The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency.

Newcastle disease virus (NDV) has been extensively studied as a promising oncolytic virus for killing tumor cells in vitro and in vivo in clinical trials. However, the viral components that regulate the oncolytic activity of NDV remain incompletely understood. In this study, we systematically compared the replication ability of different NDV genotypes in various tumor cells and identified NP protein determines the oncolytic activity of NDV. On the one hand, NDV strains with phenylalanine (F) at the 450th amino acid position of the NP protein (450th-F-NP) exhibit a loss of oncolytic activity. This phenotype is predominantly associated with genotype VII NDVs. In contrast, the NP protein with a leucine amino acid at this site in other genotypes (450th-L-NP) can facilitate the loading of viral mRNA onto ribosomes more effectively than 450th-F-NP. On the other hand, the NP protein from NDV strains that exhibit strong oncogenicity interacts with eIF4A1 within its 366-489 amino acid region, leading to the inhibition of cellular mRNA translation with a complex 5' UTR structure. Our study provide mechanistic insights into how highly oncolytic NDV strains selectively promote the translation of viral mRNA and will also facilitate the screening of oncolytic strains for oncolytic therapy.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

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.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein.

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Reintroduction of highly pathogenic avian influenza A H7N9 virus in southwestern China.

Highly pathogenic (HP) avian influenza A H7N9 virus has emerged in China since 2016. In recent years, it has been most prevalent in northern China. However, several strains of HP H7N9 reappeared in southwestern China (Yunnan Province) in 2021. As a result, we are wondering if these viruses have re-emerged in situ or been reintroduced. Here, we present phylogenetic evidence that the HP H7N9 viruses isolated in Yunnan emigrated from northern to southwestern China in 2020. The northern subregion of China has become a novel epicenter in HP H7N9 dissemination. Meanwhile, a cleavage motif re-emerged due to the T341I mutation, implying a parallel evolution. This cross-region transmission, which originated in non-adjacent provinces and traveled a great geographic distance in an unknown way, indicates that HP H7N9 dissemination did not halt in 2020, even under the shadow of the COVID-19 pandemic. Additional surveillance studies in poultry are required to determine the HP H7N9 virus's geographic distribution and spread.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses.

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.

A dominant internal gene cassette of high pathogenicity avian influenza H7N9 virus raised since 2018.

The zoonotic H7N9 avian influenza virus emerged with the H9N2-origin internal gene cassette. Previous studies have reported that genetic reassortments with H9N2 were common in the first five human H7N9 epidemic waves. However, our latest work found that the circulating high pathogenicity H7N9 virus has established a dominant internal gene cassette and has decreased the frequency of reassortment with H9N2 since 2018. This dominant cassette of H7N9 was distinct from the cocirculating H9N2, although they shared a common ancestor. As a result, we suppose that this dominant cassette may benefit the viral population fitness and promote its continuous circulation in chickens.

Foreign gene expression attenuates a virulent Newcastle disease virus in chickens.

Newcastle disease virus (NDV) is an important pathogen for poultry and is used as a vector for developing novel poultry vaccines. Previous studies showed that foreign gene insertion in NDV vector decreases virulence determined by in vitro assays; however, the impact of foreign gene expression on the pathogenicity of NDV in susceptible chickens is not fully investigated. In this study, a recombinant NDV based on a velogenic strain carrying the orange fluorescent protein (OFP) gene between the phosphoprotein (P) and matrix (M) genes was generated using reverse genetics. Biological characteristics, including virus replication, virulence, and OFP expression, and the pathogenicity in chickens were evaluated. The recombinant NDV showed comparable replication capacity in eggs and cells as the parental virus, whereas OFP insertion resulted in a mild impairment of virulence, evidenced by longer mean death time in embryos. High OFP expression was detected in the cells inoculated with the recombinant NDV. In addition, the recombinant NDV induced delayed onset of disease, lower severity of clinical signs, and lower mortality in chickens compared to the parental virus. Moreover, high titers of the parental virus were detected in the spleen, lung, and intestinal tract, while no recombinant NDV was recovered from these tissues. Our findings suggest that in vitro characteristics related to the insertion of the OFP gene in a virulent NDV do not correlate to alteration of the pathogenicity in chickens. Our results provided new information regarding assessment of the impact of foreign gene expression on the pathogenicity of NDV.

Characterization of antibody response to an epitope spanning the haemagglutinin cleavage site of H7N9 subtype avian influenza virus for the differentiation of infected and vaccinated chickens.

