Classical Swine Fever Virus RNA Research Articles

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Pathogenicity of a novel classical swine fever LOM vaccine-derived virus isolated on Jeju Island, South Korea.

Reemergent local outbreaks of classical swine fever (CSF) occurred simultaneously in multiple pig farms on CSF-free Jeju Island, South Korea, in 2014 because of inadvertent injection of a commercial CSF (LOM) vaccine into pregnant sows. The LOM virus has since spread across the island and has become endemic in Jeju herds, raising concern about possible reversion to the virulence of the LOM vaccine. We previously isolated LOM-derived field CSF virus (CSFV) strains with unique insertion-deletion (INDEL) mutations in the 3'-untranslated region (UTR), designated LOM-derived Jeju 3'-UTR INDEL variants, from CSF-recurrent swine farms on Jeju Island in 2019. The present study conducted animal experiments to investigate whether a 2019 emergent LOM 3'-UTR INDEL variant, KNU-1905, has reverted to a pathogenic form in conventional pigs (n = 10). Experimental animal infection showed that pigs inoculated with the commercial LOM vaccine strain developed no adverse effects compared to the sham-infected pigs. However, KNU-1905 displayed pathogenic characteristics in pigs, including clinical symptoms (e.g., lethargy, conjunctivitis, nasal discharge, and diarrhoea), weight loss, and gross lesions. Moreover, viremia, virus shedding in faeces and nasal fluids, and viral loads in various tissues of all the KNU-1905-infected pigs were highly significant, in contrast to those of the LOM-infected group in which CSFV RNA was detected only in the serum, nasal, and tonsil samples of one identical pig. Overall, the LOM-derived field isolate with molecular variations induced clinical adverse events in pigs, which commonly shed considerable amounts of CSFV. This study provides evidence that the genetic evolution of the LOM-derived CSFV circulating on Jeju Island might have allowed the LOM vaccine to recover its primary prototype and that these variants might have induced chronic or persistent infection in pigs that can shed CSFV in field farms leading to a risk of transmission among pigs or farms in this former CSF-free region.

A rapid and high sensitivity RNA detection based on NASBA and G4-ThT fluorescent biosensor

In recent years, various newly emerged and re-emerged RNA viruses have seriously threatened the global public health. There is a pressing need for rapid and reliable nucleic acid–based assays for detecting viral RNA. Here, we successfully developed a highly sensitive, easy-to-operate G4-ThT-NASBA system to detect viral RNA that no need for labeled primers and probes. Next, we tested the system for detecting the Classical Swine Fever Virus (CSFV), an RNA virus that causes a highly contagious disease in domestic pigs and wild boar and easily causes huge economic losses. Results showed that the system, integrated the G4-ThT fluorescent biosensor and NASBA (Nuclear acid sequence-based amplification),is capable to detect as little as 2 copies/μL of viral RNA without interfering by other swine viral RNA. Moreover, we were able to detect CSFV RNA within 2 h in serum samples taken from the field in a real-time mode. These findings indicate that the G4-ThT-NASBA system is a rapid, high sensitivity and easy-to-operate technique for RNA detection. The method also has the real-time detection capability which may be easily integrated in a highly automated system such as microfluidic chips.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Development of a fluorescent probe-based real-time reverse transcription recombinase-aided amplification assay for the rapid detection of classical swine fever virus.

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe-based real-time reverse transcription recombinase-aided amplification (rRT-RAA) assay, targeting a highly conserved position within the 5' non-translated region (5'NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50 ) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT-qPCR) assay for CSFV. The rRT-RAA assay exhibited good reproducibility, with intra- and inter-assay coefficient of variation values of<8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT-RAA and RT-qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT-RAA and RT-qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT-RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on-site testing. Our results indicate that the rRT-RAA assay is a robust diagnostic tool for the rapid detection of CSFV.

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

ABSTRACT Classical swine fever virus (CSFV), a positive-sense RNA virus, hijacks cell host proteins for its own replication. Rab18, a small Rab GTPase, regulates intracellular membrane-trafficking events between various compartments in cells and is involved in the life cycle of multiple viruses. However, the effect of Rab18 on the production of CSFV remains uncertain. In this study, we showed that knockdown of Rab18 by lentiviruses inhibited CSFV production, while overexpression of Rab18 by lentiviruses enhanced CSFV production. Subsequent experiments revealed that the negative-mutant Rab18-S22 N inhibited CSFV infection, while the positive-mutant Rab18-Q67 L enhanced CSFV infection. Furthermore, we showed that CSFV RNA replication and virion assembly, measured by real-time fluorescence quantitative PCR (RT-qPCR), indirect immunofluorescence assay (IFA), and confocal microscopy, were reduced in cells lacking Rab18 expression. In addition, co-immunoprecipitation, GST-pulldown, and confocal microscopy assays revealed that Rab18 bound to the viral protein NS5A. Further, NS5A was shown to be redistributed in Rab18 knockdown cells. Taken together, these findings demonstrate Rab18 as a novel host factor required for CSFV RNA replication and particle assembly by interaction with the viral protein NS5A.

