Newcastle Disease Virus Isolates Research Articles

Molecular analysis of Newcastle disease virus isolates reveals a novel XIId subgenotype in Vietnam.

Eight Vietnamese Newcastle disease virus field isolates from 2008-2015 and 3 vaccine specimens were genotyped based on their full F gene sequences and compared to 80 reference strains representing all 18 genotypes. Three isolates formed a novel subgenotype XIId, identified for the first time in Vietnam; while the others clustered as follows: four in subgenotypes VIId and VIIh; two in Genotype I; and two in Genotype II. Evolutionary distance calculations confirmed the Vietnamese XIId isolates were distinct from XIIa and XIIb by 0.062-0.070; and from other genotypes by 0.089-0.245. This data demonstrated that a novel XIId subgenotype emerged in Vietnam indicating considerable genetic diversity, thus highlighting the need to implement antigenic matching during vaccination against NDVs.

Potential of genotype VII Newcastle disease viruses to cause differential infections in chickens and ducks.

Newcastle disease (ND), caused by ND virus (NDV), is one of the most infectious and economically important diseases of the poultry industry worldwide. While infections are reported in a wide range of avian species, the pathogenicity of chicken-origin virulent NDV isolates in ducks remains elusive. In this study, two NDV strains were isolated and biologically and genetically characterized from an outbreak in chickens and apparently healthy ducks. Pathogenicity assessment indices, including the mean death time (MDT), intracerebral pathogenicity index (ICPI) and cleavage motifs in the fusion (F) protein, indicated that both isolates were velogenic in nature. While these isolates carried pathogenic characteristics, interestingly they showed differential pathogenicity in ducks. The chicken-origin isolate caused high (70%) mortality, whereas the duck-origin virus resulted in low (20%) mortality in 4-week-old ducks. Intriguingly, both isolates showed comparable disease pathologies in chickens. Full-genome sequence analysis showed that the virus genome contains 15192 nucleotides and carried features that are characteristic of velogenic strains of NDV. A phylogenetic analysis revealed that both isolates clustered in class II and genotype VII. However, there were several mutations in the functionally important regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins, which may be responsible for the differential pathogenicity of these viruses in ducks. In summary, these results suggest that NDV strains with the same genotype show differential pathogenicity in chickens and ducks. Furthermore, chicken-origin virulent NDVs are more pathogenic for ducks than duck-origin viruses. These findings propose a role for chickens in the evolution of viral pathogenicity and the potential genetic resistance of ducks to poultry viruses.

Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016.

Aim:The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt.Materials and Methods:The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs.Results:Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016.Conclusion:All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.

Biological characterization of wild-bird-origin avian avulavirus 1 and efficacy of currently applied vaccines against potential infection in commercial poultry.

Newcastle disease virus (NDV), the type member of the species Avian avulavirus 1 (formerly known as avian paramyxovirus serotype 1), causes a highly contagious and economically important disease in a myriad of avian species around the globe. While extensive vaccination programs have been implemented in ND-endemic countries, the disease is continuously spreading in commercial, backyard, and wild captive poultry. In order to investigate the evolution of the virus and assess the efficiency of the vaccine regimens that are currently being applied in commercial poultry, four wild-bird-origin NDV strains were characterized biologically, based on mean death time and intracerebral pathogenicity index, and genetically, based on the cleavage motif (112RRQKRF117) in the fusion (F) protein. Based on these features, all of the isolates were characterized as velogenic strains of NDV. Phylogenetic analysis based on the complete genome sequence revealed clustering of these isolates within class II, genotype VII. This class of NDV remains the predominant genotype in the Egyptian poultry industry, as well as in those of many Asian and African countries. To investigate the potential of these wild-bird-origin NDV isolates to cause infection in domesticated poultry and to assess the efficacy of currently available vaccines for protection of commercial poultry, an extensive animal challenge experiment was performed. Cumulative clinicopathological and immunological investigations of virus-challenged chickens indicate that these isolates can potentially be transmitted between chicken and cause systemic infections, and the currently applied vaccines are unable to prevent clinical disease and virus shedding. Taken together, the data represent a comprehensive evaluation of the ability of Egyptian wild-bird-origin NDV strains to cause infection in commercial poultry and highlights the need for a continuous and large-scale surveillance as well as revised vaccine approaches. These integrated and multifaceted strategies would be crucial in any efforts to control and eradicate the disease globally.

