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Newcastle disease, caused by Avian avulavirus 1 (AAvV 1), is endemic to many developing countries around the globe including Pakistan. Frequent epidemics are not uncommon even in vaccinated populations and are largely attributed to the genetic divergence of prevailing isolates and their transmission in the environment. With the strengthening of laboratory capabilities in Pakistan, a number of genetically diverse AAvV 1 strains have recently been isolated and individually characterized in comparison with isolates reported elsewhere in the world. However, there lacks sufficient comparative genomic and phylogenomic analyses of field circulating strains that can elucidate the evolutionary dynamics over a period of time. Herein, we enriched the whole genome sequences of AAvV reported so far (n = 35) from Pakistan and performed comparative genomic, phylogenetic and evolutionary analyses. Based on these analyses, we found only isolates belonging to genotypes VI, VII and XIII of AAvV 1 in a wide range of avian and human hosts. Comparative phylogeny revealed the concurrent circulation of avulaviruses representing different sub-genotypes such as VIg, VIm, VIIa, VIIb, VIIe, VIIf, VIIi, XIIIb and XIIId. We found that the isolates of genotype VII were more closely associated with viruses of genotype XIII than genotype VI. An inter-genotype comparative residue analysis revealed a few substitutions in structurally and functionally important motifs. Putative recombination events were reported for only one of the captive-wild bird (pheasant)-origin isolates. The viruses of genotype VII had a high genetic diversity as compared to isolates from genotypes VI and XIII and, therefore, have more potential to evolve over a period of time. Taken together, the current study provides an insight into the genetic diversity and evolutionary dynamics of AAvV 1 strains circulating in Pakistan. Such findings are expected to facilitate better intervention strategies for the prevention and control of ND in disease-endemic countries across the globe particularly Pakistan.

Pigeon paramyxovirus type 1 (PPMV-1) infection is enzootic in pigeon flocks and poses a potential risk to the poultry industry in China. To gain insight into the biological characteristics and transmission routes of circulating PPMV-1 in pigeons, 13 PPMV-1 isolates from domestic pigeons isolated during 2011-2015 in Guangxi province, China, were characterized using a pathogenicity assessment and phylogenetic analysis. All PPMV-1 isolates were mesogenic or lentogenic strains and had a mean death time (MDT) in 9-day-old SPF chicken embryos and a intracerebral pathogenicity index (ICPI) values of 54-154 h and 0.00-0.90, respectively. Analysis of the F and HN gene sequences of the PPMV-1 isolates and the Newcastle Disease (ND) vaccine strain La Sota, revealed that the nucleotide sequence similarity of the F and HN genes were all < 85% between the PPMV-1 isolates and La Sota, significantly lower than those > 98% among the PPMV-1 isolates. The amino acids sequence of the F protein at the cleavage site of the 13 PPMV-1 isolates was 112 RRQKR↓F 117 , characteristic of virulent Newcastle disease virus (NDV). All 13 isolates were classified as sublineage 4b by phylogenetic analysis and evolutionary distances, based on the F gene sequences. It was also found that the 13 isolates were divided into two novel sub-groups of sublineage 4b, sub-sublineages 4biig and 4biih. Since these two novel sub-sublineages had two different geographic sources, we speculated that they represent two different transmission routes of PPMV-1 in China. Phylogenetic analysis of these isolates will help to elucidate the sources of the transmission and evolution of PPMV-1 and may help to control PPMV-1 infection in the pigeon industry in China.

Five avian paramyxoviruses of serotype 8 (APMV-8) were isolated during a study monitoring wild birds in Kazakhstan in 2013 and each was further characterized. The viruses were isolated from three White-fronted geese (Anser albifrons), one Whooper swan (Cygnus cygnus), and one Little stint (Calidris minuta). Before our study, only two complete APMV-8 sequences had been reported worldwide since their discovery in the USA and Japan in the 1970s. We report the complete genome sequences of the newly detected viruses and analyze the genetic evolution of the APMV-8 viruses over four decades.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments.

A hemagglutinating virus (8KS0813) was isolated from a red-necked stint. Hemagglutination inhibition and neutralization tests indicated that 8KS0813 was antigenically related to a prototype strain, APMV-6/duck/Hong Kong/18/199/77, but with an 8- and 16-fold difference, respectively, in their titers. The full genome sequence of 8KS0813 showed 98.6 % nucleotide sequence identity to that of APMV-6/duck/Italy/4524-2/07, which has been reported to belong to an APMV-6 subgroup, and showed less similarity to that of the prototype strain (70.6 % similarity). The growth of 8KS0813 and the prototype strain in four different cell cultures was greatly enhanced by adding trypsin. Interestingly, this virus induced syncytia only in Vero cells. 8KS0813 was identified as APMV-6/red-necked stint/Japan/8KS0813/08, but it is antigenically and genetically distinguishable from the prototype strain, suggesting that variant APMV-6 is circulating in migratory birds.

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

Eight strains of pigeon paramyxovirus type 1 (PPMV-1) were isolated and identified in this study, from diseased pigeon flocks suspected to be infected with PPMV-1 in China between 2010 and 2012. These PPMV-1 isolates were purified using specific-pathogen-free (SPF) chicken embryo cells before full-length genomic sequencing. The complete genome of these isolates contained 15,192 nucleotides, similar to those of Newcastle disease virus (NDV) strains in genotypes V-XI, with the gene order 3'-NP-P-M-F-HN-L-5'. A six-nucleotide insertion was found to be located in the 5' non-coding region of the nucleoprotein gene in our eight PPMV-1 strains when compared with those of genotypes I, II, III, IV and V. The cleavage site of the fusion protein was (112)RRQKRF(117), a feature generally associated with virulent NDV strains. The structural proteins were in accordance with those of other PPMV-1 strains, with the exception of the W protein of pigeon/CHINA/LJL/100605. The length of the W protein was 227 amino acids, in common with PPMV-1 strains, whereas that of pigeon/CHINA/LJL/100605 was only 181 amino acids. Phylogenetic analysis, based on the genomic sequences and sequences of the fusion gene, revealed that our eight isolates should be classified as class II genotype VIb NDVs. To our knowledge, this is the first report to show that the strain pigeon/CHINA/LLN/110713 is similar to strains isolated abroad, but it was isolated in China, which implies that it may have been introduced to China from overseas. Differences between the Chinese and foreign strains were identified in three regions (nucleotide positions 1632-2229, 3023-3310 and 6103-6439). In addition, the values of ICPI and MDT demonstrated that PPMV-1 isolates were mesogenic or lentogenic, and virulence studies showed that these PPMV-1 strains were non-pathogenic in chickens, but they induced the generation of antibodies in vivo.