Chimeric Newcastle Disease Virus Research Articles

Spray vaccination with a safe and bivalent H9N2 recombinant chimeric NDV vector vaccine elicits complete protection against NDV and H9N2 AIV challenge

Newcastle disease virus (NDV) and H9N2 avian influenza virus (AIV) represent significant pathogenic risks to the poultry industry, leading to considerable economic losses. Vaccination is a widely used preventive measure against these pathogens, yet the lack of a live bivalent vaccine targeting NDV and H9N2 AIV imposes a heavy vaccination burden. Previously, we constructed a genotype-matched chimeric NDV vector, LX-OAI4S, in which the genotype I NDV backbone was replaced with the ectodomain of haemagglutinin-neuraminidase (HN) and modified using the attenuated F gene from the genotype VII vaccine strain A-VII. Based on the LX-OAI4S vector, we successfully generated three H9N2 recombinant viruses: LX-OAI4S-NPU-HA, LX-OAI4S-MU-HA, and LX-OAI4S-HNU-HA. These recombinants incorporated the H9N2 HA gene, flanked by untranslated regions (UTRs) from the NP, M, or HN gene of the NDV LX strain, inserted between the P and M genes of LX-OAI4S. The vaccine candidate LX-OAI4S-NPU-HA induced a more robust immune response in chickens against H9N2 AIV and NDV than the other two recombinants. This response effectively protects against virus shedding and lethal virus challenge. Furthermore, spray vaccination with LX-OAI4S-NPU-HA showed protective efficacy against H9N2 AIV and NDV. This study offers a promising strategy for comprehensive protection in regions threatened by H9N2 AIV and NDV.

Development and Evaluation of a Novel Chimeric Genotype VII Newcastle Disease Vaccine: Overcoming Maternal Antibody Interference and Spray Administration.

Newcastle disease virus (NDV) poses a significant threat to the poultry industry, with the emergence of genotype VII NDV leading to extensive outbreaks and economic losses. Vaccination is the primary means of controlling NDV, but the presence of maternal antibodies (MDAs) can interfere with the immunological effect of live virus vaccines. Thus, we constructed a chimeric NDV live virus vaccine, LX-OAI4S, by replacing the extracellular regions of the F and HN genes of the NDV LX strain with the corresponding regions of the A-VII vaccine strain. The chimeric vaccine LX-OAI4S demonstrated high genetic stability, good safety, and strong reproductive capacity in chicken embryos. The LX-OAI4S vaccine induced rapid antibody production in specific pathogen-free (SPF) and commercial chickens via the intranasal and intraocular (IN/IO) routes, with hemagglutination inhibition (HI) antibody titers reaching 4.71 ± 1.03 log2 at 7 days post-vaccination (dpv), significantly higher than those of the two classical vaccine strains La Sota and VG/GA. The LX-OAI4S vaccine group provided effective protection against the challenge of genotype VII NDV virulent strain JS2/06 and inhibited viral shedding. When administered via spray, the LX-OAI4S vaccine elicited high systemic immunity against NDV in both SPF and commercial chickens, effectively protecting against clinical disease and reducing viral shedding. The chickens were exposed to high-dose vaccine for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs in chickens, the NDV-specific antibody titers were significantly greater in the vaccinated groups than in the unvaccinated group. The vaccine exhibited high immunogenicity and the potential to overcome maternal antibody interference. The LX-OAI4S vaccine is a promising candidate for an ND vaccine. Its administration via spray can effectively prevent the occurrence of ND, making it a valuable tool for the poultry industry.

The haemagglutinin–neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death

The haemagglutinin–neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

Chimeric Newcastle Disease Virus Vectors Expressing Human IFN-γ Mediate Target Immune Responses and Enable Multifaceted Treatments.

