Diagnosis of avian metapneumovirus infection using multiplex PCR

Abstract

Avian metapneumovirus infection causes significant economic damage to poultry farming, which consists of losses from a decrease in safety and productivity, as well as the cost of health and preventive measures.The causative agent of the disease is avian metapneumovirus family Paramyxoviridae subfamily Pneumovirinae genus Metapneumovirus. The viral genome consists of 8 genes encoding 9 proteins: N-nucleoprotein, P-phosphoprotein, Mmatrix protein, F-fusion protein, M2 (M2-1,M2-2) - elongation/transcription factor, SH-(small) hydrophobic protein, Gglycoprotein, L-(large) polymerase. The virus lacks non-structural NS1 and NS2 proteins that counteract interferon.Based on the nucleotide seguence of the variable glycoprotein G gene, avian metapneumovirus was classified into four 4 subtypes of MPV are officially known, although recent publications have reported of two new pneumoviruses, GuMPV and AMPV PAR-05 isolated from the seagull and parrot in North America.The diversity of pathogen subtypes and differences in virulence properties of metapneumovirus create difficulties both in the prevention of this disease and in its diagnosis.The complexity of isolating the metapneumovirus from the test material is due to the short period of persistence of the virus in the organs of birds. It is possible to isolate the RNA of the causative agent of MPVI by the molecular biological method within 17-19 days after infection. The aim of our work was development and testing of primers and probes for the simultaneous detection and differentiation of avian metapneumovirus subtypes A and B by PCR with real-time fluorescence detection, with high specificity.Using vaccine strains 8544 and VC-03, the conditions for PCR were selected according to the composition of the PCR mixture and the annealing temperature of primers and probes. During PCR, we recorded the fluorescence signal through the FAM channel for cDNA obtained from the 8544 vaccine strain, and through the HEX channel for the VC-03 vaccine strain. The specificity of the developed primers and probes was also tested in studies with heterogeneous viruses such as Newcastle disease, classical and variant strains of chicken infectious bronchitis virus, and with bird DNA.The studies conducted to determine the specificity of the primers selected by us for multiplex PCR diagnostics make it possible to simultaneously specifically detect subtypes A and B of the causative agent of MPVI in birds in real time using different fluorescence channels, while other non-target respiratory pathogens were not observed.