Foetal Immune Response Activation and High Replication Rate during Generation of Classical Swine Fever Congenital Infection.

Classical swine fever virus (CSFV) induces trans-placental transmission and congenital viral persistence; however, the available information is not updated. Three groups of sows were infected at mid-gestation with either a high, moderate or low virulence CSFV strains. Foetuses from sows infected with high or low virulence strain were obtained before delivery and piglets from sows infected with the moderate virulence strain were studied for 32 days after birth. The low virulence strain generated lower CSFV RNA load and the lowest proportion of trans-placental transmission. Severe lesions and mummifications were observed in foetuses infected with the high virulence strain. Sows infected with the moderately virulence strain showed stillbirths and mummifications, one of them delivered live piglets, all CSFV persistently infected. Efficient trans-placental transmission was detected in sows infected with the high and moderate virulence strain. The trans-placental transmission occurred before the onset of antibody response, which started at 14 days after infection in these sows and was influenced by replication efficacy of the infecting strain. Fast and solid immunity after sow vaccination is required for prevention of congenital viral persistence. An increase in the CD8+ T-cell subset and IFN-alpha response was found in viremic foetuses, or in those that showed higher viral replication in tissue, showing the CSFV recognition capacity by the foetal immune system after trans-placental infection.

Development of functionalized gold nanoparticles as nanoflare probes for rapid detection of classical swine fever virus.

Classical swine fever (CSF) is a devastating viral disease affecting pigs that causes major economic losses worldwide. Conventional assays to identify classical swine fever virus (CSFV) face challenges, such as the required molecular amplification of the target molecules via polymerase chain reaction (PCR). We designed a gold nanoflare probe to directly detect CSFV. Gold nanoparticles (AuNPs) were conjugated with a pair of complementary DNA sequences that specifically recognized and captured CSFV RNA, resulting in a fluorescence signal to indicate the existence of CSFV. The constructed nanocomposite was then utilized in a quantitative analysis to recognize the virus sequence present at amounts as low as 50 pg/μL. The CSFV-AuNP probe enabled real-time, quantitative detection of native CSFV in response to doses of the specific RNA sequence (CSFV NS2) that indicated active viral replication of CSFV Shimen in macrophages after 12, 24, and 48 h. The potential diagnostic applications of the probe were demonstrated by measuring CSFV without nucleic acid amplification in samples from seven types of tissue samples, specifically heart, spleen, kidney, liver, lymph, intestine, and muscle samples obtained from one pig confirmed to suffer CSF. The speed, sensitivity, and versatility of this CSFV-AuNP biosensor make it an ideal candidate for further application in the prevention and control of animal epidemic diseases.

Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain.

Classical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by a de novo mechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCE Pigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genus Pestivirus, family Flaviviridae Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from −14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (<14 days) and antibody-based assays, thereafter.

Short communication: Stability and integrity of classical swine fever virus RNA stored at room temperature

Worldwide cooperation between laboratories working with classical swine fever virus (CSFV) requires exchange of virus isolates. For this purpose, shipment of CSFV RNA is a safe alternative to the exchange of infectious material. New techniques using desiccation have been developed to store RNA at room temperature and are reported as effective means of preserving RNA integrity. In this study, we evaluated the stability and integrity of dried CSFV RNA stored at room temperature. First, we determined the stability of CSFV RNA covering CSFV genome regions used typically for the detection of viral RNA in diagnostic samples by reverse transcription-polymerase chain reaction (RT-PCR). To this end, different concentrations of in vitro-transcribed RNAs of the 5’-untranslated region and of the NS5B gene were stored as dried RNA at 4, 20, and 37oC for two months. Aliquots were analyzed every week by CSFV-specific quantitative real-time RT-PCR. Neither the RNA concentration nor the storage temperature did affect CSFV RNA yields at any of the time evaluated until the end of the experiment. Furthermore, it was possible to recover infectious CSFV after transfection of SK-6 cells with dried viral RNA stored at room temperature for one week. The full-length E2 of CSFV was amplified from all the recovered viruses, and nucleotide sequence analysis revealed 100% identity with the corresponding sequence obtained from RNA of the original material. These results show that CSFV RNA stored as dried RNA at room temperature is stable, maintaining its integrity for downstream analyses and applications.

An outbreak of classical swine fever in pigs in Bangladesh, 2015.

