Genotype VII Newcastle Disease Virus Research Articles

Recombinant infectious laryngotracheitis virus expressing Newcastle disease virus F protein protects chickens against infectious laryngotracheitis virus and Newcastle disease virus challenge

In this study, we isolated and identified an infectious laryngotracheitis virus (ILTV) that was naturally avirulent in specific pathogen-free (SPF) chickens, with the aim of developing a more efficacious vaccine against ILTV and Newcastle disease virus (NDV). We constructed a US9-deleted ILTV mutant based on this avirulent ILTV, and then constructed a recombinant ILTV (designated ILTV-ΔUS9-F) expressing the fusion protein (F) of the genotype VII NDV based on the US9-deleted ILTV mutant. Expression of the F protein in ILTV-ΔUS9-F-infected cells was confirmed by indirect immunofluorescence assay and western blotting. The inserted F gene was stably expressed in ILTV-ΔUS9-F. The growth kinetics of ILTV-ΔUS9-F were comparable to those of the wild-type ILTV strain. Vaccination of SPF chickens with ILTV-ΔUS9-F produced no clinical signs but did induce low levels of NDV-specific enzyme-linked immunosorbent assay and neutralizing antibodies. A single vaccination with 104 plaque-forming units (PFU) of ILTV-ΔUS9-F provided good protection against both genotype VII and IX NDVs based on clinical signs, similar to the protection provided by the commercial live La Sota vaccine. Notably, ILTV-ΔUS9-F limited the replication and shedding of genotype VII NDV from oropharyngeal swabs more efficiently than the La Sota vaccine. In addition, vaccination with lower doses (103 and 102 PFU) of ILTV-ΔUS9-F also provided sufficient clinical protection. These results indicated that ILTV-ΔUS9-F may be a bivalent vaccine candidate against both ILTV and NDV.

Production, characterization, and epitope mapping of a monoclonal antibody against genotype VII Newcastle disease virus V protein

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152RGPAELWK159, which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays.

Effect of fusion protein cleavage site sequence on generation of a genotype VII Newcastle disease virus vaccine.

Newcastle disease (ND) causes severe economic loss to poultry industry worldwide. Frequent outbreaks of ND in commercial chickens vaccinated with live vaccines suggest a need to develop improved vaccines that are genetically matched against circulating Newcastle disease virus (NDV) strains. In this study, the fusion protein cleavage site (FPCS) sequence of NDV strain Banjarmasin/010 (Banj), a genotype VII NDV, was individually modified using primer mutagenesis to those of avian paramyxovirus (APMV) serotypes 2, 7 and 8 and compared with the recombinant Banjarmasin (rBanj) with avirulent NDV LaSota cleavage site (rBanj-LaSota). These FPCS mutations changed the in vitro cell-to-cell fusion activity and made rBanj FPCS mutant viruses highly attenuated in chickens. When chickens immunized with the rBanj FPCS mutant viruses and challenged with the virulent Banj, there was reduced challenge virus shedding observed compared to chickens immunized with the heterologous vaccine strain LaSota. Among the genotype VII NDV Banj vaccine candidates, rBanj-LaSota and rBanj containing FPCS of APMV-8 induced highest neutralizing antibody titers and protected chickens with reduced challenge virus shedding. These results show the effect of the F protein cleavage site sequence in generating genotype VII matched NDV vaccines.

A novel genotype VII Newcastle disease virus vaccine candidate generated by mutation in the L and F genes confers improved protection in chickens

Administration of vaccines combined with the good management and strict biosecurity is an effective way for Newcastle disease (ND) control. However, vaccine failure is continuously reported in some countries mainly because the antigenic difference between the used vaccine and field strains even they are of one serotype. Therefore, development of antigen-matched ND vaccines is needed to improve the vaccine efficacy in birds. In this study, we introduced four site mutations, K1756A, D1881A, K1917A and E1954Q, respectively, into the large protein gene of the virulent genotype VII Newcastle disease virus (NDV) G7 strain using reverse genetics technology. Four rescued NDVs were sharply attenuated for the pathogenicity in chickens. One of these mutants, E1954Q, was further manipulated by replacing the F cleavage site sequence of typical velogenic strains with that of the LaSota vaccine, resulting in a new mutant, G7M. Biological characterization showed that G7M was safe and genetically stable after serial passages in embryos and chickens. Vaccination of chickens with G7M induced a progressive elevation of the homologous antibodies and markedly higher CD8+ T cell percentage, T cell proliferation and IFN-γ than LaSota. G7M conferred full protection against genotype VII NDV challenge, and more importantly, it effectively reduced the challenge virus replication and shedding in chickens. Together, our data suggest that G7M is a promising genotype VII vaccine candidate, and the novel attenuation approach designed in this study could be used to develop new antigen-matched NDV vaccines.

