Newcastle Disease Virus Isolates Research Articles

Complete genome sequence of a newcastle disease virus isolate from an outbreak in northern India.

The complete genome sequence was determined for a Newcastle disease virus strain from vaccinated chicken farms in India during outbreaks in 2010. The genome is 15,192 nucleotides (nt) in length and is classified as genotype VII in class II. Compared to the available vaccine strains, the Indian strain contains a previously described 6-nt insertion in the untranslated region of the nucleoprotein gene.

Expression of full length fusion (F) protein gene of Newcastle disease virus in mammalian expression system

The present study was carried out to clone and express fusion protein gene of Newcastle disease virus in mammalian expression system. The Newcastle disease virus (2K3) isolate from pigeon was propagated in 9–11 day-old embryonated chicken eggs and infected allantoic fluid was collected for RNA isolation. The fusion (F) gene of 1.68 Kb was amplified with cDNA as template and was cloned in TOPO cloning vector. The recombinant TOPO colonies were digested with NcoI and XhoI enzymes, the insert released was further ligated into pTriEx Neo 1.1 expression vector digested with the same enzymes and was transformed in Escherichia coli. The recombinant pTriEx colonies confirmed by colony PCR and restriction digestion were induced with 1mM IPTG which showed expressed fusion protein of 55 kDa at 4 h post induction and increased in overnight induced cultures. The recombinant pTriEx plasmid was transfected into vero cells. The cell lysate collected at 48 h and 72 h post transfection showed expressed fusion protein with the molecular weight of 55 kDa in 12% SDS-PAGE. The protein was further confirmed to be NDV specific by its immunoreactivity with NDV antiserum raised in rabbits showing fusion protein of 55 kDa. The immunofluorescence assay of transfected vero cells exhibited a bright cytoplasmic fluorescence confirming the fusion protein expression.

Genetic diversity of velogenic Newcastle disease virus isolates from Ukraine

Genetic diversity of velogenic Newcastle disease virus isolates from Ukraine

English

Newcastle disease (ND) is a very contagious disease in chickens and turkeys and one of the most important diseases of poultry in the world. The infection causes sudden death with high mortality. In Indonesia, Newcastle disease in recent years showed symptoms slightly different from previous symptoms of this disease. NDV infection of genotipe VII has been reported to cause this outbreak in several commercial poultry farms in Indonesia. This study aimed to isolate and identify the recent ND virus and determine group of genotype of ND in Indonesia for the development of seed of ND vaccines expected to be more effective in the control of Newcastle disease in the field. The method used in this study includes the collection of samples from the field, ND virus isolation, RT-PCR and DNA sequencing of the F and HN genes of ND viruses. Our result showed that six isolates belong to genotype VII of ND viruses, one isolate belong to genotype VI and the other isolate belong to genotype I. The prediction of pathotypes of amino acids sequence on F gene of NDV indicated that seven isolates have motif R-R-R-K-R and R-R-Q-K-R which is a marker for pathotype for velogenic of ND viruses. The cleavage site of amino acid sequences from one isolate (RIVS isolate) has G-K-Q-G-R-L which is lentogenic pathotype of NDV. This study indicated that genotype VII viruses were predominant virus circulating in the field and we suggested the update of master seed vaccine of ND in Indonesia.   Key words: New castle disease (ND), genotype VII, phylogenetic analysis.

Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain

Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12h at a titer corresponding to the IC50 value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/H-induced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors.

Separate Evolution of Virulent Newcastle Disease Viruses from Mexico and Central America

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.

English

Unvaccinated laboratory-raised, five week old cockerels were fed V4 newcastle disease vaccine in cassava once, twice or thrice; and tested for hemagglutination inhibition (HI) antibody response. They were subsequently challenged with a local isolate of velogenic newcastle disease virus (NDV). Immune status of the chicken flocks improved with number of vaccinations. Survival from velogenic virus challenge varied significantly with pre-challenge HI antibody titer, protection being better at log2 titer of ≥3 than at log2 titers of ≤ 2 but no difference was observed between titers of 3, 4 and 5. Although HI log2 antibody titers of 3 and 4 were apparently observed to protect vaccinated chickens against ND mortality in this work, 7 out of 8 (87.5%) of the chickens that manifested torticollis (neurological disease symptom) had pre-challenge HI antibody titers within this range. Thus, it is suggested that while log2 HI antibody titer of 3 may be taken as cut-off point for sero-conversion, titers of 5 and above may protect against neurological symptoms. Key words: V4, cassava-based vaccination, velogenic challenge, HI antibody titers, chicken protection.