The therapeutic potential of Newcastle disease virus (NDV) has been reported as both an oncolytic agent and a vaccine vector against many antigens. However, in the individuals already immunized with NDVs, second and subsequent administration does not provide substantial benefits. In this study, two types of recombinant chimeric NDVs using APMV-2 F and HN genes were generated. In rNDV-2HN, the wild-type NDV HN gene was replaced with the APMV-2 HN gene, and in rNDV-2F/2HN, both wild-type F and HN genes were replaced with APMV-2 F and HN genes, respectively. We enhanced the immune responses of these chimeric viruses by inserting the human IFN-γ gene. To examine the escape from NDV antiserum, each virus was treated with diluted NDV antiserum, and HEp-2 cells were infected with these virus particles. The two constructed chimeric viruses indicated notably lower virus-neutralizing titer compared to wild-type NDV and escaped the action of NDV antiserum. These two chimeric viruses infected both respiratory and colon cancer cell lines, indicating their potential as a cancer treatment tool. Chimeric viruses with enhanced immune responses can be considered a novel therapeutic strategy in cancer treatment that can be administered multiple times and used to enhance immune cells interaction.

Construction of Novel Thermostable Chimeric Vaccine Candidates for Genotype VII Newcastle Disease Virus.

Genotype VII Newcastle Disease Virus (NDV) has caused a pandemic in many countries and usually causes fatal consequences in infected chickens. Although current commercial attenuated NDV vaccines can provide an ideal protection against genotype VII NDV, they cannot completely prevent the infection and viral shedding, and the genotype of some vaccine strains cannot match with the prevalent strain. In this study, in order to construct a thermostable and genotype VII-matched live attenuated vaccine, we used a thermostable genotype VIII virulent HR09 strain as the backbone and replaced its F gene with that of the genotype VII DT-2014 strain. Meanwhile, the cleavage site of F gene of DT-2014 was mutated to that of class I F protein and avirulent class II F protein, respectively. The results showed that the two chimeric viruses, designated rcHR09-CI and rcHR09-CII, shared a similar growth kinetics and thermostability with their parental HR09 strain. Mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests showed that the two chimeric viruses were highly attenuated. Though both chimeric NDVs and La Sota vaccine strain could provide complete protection to immunized chickens against the challenge of virulent genotype VII ZJ1 strain, the two chimeric NDVs could induce a higher level of antibody response against ZJ1 strain and could significantly reduce the viral shedding compared with La Sota vaccine strain. In conclusion, our study constructed two chimeric thermostable genotype VII-matched NDV vaccine candidates, which provided complete protection against the challenge of virulent genotype VII NDV.

Fusion Protein Cleavage Site Containing Three Basic Amino Acids Attenuates Newcastle Disease Virus in Chicken Embryos: Use as an in ovo Vaccine.

In ovo vaccination is an attractive immunization strategy for the poultry industry. However, although most live Newcastle disease virus (NDV) vaccine strains, such as LaSota and V4, can be used after hatching, they are pathogenic to chicken embryos when administered in ovo. We have previously reported that NDV strain TS09-C is a safe in ovo vaccine in specific-pathogen-free and commercial chicken embryos because it is attenuated in chicken embryos. However, the molecular basis of its attenuation is poorly understood. In this study, we firstly evaluated the safety of chimeric NDV strains after exchanging genes between strains TS09-C and LaSota as in ovo vaccines, and demonstrated that the attenuation of NDV in chicken embryos was dependent upon the origin of the fusion (F) protein. Next, by comparing the F protein sequences of TS09-C strain with those of LaSota and V4 strain, the R115 in cleavage site and F379 were found to be unique to TS09-C strain. The mutant viruses were generated by substituting one or two amino acids at position 115 and 379 in the F protein, and their safety as in ovo vaccine was evaluated. Mutation in residue 379 did not affect the viral embryonic pathogenicity. While the mutant virus rTS-2B (R115G mutation based on the backbone of TS09-C strain) with two basic amino acids in F cleavage site, was pathogenic to chicken embryos and similar with rLaSota in its tissue tropism, differing markedly from rTS09-C with three basic amino acids in F cleavage site. Together, these findings indicate that the F protein cleavage site containing three basic amino acids is the crucial determinant of the attenuation of TS09-C in chicken embryos. This study extends our understanding of the pathogenicity of NDV in chicken embryos and should expedite the development of in ovo vaccines against NDV.