Oncolytic effect of wild-type Newcastle disease virus isolates in cancer cell lines in vitro and in vivo on xenograft model.

Oncolyic virotherapy is one of the modern experimental techniques to treat human cancers. Here we studied the antitumor activity of wild-type Newcastle disease virus (NDV) isolates from Russian migratory birds. We showed that NDV could selectively kill malignant cells without affecting healthy cells. We evaluated the oncolytic effect of 44 NDV isolates in 4 histogenetically different human cell lines (HCT116, HeLa, A549, MCF7). The safety of the isolates was also tested in normal peripheral blood mononuclear (PBMC) cells. The viability of tumor cell lines after incubation with NDV isolates was evaluated by MTT. All cell lines, except for normal PBMC primary cells, had different degrees of susceptibility to NDV infection. Seven NDV strains had the highest oncolytic activity, and some NDV strains demonstrated oncolytic selectivity for different cell lines. In vivo, we described the intratumoral activity of NDV/Altai/pigeon/770/2011 against subcutaneous non-small cell lung carcinoma using xenograft SCID mice model. All animals were responsive to therapy. Histology confirmed therapy-induced destructive changes and growing necrotic bulk density in tumor tissue. Our findings indicate that wild-type NDV strains selectively kill tumor cells with no effect on healthy PBMC cells, and intratumoral virotherapy with NDV suppresses the subcutaneous tumor growth in SCID mice.

Phylogenetic and pathotypic characterization of newcastle disease virus in Tibetan chickens, China

ABSTRACT: Chickens are considered to be potential reservoirs of Newcastle disease virus (NDV). In this study, six Newcastle disease virus strains were isolated and characterized in Tibetan chickens. The HN gene was sequenced, and phylogenetic relationship to reference strains was studied. The phylogenetic analysis demonstrated that these six isolated strains were closely related to NDV isolates of the reference strains GQ245823, KT002186, KU527561, KJ563939, AY225110, EU305607, KM056357, Y18898, GQ245832, AF077761 and lasota strain. Among them, EU305607, KJ563939 and KM056357 were isolated from India, while lasota strain came from attenuated vaccine widely used in China. Then, mean death time (MDT) and intracerebral pathogenicity index (ICPI) were used to estimate the pathogenicity of the isolates. Pathogenicity experiment showed HNH1 and HN17 to be virulent. Our results indicated that genetically diverse viruses circulate in Tibetan chickens, and based upon the phlogeographic analysis, we estimated the origin of ancestral viruses of the isolates and its sister strains located in India and China (lasota strain). It indicates the importance of continuous surveillance to enhance current understanding of the genetic evolution of the NDV strains.

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

BackgroundThe 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.ResultsHere, 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.ConclusionsWe 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.

Spillover of Newcastle disease viruses from poultry to wild birds in Guangdong province, southern China

Despite intensive vaccination programs in many countries, including China, Newcastle disease has been reported sporadically and is still a significant threat to the poultry industry in China. Newcastle disease virus (NDV) is infectious for at least 250 bird species, but the role of wild birds in virus epidemiology remains largely unknown. Fourteen NDV isolates were obtained from 2040 samples collected from wild birds or the environment in Guangdong province, southern China, from 2013 to 2015. The isolation rate was the highest in the period of wintering and lowest during the periods of spring migration, nesting, and postnesting. A maximum clade credibility phylogenetic analysis revealed that at least four genotypes circulate in southern China: three class II genotypes (II, VI, and IX) and one class I (1b). We also demonstrated that most isolates from wild birds were highly similar to isolates from poultry, and two isolates were linked to viruses from wild birds in northern China. These data suggested that wild birds could disseminate NDV and poultry-derived viruses may spillover to wild birds. Accordingly, vaccine development and poultry management strategies should be considered to prevent future NDV outbreaks, particularly given the strength of the poultry industry in developing countries, such as China.

