Classical Swine Fever Virus RNA Research Articles

English

TRIzol ® , TRI Reagent ® , and RNAzol ® are widely used commercial reagents for the extraction of cellular or viral RNA. Several other brand name products, some of which are advertised for the processing of specific sample types such as blood, are also available. Here, we compare the efficiency of these products for classical swine fever virus RNA extraction from cell culture supernatant, serum, and tonsil tissue, assessed by quantitative RT-PCR. Furthermore, the detection of a synthetic RNA transcript used as an internal positive control for extraction and RT-qCR was compared as well. Most tested products showed a similar extraction efficiency, and none of the products recommended for specific sample types performed better than the all-purpose reagents. We also show that the homogenization method for tissue samples has a significant impact on the detection efficiency of the RNA after extraction from the homogenized tissue. Homogenization of 100 mg tissue in 5 ml cell culture medium and using an UltraTurrax ® tissue grinder yielded the best results, whereas TissueLyser ® -mediated homogenization in 1 ml cell culture medium or direct homogenization in RNA extraction medium proved to be less efficient. Keywords: RNA extraction, homogenization, comparison, TRIzol, TRI reagent, reverse transcription-quantitative PCR (RT-qPCR) African Journal of Biotechnology , Vol 13(42) 4100-4104

Analysis of classical swine fever virus RNA replication determinants using replicons

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.

Co-expression of Erns and E2 genes of classical swine fever virus by replication-defective recombinant adenovirus completely protects pigs against virulent challenge with classical swine fever virus

The objective of this study was to construct a recombinant adenovirus for future CSFV vaccines used in the pig industry for the reduction of losses involved in CSF outbreaks. The Erns and E2 genes of classical swine fever virus (CSFV), which encode the two main protective glycoproteins from the “Shimen” strain of CSFV, were combined and inserted into the replication-defective human adenovirus type-5 and named the rAd-Erns-E2. Nine pigs were randomly assigned to three treatment groups (three pigs in each group) including the rAd-Erns-E2, hAd-CMV control and DMEM control. Intramuscular vaccination with 2×106 TCID50 of the rAd-Erns-E2 was administered two times with an interval of 21days. At 42days post inoculation, pigs in all groups were challenged with a lethal dose of 1×103 TCID50 CSFV “Shimen” strain. Observation of clinical signs was made and the existence of CSFV RNA was detected. Animals in the hAd-CMV and DMEM groups showed severe clinical CSF symptoms and were euthanized from 7 to 10days after the challenge. However, no adverse clinical CSF signs were observed in vaccinated pigs after the administration of rAd-Erns-E2 and even after CSFV challenge. Neither CSFV RNA nor pathological changes were detected in the tissues of interest of the above vaccinated pigs. These results implied that the recombination adenovirus carrying the Erns-E2 genes could be used to prevent swine from classical swine fever.

Modulation of Translation Initiation Efficiency in Classical Swine Fever Virus

Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild-type (wt) or mutant forms of the IRES of CSFV strain Paderborn were amplified and inserted into dicistronic reporter plasmids encoding Fluc and Rluc under the control of a T7 promoter. The mutations were within domains II, IIId(1), and IIIf of the IRES. The plasmids were transfected into baby hamster kidney (BHK) cells infected with recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase. IRES mutants with different levels of IRES activity were identified and then introduced by homologous recombination into bacterial artificial chromosomes (BACs) containing CSFV Paderborn cDNA downstream of a T7 promoter. From the wt and mutant BACs, full-length CSFV RNA transcripts were produced in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of the wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced changes within the IRES. The growth characteristics of each rescued mutant virus were compared to those of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES have reduced growth in cell culture compared to the wt virus.

The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation

The 3′ untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3′ UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5′ and 3′ UTRs, we found that the 3′ UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3′-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3′-ends such as the poly(A) or the 3′ UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3′ UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5′ UTR. In conclusion, we have found that the 3′-end terminal sequence can play a role in regulating the translation of CSFV.

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.

Influence of the 5′-proximal elements of the 5′-untranslated region of classical swine fever virus on translation and replication

The 5'-terminal sequence spanning nt 1-29 of the 5'-untranslated region of classical swine fever virus (CSFV) forms a 5'-proximal stem-loop structure known as domain Ia. Deletions and replacement mutations were performed to examine the role of this domain. Deletion of the 5'-proximal nucleotides and disruption of the stem-loop structure greatly increased internal ribosome entry site-mediated translation but abolished the replication of the replicons. Internal deletions resulting in a change in the size of the loop of domain Ia, and even removal of the entire domain, did not substantially change the translation activity, but reduced the replication of CSFV replicons provided the replicons contained the extreme 5'-GUAU terminal sequence. Internal replacements leading to a change in the nucleotide sequence of the loop did not alter the translation and replication activities of the CSFV RNA replicon, and did not influence the rescue of viruses and growth characteristics of new viruses. These results may be important for our understanding of the regulation of translation, replication and encapsidation in CSFV and other positive-sense RNA viruses.

Development and evaluation of rapid detection of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of classical swine fever virus (CSFV) and a set of primers designed based on the E2 gene reference sequences of the CSFV. The assay was optimized to amplify CSFV RNA by incubation at 63 °C for 50 min. The RT-LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. The RT-LAMP assay showed higher sensitivity than the conventional RT-PCR, and no cross-reactivity appeared with other related porcine viruses including porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). The positive predictive value (PPV) of CSFV RT-LAMP for 227 field tissue samples was 94.7%, the negative predictive value (NPV) was 30.8% and showed better for the conventional RT-PCR method. Thus, the RT-LAMP assay is extremely rapid, sensitive, and specific and has potential usefulness for rapid laboratory diagnosis and pen-side detection for CSFV detection in pigs.

Classical swine fever virus strain “C” protects the offspring by oral immunisation of pregnant sows

The aim of this study was to evaluate if oral immunisation of wild sows protects the fetuses from transplacental infection. Two experiments were carried out with gilts vaccinated orally with C-strain virus approximately 5 weeks after insemination. They were challenged at mid-gestation with highly virulent classical swine fever virus (CSFV) or moderately virulent field virus. The results revealed that oral vaccination has no negative impact on the pregnancy, and all vaccinated sows developed neutralising antibodies. After infection no symptoms were detected in the six vaccinated–infected sows. Challenge virus could neither be found in blood, nasal and fecal swabs or saliva nor in organs sampled at necropsy. Likewise, all fetuses originating from vaccinated sows were virologically and serologically negative. In contrast, the controls developed a short viremia and as a result of the transplacental infection all fetuses were CSFV positive. In addition, 22 serologically positive wild sows of an endemically infected area, where oral vaccination had also been carried out, and their offspring were free from CSFV or viral RNA. Our results confirm that oral immunisation of pregnant wild sows with C-strain vaccine may protect the fetuses against CSF.

A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.

Classical Swine Fever (CSF) in Wild Boar: the Role of the Transplacental Infection in the Perpetuation of CSF

Thirty-four pregnant wild sows and their unborn progeny derived from an endemically infected population in the district of Nordvorpommern (Mecklenburg-Western Pomerania) were investigated for classical swine fever virus (CSFV) and antibodies. During the last 2.5 years of the epidemic, 20 out of 34 pregnant wild sows investigated were serologically positive. No CSFV or viral RNA was detected in organs derived from these animals and their progeny. This indicates that young wild boars persistently infected by transplacental virus transmission do not play a crucial role in the perpetuation of CSFV in wild boar. Other factors seem to be more important for the establishment of CSF as well as for virus perpetuation in the population.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly–Asp–Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase.

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5Bdelta24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus- or minus-strand viral RNA synthesis de novo in a primer-independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA-dependent RNA polymerase in vitro. Both Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm GTP, and a 3'-terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North-Western blot analysis, to specifically interact with the 3'-end of both plus- and minus-strand viral RNA templates.

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry.

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.

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.

Classical swine fever virus (C strain) distribution in organ samples of inoculated piglets

Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E RNS and protein NS2-3 using commercially available ELISA kits. E RNS and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E RNS and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5′ non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.

Characterization of Classical Swine Fever Virus Associated with Defective Interfering Particles Containing a Cytopathogenic Subgenomic RNA Isolated from Wild Boar.

Classical swine fever virus (CSFV) strain WB82, isolated from a wild boar in 1982, induced a distinct cytopathic effect (CPE) in primary swine testicle cell culture and in most of the porcine cell lines. This strain of CSFV was found to be composed of two biotypes. cytopathogenic (cp) CSFV, as a minor population, and noncytopathogenic (noncp) CSFV, as a major population. The noncp CSFV (designated strain WB82/E+) was obtained by biological cloning, and it showed the exaltation of Newcastle disease virus phenomenon. In Northern blot analysis and RT-PCR assay, CSFV RNA with a subgenomic (sg) length was detected in addition to full-length viral RNA only in the cells in which a CPE had been revealed. These RNAs represent the genomes of typical defective interfering (DI) particles because of the strict dependence on a complementing helper virus and interference with replication of the helper virus. The sg RNA, which exhibits the genomes of the DI particles, lacked the nucleotides of the viral genomic region from Npro to NS2 (4764 bases). When extracted sg RNA was transfected to the cells infected with the WB82/E+ strain, a distinct CPE was observed. Interestingly, the CPE was observed in cells infected with other heterologous noncp CSFV ALD and GPE- strains by sg RNA transfection. The results suggested that these noncp CSFVs act as helper viruses for the replication of sg RNA (DI particles). It was also shown that the cytopathogenicity of strain WB82 is caused by apoptosis.

A recombinant classical swine fever virus with a marker insertion in the internal ribosome entry site.

Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.

A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan)

Detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation (VI) in cell cultures, antigen detection, or molecular analysis. To simplify the latter, a 5′-nuclease assay (TaqMan) was developed for the rapid and specific detection of CSFV with the minimum of downstream PCR processing. A pair of 5′-non-coding region, panpestivirus-specific PCR primers were assessed in a one-step reverse transcription-PCR with each of 36 diverse pestiviruses. The PCR products were subsequently reamplified, in conjunction with a CSFV-specific fluorogenic probe, in a nested-PCR with a second set of panpestivirus PCR primers. During nested PCR, when the target of interest was present, the CSFV probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5′-3′ nucleolytic activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. Each PCR tube was then placed directly into a luminescence spectrometer to monitor for any increase in fluorescence due to cleavage of the probe. This assay detected representatives of all genetic sub-groups of CSFV, but gave negative results for other pestiviruses. A preliminary assessment showed that the method could be used to detect CSFV RNA extracted from infected pig blood with a sensitivity greater than that of VI.