Single-Dose Vaccination of Recombinant Chimeric Newcastle Disease Virus (NDV) LaSota Vaccine Strain Expressing Infectious Bursal Disease Virus (IBDV) VP2 Gene Provides Full Protection against Genotype VII NDV and IBDV Challenge.

Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) are the two most important and widespread viruses causing huge economic losses in the global poultry industry. Outbreaks of genotype VII NDV and IBDV variants in vaccinated poultry flocks call for genetically matched vaccines. In the present study, a genetic matched chimeric NDV LaSota vaccine strain expressing VP2 gene of IBDV variant, rLaS-VIIF/HN-VP2 was generated for the first time, in which both the F and HN genes of LaSota were replaced with those of the genotype VII NDV strain FJSW. The cleavage site of the FJSW strain F protein in the rLaS-VIIF/HN-VP2 genome was mutated to the avirulent motif found in LaSota. Expression of IBDV VP2 protein was confirmed by western blot. The rLaS-VIIF/HN-VP2 maintained the efficient replication ability in embryonated eggs, low pathogenicity and genetic stability comparable to that of parental LaSota virus. One dose oculonasal vaccination of one-week-old SPF chickens with rLaS-VIIF/HN-VP2 induced full protection against genotype VII NDV and IBDV lethal challenge. These results indicate that the rLaS-VIIF/HN-VP2 is a promising bivalent vaccine to prevent infections of IBDV and genotype VII NDV.

Matrix metalloproteinase-14 regulates collagen degradation and migration of mononuclear cells during infection with genotype VII Newcastle disease virus.

Upregulation of matrix metalloproteinase (MMP)-14, a major driven force of extracellular-matrix (ECM) remodelling and cell migration, correlates with ECM breakdown and pathologic manifestation of genotype VII Newcastle disease virus (NDV) in chickens. However, the functional relevance between MMP-14 and pathogenesis of genotype VII NDV remains to be investigated. In this study, expression, biofunction and regulation of MMP-14 induced by genotype VII NDV were analysed in chicken peripheral blood mononuclear cells (PBMCs). The results showed that JS5/05 significantly increased expression and membrane accumulation of MMP-14 in PBMCs, correlating to enhanced collagen degradation and cell migration. Specific MMP-14 inhibition significantly impaired collagen degradation and migration of JS5/05-infected cells, suggesting dependence of these features on MMP-14. In addition, MMP-14 upregulation correlated with activation of the extracellular signal-regulated kinase (ERK) pathway upon JS5/05 infection, and blockage of the ERK signalling significantly suppressed MMP-14-mediated collagen degradation and migration of JS5/05-infected cells. Using a panel of chimeric NDVs derived from gene exchange between genotype VII and IV NDV, the fusion and haemagglutinin-neuraminidase genes were identified as the major viral determinants for MMP-14 expression and activity. In conclusion, MMP-14 was defined as a critical regulator of collagen degradation and cell migration of chicken PBMCs infected with genotype VII NDV, which may contribute to pathology of the virus. Our findings add novel information to the body of knowledge regarding virus-host biology and NDV pathogenesis.

Genotype-matched Newcastle disease virus vaccine confers improved protection against genotype XII challenge: The importance of cytoplasmic tails in viral replication and vaccine design.