In a group of 22 healthy pigs aged between 4 and 6 months, 2 pigs became ill with high fever, complete anorexia, cough and abnormal swaying movements on 22 June 2015. One of them died on June 24 and the second died on July 3. Shortly after, the remaining pigs also fell ill and died from the same illness by 10 August 2015. We investigated the aetiology, epidemiological and clinical features of the outbreak. We recorded the clinical signs and symptoms for each pig with the date of onset of illness. Veterinarians conducted post‐mortem examinations on the 12 dead pigs, they collected tissue samples from the dead pigs and placed them in a tube containing 1 mL of nucleic acid extraction buffer (lysis buffer). We tested all the tissue samples by real‐time reverse transcription polymerase chain reaction (rRT‐PCR) to detect classical swine fever virus (CSFV) because the animals’ symptoms matched those of this disease. We also conducted a phylogentic analysis of the nucleotide sequence of the E2 gene segment of CSFV detected in a lung tissue sample. The attack rate (22/22) and the case fatality were 100%. The predominant symptoms of the disease included high fever, cough, diarrhoea and swaying movements of the hind legs prior to death. Of the 12 pigs tissue samples tested, all had evidence of the presence of CSFV RNA by rRT‐PCR. The phylogenetic analysis indicated that the virus belongs to genotype 2.2, which is closely related to CSFV genotype 2.2 reported in India. Our investigation suggests that CSF is circulating in pigs, posing a risk for communities in Bangladesh that rely on pigs for economic income and dietary protein. Future research could focus on estimating the disease and economic burden of CSFV in pig rearing areas to determine if interventions might be warranted or cost‐effective.

African swine fever virus infection in Classical swine fever subclinically infected wild boars

BackgroundRecently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.ResultsAfter ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.ConclusionsOur results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells

BackgroundClassical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells.MethodsWe designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2).ResultsThe lowest detection threshold of the ViewRNA ISH method was 10−8, while the sensitivity of FAT and IHC were 10−5 and 10−4, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3–9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24–48 hpi and reached the peak at 72hpi.ConclusionsTo our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6–9 hpi and CSFV RNA has ever existed in nucleus.

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus.

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.

Safety of classical swine fever virus vaccine strain LOM in pregnant sows and their offspring

The present study aimed to evaluate the safety of the classical swine fever virus (CSFV) vaccine strain LOM in pregnant sows. Pregnant sows with free CSFV antibody were inoculated with a commercial LOM vaccine during early pregnancy (day 38; n=3) or mid-pregnancy (days 49–59; n=11). In pregnant sows vaccinated during the early stages of gestation, abortion (day 109) was observed in one case, with two stillbirths and seven mummified fetuses. The viability of live-born piglets was 34.9% in sows vaccinated during mid-pregnancy compared with 81.8% in the control group. Post-mortem examination of the organs of the sows and piglets did not reveal any pathological lesions caused by CSFV; however, CSFV RNA was detected in the organs of several vaccinated sows and their litters. The LOM strain was transmitted from sows with free CSFV antibody to their fetus, but did not appear to induce immune tolerance in the offspring from vaccinated pregnant sows. Side effects were not observed in pregnant sows with antibody to the LOM strain: transmission from sow to their litters and stillbirth or mummified fetuses. The LOM strain may induce sterile immunity and provide rapid, long-lasting, and complete protection against CSFV; however, it should be contraindicated in pregnant sows due to potential adverse effects in pregnant sows with free CSFV antibody.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs.

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

Insulated Isothermal Reverse Transcriptase PCR (iiRT-PCR) for Rapid and Sensitive Detection of Classical Swine Fever Virus.

SummaryClassical swine fever (CSF) is an OIE‐listed disease that can have a severe impact on the swine industry. User‐friendly, sensitive, rapid diagnostic tests that utilize low‐cost field‐deployable instruments for CSF diagnosis can be useful for disease surveillance and outbreak monitoring. In this study, we describe validation of a new probe‐based insulated isothermal reverse transcriptase PCR (iiRT‐PCR) assay for rapid detection of classical swine fever virus (CSFV) on a compact, user‐friendly device (POCKIT ™ Nucleic Acid Analyzer) that does not need data interpretation by the user. The assay accurately detected CSFV RNA from a diverse panel of 33 CSFV strains representing all three genotypes plus an additional in vitro‐transcribed RNA from cloned sequences representing a vaccine strain. No cross‐reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, HoBi atypical pestivirus), African swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. The iiRT‐PCR assay accurately detected CSFV as early as 2 days post‐inoculation in RNA extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. The limit of detection (LOD 95%) calculated by probit regression analysis was 23 copies per reaction. The assay has a sample to answer turnaround time of less than an hour using extracted RNA or diluted or low volume of neat serum. The user‐friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of CSF in a disease outbreak.

Annexin A2 is involved in the production of classical swine fever virus infectious particles.

Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses. To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus (CSFV), an RNA interference (RNAi) approach was utilized. Knockdown of ANXA2 did not impair CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of NS5A were revealed to be pivotal for the ANXA2-NS5A interaction. Substitutions of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production, which might partly be due to these mutations destroying the ANXA2-NS5A interaction. These results suggested that ANXA2 might participate in CSFV production process by binding NS5A.