Infection of domestic pigs with a genotype II potent strain of ASFV causes cytokine storm and lymphocyte mass reduction.

The whole-genome sequence of an African swine fever virus (ASFV) strain (HuB/HH/2019) isolated from Hubei, China, was highly similar to that of the Georgia 2007/1 strain ASFV. After infection with strong strains, domestic pigs show typical symptoms of infection, including fever, depression, reddening of the skin, hemorrhagic swelling of various tissues, and dysfunction. The earliest detoxification occurred in pharyngeal swabs at 4 days post-infection. The viral load in the blood was extremely high, and ASFV was detected in multiple tissues, with the highest viral loads in the spleen and lungs. An imbalance between pro- and anti-inflammatory factors in the serum leads to an excessive inflammatory response in the body. Immune factor expression is suppressed without effectively eliciting an immune defense. Antibodies against p30 were not detected in acutely dead domestic pigs. Sequencing of the peripheral blood mononuclear cell transcriptome revealed elevated transcription of genes associated with immunity, defense, and stress. The massive reduction in lymphocyte counts in the blood collapses the body's immune system. An excessive inflammatory response with a massive reduction in the lymphocyte count may be an important cause of mortality in domestic pigs. These two reasons have inspired researchers to reduce excessive inflammatory responses and stimulate effective immune responses for future vaccine development.

A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains.

BACKGROUND: Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease. METHODS: Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains. RESULTS: After optimization, the calibration curve showed good linearity (R2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 102 HAD50/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 106, 1 × 105, and 1 × 104 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible. CONCLUSIONS: The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.

Molecular evolution, diversity, and adaptation of foot-and-mouth disease virus serotype O in Asia.

Foot-and-mouth disease (FMD) is highly contagious and affects the economy of many countries worldwide. Serotype O is the most prevalent and is present in many regions of Asia. Lineages O/SEA/Mya-98, O/Middle East-South Asia (ME-SA)/PanAsia, O/Cathay and O/ME-SA/Ind-2001 have been circulating in Asian countries. Low antigenic matching between O/Cathay strains and current vaccine strains makes the disease difficult to control, therefore, analyzing the molecular evolution, diversity, and host tropisms of FMDV Serotype O in Asia may be helpful. Our results indicate that Cathay, ME-SA, and SEA are the predominant topotypes of FMDV serotype O circulating in Asia in recent years. Cathay topotype FMDV evolves at a higher rate compared with ME-SA and SEA topotypes. From 2011 onwards, the genetic diversity of the Cathay topotype has increased substantially, while large reductions were found in the genetic diversity of both ME-SA and SEA topotypes, suggesting a trend that infections sustained by the Cathay topotype were becoming a more severe epidemic in recent years. Analyzing the distributions of host species through time in the dataset, we found that the O/Cathay topotype was characterized by a highly swine-adapted tropism in contrast with a distinct host preference for O/ME-SA. The O/SEA topotype strains identified in Asia were isolated mainly from cattle until 2010. It is worth noting that there may be a fine-tuned tropism of the SEA topotype viruses for host species. To further explore the potential molecular mechanism of host tropism divergence, we analyzed the distribution of structure variations on the whole genome. Our findings suggest that deletions in the PK region may reflect a common pattern of altering the host range of serotype O FMDVs. In addition, the divergence of host tropism may be due to accumulated structural variations across the viral genome, rather than a single indel mutation.

Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus.

BACKGROUND: Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. METHODS: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. RESULTS: This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875-2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. CONCLUSION: This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

[Characterization of the antigens in inactivated porcine circovirus type 2 vaccines and virus-like particle vaccines by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering].

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

Size exclusion chromatography using large pore size media induces adverse conformational changes of inactivated foot-and-mouth disease virus particles.