Although typical Newcastle disease virus (NDV) vaccines can prevent mortality, they are not effective in preventing viral shedding. To overcome this, genotype-matched vaccines have been proposed. To date, this approach has never been tested against genotype XII strains. In this study, we generated and assessed the protection against genotype XII challenge of two chimeric NDV vaccine strains (rLS1-XII-1 and rLS1-XII-2). The rLS1-XII-1 virus has the complete fusion protein (F) and the hemagglutinin-neuraminidase (HN) open reading frames replaced with those from genotype XII strain NDV/peacock/Peru/2011 (PP2011) in a recombinant LaSota (rLS1) backbone. In rLS1-XII-2 virus, cytoplasmic tails of F and HN proteins were restored to those of rLS1. In vitro evaluation showed that rLS1-XII-2 and the parental rLS1 strains replicate at higher efficiencies than rLS1-XII-1. In the first vaccine/challenge experiment, SPF chickens vaccinated with rLS1-XII-1 virus showed only 71.3% protection, whereas, rLS1 and rLS1-XII-2 vaccinated chickens were fully protected. In a second experiment, both rLS1-XII-2 and the commercial vaccine strain LaSota induced 100% protection. However, rLS1-XII-2 virus significantly reduced viral shedding, both in the number of shedding birds and in quantity of shed virus. In conclusion, we have developed a vaccine candidate capable of fully protecting chickens against genotype XII challenges. Furthermore, we have shown the importance of cytoplasmic tails in virus replication and vaccine competence.

Newcastle disease virus vectors expressing consensus sequence of the H7 HA protein protect broiler chickens and turkeys against highly pathogenic H7N8 virus

Continuous outbreaks of highly pathogenic avian influenza (HPAI) viruses in commercial poultry have caused devastating losses to domestic poultry with a raising public health concern. The outbreaks of HPAI viruses have increased worldwide, including the North America. Therefore, vaccination has been considered as an alternative strategy for an efficient control of HPAI viruses. In this study, we aimed to generate Newcastle disease virus (NDV) vectored H7 serotype-specific vaccines by expressing the consensus sequence of the HA protein. Conventional NDV strain LaSota vector and a chimeric NDV vector containing the avian paramyxovirus type-2 F and HN protein were able to express the consensus sequence of HA protein. The protective efficacy of vaccines was evaluated in broiler chickens and in turkeys. One-day-old poults were prime immunized with the chimeric vector expressing the HA protein followed by boost immunization with LaSota vector expressing the HA protein or co-expressing the HA and NA proteins. Our vaccine candidates provided complete protection of broiler chickens from mortality and shedding of H7N8 HPAI challenge virus. Turkeys were better protected by boosting with the LaSota vector co-expressing the HA and NA proteins than the LaSota vector expressing only the HA protein. Our study demonstrated a potential use of heterologous prime and boost vaccination strategy to protect poultry against H7 HPAI viruses.

Innovation in Newcastle Disease Virus Vectored Avian Influenza Vaccines.

Highly pathogenic avian influenza (HPAI) and Newcastle disease are economically important avian diseases worldwide. Effective vaccination is critical to control these diseases in poultry. Live attenuated Newcastle disease virus (NDV) vectored vaccines have been developed for bivalent vaccination against HPAI viruses and NDV. These vaccines have been generated by inserting the hemagglutinin (HA) gene of avian influenza virus into NDV genomes. In laboratory settings, several experimental NDV-vectored vaccines have protected specific pathogen-free chickens from mortality, clinical signs, and virus shedding against H5 and H7 HPAI viruses and NDV challenges. NDV-vectored H5 vaccines have been licensed for poultry vaccination in China and Mexico. Recently, an antigenically chimeric NDV vector has been generated to overcome pre-existing immunity to NDV in poultry and to provide early protection of poultry in the field. Prime immunization of one-day-old poults with a chimeric NDV vector followed by boosting with a conventional NDV vector has shown to protect broiler chickens against H5 HPAI viruses and a highly virulent NDV. This novel vaccination approach can provide efficient control of HPAI viruses in the field and facilitate poultry vaccination.