Comparison of Use of Agglutination of Mammalian Erythrocytes Plus Heat Stability Test and Use of Agglutination of Mammalian Erythrocytes Plus Erythrocyte Elution Time to Characterize Isolates of Newcastle Disease Virus

Aim: For a simpler, more rapid and more accurate method of characterizing new isolates of Newcastle disease virus (NDV), agglutination of mammalian erythrocytes (HA) plus heat stability test and agglutination of mammalian erythrocytes plus erythrocytes elution time (EET) were compared with use of Intra-cerebral pathogenicity index (ICPI) to characterize the isolates. Materials and Methods : NDV isolates characterized by their ICPI were re-characterized by HA of mammalian erythrocytes plus heat stability test and by HA of mammalian erythrocytes plus EET. Rate of agreement of each of the two combinations with ICPI was calculated. Results: HA of mammalian erythrocytes plus heat stability agreed with ICPI in

Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

Pathotypic characterization of the Newcastle disease virus isolated from commercial poultry in northwest Iran

Despite vaccination, outbreaks of Newcastle disease in commercial poultry flocks in northwest Iran have been regularly reported in recent years. This research was proposed in order to characterize, through a molecular approach, the genetic nature of strains of Newcastle disease virus isolated from commercial poultry flocks in northwest Iran. Ten Newcastle disease virus isolates were obtained from various districts of northwest Iran in 2010. The isolates were screened with F gene-targeting reverse transcription polymerase chain reaction (RT-PCR). The amplified products of virulent strains were sequenced. The deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif ^{112}R-R-Q-R-R.F^{117}, typical for velogenic strains of NDV. These results confirmed that virulent NDVs are circulating in the region, causing economic losses within the poultry industry.

Newcastle Disease Virus Isolation and Its Prevalence in Uganda Poultry Farms

The present research work was carried out to isolate and identify Newcastle disease virus (NDV) by using haemagglutination inhibition (HI) test and HA-HI virus isolation, embryonated eggs (EE) and chicken embryo fibroblasts (CEF). A total of 95 clinical (blood, tracheal and cloacal swabs) and post-mortem (brain, lung, colon and spleen) samples were collected from chickens of field outbreaks of suspected Newcastle disease virus (NDV). The HI and HA-HI were employed to detect NDV in tissue homogenates of all the clinical and post-mortem samples as well as laboratory samples (AF and ICF). Among the four different types of post-mortem samples, virus isolation rate was found to be low in body organs. In CEF cell culture system, the rate of virus isolation from all the aforesaid samples was found to be at 100% with the exception of serum samples; while in tracheal and cloacal swabs, it was at 90%; while in serum, it was at 10%, in all clinical cases. The isolation rate of NDV was higher in CEF culture system (66.7%) compared to that of avian embryos (33.3%). Samples were inoculated and the allantoic fluid (AF) of the dead embryos and the infected culture fluid (ICF) of the CEF were harvested at 24 to 96 hours of the post-infection, respectively, which revealed that the virulent strain of NDV is highly prevalent in the region. The prevalence of NDV was established at 1.1%, 2.1% and 4.2% using HA-HI, EE, and CEF methods. Rapid detection and identification of the virus are crucial for the effective control of the disease as conventional diagnostic methods such as virus isolation on embryonated eggs followed by serological identification in haemagglutination-inhibition test are laborious and time-consuming. The speed of the diagnosis can be considerably increased by using methods based on molecular biology, e.g. reverse transcription—polymerase chain reaction. However, the genetic variability of APMV-1 isolates should be considered carefully as the potential cause for false negative results of genetic-based laboratory tests.

Molecular characterization of chicken-derived genotype VIId Newcastle disease virus isolates in China during 2005–2012 reveals a new length in hemagglutinin–neuraminidase

Newcastle disease (ND) is one of the most important diseases of poultry, and causes severe economic losses in the global poultry industry. Although all Newcastle disease virus (NDV) isolates belong to a single serotype, significant genetic diversity has been described between different NDV isolates. Here, we report the molecular characterization of 23 virulent genotype VIId NDV isolates of class II circulating in China. Phylogenetic construction and analysis revealed the existence of distinctly genomic and amino acid differences that clearly distinguished these isolates from other typical NDV genotypes and vaccine strains. We also report a new 582-amino-acid hemagglutinin–neuraminidase in genotype VII NDV strains. This is believed to be the first study to investigate systematically the most predominant NDV strains, and provides more information on the genetic nature of genotype VIId NDV of class II circulating in China.

Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran

The pathogenicity and genetic characterizations of six Newcastle disease virus (NDV) isolates obtained from chicken farms in six different regions in Iran were carried out using conventional and molecular techniques. Based on the pathogenicity indices (MDT, ICPI, and IVPI), all of these isolates were found to be velogenic (highly virulent) strains. A sequence analysis of the full-length mRNA encoding the fusion glycoprotein precursor (F0) of the NDV's fusion proteins F1 and F2 in these six isolates showed the presence of point mutations in form of nucleic acid substitutions at positions 82((C→T)), 83((T→C)), 736((A→G)), and 1,633((G→A)). However, the nucleic acid residues at positions 330-347 of the precursor F0 gene, corresponding to the cleavage site of the F0 protein, were found to have remained conserved among the six NDV isolates. A phylogenetic comparison between the six Iranian isolates and the NDVs whose F0 gene sequences were previously deposited in GenBank Database showed that all of the newly characterized Iranian NDV isolates belonged to genotype VII.

Molecular epidemiology of Newcastle disease virus isolates from vaccinated commercial poultry farms in non-epidemic areas of Japan

BackgroundNewcastle Disease (ND) is a highly contagious and economically devastating disease of poultry. At present, limited molecular epidemiological data are available regarding the causes of ND outbreaks in vaccinated commercial poultry farms. Knowing the genomic characteristics of Newcastle disease virus (NDV) infecting commercial poultry operations in spite of vaccination might give important insights on the infection dynamics of these viruses. In addition, molecular analyses at the subgenotype level and studies on the relationship of Japanese NDVs with other isolates from around the world are lacking. Therefore, in the present study, a molecular epidemiological investigation was conducted to characterize nine NDVs isolated from vaccinated commercial poultry flocks in five different Prefectures in non-epidemic areas of Japan between 1969 and 2002.MethodsNucleotide sequencing and phylogenetic studies were performed to characterize the complete fusion (F)-protein gene, 3-prime end of the nucleoprotein (NP)-gene and 5-prime end of the RNA dependent RNA polymerase (L)-gene. Sequence data were compared with 180 NDV strains from GenBank representing different NDV genotypes and subgenotypes from different regions of the world at different time periods. Deduced amino acids were analyzed for homologies, recombination and mutation. Recombination events were estimated using Recombination Detection Program (RDP) version 3.44. Phylogenetic trees were constructed to determine evolutionary relationships among strains.ResultsMean death time (MDT: 48-56 hr), Intracerebral Pathogenicity Index (ICPI: 1.7-1.9) and deduced amino acid sequences of the F0 proteolytic cleavage site (112RRQKR116) revealed that all nine field isolates were velogenic. Phylogenetic analysis showed that these isolates could be classified into two genetic lineages and three sublineages namely genotypes VIa (lineage 4a), VId (lineage 4d) and VIId (lineage 5d). No recombination events were observed but a point mutation in one of the neutralizing epitope of the F-protein was identified in the field isolates from Japan.ConclusionsAll field isolates from vaccinated commercial poultry in non-epidemic areas of Japan were part of much bigger outbreaks in provinces and regions and, in some cases, continents. In general, four ND panzootics occurred in Japan and that these outbreaks were mostly characterized by co-circulation of genetically distinct virus lineages due to involvements of infected wild birds. The point mutation identified in the field isolates from Japan may be due to escape from vaccine pressure. The identification of such mutation may be useful for future site-directed mutagenesis to understand the dynamics of NDV infection in vaccinated chickens.

Inactivation of the infectivity of two highly pathogenic avian influenza viruses and a virulent Newcastle disease virus by ultraviolet radiation

Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm2, which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.

