Development Of Universal Antigen And Antibody Detection Methods For Avain Metapneumovirus

Avian metapneumovirus(aMPV)causes turkey rhinotracheitis(TRT),an acute upper respiratory tract infection in turkeys and chickens,and is associated with swollen head syndrome(SHS)in chickens.In laying or breeding turkeys,the same virus may also cause substantial falls in egg production and poor shell qualit.aMPV is now considered a major disease threat in both turkeys and chickens in many parts of the world.Australia is probably the only large area of the world currently free from infection,resulting in substantial economic losses to the poultry industry.Isolates of aMPV have been classified into four subtypes,A,B,C,and D,based on the level of genetic variations and antigenic differences?The disease has a high infection rate and is highly communicable.It is easy to cause mixed infection with other bacteria or viruses to increase the mortality rate.The diagnosis of APV infection is accomplished by virus isolation,the demonstration of viral nucleic acid by reverse transcription-polymerase chain reaction,or serology.Because of the fastidious nature of the virus,the isolation of TRTV is very difficult and time consuming;molecular biology methods have been widely used for laboratory detection of pathogens.However,all of these assays were reported to allow the detection of only one or two subtypes and not all four subtypes(A,B,C,and D).serological methods are currently the most popular method.This is by far the most commonly used method,and a number of commercial kits are now available.N is the most highly expressed of all pneumovirus proteins.them used synthetic peptides derived from the consensus N amino acid sequence to develop a universal Metapneumovirus ELISA antigen capable of detecting antibodies in sera from infected hosts that has been adapted for use with turkey sera.But it has not been widely used due to its high cost.Based on the existing studies on aMPV,this study development of a universal Eva Green qRT-PCR method for detection of Avian Metapneumovirus and based on the highly conserved region of the amino acid sequence of the N protein gene cloned into E.coli.The target protein was expressed in order to development a universal indirect ELISA method using recombinant N protein as antigen.1.Development of a Universal Eva Green qRT-PCR Method for Detection of Avian MetapneumovirusIn order to establish a rapid assay which can detect all subtypes of avian metapneumovirus(aMPV),and can be used for epidemiological investigation of aMPV infection in China,a universal Eva Green quantitative RT-PCR(qRT-PCR)assay was developed by using primers targeting the conserved region of N genes of aMPV subtypes.The standard curve for each subtype of aMPV showed a good linear relationship between cycle threshold(Ct)and template concentration ranging from 103copies/ul to 108copies/ul.The detection limit of the developed Eva Green qRTPCR was 103copies/ul,which was ten–fold more sensitive than conventional RT-PCR.The developed Eva Green qRT-PCR had no cross reactivity with other common avian pathogens and had high reproducibility with coefficient variation of 0.89~1.34% and 2.01~3.29% in intra-and inter-assay,respectively.aMPV surveillance in partial poultry farms in Henan province was determined with the developed Eva Green qRTPCR.The average positive rate was 46.6%.The coincidence rate of the developed Eva Green qRT-PCR with conventional RT-PCR is 94.7%.The developed universal Eva Green qRT-PCR can be used for detecting all subtypes of aMPV and will provide technical support for aMPV epidemiological investigation.2.Prokaryotic expression and immunogenicity analysis of avian PPV(aMPV)N proteinBased on the amino acid sequence analysis of N protein of 4 subtypes of aMPV,a highly conserved region of N protein was selected and cloned into the p ET-32a(+)expression vector according to E.coli codon bias to obtain the recombinant plasmid p ET32-N.Through optimization of a series of conditions,the optimal condition for the expression of aMPV-N protein is at 37°C,and the IPTG concentration is 1 mmol/L.After induction for 6 h,the recombinant protein was expressed in the form of inclusion bodies,facilitating the purification and preparation of polyclonal antibodies.Western blot results showed that N protein has good immunoreactivity.For the aMPV diagnostic method provides a certain reference value.3.Development of a universal indirect ELISA based on avian metapneumovirus N protein for antibody detectionELISA was developed using the purified recombinant aMPV N protein as coating antigen.The optimal reaction conditions were as follow: amount of coating antigen was 1?g/well,and coating condition was at 37? for 1h then 4? overnight;dilution of serum sample was 1:400,and incubated for 2h;working dilution of second antibody was 1:8000,incubation time was 1.5h;TMB incubation condition was 15 min at room temperature.Serum was determined as positive when its S/P?0.234 and negative when its S/P<0.194,respectively.There was no cross-reactivity between aMPV antigen and anti-NDV,-H9/H5 subtype AIV and-EDSV serum.,indicating that the method had a good specificity.The maximal intra-and inter-assay coefficient of variation was5.33% and 5.34%,indicating that the method had a good reproducibility.Fifty serum samples from clinical suspected aMPV-infected chicken were detected for aMPV-specific antibody,the results showed the coincidence with those of aMPV antigen detection was 98%.This indirect ELISA can be used for surveillance of aMPV infection in chicken flocks.