A novel recombinant attenuated Newcastle disease virus expressing H9 subtype hemagglutinin protected chickens from challenge by genotype VII virulent Newcastle disease virus and H9N2 avian influenza virus

Newcastle disease virus (NDV) and H9 subtype avian influenza virus (AIV) are two avian pathogens across the globe. Inasmuch as most poultry flocks worldwide are vaccinated with a live low-virulence or attenuated NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificities and would allow a single immunization against both avian influenza (AI) and Newcastle disease (ND). Therefore, in the present work, a cloned full-length copy of the genome of the lentogenic NDV strain rmNA-1 was selected as a backbone vector to construct three chimeric NDVs that expressed (i) the ORF encoding the HA, (ii) the ectodomain of HA fused with the transmembrane domain and cytoplasmic tail regions derived from the NDV F protein and (iii) the ectodomain of HA fused with a short GS linker and the GCN4 sequences, and designated as rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO), respectively. rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO) stably expressed the modified HA gene for 10 egg passages and the three recombinants were found innocuous to chickens. The insertion of the chimeric HA-F, rather than HA-ECTO or ORF of HA, resulted in a recombinant virus with enhanced incorporation of the HA protein into the viral surface. A single immunization of SPF chickens with the three recombinants induced NDV- and AIV H9-specific antibodies, and protected chickens against a challenge with a lethal dose of velogenic NDV or AIV H9N2. Remarkably, non-shedding of influenza virus and higher levels of H9 subtype HI titers were observed 7 days post challenge (dpc) in rmNA-H9F vaccinated chickens, than other recombinants. Furthermore, a prime-boost vaccination of chickens with rmNA-H9F induced higher levels of NDV- and H9- HI and secretory IgA, as well as reduced viral shedding and virus-induced gross lesions, compared with the commercial vaccine. Therefore, the recombinant rmNA-H9F is a promising bivalent vaccine candidate against NDV and H9 subtype AIV in chickens.

Co-expression of the Hemagglutinin and Neuraminidase by Heterologous Newcastle Disease Virus Vectors Protected Chickens against H5 Clade 2.3.4.4 HPAI Viruses

Avian influenza remains an important zoonotic disease with a significant global impact. The spread of H5 highly pathogenic avian influenza (HPAI) viruses (clade 2.3.4.4) by migratory birds has caused outbreaks in wide geographic regions (Asia, Europe, and North America) with great economic losses during 2014–2015. Efficient vaccines and vaccination approaches are needed to enhance protective immunity against HPAI viruses. Although several vaccination strategies have been developed, none has been satisfactory. Our strategy has been to use avirulent vaccine strain of Newcastle disease virus (NDV) as a vaccine vector for HPAI viruses. For poultry vaccination, we previously generated a new platform of chimeric NDV vector to overcome preexisting maternal antibodies to NDV in poultry. In this study, we have generated vaccine candidates targeting H5 clade 2.3.4.4 HPAI viruses by using our chimeric NDV and conventional NDV strain LaSota vectors for a heterologous prime-boost immunization approach. Co-expression of the HA and NA proteins by our vaccine vectors induced enhanced HPAI virus specific immune responses in specific-pathogen free and broiler chickens prior to challenge. Further, these vaccine candidates efficiently protected broiler chickens from mortality, clinical signs, and shedding of homologous and heterologous H5 HPAI viruses and highly virulent NDV, thus providing a dual vaccination approach in the field.

Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity.

A chimeric Newcastle disease virus (NDV) vector (NDV/AI4-TFHN) was constructed with the replacement of the ectodomains of the fusion and hemagglutinin-neuraminidase proteins by those from avian paramyxovirus type 2. The chimeric virus induced high antibody response in chickens pre-immunized with NDV. A recombinant vaccine candidate, NDV/AI4-TFHN-H9, expressing the hemagglutinin of H9N2 avian influenza virus, was generated, on the basis of the chimeric NDV vector mentioned above. The NDV/AI4-TFHN-H9 vaccine elicited H9-specific hemagglutination inhibition antibodies in chickens pre-immunized with NDV vaccine, and reduced the numbers of chickens shedding virus after H9N2 challenge. NDV/AI4-TFHN-H9 could serve as an alternative vaccine for the prevention of H9N2 infection in commercial poultry flocks.