Size exclusion chromatography (SEC) of biomacromolecules using large pore size media and long column are usually necessary to obtain a satisfactory separation. However, the SEC separation of inactivated foot and mouth disease virus (FMDV) was found to induce some subtle but important conformational changes of FMDV in a pore-size and column length dependent manner. Here three Sephacryl media including S-300 HR, S-400 HR, and S-500 HR were tested, whose pore sizes were smaller than, similar to, and larger than the FMDV particles, respectively. High performance size exclusion chromatography (HPSEC) analyses showed that the FMDV after all these three SEC processes had earlier retention time, compared with that before SEC, but had no detectable difference in particle integrity. Longer SEC column led to more significant peak shifting in subsequent HPSEC analysis of FMDV. Further analyses indicated the SEC using larger pore size media induced more remarkable conformational changes and decrease in thermostability of FMDV, as well as decrease in immunogenicity in animal test. Fluorescence probe diffusion study suggested compared to SEC by S300, the compactness of the viral capsid after SEC by S400 and S500 was decreased, possibly due to more shear-induced FMDV particle rotation and inter-particle collision inside the media pores, as well as their interactions with the pore walls of the media during flowing through the column. Finally, a stabilization strategy by appending 5 mM CaCl2 in mobile phase of SEC separation was proposed and proved to efficiently maintain the conformation of the FMDV.

On-line separation and quantification of virus antigens of different serotypes in multivalent vaccines by capillary zone electrophoresis: A case study for quality control of foot-and-mouth disease virus vaccines.

Accurate quantification of effective antigens of different serotypes is crucial for quality control of multivalent vaccines but challenging. A simple and rapid capillary zone electrophoresis (CZE) method was developed for on-line separation and quantification of foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent FMDV vaccines. The FMDV peak identity in CZE was demonstrated by the study of FMDV dissociation combined with high performance size exclusion chromatography (HPSEC) analysis. After optimizing CZE conditions including UV detecting wavelength, injection volume, and separation voltage, both serotype A and O FMDV showed good reproducibility (RSD <5%) and linear responses (R2=0.999) between the peak area and FMDV content in the concentration range of 15-400 µg/mL. The two serotypes of FMDV with similar size had different migration time in CZE according to their different zeta potential, which allows them to be separated and quantified, with accuracy of <10% relative error. CZE was then successfully applied for antigen quantification of commercial O monovalent and A/O bivalent FMDV vaccines. Compared with HPSEC, CZE was not only able to quantify each serotype of FMDV, but also free from interference of nucleic acids impurities. In summary, the CZE can be a simple, rapid, and reliable tool for quality control of monovalent and bivalent FMDV vaccines. The CZE method can also be further extended to the quality control of other multivalent virus and virus like particle vaccines.

Mechanism of bio-macromolecule denaturation on solid-liquid surface of ion-exchange chromatographic media - A case study for inactivated foot-and-mouth disease virus.

Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (Tm1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest Tm1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and Tm1, three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery.

[Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography].

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 µg/mL by ultracentrifugation, 9.7 and 10.4 µg/mL by PEG precipitation, and 10.5 and 10.4 µg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 µL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.

Recombination of host cell mRNA with the Asia 1 foot-and-mouth disease virus genome in cell suspension culture.

Foot-and-mouth disease virus (FMDV) exhibits high mutation rates during replication. In this study, an isolate of FMDV serotype Asia-1 was serially passaged in a BHK-21 cell monolayer and then adapted to serum-free BHK-21 cell suspension culture to produce a seed virus for production of an inactivated vaccine. Analysis of the sequence encoding the structural proteins of the virus at various passages showed the presence of overlapping peaks in sequencing electropherograms after nucleotide 619 of VP1 in viruses recovered from the fourth passage in suspension culture, suggesting the possible introduction of an insertion or deletion into this portion of the viral genome of our seed virus stock. To evaluate this phenomenon, a virus designated "Vac-Asia1-VDLV", was isolated by plaque purification from the tenth passage in suspension culture. Sequencing results showed that a 12-nt-long exogenous sequence was inserted into the 3' end of the VP1 coding region at the position where the original overlapping peaks were identified. Analysis of the host cell transcriptome showed that the 12-nt sequence was identical to a highly expressed sequence in BHK-21 cells, strongly suggesting that recombination between the FMDV genome and host cell mRNA produced the recombinant virus. A growth curve showed that the virus with the 12-nt insertion reached a peak earlier than the parental strain and that this virus had acquired the ability to bind to the cell surface by a mechanism that was not dependent on integrin or the heparan sulfate receptor. This novel pathogen-host cell recombination event is discussed in terms of the mechanism of viral RNA replication and the phenotypic constraints of FMDV biology and evolution.

