RESUMEN
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.
RESUMEN
At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50-100 µM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.
RESUMEN
Porcine circovirus type 2 (PCV2) is one of the smallest known animal viruses and is the main pathogen of PCV-associated diseases (PCVAD). Epidemiological surveillance results have shown that the PCV2 infection rate is on the rise in China, thus, PCV2 disease prevention and control has become a huge challenge for the Chinese swine industry. We collected clinical samples from multiple different provinces in China from 2018 to 2020 and found that the positive rate of PCV2 was 53% (3619/6872), identity between the cloned 62 ORF2 genes was 84.4-100% and identity between the cloned 62 ORF2 sequences and reference sequence was 72.9-99.8%. Genetic evolution analysis found that PCV2d accounted for 79% (49/62 samples), PCV2a for 12.9% (8/62 samples), PCV2b for 8% (5/62 samples), and PCV2c and PCV2e genotypes were not found. However, most commercial PCV2 subunit vaccines are based on the PCV2a genotype, and there are very few vaccines based on PCV2b or PCV2d. Therefore, the homologous and heterologous protection ability of PCV2b and PCV2d Cap proteins based on the baculovirus against the PCV2b and PCV2d infections was evaluated, which is expected to design and develop excellent PCV2 protein vaccine candidates. This study found that both PCV2b and PCV2d Cap proteins can increase the level of humoral immunity and cellular immune response in mice. Importantly, both PCV2b and PCV2d cap proteins can provide homologous and heterologous protection against the PCV2b and PCV2d viruses. Overall, this study provides a reference for the prevention and control of PCVAD in mainland China and the development of PCV2 vaccines.