Evaluation of Moisture percentage and pH Scale in Relation to Survivability of Vaccine Escape Mutant Newcastle Disease Virus Genotype VII in Poultry Manure

Evaluation of Moisture percentage and pH Scale in Relation to Survivability of Vaccine Escape Mutant Newcastle Disease Virus Genotype VII in Poultry Manure

The positive expression of genotype VII Newcastle disease virus (Malaysian isolate) in Japanese quails (Coturnix coturnix japonica).

Aim:Genotype VII Newcastle disease virus (NDV) is the most predominant NDV strains that circulating in Malaysia; thus, this study was aimed to determine the susceptibility of Japanese quails toward genotype VII NDV. Clinical signs, gross pathological lesions of organs, positive detection of virus in organs and cloacal swabs, as well as the expression of the antibody titer, were used as parameters to assess the susceptibility of Japanese quails following infection of genotype VII NDV.Materials and Methods:About 20 quails were divided into three groups (n=8 for Groups A and B; n=4 for the control group). The quails in the Groups A and B were infected via intraocular route with 0.03 ml of 103.5 ELD50 and 107.0 ELD50 of NDV strain IBS 002, respectively, while the control group received 1× phosphate-buffered saline. Cloacal swabs and necropsy were taken on day 7 post-infection for all quails were subjected to one-step reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) for detection of virus and examination for gross pathological lesion, respectively. Blood serums of infected quails were taken on day 10, 14, and 21 post-day infections and were subjected for hemagglutination inhibition (HI) assay.Results:Depression and ruffled feathers, trachea rales, leg paralysis, and torticollis were shown in some of the quails in both infected groups. Based on statistical analysis, there was no significant difference (p>0.05) in clinical signs between the infected groups. The results for RT-qPCR were found to be negative for all groups, and no gross pathological lesions of organs observed for quails in both infected groups. Trachea, proventriculus, and cecal tonsil were taken for the detection of NDV by RT-qPCR, and some of the organ samples showed positive detection of virus in both infected groups. HI assay showed an increase in mean titers of antibody across time and between infected groups.Conclusion:In summary, Japanese quails are susceptible to genotype VII NDV based on parameters assessed.

Genetic diversity of the genotype VII Newcastle disease virus: identification of a novel VIIj sub-genotype.

Newcastle disease (ND) is a highly contagious disease of poultry caused by Newcastle disease virus (NDV). Multiple genotypes of NDV have been circulating worldwide and NDV is continuously evolving, resulting into more diversity. Of multiple viral genotypes, VII is particularly important given that it had been associated with most recent ND outbreaks worldwide. In this study, an epidemiological investigation performed in northeastern China during 2014-2015 showed that 11 genotype VII isolates amounted to 55 percent in a total number of NDV isolates. Therefore, to evaluate the genetic diversity worldwide and epidemiological distribution in China of genotype VII NDV, a phylogenetic analysis based on the 1255 complete F gene sequences showed that VII is the most predominant genotype worldwide. A further detailed characterization on genotype VII was conducted based on the 477 complete F gene sequences from 11 isolates and 466 reference viruses available in GenBank. The results demonstrated that VII can be further divided into 8 sub-genotypes (VIIb, VIId-VIIj), indicating its complex genetic diversity. It is worthy of note that the isolation rate of VIIj is increasing recently. It emphasizes the necessity to pay close attention to the epidemiological dynamic of genotype VII NDV and highlights the importance of vaccination program.

Rescue of duck-origin virulent Newcastle disease virus from cloned cDNA and stable expression of the red fluorescent protein

Ducks are generally considered as potential reservoirs for different genotypes of Newcastle disease virus (NDV) and to be resistant even to velogenic NDV strains. However, outbreaks of highly virulent genotype VII NDV lethal to ducks have been frequently reported in China in recent years. But until now, the pathogenesis and potential vaccine of duck-origin genotype VII NDV are not known. In this study, a reverse genetics system using the prevalent high virulence genotype VIId isolate SS1 was constructed. Based on this system, a red fluorescent protein (RFP)-expressing virus was generated by inserting an additional transcription cassette coding for the RFP between the noncoding region of P and M genes. The rescue of the recombinant viruses was confirmed by western blotting, fluorescence microscopy and genetic marker detection. In addition, the replication kinetics, biological characteristics and pathogenicity of the rescued viruses were indistinguishable from the parental wild-type virus. Moreover, the recombinant virus rSS1-RFP could efficiently replicate in most of the duck tissues, especially in duck immune organs. The results obtained suggest that this reverse genetics system will provide a useful tool for the analysis of duck-origin NDV pathogenesis and dissemination, as well as preparation for novel vaccine vector or genotype-matched NDV attenuated vaccines used in ducks.