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs.

A novel chimeric Newcastle disease virus vectored vaccine against highly pathogenic avian influenza virus

Avian influenza (AI) is an economically-important disease of poultry worldwide. The use of vaccines to control AI has increased because of frequent outbreaks of the disease in endemic countries. Newcastle disease virus (NDV) vectored vaccine has shown to be effective in protecting chickens against a highly pathogenic avian influenza virus (HPAIV) infection. However, preexisting antibodies to NDV vector might affect protective efficacy of the vaccine in the field. As an alternative strategy, we evaluated vaccine efficacy of a chimeric NDV vectored vaccine in which the ectodomains of F and HN proteins were replaced by those of avian paramyxovirus serotype-2. The chimeric NDV vector stably expressed the HA protein in vivo, did not cross-react with NDV, was attenuated to be used as a safe vaccine, and provided a partial protection of 1-day-old immunized chickens against HPAIV subtype H5N1challenge, indicating its potential use for early protection of chickens.

Molecular basis for the thermostability of Newcastle disease virus.

Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease, due to their decreased dependence on cold chain for transport and storage. However, the genetic basis underlying the NDV thermostability is poorly understood. In this study, we generated chimeric viruses by exchanging viral genes between the thermostable TS09-C strain and thermolabile LaSota strain using reverse genetics technology. Evaluations of these chimeric NDVs demonstrated that the thermostability of NDV was dependent on the origin of HN protein. Chimeras bearing the HN protein derived from thermostable virus exhibited a thermostable phenotype, and vice versa. Both hemagglutinin and neuraminidase activities of viruses bearing the TS09-C HN protein were more thermostable than those containing LaSota HN protein. Furthermore, the newly developed thermostable virus rLS-T-HN, encoding the TS09-C HN protein in LaSota backbone, induced significantly higher antibody response than the TS09-C virus, and conferred complete protection against virulent NDV challenge. Taken together, the data suggest that the HN protein of NDV is a crucial determinant of thermostability, and the HN gene from a thermostable NDV could be engineered into a thermolabile NDV vaccine strain for developing novel thermostable NDV vaccine.

The Signal for Clathrin-Mediated Endocytosis of the Paramyxovirus SV5 HN Protein Resides at the Transmembrane Domain–Ectodomain Boundary Region

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is internalized from the cell surface via clathrin-coated pits. However, the cytoplasmic domain of SV5 HN does not contain a previously characterized internalization motif. A cell-surface-expressed chimeric protein (APK), consisting of the cytoplasmic tail, transmembrane (TM) domain, and 12 residues of the ectodomain of HN joined to the cytoplasmic protein pyruvate kinase is internalized, indicating that the N-terminal region of HN contains an internalization signal. Although SV5 HN is internalized at a rate similar to that of influenza virus hemagglutinin (HA) mutant Y543, which contains a degenerate tyrosine-based signal in its cytoplasmic tail, the elimination of the majority of the HN cytoplasmic tail, or substitution of the HN TM domain with leucine residues, did not affect the rate of HN internalization. The HN protein of the closely related virus, Newcastle disease virus (NDV), is not internalized from the cell surface. Working under the usual convention that the TM domain consists of the hydrophobic residues bounded by two charged residues, analysis of internalization of mutant and chimeric NDV HN molecules indicates that the first seven SV5 HN ectodomain residues are critical for internalization of HN. A glutamic acid residue (E37) that abuts this presumptive HN TM domain/ectodomain boundary is important for SV5 HN internalization.