Lack of phylogenetic evidence that the Shimen strain is the parental strain of the lapinized Chinese strain (C-strain) vaccine against classical swine fever

The Chinese hog cholera lapinized virus (HCLV), also called the “Chinese strain” or “C-strain” of classical swine fever virus (CSFV), was developed in China in the 1950s. There are uncertainties about the genetic heterogeneity and origin of this vaccine virus. The objectives of this study were to investigate the genetic heterogeneity of the C-strain, for which nucleotide sequences have been submitted to GenBank from different laboratories, and to determine whether there is any evidence to support the hypothesis that the C-strain originated from the Shimen strain. Analysis of 150 nearly complete E2 gene sequences revealed that the C-strain clade includes several HCLV vaccine strains, cell-culture-adapted Riems strains, and viruses isolated from diseased pigs. The whole-genome phylogeny indicated that the ancestor of the C-strain was only distantly related to the Shimen strain. Therefore, there was no phylogenetic evidence to support the Shimen-origin hypothesis.     

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.     

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003–2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.     

Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection

The diagnostic properties of several assays on live animals were studied using data from different experiments. These experiments involved 128 classical swine fever virus (CSFV) infected pigs (weaner pigs, fatteners and sows). Since all pigs in the study were infected with CSFV, only the proportion of test positive results and the time until a test positive result is obtained were evaluated. The RT-nPCR detected the highest proportion of infected pigs (98.9%), whereas the Antigen ELISA gave the worst detection results (74.7%). Within the group of test positive animals, infection was detected earliest using the leukocyte count and latest using Antigen ELISA. Using the virus neutralisation test, antibodies against CSFV were detectable on average 7.6 days after the onset of viraemia in virus isolation in whole blood. Using survival analysis, the time until the first positive diagnosis and the proportion of detected animals were combined in one test. Results showed that RT-nPCR performed significantly better than either virus isolation in different blood fractions or antigen ELISA. It is concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a classical swine fever infection.     

A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451bp (PRRSV), 343bp (CSFV), or 163bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 x 102, 2.90 x 103, and 6.16 x 103 copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology.     

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.     

Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C

Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer (sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).     

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).     

Comparisons of envelope through 5B sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes

The function of a 12-nt insertion 'CTTTTTTCTTTT' in the 3' untranslated region (3'UTR) of the HCLV strain, a vaccine strain derived from the Shimen strain of classical swine fever virus (CSFV), was examined in vitro. RNA synthesis increased when the 12-nt insertion was deleted from the 3'UTR of the HCLV strain. RNA synthesis also decreased when the 12-nt insertion was introduced into the 3'UTR of the CSFV Shimen, virulent strain. Therefore, the 12-nt insertion present in the 3'UTR of the HCLV strain may be a marker for avirulence. It was also found that the predicted secondary structure of the 3'UTR of the HCLV strain was more stable when the 12-nt insertion was deleted, and that the 3'UTR of Shimen strain was more unstable when the 12-nt insertion was introduced. Replacement of the 12-nt insertion 'CTTTTTTCTTTT' by another 12-nt fragment 'ATTATTATTTAT' in the 3'UTR of HCLV strain facilitated RNA synthesis and stabilized the predicted secondary structure, which was also observed in the 3'UTR of the Shimen strain.     

Simple and rapid detection of swine hepatitis E virus by reverse transcription loop-mediated isothermal amplification

Hepatitis E virus (HEV) is an enteric pathogen of humans and animals, and pigs have been considered an important reservoir of this virus. Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. In this study, we have developed a one-step RT-LAMP assay for rapid detection of swine HEV. Specific primer sets targeting the ORF3 gene were designed. The sensitivity of the RT-LAMP assay was 10¹ copies/??l of RNA template, which was tenfold higher than that of RT-nPCR. The specificity of this assay was demonstrated by the lack of amplification of DNA/RNA from other swine viruses. Furthermore, a total of 41 bile samples were subjected to RT-LAMP and RT-nPCR. Eighteen positive samples were detected by RT-nPCR, while 36 positive samples were detected by RT-LAMP, indicating that the sensitivity of the RT-LAMP assay was higher than that of the conventional RT-nPCR assay. The RT-LAMP assay reported here may be used for diagnosis of swine HEV, not only in laboratories but also under field conditions.     

Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N^pro gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.     

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles

Summary.  To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E⁺ strain), WB82 and WB82/E⁺ strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E⁺ strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E⁺ strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV. Received January 14, 2002; accepted August 19, 2002     

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.     

Phylogenetic comparison of classical swine fever virus in China.

An N-terminal fragment of the E2 gene of classical swine fever (CSF) virus encoding major immunogenic sites was amplified by RT-PCR directly from 110 clinical specimens representing 109 epizootic sites during the last decade in China. Phylogenetic relationships between these viruses as well as 20 reference strains were determined by comparison of their nucleotide sequences. A phylogenetic tree showed that 103 of the 110 field viruses (93.6%) were clustered within group 2 and subdivided into three subgroups, while the remaining seven viruses (6.4%), along with two Chinese reference strains, Shimen and HCLV (attenuated vaccine strain), were clustered into subgroup 1.1 within group 1. However, none of the Chinese CSF viruses were members of subgroup 1.2 (represented by reference strain Brescia). This is the first report on the distribution of CSF virus genotypes in China. Results indicated that CSF viruses predominating in recent epizootics within China are genetically divergent from the reference strain Shimen and the vaccine strain HCLV.     

Development of a reverse transcription-PCR assay to detect porcine circovirus type 2 transcription as a measure of replication

Porcine circovirus type 2 (PCV2) is a non-enveloped, single-stranded, circular DNA virus. In situ hybridization and PCR assays have detected PCV2 DNA in multiple organs and cell types from infected pigs; however, it is not clear if this represents replicating virus or virion DNA. We describe the development of a single-tube RT-PCR assay to differentiate PCV2 replication products and virus DNA. Primers targeted to the open-reading frame 2 (ORF2) of PCV2 were designed to amplify both virus DNA (984 bp) and the spliced Cap mRNA (594 bp). The 984 bp fragment, but not the 594 bp fragment, was amplified from PCV2 stock, confirming that the spliced Cap mRNA was not present in the PCV2 stock. The 594 bp fragment was amplified from DNase-treated RNA extracted from PCV2-infected PK-15 cells, and was detected as early as 14 h post-infection. No products were amplified from either the PCV1 stock or PCV1-infected PK-15 cells, or from cells infected with UV-inactivated PCV2. Therefore, the presence of the 594 bp fragment is specific for PCV2 replication. This assay will be useful in assessing cell populations that support PCV2 replication in vivo or in vitro and advance the understanding of PCV2 replication and pathogenesis.     

Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase

A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.     

12-nt insertion in 3' untranslated region leads to attenuation of classic swine fever virus and protects host against lethal challenge

We report here the discovery of an attenuation mechanism of classic swine fever virus (CSFV) induced by introduction of a continuous 12-nt (CUUUUUUCUUUU) insertion in viral 3′ UTR. The 12-nt insertion sequence was first found in one attenuated vaccine strain HCLV (Hog Cholera Lapinized Virus) which did not exist in other CSFV strains. To address the function of the 12-nt insertion in viral replication and attenuation, we constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3′ UTR or the replacement of viral 3′ UTR by the 3′ UTR of HCLV. We found that the two chimeras' maximum titers declined approximately 100-fold than their parental strain Shimen in PK15 cells. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs. All the chimera-infected pigs survived infection and remained clinically normal with the exception of a transient fever while the 100% mortality was observed for the Shimen-infected pigs. In addition, the two chimeras can induce neutralization antibody to completely protect the pigs against lethal challenge with highly virulent CSFV, which was similar to the vaccine strain HCLV. These data demonstrate that the 12-nt insertion in 3′ UTR is sufficient for the attenuation of CSFV. Taken together, a novel attenuation mechanism of CSFV is found and may pave a way to further research for new attenuated vaccine.