Abstract
BackgroundMorbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.ResultsA specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34–0.53% and 1.38–2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.ConclusionsThe TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.
Highlights
Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep
Optimization of the TaqMan-based real-time Reverse transcription polymerase chain reaction (RT-PCR) targeting N gene of Feline morbillivirus (FeMV) The TaqMan-based Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was optimized by determining the optimal primers and probe concentration, in which 0.8 μL of 10 μM primers and 0.2 μL of 10 μM probe were used
Sensitivity of TaqMan-based real-time RT-PCR targeting N gene of FeMV The concentration of the complementary RNA (cRNA) was 12 ng/μL, which was equivalent to 1.74 × 1011 copies/μL
Summary
Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is a novel virus under the subfamily Orthoparamyxovirinae, which has been associated with the occurrence of chronic kidney disease in cats. A putative FeMV recombination between a Japan isolate (ChJ073) with Hong Kong isolate (776 U) involving fusion (F) and hemagglutinin (H) genes has been documented for a Japanese strain (MiJP003) [12]. This type of recombination may occur when two different virus strains infect the same cats, which raise a speculation of the possibility that the FeMV strain closely related to 776 U may be circulating in Japan
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