EFFECT ON IMMUNE RESPONSE AND VIRUS SHEDDING IN THE CHICKEN VACCINATED AGAINST INACTIVATED LOCAL STRAIN OF NEWCASTLE DISEASE VIRUS GENOTYPE VII

The effectiveness of the new inactivated vaccine develop from local velogenic strain of Newcastle disease virus (NDV) Genotype VII and commercial vaccines LaSota were compared by determining the immune response and virus shedding of vaccinated chickens. Ten different groups of chicken consists of ten each, were vaccinated with and without adjuvant of the inactivated vaccine via intramuscular and subcutaneous, respectively. Three different adjuvants were used which include Oil-emulsion Complete Freund’s, Oil-emulsion Squalene-based, and aluminium hydroxide gel (Alum). As a comparison, a group of chicken was vaccinated with commercial vaccine and control group was not vaccinated. With 100% survival rate and highest mean haemaglutination-inhibition (HI) titre of log2 6, the inactivated vaccine with adjuvant Alum and Oil-emulsion Complete Freund’s surpassed the LaSota. In addition, the virus shedding was significantly reduced and comparable to LaSota vaccinated chicken. Whereas, without adjuvant, the chicken HI antibody titre is below log2 4 after vaccination and only 20-30% were survived. Based on the post-mortem findings on the survived chicken from each vaccinated group, their internal organs appeared normal and no sign of haemorrhage or pathognomonic signs of Newcastle disease (ND). Conclusively, vaccinated chicken are effectively protected from morbidity and mortality against virulent genotype VII challenge with the addition of adjuvant into inactivated local strain of NDV genotype VII vaccine. Thus, the development of inactivated local NDV genotype VII vaccine is a promising candidate to control the current ND endemic in Malaysia.

Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings

Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds.

Generation by reverse genetics of an effective attenuated Newcastle disease virus vaccine based on a prevalent highly virulent Chinese strain.

To investigate whether the differences between the circulating Newcastle disease virus (NDV) isolates and the used vaccine might account for the current ND outbreaks in vaccinated poultry flocks. A reverse genetics system using prevalent genotype VIId isolate SG10 was constructed and a mutant virus, named aSG10, was developed by changing the virulent F protein cleavage site motif "(112)RRQKR↓F(117)" into an avirulent motif "(112)GRQGR↓L(117)". The attenuated pathogenicity of aSG10 was confirmed from the mean death time and intracerebral pathogenicity index. aSG10 and LaSota both protected vaccinated birds from death after challenge with highly virulent genotype VII NDV, strain SG10. However, aSG10 significantly reduced the challenge virus shedding from the vaccinated birds compared to LaSota vaccine. We also generated a recombinant virus, aSG10-enhanced green fluorescent protein (EGFP), which expresses EGFP. aSG10-EGFP stably expressed EGFP for at least 10 passages. The mutant, aSG10, can be safely used as a vaccine vector and is a potential vaccine candidate in increasing the protective efficacy for the control of current ND epidemic in China.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009–2010), Malaysia (2011), China (2011), and Cambodia (2011–2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009–2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.

Development of a reverse genetics system based on RNA polymerase II for Newcastle disease virus genotype VII.

Newcastle disease virus (NDV) has only a single serotype but diversified genotypes. Genotype VII strains are the prevalent currently circulating genotype worldwide, and in particular, these strains cause outbreaks in waterfowl. In this study, a reverse genetics system for highly virulent NDV isolated from goose flocks was developed independent of conventional T7 RNA polymerase. Infectious virus was successfully generated by an RNA polymerase II promoter to drive transcription of the full-length virus antigenome. A green fluorescent protein (GFP)-expressing virus was generated by inserting an additional transcription cassette coding for the enhanced GFP between the P and M genes of the genome. The expression of GFP was confirmed by western blotting and fluorescence microscopy. The replication kinetics and pathogenicity of the recombinant viruses are indistinguishable from the parental wild-type virus. This reverse genetics system will provide a powerful tool for the analysis of goose-origin NDV dissemination and pathogenesis, as well as preparation for genotype-matched NDV attenuated vaccines.

Complete Genome Sequence of a Newcastle Disease Virus from a Coturnix coturnix japonica (Japanese Quail) Covey in India.