Molecular evolution and epidemiological links study of Newcastle disease virus isolates from 1995 to 2016 in Iran.

In the case of Newcastle disease virus, multiple factors such as host adaptation, immune response evasion, and selective pressures have been suggested to result in evolution of viruses and the emergence of genetic variants. Multiple studies on virus classification and global epidemiological links have yielded consistent data. Here, we have performed a molecular analysis study of circulating Newcastle disease viruses in Iran (1995-2016). According to evolutionary divergences, subgenotype VIg, VIj, VIIj, VIId, XIIIa and XIIId isolates have been circulating in the country during a 21-year period. Based on data analysis, VIg isolates shared highest sequence identity with Russian and Polish isolates of the VIg subgenotype, while VIj subgenotype isolates (2012) were most similar to a virus isolated in 2015 in India. Analysis of the evolutionary divergence of subgenotype VIIj suggests that Chinese and Ukrainian viruses may have played a crucial role in the emergence of VIIj isolates. Evolutionary difference studies also indicated that XIIIa isolates circulating in Iran may have caused the emergence of adapted variants of subgenotype XIIId. Therefore, we propose that the evolutionary and epidemiological study of virulent Newcastle disease viruses could help to provide accurate molecular data about variants circulating in the region, thus aiding in the design of more efficient recombinant vaccines.

Detection of Newcastle disease virus of poultry by real time reverse transcription-polymerase chain reaction

A real-time reverse transcription - polymerase chain reaction (rRT-PCR) was used for the detection of Newcastle disease virus (NDV) of poultry. A panel of seven known isolates of NDV in the form of allantoic fluid, obtained from a laboratory repository, was used for the development of the test. RNA was extracted from the allantoic fluid with a magnetic processor based automated RNA extraction system. The identity of the reference virus was first reconfirmed by a conventional RT-PCR specific for the Fusion (F) protein gene. Using these RNA, the rRT-PCR protocol was optimized with regard to the reaction mix and thermal profile using published primers and probes specific for M gene. The sensitivity of standardized rRT-PCR was compared to that of the conventional RT-PCR using serial 10-fold dilutions of the RNA of a selected sample. The thermal profile was modified from the published one; the annealing and extension steps were combined to a single step performed at 60ºC. The adopted rRT-PCR successfully amplified M gene from all the seven reference samples with a CT value ranging from 15.28 to 32.68. The rRT-PCR for M gene was 100-fold more sensitive than the conventional RT-PCR for F gene. This is the first report of the use of rRT-PCR for the detection of NDV in Bangladesh. This test will be useful for virological surveillance, particularly for screening NDV in respiratory infections.Bangl. vet. 2016. Vol. 33, No. 1, 16-22

Phylogenetic analysis of Newcastle disease viruses from Bangladesh suggests continuing evolution of genotype XIII.

A total of 23 Newcastle disease virus (NDV) isolates from Bangladesh taken between 2010 and 2012 were characterized on the basis of partial F gene sequences. All the isolates belonged to genotype XIII of class II NDV but segregated into three sub-clusters. One sub-cluster with 17 isolates aligned with sub-genotype XIIIc. The other two sub-clusters were phylogenetically distinct from the previously described sub-genotypes XIIIa, XIIIb and XIIIc and could be candidates of new sub-genotypes; however, that needs to be validated through full-length F gene sequence data. The results of the present study suggest that genotype XIII NDVs are under continuing evolution in Bangladesh.

Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.

Newcastle disease virus (NDV) is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins (haemagglutin-neuraminidase and fusion proteins) but not to internal proteins (nucleoprotein or matrix protein) of NDV strains.

An Australian Newcastle Disease Virus With a Virulent Fusion Protein Cleavage Site Produces Minimal Pathogenicity in Chickens.

Newcastle disease is an important disease of poultry caused by virulent strains of Newcastle disease virus (NDV). During the 1998 to 2002 outbreaks of Newcastle disease in Australia, it was observed that the mild clinical signs seen in some chickens infected with NDV did not correlate with the viruses' virulent fusion protein cleavage site motifs or standard pathogenicity indices. The pathogenicity of 2 Australian NDV isolates was evaluated in experimentally challenged chickens based on clinical evaluation, histopathology, immunohistochemistry, and molecular techniques. One of these virus isolates, Meredith/02, was shown to induce only very mild clinical signs with no mortalities in an experimental setting, in contrast to the velogenic Herts 33/56 and Texas GB isolates. This minimal pathogenicity was associated with decreased virus replication and antigen distribution in tissues. This demonstrates that the Australian Meredith/02 NDV, despite possessing a virulent fusion protein cleavage site, did not display a velogenic phenotype.

First genetic characterization of newcastle disease viruses from Namibia: identification of a novel VIIk subgenotype.

The complete sequences of the fusion (F) protein genes of six Newcastle disease virus (NDV) isolates from backyard poultry in Namibia in 2016 have been determined. The F gene cleavage site motif for all of the isolates was 112RRQKRF117, indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses belong to a novel subgenotype, VIIk. This is the first genetic characterization of NDV isolates from Namibia, and the findings have important implications for Newcastle disease management and control in the region.

Full Genomic Characterization of a Lentogenic Newcastle Disease Virus Isolated from Farm-Reared Ostriches (Struthio camelus) in Northwest China.

ABSTRACTTo our knowledge, our study is the first to report the whole-genome sequence of an ostrich-origin Newcastle disease virus (NDV) isolate, abbreviated as Ostrich/SX-01/06. Phylogenetic analysis revealed that this isolate belongs to the subgenotype c in class II. The identification of the complete genome will provide useful information regarding ostrich diseases, especially NDV.

Molecular screening and isolation of Newcastle disease virus from live poultry markets and chickens from commercial poultry farms in Zaria, Kaduna state, Nigeria

Newcastle disease is one of the major economic threats to poultry population because of its high morbidity and mortality varying from 90-100%. It is caused by Avian Paramyxovirus-1 (APMV-1). This research work was carried out to identify Newcastle disease virus (NDV) by using reverse transcription-polymerase chain reaction (RT-PCR) assay and further isolate the virus in embryonated chicken eggs. A total of 127 cloacal swabs were collected from local chickens in live bird market and exotic chickens in commercial poultry farms in Zaria and environs, Nigeria between November, 2014 and January, 2015. Five commercial poultry farms and four live bird markets were purposively sampled. Molecular screening of NDV Matrix-gene (M-gene) was performed on all the samples using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Newcastle disease positive samples were further inoculated into embryonated chicken eggs for isolation of Newcastle disease virus. Isolates were confirmed as Newcastle disease virus by haemaggulitination inhibition (HI) test. Newcastle disease virus Matrix-gene was detected in 16 (12.5%) out of 127 cloacal swabs; 13 (10.2%) from live bird markets and 3 (2.3%) from commercial poultry farms. However, only 10 Newcastle disease viruses were isolated in embryonated chicken eggs as confirmed by Haemagglutination inhibition (HI) test. Due to the higher detection rate recorded by reverse transcriptase-polymerase chain reaction (RT-PCR), it is therefore important that molecular technique be made easily accessible so that samples from each suspected outbreaks of NDV be screened so that rapid and confirmatory diagnosis can be achieved.Keywords: Embryonated chicken eggs, Haemagglutination inhibition test, Newcastle disease virus, RT-PCR

