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.

Establishment of a rescue system for porcine parvovirus using a seamless cloning method.

In this study, we describe a novel and rapid method for the construction of a full-length infectious clone (pPPV). The constructed clone contained an engineered EcoRv site that served as a genetic marker and was shown to be infectious when transfected into a monolayer of PK-15 cells. The rescued virus (rPPV) of the infectious clone was found to be indistinguishable from wild-type virus BQ in terms of its biological properties. The generation of this PPV infectious clone provides a potentially powerful tool with which to elucidate the molecular pathogenesis of PPV.

The radiation damage of crystalline silicon PN diode in tritium beta-voltaic battery.

A tritium beta-voltaic battery using a crystalline silicon convertor composed of (100)Si/SiO2/Si3N4 film degrades remarkably with radiation from a high intensity titanium tritide film. Simulation and experiments were carried out to investigate the main factor causing the degradation. The radiation damages mainly comes from the x-ray emitted from the titanium tritide film and beta particle can relieve the damages. The x-ray radiation induced positive charges in the SiO2 film destroying the output property of the PN diode with the induction of an electric field.

The survey of porcine teschoviruses in field samples in China with a universal rapid probe real-time RT-PCR assay.

A real-time reverse transcription polymerase chain reaction (RT-PCR) based on TaqMan was established and evaluated for quantitative detection of porcine teschoviruses (PTVs). A pair of primers and a TaqMan probe targeting on the highly conserved sequence of the 5'-untranslated region (5'-UTR) of one to 11 serotypes of PTV were designed. Standard plasmid DNA containing PCR amplification of the 5'-UTR were constructed and used to develop the real-time RT-PCR. The results indicated that the real-time RT-PCR was specific for detection of PTV with a detection limit of 10 copies/µL, but not for porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus. The coefficient of variation of inter-assay and intra-assay were less than 3 %. A total of 91 clinical samples were tested by the real-time RT-PCR and virus isolation (OIE 2008) and positive rates were 79.12 % (72/91) and 57.14 % (48/91), respectively. In conclusion, the developed real-time RT-PCR assay was an effective method for detection and quantification of PTV in fields or organs of infected pigs.

Simulations about self-absorption of tritium in titanium tritide and the energy deposition in a silicon Schottky barrier diode.

Simulations on the self-absorption of tritium electrons in titanium tritide films and the energy deposition in a silicon Schottky barrier diode are carried out using the Geant4 radiation transport toolkit. Energy consumed in each part of the Schottky radiovoltaic battery is simulated to give a clue about how to make the battery work better. The power and energy-conversion efficiency of the tritium silicon Schottky radiovoltaic battery in an optimized design are simulated. Good consistency with experiments is obtained.

Interclade recombination in porcine parvovirus strains.

A detailed analysis of the Ns1/Vp1Vp2 genome region of the porcine parvovirus (PPV) strains isolated from vaccinated animals was performed. We found many inconsistencies in the phylogenetic trees of these viral isolates, such as low statistical support and strains with long branches in the phylogenetic trees. Thus, we used distance-based and phylogenetic methods to distinguish de facto recombinants from spurious recombination signals. We found a mosaic virus in which the Ns1 gene was acquired from one PPV clade and the Vp1Vp2 gene was acquired from a distinct phylogenetic clade. We also described the interclade mosaic structure of the Vp1Vp2 gene of a reference strain. If recombination is an adaptive mechanism over the course of PPV evolution, we would likely observe increasing numbers of chimeric strains over time. However, when the PPV sequences isolated from 1964 to 2011 were analysed, only two chimeric strains were detected. Thus, PPV recombination is an independent event, resulting from close contact between animals housed in high-density conditions.

A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets.

A real-time polymerase chain reaction with SYBR Green was developed for the detection and quantification of encephalomyocarditis virus (EMCV) in porcine tissues; the method uses two primers specific for the 3D gene. The detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001 TCID(50)/ml. The assay was linear over a 10(7) dilution range of template concentrations and was specific for EMCV; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductive and respiratory virus, classical swine fever virus, pseudorabies virus, or porcine teschovirus). This assay detected EMCV titers at least 10(4) smaller than the routine PCR assay. To increase our understand of EMCV pathogenesis, the new method was used to quantify levels of EMCV genome in various tissues of artificially challenged sows and piglets. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. The real-time PCR method described here should be useful for the study of EMCV infection and distribution in pigs.

Development of a loop-mediated isothermal amplification method for rapid detection of caprine arthritis-encephalitis virus proviral DNA.

A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0.0001 TCID(50), reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.

Isolation, molecular characterization, and phylogenetic analysis of porcine encephalomyocarditis virus strain HB10 in China.

Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and mortality in pregnant sows, fetuses, and ablactating piglets. Diseases caused by EMCV currently affect the swine industry worldwide. A virus was isolated from organs of dead piglets that presented with acute myocarditis in northern China. The production of a specific cytopathic effect on susceptible cells and the results of hemagglutination inhibition (HI) assay, PCR, electron microscopy (EM), and sequencing indicated that the pathogen was EMCV; the strain was named HB10. Other pathogenic agents causing myocarditis and death were excluded as possible pathogenic agents. Phylogenetic analyses of the capsid coding region and the VP3/VP1 genes using the neighbour-joining method revealed that EMCV isolates cluster into two groups (groups 1 and 2) with two sub-clusters within group 1 (group 1a and b). HB10 belongs to group 1a, along with strains CBNU, GX0601, BJC3, NJ08, and BEL-2887A/91. Five strains isolated from Sus scrofa belong to group 2. The results of this and previous studies indicate that HB10 and other EMCV strains cause myocarditis of pigs in China.

Investigation on a radiation tolerant betavoltaic battery based on Schottky barrier diode.

An Au-Si Schottky barrier diode was studied as the energy conversion device of betavoltaic batteries. Its electrical performance under radiation of Ni-63 and H-3 sources and radiation degradation under Am-241 were investigated and compared with those of the p-n junction. The results show that the Schottky diode had a higher I(sc) and harder radiation tolerance but lower V(oc) than the p-n junction. The results indicated that the Schottky diode can be a promising candidate for energy conversion of betavoltaic batteries.

Rapid and sensitive detection of PRRSV by a reverse transcription-loop-mediated isothermal amplification assay.

A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10(-4) dilution of template RNA extracted from 250 µL of 10(5) of the 50% tissue culture infective dose (TCID(50)) of PRRSV-containing cells, and no cross-reactivity was observed with other related viruses including porcine circovirus type 2, swine influenza virus, porcine rotavirus and classical swine fever virus. From forty-two field samples, 33 samples in the RT-LAMP assay was detected positive, whereas three of which were not detected by RT-PCR. Furthermore, in 33 strains of PRRSV, an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages. These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates, especially in developing countries.

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2.

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10(-5) ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.