The genome of a Newcastle disease virus isolated from a Japanese quail in 2003 is reported here. The genome is 15,192 nucleotides (nt) long, as found in the recent genotypes, and grouped as genotype VIIb, with a 6-nt insertion. This is the first report on the sequence of a genotype VII Newcastle disease virus (NDV) from India.

Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: efficacy of field vaccination.

Here we report the biological and molecular characterization of a virulent genotype VII Newcastle disease virus (NDV) circulating in Venezuela and the assessment of the vaccination efficacy under field conditions compared to controlled rearing conditions. Biological pathotyping showed a mean embryo dead time of 50 h and an intracerebral pathogenicity index of 1.86. Sequence-based phylogenetic analysis demonstrated that the virus belongs to genotype VII in class II (a genotype often found in Asia and Africa), representing the first report of the presence of this genotype in the continent of South America. A vaccine-challenge trial in commercial broilers reared in fields or in a experimental setting included dual (live/killed) priming of 1-day-old chicks plus two live NDV and infectious bursal disease virus (IBDV) field vaccinations at days 7 and 17, followed by a very stringent genotype VII NDV challenge at day 28. Serology for NDV and IBDV, bursal integrity, and protection against NDV lethal challenge were assessed. At 28 days, field vaccinates showed significantly lower NDV (1,356 versus 2,384) and higher IBD (7,295 versus 1,489) enzyme-linked immunosorbent assay (ELISA) antibody titers than the experimentally reared birds. A lower bursal size and bursa-body weight ratio (P < 0.05) and higher bursa lesion score were also detected in the field set. Only 57.1% of field vaccinates survived the lethal challenge, differing (P < 0.05) from 90.5% survival in the experimental farm. Overall, results confirmed the presence of the genotype VII viruses in South America and suggest that field-associated factors such as immunosuppression compromise the efficacy of the vaccination protocols implemented.

Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs

To generate a genotype VII Newcastle disease virus (NDV) vaccine with high yield in embryonated chicken eggs, we selected genotype VII NDV strain JS5/05, which possesses a high virus titer in embryos as the parental virus. Using reverse genetics, we generated a genetically tagged derivative (NDV/AI4) of JS5/05 by changing the amino acid sequence of the cleavage site of the F0 protein. Pathogenicity tests showed that NDV/AI4 was completely avirulent. NDV/AI4 was genetically stable and replicated efficiently during 10 consecutive passages in embryos. More importantly, serologic assays showed that oil-emulsion NDV/AI4 induced higher hemagglutination inhibition (HI) titers against the prevalent virus than oil-emulsion LaSota vaccine in chickens and geese. Moreover, NDV/AI4-induced HI titers rose faster than those elicited by LaSota in chickens. Both NDV/AI4 and LaSota provided protection against clinical disease and mortality after the challenge with the genotype VII NDV strain JS3/05. However, NDV/AI4 significantly reduced virus shedding from the vaccinated birds compared to LaSota. Taken together, these results suggest that NDV/AI4 can provide better protection than LaSota and is a promising vaccine candidate against genotype VII NDV.

Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs

Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs

A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics

Genotype VII Newcastle disease virus (NDV) has been documented as the predominant epidemic genotype in China and some other Asian countries since 1990s. Recent work has demonstrated that NDV vaccines phylogenetically closer to epidemic viruses provide better protection than conventional vaccines in terms of reducing virus shedding and transmission. Since there is currently no available vaccine which possesses a close antigenic relationship to the prevalent virulent NDV, a new vaccine to protect against the infection of this genotype NDV is in urgent need. Here, we describe the generation of a pathogenicity-attenuated genotype VII NDV (NDV/ZJ1HN) from a velogenic NDV by mutating the velogenic amino acid motif at the F protein cleavage site using reverse genetics techniques. The attenuated-pathogenicity of NDV/ZJ1HN was confirmed by examination of mean death time (MDT) in embryonated eggs and intracerebral pathogenicity index (ICPI) in day-old chickens. Subsequently, 2 weeks old birds were immunized with live and inactivated NDV/ZJ1HN-based vaccines and challenged 3 or 4 weeks post-immunization with a lethal dose of a virulent genotype VII NDV strain. Results showed that NDV/ZJ1HN effectively protected the vaccinated birds from morbidity and mortality against genotype VII virus challenge and significantly reduced virus shedding from the vaccinated birds when compared with La Sota vaccinated animals, suggesting that NDV/ZJ1HN is a promising vaccine candidate for the control of current ND epidemic in China.

F gene recombination between genotype II and VII Newcastle disease virus

A velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense RNA viruses.