Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe, Asia, and Africa.

BACKGROUND: The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. RESULTS: Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014-2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. CONCLUSIONS: We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions.

Complete Genome Sequence of a Virulent Newcastle Disease Virus Strain Isolated from a Clinically Healthy Duck (Anas platyrhynchos domesticus) in Pakistan.

Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks (Anas platyrhynchos domesticus) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II.

Complete Genome Sequence of a Recent Panzootic Virulent Newcastle Disease Virus from Pakistan.

The genome sequence of a new strain of Newcastle disease virus (NDV) (chicken/Pak/Quality Operations Lab/SFR-611/13) is reported here. The strain was isolated from a vaccinated chicken flock in Pakistan in 2013 and has panzootic features. The genome is 15,192 nucleotides in length and is classified in subgenotype VIIi of genotype VII, class II.

Characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008.

Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006 to 2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of the HA genes, these isolates belong to clade 2.2 and both the HA and NA are closely related to each other (nucleotide identities above 99.0%) and to other Middle Eastern H5N1 AIV isolates (nucleotide identities above 98.0%). The phylogenetic data suggest that the virus in both epornitics of H5N1 HPAIV in commercial poultry in the Karachi region of Pakistan between 2006 and 2008 were from a very closely related source, however, there is inadequate epidemiological data to determine what the reservoir was for the virus between the 2006 and 2007 outbreaks other than that there was a single introduction into the region.