[Development and verification of reference nucleic acid materials of H9N2 influenza viruses by real-time RT-PCR].

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 109 copies/µL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.

[Using size-exclusion chromatography to quantify the 146S antigen in inactivated foot-and-mouth disease vaccine].

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 µL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 µg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.

Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

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

BACKGROUND: Classical 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. METHODS: We designed several specific probes of CSFV RNA and reference gene ß-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). RESULTS: The 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. CONCLUSIONS: To 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.

Stabilization study of inactivated foot and mouth disease virus vaccine by size-exclusion HPLC and differential scanning calorimetry.

The inactivated foot and mouth disease virus (FMDV), which has a sedimentation coefficient of 146S, is crucial to the efficacy of vaccine preparations, but extremely unstable in vitro. It is prone to dissociate into smaller particles referred to as 12S with a concomitant decrease in immunogenicity; therefore, it is of great importance to find the best condition for stabilizing the FMDV. In the present work, the effects of solution pH and temperature on the dissociation of 146S was investigated and potential stabilizers were screened, with aid of high-performance size-exclusion chromatography (HPSEC) for rapid and quantitative determination of 146S, together with differential scanning calorimetry (DSC) technology for thermal stability analysis. The most stable pH was found between 7.5 and 8.0. Among excipients tested, sucrose and glycerol provided the best protection, such that the half-life of 146S in solution at 45°C could be prolonged from less than 30min to more than 3days by adding 20% sucrose. The stabilization mechanism was confirmed using DSC analysis, which showed that the transition temperature related to 146S dissociation was increased by 5.4°C in the presence of 20% sucrose. The physical stabilization effects afforded by these stabilizers would allow for the retaining of effective 146S antigens during transportation and storage under relative harsh condition.

[Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold purification, with 92% recovery. The columns were coupled through an intermediate ultrafiltration unit to concentrate the virus. The entire process was completed in about 3.5h, with 75% final FMDV recovery, and 247-fold purification. The final product had purity above 98%, with over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC), Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that the purified virus contained the required antigen, and was structurally intact with a spherical shape and a particle size of 28 nm.

Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs.

BACKGROUND: Classical swine fever (CSF), caused by the Classical swine fever virus (CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand the distribution, tissue tropism, pathogenesis, replication and excretion of CSFV in pigs. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of the experimentally infected pigs using real-time RT-PCR and immunohistochemistry (IHC). RESULTS: A relative quantification real-time PCR was established and used to detect the virus load in internal organs of the experimentally infected pigs. The study revealed that the virus was detected in all 21 of the internal organs and blood collected from pigs at day 1 to day 8 post infections, and had an increasing virus load from day 1 to day 8 post infections. However, there was irregular distribution virus load in most internal organs over the first 2 days post infection. Blood, lymphoid tissue, pancreas and ileum usually contain the highest viral loads, while heart, duodenum and brain show relatively low viral loads. CONCLUSIONS: All the data suggest that CSFV had an increasing virus load from day 1 to day 8 post infections in experimentally infected pigs detected by real-time RT-PCR, which was in consistent with the result of the IHC staining. The data also show that CSFV was likely to reproduce in blood, lymphoid tissue, pancreas and the ileum, while unlikely to replicate in the heart, duodenum and brain. The results provide a foundation for further clarification of the pathogenic mechanism of CSFV in internal organs, and indicate that blood, lymphoid tissue, pancreas and ileum may be preferred sites of acute infection.

A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus.

A simple and rapid assay for the detection of Classical swine fever virus (CSFV) was established using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This study describes the amplification of the genomic RNA of CSFV under isothermal conditions (63 °C) within one hour, using a set of six primers (two outer primers, two inner primers and two loop primers). This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV. PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries.

[Construction of an infectious cDNA clone derived from foot-and-mouth disease virus O/QYYS/s/06].

After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain.