Newcastle Disease Virus Matrix Research Articles

Virus-Like Particles Assembled Using Respiratory Syncytial Virus Matrix Protein Elicit Protective Immunity in Mice.

Heterologous virus-like particle (VLP) assembly involving influenza or the Newcastle disease virus matrix protein (M) has been extensively used to explore the efficacies of VLP vaccines against the respiratory syncytial virus (RSV). Here, we attempted to generate homologous RSV VLPs by expressing the pre-fusion (pre-F) or the glycoprotein (G) on the RSV M protein and evaluated their protective efficacy in mice. We generated VLPs using the baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Recombinant baculoviruses expressing the RSV pre-F, G, and M antigens were inoculated into Sf9 cells, and particles were self-assembled. Mice were immunized with either pre-F or G-expressing VLPs, and immune parameters were assessed to determine protection. Our findings show that successful VLP assembly can be achieved by utilizing recombinant baculoviruses expressing the RSV pre-F or G proteins with the native matrix protein. Mice immunized with either pre-F or the G antigen-expressing VLPs elicited robust serum-mediated virus neutralization. VLP immunization evoked Th1-biased RSV-specific antibody responses in the sera of mice. Following challenge infection with the RSV A2 strain, immunized mice experienced lesser eosinophil and IL-4 accumulation in the lungs, though a substantial increase in TNF-α secretion was observed from CD4+ T cells. Interestingly, splenic antibody-secreting cell responses were substantially enhanced against RSV F antigen, but not against the RSV G antigen following immunization and challenge infection. Immunizing mice with the VLPs significantly inhibited pulmonary histopathology development, as indicated by the diminished inflammatory immune cell influx and mucin secretion. Combined, these vaccine-induced immune responses contributed to successfully inhibiting the RSV replication in the lungs of mice and demonstrated that RSV VLP assembly using insect cell-derived homologous RSV matrix protein is a feasible approach.

Optimization of infectious bronchitis virus-like particle expression in Nicotiana benthamiana as potential poultry vaccines.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 μg or 20 μg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B.

The matrix (M) protein FPIV L-domain is conserved among multiple paramyxoviruses; however, its function and the associated mechanism remain unclear. In this study, the paramyxovirus Newcastle disease virus (NDV) was employed to study the FPIV L-domain. Two recombinant NDV strains, each carrying a single amino acid mutation at the Phe (F23) or Pro (P24) site of 23FPIV/I26 L-domain, were rescued. Growth defects were observed in only the recombinant SG10-F23A (rSG10-F23A) strain. Subsequent studies focused on rSG10-F23A revealed that the virulence, pathogenicity, and replication ability of this strain were all weaker than those of wild-type strain rSG10 and that a budding deficiency contributed to those weaknesses. To uncover the molecular mechanism underlying the rSG10-F23A budding deficiency, the bridging proteins between the FPIV L-domain and endosomal sorting complex required for transported (ESCRT) machinery were explored. Among 17 candidate proteins, only the charged multivesicular body protein 4 (CHMP4) paralogues were found to interact more strongly with the NDV wild-type M protein (M-WT) than with the mutated M protein (M-F23A). Overexpression of M-WT, but not of M-F23A, changed the CHMP4 subcellular location to the NDV budding site. Furthermore, a knockdown of CHMP4B, the most abundant CHMP4 protein, inhibited the release of rSG10 but not that of rSG10-F23A. From these findings, we can reasonably infer that the F23A mutation of the FPIV L-domain blocks the interaction between the NDV M protein and CHMP4B and that this contributes to the budding deficiency and consequent growth defects of rSG10-F23A. This work lays the foundation for further study of the FPIV L-domain in NDV and other paramyxoviruses. IMPORTANCE Multiple viruses utilize a conserved motif, termed the L-domain, to act as a cellular adaptor for recruiting host ESCRT machinery to their budding site. Despite the FPIV type L-domain having been identified in some paramyxoviruses 2 decades ago, its function in virus life cycles and its method of recruiting the ESCRT machinery are poorly understood. In this study, a single amino acid mutation at the F23 site of the 23FPIV26 L-domain was found to block NDV budding at the late stage. Furthermore, CHMP4B, a core component of the ESCRT-III complex, was identified as a main factor that links the FPIV L-domain and ESCRT machinery together. These results extend previous understanding of the FPIV L-domain and, therefore, not only provide a new approach for attenuating NDV and other paramyxoviruses but also lay the foundation for further study of the FPIV L-domain.

In silico Prediction of Molecular Docking between Chicken BRD2 and Newcastle Disease Virus Matrix Protein to Elucidate Host-pathogen Interaction

Background: Newcastle disease is one of the most contagious viral diseases caused by the Newcastle disease virus (NDV) which causes a huge loss in the terms of economy as well as mortality level in the poultry sector. The viral matrix protein is one of the proteins which helps in the attachment of virus, budding and assembly of progeny virions in the host cell. Host-pathogen interaction plays a very important role in the changeover of genetic diversity in organisms. Methods: The amino acid sequences of chicken BRD2 gene and matrix protein of NDV were used for structure prediction and visualization of protein-protein interaction between BRD2 and matrix protein by using Discovery Studio software. The nucleotide sequences of both genes were used for evolutionary analysis using MEGA7 software followed by selection pressure analysis using online Datamonkey server. Result: The results indicated that the ligand was attached with the matrix protein, which acts as a receptor on the virus surface. The purifying selection has been evident for the viral matrix protein gene. The docking of viral protein with host BRD2 protein has been detected in the study which further warrants experimental validation. In conclusion, the viral matrix protein interacts at amino acid position Tyrosine-148, Valine-339, Alanine-341 with ligand present on the chicken BRD2 protein. The evolutionary analysis revealed that the matrix protein of NDV R2B strain was the closest to that of Peacock, pigeon, duck and quail however, it exhibited maximum distance with the chicken NDV from Ireland, Vietnam and Haryana state of India.

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID50) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.

Presence of Newcastle Disease Virus in Vaccinated Indigenous Chicken in Selected Regions in Kenya —A Cross-Sectional Study

Vaccination of flocks against Newcastle disease virus (NDV) outbreaks is the main approach for controlling the spread of Newcastle disease (ND). Nevertheless, NDV outbreaks have been reported in vaccinated chickens. In this study, we determined the prevalence of NDV among vaccinated indigenous chickens (ICs) and examined the relationship of the disease with the weather (temperature, rainfall, humidity, and wind speed) at the time of sample collection, production system, and the presence of other species. The genetic diversity of the NDV matrix and fusion genes was also inferred. A total of 1,210 swabs were collected between 2017 and 2018 from ICs that were vaccinated or unvaccinated against NDV in free-range and semi-free-range production systems. We collected 650 swabs each from the oropharynx and cloaca of ICs in 68 households within the Bomet, Baringo, Kilifi, Nakuru, Kakamega, and Machakos counties in Kenya. NDV matrix genes were detected using reverse transcription-polymerase chain reaction, and amplicons of matrix and fusion genes were sequenced using a capillary sequencer from the pooled samples. Among the vaccinated ICs, the prevalence of NDV was 78.5% (p=0.045). There were significant relationships between the presence of NDV and vaccination history of the ICs (p=0.034), the type of production system for ICs (p=0.004) and the months of sample collection (p < 0.0001). However, no significant relationship was found between the presence of NDV and the interaction between ICs and other birds. The presence of matrix and fusion genes in samples from vaccinated flocks indicated the presence of both virulent and low-virulence strains of NDV. These findings highlight the significant presence of NDV among vaccinated ICs and suggest the possibility of inadequate vaccination and viral shedding post-vaccination as the drivers of infections.

Chicken bromodomain-containing protein 2 interacts with the Newcastle disease virus matrix protein and promotes viral replication

Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264–313) of the M protein and the extra-terminal (ET) domain (aa 619–683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.

Seroprevalence and detection of newcastle disease virus matrix gene in domestic local breed of chickens from eight communities in Bauchi State, Nigeria

Indigenous breed of domestic chickens have been identified as an appropriate tool for eradication poverty and hunger because of the ease at which poor people can acquire, grow and consume or sale the meat and eggs of these animals. The production of these chickens is largely constrained by disease especially, velogenic Newcastle disease (ND) which have the potential of wiping out an entire susceptible chicken population. Foundational to the control of ND in local breed of chickens in every community is the need for baseline information. Such information is scanty in Bauchi State even though the state is one of the major producers of these chickens in Nigeria. The aim of this study is to determine the seroprevalence of ND and also detect Newcastle disease virus (NDV) for the purpose of understanding the presence and distribution of the disease in the State. This study was conducted among 1085 village chickens from nine (9) randomly selected communities in Bauchi State, Nigeria. The seroprevalence of ND by Haemagglutination inhibition test was 36.4%. Matrix gene of ND virus (NDV) was also detected from 29.9% of 281 pooled cloacal swabs of the same chickens. The result indicates that ND virus (NDV) and its antibodies were widespread among village chickens in these communities. Vaccination is suggested as an appropriate control measure to protect chickens against a possible attack by a velogenic strain of NDV in chickens from these communities.Keywords: Newcastle disease, seroprevalence, molecular detection, domestic breed of local chickens, Bauchi State

Nuclear localization of Newcastle disease virus matrix protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription

Nuclear localization of paramyxovirus proteins is crucial for virus life cycle, including the regulation of viral replication and the evasion of host immunity. We previously showed that a recombinant Newcastle disease virus (NDV) with nuclear localization signal mutation in the matrix (M) protein results in a pathotype change and attenuates viral pathogenicity in chickens. However, little is known about the nuclear localization functions of NDV M protein. In this study, the potential functions of the M protein in the nucleus were investigated. We first demonstrate that nuclear localization of the M protein could not only promote the cytopathogenicity of NDV but also increase viral RNA synthesis and transcription efficiency in DF-1 cells. Using microarray analysis, we found that nuclear localization of the M protein might inhibit host cell transcription, represented by numerous up-regulating genes associated with transcriptional repressor activity and down-regulating genes associated with transcriptional activator activity. The role of representative up-regulated gene prospero homeobox 1 (PROX1) and down-regulated gene aryl hydrocarbon receptor (AHR) in the replication of NDV was then evaluated. The results show that siRNA-mediated knockdown of PROX1 or AHR significantly reduced or increased the viral RNA synthesis and viral replication, respectively, demonstrating the important roles of the expression changes of these genes in NDV replication. Together, our findings demonstrate for the first time that nuclear localization of NDV M protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription, improving our understanding of the molecular mechanism of NDV replication and pathogenesis.

Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts

ABSTRACTThe matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Survey for Newcastle disease viruses in poultry and wild birds in Kogi state, Nigeria

Newcastle disease (ND) outbreak even in the face of vaccination is a common problem in Nigeria. A survey was carried out between June, 2012 to February, 2013 to detect ND viruses in poultry and wild birds in 12 Local Government Areas (LGAs) of Kogi state, Nigeria. Oropharyngeal swabs from 710 poultry and cloacal swabs from 100 species in eight families of wild birds were tested using reverse transcription polymerase chain reaction with set of primers targeting the ND virus matrix protein with 60.5% swabs being positive. The prevalence of ND viruses was highest in live bird market with 62.5% and lowest in backyard farm with 57.7%, while based on species of birds, Swallow (Hirundo spp.) had the highest prevalence with 83.3% and zero in Laughing dove (Streptopelia senegalensis). The χ2 value of the prevalence of ND viruses in contiguous areas A against contiguous areas B was significant (χ2 =6.59, p≤ 0.01, OR = 4.10 at 95% CI = 1.34 – 12.28). The study revealed the circulation of ND viruses in poultry and wild birds in Kogi state. This is the first report of ND viruses in Hirundo spp and Swift (Apus spp) in Kogi state, Nigeria. There is need for sequencing of the F-protein of the ND viruses circulating in Kogi state to compare with already known strains in Nigeria. Vaccination against ND should be stepped up in backyard poultry and instituted in rural poultry for effective control. Keywords : Backyard poultry, Newcastle disease viruses, RT-PCR, Swallow and Swift, Wild birds

A single amino acid mutation, R42A, in the Newcastle disease virus matrix protein abrogates its nuclear localization and attenuates viral replication and pathogenicity

The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 aa of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted M's nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30–60 of M and amino acids 188–245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process.

