Isolation And Identification Of Avian Metapneumovirus And The Development Of Nested RT-PCR Detection Method

Avian metapneumovirus (aMPV) is one of the commen respiratory diseases in chickensand turkeys. The clinical diseases that may result from aMPV infection of chickens or turkeyshave been terned turkey rhinotracheitis (TRT), swollen head syndrome (SHS) based onclinical signs and lesions. The drop in egg production invariably accompanies the infection ofaMPV.Since aMPV was first described in South Africa in1978, the virus has been wildlyreported in other parts of Europe and the Middle East. aMPV has been classified into foursubtypes, A, B, C and D, respectively. Subtype A and B have been reported all through theworld. Subtype C has been reported in America, and subtype D has been reported in France.There has been only one aMPV isolation from broilers in China Mainland. Theserosurvey of aMPV in the years2007-2008revealed that the infection rate of aMPV was ashigh as100%among breeder flock in some regions of Shandong province. In Sun’s detectionduring the years2009-2010by using nucleic acid probe, the aMPV infection rate was36.87%among clinically healthy broilers in some regions of Shandong province. And the co-infectionrates were very high. We used the established Nested RT-PCR method in the detection ofaMPV, and successfully isolated one aMPV strain, which was used in the characteristicresearch. The main objective of this research was to provide a theoretical basis for theprevention and control of this disease. Our research was described in the following two parts.1. Development and application of a nested RT-PCR assay for detection of aMPV subtype B.We develop a nested RT-PCR method for the rapid detection of avian metapneumovirus.According to the F gene of the avian metapneumovirus published in GenBank, we designedtwo pairs of primers and synthesized. We amplified a specific415bp fragment from the RNAtemplates of avian metapneumovirus strait isolated from Shandong province. While theamplification results of H9subtype avian influenza virus (AIV), Newcastle disease virus(NDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV) werenegative. The sensitivity results of the amplifications by the nested RT-PCR assay were1×107copies/μL and1×102copies/μL, respectively. The sensitivity of the second amplification wasincreased by105times, which leading to a higher detection rates of clinical samples. In the clinical tests, the ordinary RT-PCR detected sixteen positive samples from the one hundredand forty two samples, while the nested RT-PCR assay could detect seventy five positivesamples. The newly established nested RT-PCR assay was successfully applied to the clinicaltests, which demonstrated it was more sensitive than the ordinary PCR and able to adjust thepseudo-negative results of the conventional PCR detection method.2. Isolation, identification and characterization of subtype B avian metapneumovirus.We collected samples suspected affecting with avian metapneumovirus from Shandongprovince. The samples were detected by RT-PCR method and some of the positive shareswere used for the isolation of aMPV. Six to eight day old specific pathogen free embryonatedchicken eggs were used to isolate the virus following inoculation by the yolk-sac route. Afterseven passages, embryo haemorrhages could be seen and the bodies were small. Then the eggallantoic fluid was inoculated onto chicken embryo fibroblast cells. After a few passages,scattered focal areas of cell rounding and syncytial formation could be seen, which wereregarded as characteristic CPE of aMPV. The isolated virus was named as SDWF Strain. TheCPE also could be seen in a range of avian and mammalian cells like Vero cells and DF-1cells.The SDWF strain can also cause cytopathic effect (CPE) on Vero cells. The inoculated Verocells became shrinkage and turned round, some cells were floated about48h post-infection.The number of rounding cells increased over time, and typical syncytium could be observedabout72h post-infection. The virus was identified by RT-PCR using specific primers andthen the PCR products were cloned and sequenced. The phylogenetic tree analysis indicatedthat there was a close genetic relationship between SDWF Strain and subtype B aMPV strainsisolated from Europe as well as some other regions. The genotyping research showed thatSDWF Strain belonged to subtype B aMPV.The type of nucleic acid of the SDWF Strain was RNA. The hemagglutination test (HA)revealed that the SDWF strain could not agglutinate the red blood cells of healthy chicken andducks. The research of the physical chemistry characteristics showed that SDWF strain wassensitive to fat-soluble solvents, such as diethyl ether and chloroform, this research as well asthe analysis test for virus nucleic acid confirmed that the isolated strain was RNA virus withenvelope. What’s more, the virus was inactivated at60°C for1h. We measured the TCID50ofthe strain on CEF according to the Reed-Muench method, the result showed that the TCID50of the strain was10-3.3/0.1mL. The result of animal regression experiment showed that thepathogenicity of the SDWF Strain was characterized by respiratory symptom. The lungs andtracheas of each groups were prepared for paraffin sections. The congestion and bleeding ofthe lungs as well as the infiltration of large amount of inflammation cells could be observed under optical microscope. The tracheal epithelial cells were found degeneration and swelling.The lamina propria of the epithelial was found bleeding and congestion as well as theinfiltration of large amount of inflammation cells.