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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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.
Livestock and poultry contribute more than 49% of the agricultural GDP of Pakistan and play an important part in the alleviation of poverty and improvement in quality of life. Poultry industry is the ...second largest and organized industry of the country, progressing with an average rate of 8-10% yearly. However, viral and bacterial diseases are the major constraints in its growth. Respiratory infections are of paramount importance as high mortality may occur in poor management. Therefore, the etiology of respiratory disease is complex, often involving more than one pathogen at the same time. A wide variety of pathogens have been associated with respiratory infection in poultry, including Avian Pneumovirus (APV), Avian Influenza Virus (AIV), Infectious Bronchitis (IBV), Newcastle Disease Virus (NDV), Infectious Laryngotracheal virus (ILTV) and Mycoplasma gallisepticum (MG). As the Avian Influenza and Newcastle disease are endemic in the country,
therefore, this study was planned to evaluate their status during the winter outbreaks. Tracheal samples were collected from poultry farms in southern region of Pakistan, experiencing heavy mortality of poultry flock. The samples were subjected to RNA extraction followed by RT-PCR. Out of 50 samples, 20 samples were positive for NDV, 28 for AIV and 2 were negative for both. Further, serotyping of 28 AIV isolates showed that, 6 were positive for H9, 20 for H5 and 2 for H7. Thus, it can be concluded that molecular techniques help in rapid identification of the agents causing infections. Further, the southern region of Pakistan had major infection resulting from AIV (H5) during October 2007 to February 2008.
Successful oral vaccination of chickens with Newcastle disease (ND) depends on the survival of vaccine virus on the grains that are used as carriers. Some interactions between grains and the V4 ...strain of ND virus (NDV) were studied. Crude saline washings were prepared from several grains — rice (unhusked, brown, white and boiled white), sorghum, millet, wheat, maize and barley — and tested for lectin activity, as indicated by agglutination of chicken erythrocytes. Only washings from unhusked rice, sorghum and millet failed to haemagglutinate. None of the crude washings antagonised the haemagglutinating activity of NDV, and the washing from white rice produced an 8-fold enhancement. The presence of lectins in the washings from rice, wheat and barley was confirmed by purifying a substance with N-acetyl-D-glucosamine specificity. Only the crude extract from white rice had any profound effect on infectivity, reducing the infectivity titre by 99.99%. It is not known if the viricidal substance is identical with the lectin. Of 9 commercial lectins tested, only ConA bound the V4 virus.
Twelve-day-old chickens were vaccinated once with different Newcastle disease (ND) vaccines ( F, La Sota and Mukteswar) by two different routes (intraocular and drinking water). Chickens from a ...seventh group were uninoculated controls. At weekly intervals for 7 weeks after vaccination, 20 chickens from each vaccinated group and 20 chickens from the control group were examined for the production of haemagglutination-inhibition (HI) antibodies and for protection as assessed after challenge with velogenic, viscerotropic ND virus.
La Sota ND vaccine used intraocularly ranked the best and Mukteswar vaccine by the drinking water route the worst for their HI antibody titres prior to challenge. Differences between the treatments in protection were examined. For all three vaccines intraocular vaccine produced higher protection than drinking water vaccine. An inverse relationship between prechallenge and postchallenge HI titres was also recorded.
Enterocytes were detached from various parts of the digestive tract of chickens by treatment with DTT or with hyaluronidase. Isolated enterocytes were exposed to suspensions of the V4 strain of ...Newcastle disease virus (NDV). Removal of virus from the supernatant fluid was taken as evidence of binding of virus to enterocytes and residual virus was measured both by infectivity assay and by ELISA. Enterocytes from duodenum, jejunum, ileum, caecum, and rectum bound the virus; enterocytes from oesophagus, crop and proventriculus did not.
Living V4 strain Newcastle disease vaccine was given to chickens orally. The inclusion of DEAE-dextran, Quil A or TiterMax® in the vaccine, or delivering the vaccine as Iscoms, did not enhance the ...serological response. The immediate serological response to living V4 vaccine was enhanced in the presence of Avridine. Chickens produced a low serological response to oral administration of inactivated V4 vaccine. This response was not enhanced in the presence of Avridine.
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