Molecular characterization of Newcastle disease viruses isolated from rural chicken in northwest Ethiopia reveals the circulation of three distinct genotypes in the country

Newcastle disease (ND) is a highly contagious disease that affects many species of birds and causes significant economic losses to the poultry industry worldwide. Fifteen Newcastle disease virus (NDV) isolates obtained from rural chickens in northwest Ethiopia in 2011 and 2012 were characterized genotypically. The main functional region of the F gene was amplified and sequenced (260 nucleotides). Among the Ethiopian NDV isolates, 2 isolates had the virulent motif (112)R-R-Q-K-R-F(117) at the cleavage site of the fusion protein while 13 isolates contained the lentogenic motif (112)G-G/R-Q-G-R-L(117). Phylogenetic analysis based on the variable region of the F gene indicated that the two isolates exhibiting the virulent motif belonged to lineage 5 (genotype VII) subgenotype d and the remaining 13 isolates were grouped into lineage 2 (genotype II). The nucleotide sequences of lineage 5 isolates were genetically related to the Sudanese NDV isolates, suggesting potential epidemiological link of ND outbreaks between neighbouring countries. The lentogenic strains shared similarities with La Sota vaccine strain and probably originated from the vaccine strain either through direct exposure of birds to the live vaccine or to infectious La Sota-like strains circulating in rural poultry. This study provides genetic evidence on the existence of different NDV genotypes circulating in the rural poultry in Ethiopia. The virulent NDV continues to be a problem in poultry sector in Ethiopia, and their continuous circulation in rural and commercial poultry calls for improved surveillance and intensified vaccination and other control measures.

Characterization of genotype IX Newcastle disease virus strains isolated from wild birds in the northern Qinling Mountains, China

This study was conducted to evaluate the virulence and evolution of genotype IX Newcastle disease virus (NDV) isolates obtained from wild birds in the northern Qinling Mountains of China. Five isolates were obtained from 374 larynx and cloacae swabs, which were collected from multiple asymptomatic wild bird species from August 2008 to July 2011, and were subsequently characterized by pathotype and genotype. Deduced amino acid sequences revealed that all five NDV isolates exhibited velogenic fusion protein cleavage sites motif (112)R-R-Q-R-R-F(117), shared as high as 99.8-99.9 % homology with each other, and varied in pathotype by intracerebral pathogenicity indices (ICPI) of 0.425-1.638. Phylogenetic analysis showed that all five isolates were clustered to genotype IX NDV. This is the first study to confirm multiple asymptomatic wild bird species as natural carriers of virulent genotype IX NDV. A novel NDV isolate from the Spotted-necked Dove (family Columbidae) exhibited discordance between its lentogenic ICPI and its virulent proteolytic cleavage site motif (112)R-R-Q-R-R-F(117). Although the five isolates underwent several amino acid mutations in the fusion protein, evidence of continuous evolutionary divergence did exist in the genotype IX NDV, which was always regarded as a conservative genotype.

Molecular epidemiology of Newcastle disease viruses in Vietnam

Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.

IMMUNE RESPONSE CAPABILITY OF `SHUVRA’ CHICKEN

The study was carried out to determine the humoral immune response to Newcastle Disease Virus (NDV) and Bangladesh Agricultural University Fowl cholera (BAUFC) vaccines in Shuvra chicken, a newly develop chicken strain by BLRI (Bangladesh Livestock Research Institute). Ten Shuvra chickens were vaccinated with Baby Chick Ranikhet Disease Vaccine (BCRDV) at day 7 through intra ocular route (i/o) and with Ranikhet Disease Vaccine (RDV) at day 35 through intramuscular (i/m) route. Vaccine induced serum Haemagglunination Inhibition (HI) antibodies were measured by HI test. Two weeks after final immunization all vaccinated and control Shuvra chickens were challenged with virulent field isolates of NDV where all the vaccinated birds survived without showing any typical signs of NDV during the period of ten days observation period and all the control chickens died. Another 10 Shuvra were vaccinated twice with BAUFC vaccine through intramuscular route at day 42 and 70, and 10 Shuvra chickens were kept as unvaccinated control. This vaccine also induced significantly higher level of antibody titre as determined by Passive Haemagglutination (PHA) test. Vaccinated chickens showed significantly higher survival (80%) following challenge with virulent fowl cholera isolate and all the control birds died within 10 days of observation period. DOI: http://dx.doi.org/10.3329/bjvm.v10i1-2.15639Bangl. J. Vet. Med. (2012). 10 (1&2): 1-7

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs)

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5–10ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R2=0.990, which was comparable to the value of R2=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.