Isolation, Identification And Preliminary Characterization Of Three Avian Paramyxovirus Type 2 Isolates

Paramyxoviruses are widely presented in humans and animals, some of them can cause severe diseases in human beings and animals, and led to public health events and significant economic losses. Three samples, which were positive in hemagglutination but without Avian inf luenza viruses and Newcastle disease virus, were collected from Daxing’anling and Suiling during surveillance of avian influenza viruses in wild birds in Heilongjiang Province, in 2013. To identify the potential causative agents in these samples, experiments were carried out as following:First, allantoic fluids of these three samples were collected and treated with differential centrifugation, then 100 μL allantoic fluid of each sample was negative stained and detected using electron microscopy. From each of samples, paramyxovirus-like particles were observed and characterized by the morphology of 150~200 nm in diameter, enveloped and visible linear nucleic acids inside the partic les. Subsequently, the nucleic acids of these samples were extracted and amplif ied using random primers for sequencing, and the result showed that nucleic acid sequences of fragments obtained from each of samples had high identities with Avian paramyxovirus type 2 strains, indicating that these viruses were homologous with the APMV-2 strains(71~92%). To further identify these viruses, the hemagglutination inhibition test of each of samples of was made with polyclonal antibodies of NDV and APMV-2 respectively. And the results showed that positive NDV serum had a lower inhibition against these samples(23), while polyclonal antibody of APMV-2 had a higher inhibition of hemagglutination activity(29). Taking all the together, these three virues were tentatively designated as avian paramyxovirus virus type 2 and named respectively as APMV-2 / Emberiza spodocephala/China/Daxing’anling/974/2013, AP MV-2 /Procarduelis nipalens is/China/Suiling/53/2013, and APMV-2/Anthus rufulus/China/Suiling/106/2013.Because there is no report about avian paramyxovirus type 2 isolated from wild birds in China, the genomes of these three viruses were determined. Primers for sequencing were designed based on genomes of representatives of the APMV-2 family downloaded from Gen Bank and the sequences obtained from the previous random amplif ication. The result of genome sequencing showed that the genome of these three viruses is 15000 nt in length. The complete genome of these isolates consists of six non-overlapping genes in the order of 3’-N-P-M-F-HN-L-5’. The genome length of them is consisted with the other APMV-2 strains, abiding the “rule of six”. These isolates have 75.8~80.2% complete genome sequence identity with other avian paramyxovirus serotype 2 strains and are most closely related to AP MV-2 strain Bangor. They have the same sequences of 55 nt in the 3’leader and 161 nt in the 5’trailer. The m RNA editing sites of P gene of these three strains are all the same sequence "A5G4", and by the insertion of 1 G or 2 G in the editing site, the P gene encodes the V protein or W protein respectively. The N, P, M, F, and L genes of these three viruses have the highest corresponding gene similarity with that of APMV-2/Bangor, while the HN genes of these three strains virus have highest similar ity with APMV-2/Kenya. These three strains and the other 6 strains of APMV-2 were in the same branch in the phylogenetic tree generated with complete genome sequences.To further understand the pathogenicity of these three viruses, APMV-2/Suiling/53 was chose to be studied in SPF chickens. The pathogencity index of mean death time(MDT) of APMV-2/Suiling/53 was 120 h and the Intra Cerebral Pathogenicity Index(ICPI) of which was zero. The animal infection experiments showed that APMV-2/Suiling/53 was low pathogenic in SPF chickens and had no infection in BALB/c mice.In conclusion, three avian paramyxovirus type 2 viruses were identified in this study with the morphology, analysis of nucleic acids and the hemagglutination inhibition assay, and the genomes of these three isolates and pathogenicity of isolate Suiling/53 were preliminar ily analyzed. The result provides the basic data for the study on the genetic diversity and pathogenicity of avian paramyxovirus.