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.

Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein

Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein.

Characterization of signal sequences determining the nuclear export of Newcastle disease virus matrix protein

The Newcastle disease virus (NDV) matrix (M) protein has been demonstrated to be a nuclear-cytoplasmic trafficking protein. Previous studies have shown that the M protein localizes in the nucleus through a bipartite nuclear localization signal. Here, we report that the ability of the M protein to shuttle to the cytoplasm is mediated by three nuclear export signal sequences (NESs). Using leptomycin B (LMB), a specific inhibitor of CRM1, we found that the nuclear export of the three NESs was LMB insensitive and thus was CRM1 independent. In addition, inactivation of these NESs led to nuclear accumulation of the M protein. Our results highlight the significance of these NESs to the nuclear export of the NDV M protein.

Engagement of new castle disease virus (NDV) matrix (M) protein with charged multivesicular body protein (CHMP) 4 facilitates viral replication

Newcastle disease virus (NDV) causes heavy economic losses to poultry industry across the globe every year. Although NDV matrix (M) protein is involved in virus budding and our previous data indicate that in ovo expression of M protein facilitates NDV replication, the underlying mechanism for the role of M protein in NDV replication is not clear. Using yeast two-hybrid system and immunoprecipitation approaches, we found that M protein interacted with host vacuolar sorting protein charged multivesicular body protein (CHMP) 4B and 4C. In addition, the colocalization of M protein and CHMP4B/C could be observed using a laser confocal scanning microscope. Knockdown of CHMP4B by siRNA or transient expression of CHMP4B/C dominant negative forms markedly inhibited NDV growth in DF-1 cells. Thus, cellular CHMP4s play a critical role in NDV replication.

Detection of Newcastle Disease Virus RNA by Reverse Transcription-Polymerase Chain Reaction Using Formalin-Fixed, Paraffin-Embedded Tissue and Comparison with Immunohistochemistry and in situ Hybridization

The usefulness of reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of 2 NDV isolates: a low virulent virus (LaSota) and a virulent virus (from the 2002-2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just before euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By seminested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-microm sections and to those of virus isolation from swabs. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens that were no longer shedding virus detectable by virus isolation. The RT-PCR was an effective and sensitive method to detect NDV in FFPE tissues. To the authors' knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.

Virulence of Pigeon-Origin Newcastle Disease Virus Isolates for Domestic Chickens

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.

Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae

Matrix (M) gene sequences for recent field isolates and older reference Newcastle disease viruses (NDV) were examined to determine phylogenetic relationships and population trends among these viruses. Overall, the M gene has a majority of synonymous nucleotide sequence substitutions occurring among NDV isolates. However, several predicted amino acid changes in the M protein of specific NDV isolates have occurred that correlate to phylogenetic relationships. Nucleotide substitutions in these codons have a greater number of nonsynonymous base changes. The NDV isolates arising since the 1970s belong to a population of viruses that expanded worldwide at an exponential rate. These viruses may have their origins in free-living birds, are present worldwide, and continue to circulate causing disease in poultry. A specific NDV lineage composed of virulent isolates obtained in the US prior to 1970 appears to no longer exists among free-living birds or commercial poultry. However, “vaccine-like” viruses are common in the US and continue to circulate among commercial poultry. Based on M protein amino acid sequences, NDV separates as a clade most closely related to morbilliviruses and not with their current designated category, the rubulaviruses among the Paramyxoviridae. Consequently, avian paramyxoviruses should have their own taxonomic subfamily among the Paramyxovirinae.