Nano česticama potpomognuta lančana reakcija polimerazom za dokaz virusa encefalomiokarditisa.

The encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. Diseases caused by EMCV currently affect the swine industry worldwide. In this study, a nano polymerase chain reaction (nanoPCR) assay, targeting the 3D gene of EMCV, was developed and its sensitivities and specificities were investigated. The results indicated that the nanoPCR assay is highly sensitive and able to detect 1.2×102 copies/μL of EMCV RNA, as no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nanoPCR assay for the detection of EMCV. The sensitive, and specific nanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of EMCV-infection.Virus encefalomiokarditisa može uzrokovati akutni miokarditis u prasadi ili reprodukcijske poremećaje u krmača. Poremećaji uzrokovani virusom encefalomiokarditisa prošireni su u svinja u industrijskoj proizvodnji diljem svijeta. U ovom je istraživanju razvijena nano-lančana reakcija polimerazom (nanoPCR) za dokaz gena 3D virusa encefalomiokarditisa te je istražena njezina osjetljivost i specifičnost. Rezultati su pokazali da je nanoPCR visoko osjetljiva metoda kojom se može dokazati 1,2×102 kopija RNA virusa encefalomiokarditisa na μL te da nisu dokazane križne reakcije s drugim virusima. Ovo je prvo izvješće u kojem je dokazana primjena nanoPCR-a za dokaz virusa encefalomiokarditisa. Njezina osjetljivost i specifičnost upućuju na zaključak da se može naširoko rabiti u kliničkoj dijagnostici i terenskim istraživanjima encefalomiokarditisa svinja

Development and evaluation of a real-time recombinase-aid amplification assay for rapid detection of Pseudomonas aeruginosa

Objective To establish a real-time recombinase-aid amplification (RAA) method for rapid detection of Pseudomonas aeruginosa. Methods Specific primers and exo probes based on ecfX gene of P. aeruginosa were designed in this study, and the validity of the method was evaluated by sensitivity, specificity and suspected strains detection. Results Real-time RAA was performed successfully at 39℃ for 20 min. Only the P. aeruginosa strains but not other bacteria were amplified, showing the good specificity. The limit of detection was 3.0×103 fg genomic DNA per reaction, and 1.0×103 CFU P. aeruginosa pure culture per reaction. The developed real-time RAA was further evaluated on 36 suspected of P. aeruginosa, which were identified successfully to be P. aeruginosa.The detection result were the same with those of a real-time PCR assay and the VITEK 2 Compact. Conclusion The developed real-time RAA assay is a rapid, simple and reliable tool for accurate detection of P. aeruginosa of diverse origins

Complete Genomic Characterization of Porcine Reproductive and Respiratory Syndrome Virus Strain HB-XL

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causal agent of a serious disease of swine. Here, we report the genome sequence of PRRSV strain HB-XL isolated from a pig farm with a clinical outbreak of porcine reproductive and respiratory syndrome. The genome is 15,323 bp long and has nine open reading frames (GenBank: KP162169). Comparative and phylogenetic analysis showed that HB-XL belongs to the highly pathogenic PRRSV (HP-PRRSV) subfamily in the family PRRSV. The viral nonstructural protein 2 (Nsp2) of the HB-XL strain contained 30 discontinuous amino acid (AA) deletions relative to that of the Nsp2 of the VR2332 strain. The AA substitutions R13 and R151 suggested high virulence of the HB-XL strain. The unique mutations in glycoprotein 5 (GP5) and Nsp2 revealed that HB-XL might be a novel variant PRRSV strain recombined with vaccine strains. However, the low morbidity and mortality in the pig herd from which HB-XL was isolated indicate that the virulence of the virus was weak, so it has potential as a future vaccine strain

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1.

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification

Abstract Background Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. Methods A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. Results The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min–12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R2 value of the positive results was 0.947. Conclusions The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings

Pathogenicity and molecular characterization of a fowl adenovirus 4 isolated from chicken associated with IBH and HPS in China

Abstract Background Since July in 2015, an emerging infectious disease, Fowl adenovirus (FAdV) species C infection with Hepatitis-Hydropericardium syndrome was prevalent in chicken flocks in China. In our study, one FAdV strain was isolated from commercial broiler chickens and was designated as SDSX1.The phylogenetic information, genetic mutations and pathogenicity of SDSX1 were evaluated. Results The phylogenetic analysis indicated that SDSX1 is a strain of serotype 4, FAdV-C. The amino acid analysis of fiber-2 showed that there were more than 20 mutations compared with the non-virulent FAdV-C strains. The pathogenic evaluation of SDSX1 showed that the mortality of one-day-old chickens inoculated SDSX1 was 100%. The typical histopathological changes of SDSX1 were characterized by the presence of basophilic intranuclear inclusion bodies in hepatocytes. The virus copies in different tissues varied from107 to 1011 per 100 mg tissue and liver had the highest virus genome copies. Conclusion In conclusion, the isolate SDSX1, identified as FAdV-4, could cause one-day-old chicks’ typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were the highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1

A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine

ABSTRACT: Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines