements are therefore necessary before marker vaccines can be considered for emergency use in EU Member States. Prevention of CSF remains the main objective within the EU.     

Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus

The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.     

Genetic typing of German isolates of classical swine fever virus]

During the last decade several outbreaks of classical swine fever (CSF) occurred in Germany in domestic pigs and in wild boar, respectively. Two major epidemics which also affected other EU Member States were recorded. To support epidemiological investigations genetic typing was applied and virus isolates originating from different outbreaks in Germany were assigned to groups and virus types. Two genomic regions were selected for the phylogenetic analysis, namely 150 nucleotides from the 5' non-translated region (5'-NTR) and 190 nucleotides from the E2 glycoprotein gene. All German CSF virus isolates of the nineties (Group 2) were distinct from former reference strains (Group 1). Within Group 2 both genomic regions allowed to distinguish three subgroups, namely 2.1, 2.2 and 2.3. Within subgroup 2.3 five virus types could be discriminated using the 5'-NTR sequences. These are 2.3*Uelzen and 2.3*Spreda, mainly with isolates from Lower Saxony, as well as 2.3*Rostock, 2.3*Güstrow and 2.3*Spante, mainly with isolates from Eastern Germany. Analysis of the E2 gene fragment allowed a better discrimination between single isolates, but only two virus types could be defined: 2.3*MV/BB, comprising the isolates from Eastern Germany, and 2.3*NI, with the isolates from Lower Saxony. Genetic typing allowed to discriminate between isolates involved in different CSF epidemics, and was useful for tracing the origin and spread of CSF viruses. Due to the close relationship between German CSF virus isolates, epidemiological data are a prerequisite for the interpretation of the results obtained by genetic typing. In addition, at least both genomic regions suggested here should be analysed to determine the identity of a new isolate.     

Molecular characterization of African swine fever virus isolates originating from outbreaks in the Russian Federation between 2007 and 2011

African swine fever is one of the most important viral diseases of pigs and which caused significant economic damage on the pig production worldwide. Nowadays, it is still present on the African continent, in Transcaucasus countries (TCC), on Island of Sardinia and in Russia. Outbreaks of the disease have been reported in Russia for the last four years, affected especially the Southern Federal District of the country. Since 2010, a new outbreak area has been observed in the Northwestern Federal District. In order to study the evolution of African swine fever virus (ASFV) isolates, strains were collected in the Russian Federation from 2007 to 2011 and investigated by means of partial sequencing and fragment length polymorphism. In detail, 7 variable regions, namely B646L, E183L, I196L, B602L, I73R/I329R, I78R/I215L and KP86R were investigated. Phylogenetic analyses revealed 100% nucleotide identity of B646L and E183L gene sequences of all examined isolates. All isolates formed one genetic cluster within genotype II. Moreover, no amplified fragment length polymorphism (AFLP) was observed for B602L, I196L, I73R/I329R, and I78R/I215L genes. The flanking primers used to amplify the KP86R gene failed to amplify a product in all the isolates. The obtained data strongly suggests that only one ASFV virus variant caused the outbreaks from 2007 to 2011 in the territory of the Russian Federation.     

Comparison of viraemia- and clinical-based estimates of within- and between-pen transmission of classical swine fever virus from three transmission experiments

Analyses of recent classical swine fever (CSF) epidemics in the European Union have shown that silent circulation of CSF virus (CSFV) occurs before the first outbreak is detected and this may lead to a large epidemic. However, severity of CSF disease signs may be linked with efficacy of disease transmission, the most severely affected animals having a higher infectivity than the less affected ones. The purpose of this study was to combine disease transmission quantification methods with CSF clinical signs quantification tools to investigate whether clinical signs, considered as infectivity markers, may allow us to calculate reliable estimates for disease transmission parameters. Data from three transmission experiments were used, varying according to the viral strain (Eystrup or Paderborn) and to the contact structure between experimentally inoculated and contact animals (direct or indirect contact). Within- and between-pen basic reproduction ratios (R0) were compared using viraemia data or clinical data. Between-pen R0 estimates were close and not significantly >1, with either strain or computation mode (using viraemia or clinical data). Conversely, within-pen R0s (Paderborn strain) computed using clinical data appeared higher than the estimates obtained using viraemia data. A models comparison (Bayes information criterion) showed a better fit of the clinical-based models, for both strains. This suggests that, in affected herds, the most severely affected animals could play a prominent role in CSFV transmission.     

Replication of classical swine fever virus strains and isolates in different porcine cell lines

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.     

Disease severity declines over time after a wild boar population has been affected by classical swine fever--legend or actual epidemiological process?

Classical swine fever (CSF) is a severe multi-systemic disease that can affect both domestic pigs and wild boar. Past outbreaks in European wild boar involved high-virulent CSF virus (CSFV) strains and were mostly self-limiting. In these cases, morbidity and mortality rates were high in the affected regions. In contrast, endemic infections have been observed in several European wild boar populations in recent decades. Morbidity and mortality rates were much lower despite the fact that outbreaks were still detected via diseased or fallen animals. The virus strains involved were mostly classified as genotype 2.3 strains of moderate virulence causing age-dependent disease outcomes. The mechanisms leading to the establishment and perpetuation of endemicity are still not fully understood, but the factor "moderate virulence" seems to be of considerable importance. In this study, we aim to clarify whether the perception of declined 'CSF severity' could hypothetically reflect the adaptation of an initially high-virulent virus or whether this might be better explained as a misinterpretation of observations. A mechanistic eco-epidemiological model was employed to follow up a highly virulent strain of CSFV introduced into large connected wild boar populations. In the model, the virulence of the CSF virus is represented by case mortality and life expectancy after lethal infection. Allowing for small stochastic variation, these two characteristics of the virus are passed on with every new simulated infection that occurs. Model analysis revealed a decrease from high to moderate case mortality within a few years of simulated perpetuation of the virus. The resulting mortality corresponded to the level where the population average of the infectious period and the basic reproduction number of the disease were maximal. This shift in virulence was sufficient to prolong virus circulation considerably beyond the epidemic phase of the simulated outbreaks. Alternative mechanistic explanations for the decrease in disease severity in a CSF-affected wild boar population were evaluated in the light of the simulation experiments and the available epidemiological or virological evidence. In conclusion, the current virus isolates of subgroup 2.3 might be the ideally adapted variants of the CSF virus for long-term perpetuation in wildlife and indeed may have evolved (once) during past outbreaks in large populations. A repeated perception of a declining severity of disease pattern during the course of a CSF outbreak, however, favours the explanation based on monitoring and detection biases rather than repeated observation of selection against highly virulent virus during the time of virus perpetuation.     

Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human, and avian lineage genes

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.     

Emergence of classical swine fever virus in Israel in 2009

Classical swine fever (CSF) re-emerged in Israel in February 2009 after an absence of 62 years. The outbreak occurred on a domestic pig farm in northern Israel and affected domestic pigs and wild boar. On the basis of phylogenetic analysis of a 190 base pair fragment of the E2 glycoprotein gene, the Israeli CSF virus strain belonged to genotype 2.1 and was genetically most similar to a Chinese CSF virus strain.     

Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU).     

The potential negative impacts of the classical swine fever virus on wild boar population in Gifu prefecture,Japan

There is a possibility that classical swine fever (CSF) virus outbreak has negative impacts on wild boar. To adequately manage native wild boar populations, wildlife managers need to gather the field data on wild boar and implement population management practices. We aimed to report the relative abundance index of wild boar before and after this outbreak. Our results showed that relative abundance index declined from 2017 (8.88 wild boars/100 trap days) to 2019 (2.03 wild boars/100 trap days), because of the negative impact of this virus and continuous culling programs. Although the eradication risk from the synergistic effect is low, wildlife managers need to consider the relationship between the trade-off between the risk of CSF and the conservation ecology risk of native species eradication.     

Genetic typing of recent classical swine fever virus isolates from Croatia

During a period of 5 years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5'-non-translated region (5'-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.