Molecular Detection, Biological Characterization and Evaluation of Protective Potentiality of a Velogenic Strain of Newcastle Disease Virus Isolate of Bangladesh

Molecular Detection, Biological Characterization and Evaluation of Protective Potentiality of a Velogenic Strain of Newcastle Disease Virus Isolate of Bangladesh

Molecular Survey of Respiratory and Immunosuppressive Pathogens Associated with Low Pathogenic Avian Influenza H9N2 Subtype and Virulent Newcastle Disease Viruses in Commercial Chicken Flocks.

The study was carried out in 48 poultry flocks to elucidate the roles of various complicating pathogens involved along with Newcastle disease (ND)/ low pathogenic avian influenza (LPAI) outbreaks. Necropsy was conducted and samples were collected for the isolation of Newcastle disease virus (NDV), Influenza A virus, infectious bronchitis virus (IBV), pathogenic bacteria; molecular detection of infectious laryngotracheitis virus (ILTV), fowl adeno virus (FAV), chicken anaemia virus (CAV), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). The isolation results confirmed that 18/48 flocks (37%) were positive for the presence of hemagglutinating agents. Out of 18 hemagglutination (HA) positive flocks, 11 flocks (61%) were positive for both avian influenza virus (AIV) and NDV; 4 flocks (22%) were positive for NDV; and 3 flocks (17%) were positive for AIV. Sequence analysis of hemagglutinin and neuraminidase genes of AIV revealed that all were belonging to LPAI-H9N2 subtype. Sequence analysis of F gene of NDV revealed that they belong to virulent type. The PCR results confirmed the presence of three to seven etiological agents (CAV, FAV, ILTV, MG, MS and avian pathogenic E. coli along with LPAI/NDV from all the 18 HA-positive flocks. The detection rate of triple, quadruple, quintuple, sextuple and sevenfold infections was 17% (3 flocks), 28% (5 flocks), 11%, (2 flocks) 28% (5 flocks) and 17% (3 flocks), respectively. In conclusion, the disease complex involved more than one pathogen, primarily resulting from the interplay between LPAI-H9N2 and NDV; subsequently this could be exacerbated by co-infection with other agents which may cause exacerbated outbreaks that may otherwise go undetected in field.

Identification of novel Newcastle disease virus sub-genotype VII-(j) based on the fusion protein

Newcastle disease virus (NDV) is believed to be the cause of fatal poultry disease worldwide. The fusion (F) protein plays a key role in virus pathogenesis, and it is also used for Newcastle disease virus classification. In this study, we determined the complete coding sequence of the F gene in new velogenic NDV isolates with an intravenous pathogenicity index (IVPI) of 1.8 and a mean death time (MDT) of 72 or 48h. Complete sequences of the F genes of new Iranian isolates were amplified and sequenced in both directions. These isolates were compared to 195 nucleotide sequences from GenBank (available as of 07/17/2016). A phylogenetic tree was constructed for the F gene, using MEGA6 software with statistical analysis based on 500 bootstrap replicates. Evolutionary distances revealed that the new virulent isolates from Iran belonged to genotype VII in a new distinct sub-genotype named VII-(j). This new sub-genotype showed 3% divergence from genotype VIId. Recombination analysis showed that these new isolates were not recombinant NDVs.

Complete Genome Sequences of Two Newcastle Disease Virus Strains Isolated from a Wild Duck and a Pigeon in Russia.

Here, we report the complete genome sequences of two Newcastle disease virus (NDV) isolates, Adygea/duck/12/2008, from a wild duck in Russia, and Altai/pigeon/777/2010, from a pigeon in Russia. Based on comparative sequence analysis of the F gene, these strains were classified as NDV class II, genotypes VIId and VIb/2, respectively.