H7N9 subtype avian influenza virus (AIV) is endemic in poultry in China, and vaccination is used as the primary strategy for disease control. However, by current serological tests, monitoring H7N9 virus infection in vaccinated poultry is difficult because vaccine-induced antibodies are not readily distinguishable from field viruses. Therefore, a test differentiating infected and vaccinated animals (DIVA) is critical for monitoring H7N9 virus. However, no DIVA test is available for the H7N9 subtype AIV. This study investigated the potential of an epitope (peptide 11) spanning the haemagglutinin (HA) cleavage site as a DIVA antigen for the H7N9 virus. The results showed that the H7N9 virus infection sera and post-challenge sera obtained from H7N9-vaccinated chickens reacted with peptide 11, whereas the sera elicited by inactivated and viral-vectored H7N9 vaccines had no reactivity with this peptide. Peptide 11 was further split into two peptides at the HA cleavage site, and the truncated peptides failed to discriminate H7N9 infected and vaccinated chickens. Peptide 11 is located in a main surface loop in the HA protein, and contains highly conserved residues in the HA cleavage site among the H7N9 subtype and different subtypes of groups 1 and 2, suggesting the potential of this peptide as a broad DIVA antigen for influenza viruses. Our study highlighted that peptide 11 is a promising DIVA antigen, and serological tests based on this peptide may serve as useful tools for monitoring H7N9 virus infection in vaccinated poultry. RESEARCH HIGHLIGHTSThe epitope spanning the HA cleavage site is a potential DIVA antigen for H7N9 AIV.The epitope reacted with LP and HP H7N9 viruses.The epitope has potential as a broad DIVA antigen for influenza viruses.

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.

N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses.

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Activation of the extracellular signal-regulated kinase pathway is required for replication of Newcastle disease virus.

Replication of Newcastle disease virus (NDV) is regulated by various host mechanisms, but the role of the extracellular signal-regulated kinase (ERK) pathway in regulating NDV replication is an open question. In this study, the relationship between the ERK pathway and NDV replication was investigated. NDV activated the ERK signaling in chicken embryo fibroblasts at the late stage of infection, correlating to expression of viral proteins. Specific blockage of the ERK pathway activation significantly decreased the transcription and translation levels of viral genes as well as virus replication and the cytopathogenic effect caused by NDV. Our results demonstrate that activation of the ERK pathway is required for NDV replication.

Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay.

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/µL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11 years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

G1-like M and PB2 genes are preferentially incorporated into H7N9 progeny virions during genetic reassortment.

BACKGROUND: Genotype S H9N2 viruses have become predominant in poultry in China since 2010. These viruses frequently donate their whole internal gene segments to other emerging influenza A subtypes such as the novel H7N9, H5N6, and H10N8 viruses. We recently reported that the PB2 and M genes of the genotype S H9N2 virus, which are derived from the G1-like virus, enhance the fitness of H5Nx and H7N9 avian influenza viruses in chickens and mice. However, whether the G1-like PB2 and M genes are preferentially incorporated into progeny virions during virus reassortment remains unclear; whether the G1-like PB2 and M genes from different subtypes are differentially incorporated into new virion progeny remains unknown. RESULTS: We conducted a reassortment experiment with the use of a H7N9 virus as the backbone and found that G1-like M/PB2 genes were preferentially incorporated in progeny virions over F/98-like M/PB2 genes. Importantly, the preference varied among G1-like M/PB2 genes of different subtypes. When competing with F/98-like M/PB2 genes during reassortment, both the M and PB2 genes from the H7N9 virus GD15 showed an advantage, whereas only the PB2 gene from the H9N2 virus CZ73 and the M gene from the H9N2 virus AH320 displayed the advantage. CONCLUSION: Our findings highlight the preferential and variable advantages of H9N2-derived G1-like M and PB2 genes in incorporating them into H7N9 progeny virions over SH14-derived F/98-like M/PB2 genes.

Early risk factors of the exacerbation of coronavirus disease 2019 pneumonia.

The purpose of this study was to investigate the early risk factors for the exacerbation of coronavirus disease 2019 (COVID-19) pneumonia. Restrospective analysis of clinical data of 85 patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including gender, age, comorbidities, symptoms, blood routine, clotting profile, biochemical examination, albumin, myocardial enzyme profile, inflammatory markers, and chest computed tomography (CT). All laboratory examinations were measured within first 24 hours after admission, and chest CT was performed before admission. A total of 56 (65.9%) patients had a history of exposure to the Huanan seafood market in Wuhan. Fever and dry cough accounted for the highest percentage of all symptoms. Male COVID-2019 patients were more likely to develop severe pneumonia. Patients with severe and critical conditions are older and have higher rates of hypertension (P = .003) and coronary heart disease (P = .017). All severe and critical patients infected with SARS-CoV-2 showed bilateral lung involvement and have more multiple lobes involvement than common patients (P < .001). Severe and critical patients showed higher white blood cell count (P = .006), neutrophil (NEU) count (P = .001), NEU% (P = .002), procalcitonin (P = .011), C-reactive protein (P = .003), prothrombin time (P = .035), D-dimer (P = .025), aspartate aminotransferase (P = .006), and lower lymphocyte (LYM) count (P = .019), LYM% (P = .001), albumin (P < .001). Logistic regression analysis showed that NEU count is an independent risk factor for deterioration, with the threshold of 6.5 × 109 ·L-1 . We concluded that the laboratory independent risk factor for the progression of COVID-19 pneumonia is NEU count. In addition, COVID-19 patients with bilateral lung involvement or multiple lobes involvement should be taken seriously and actively treated to prevent deterioration of the disease.

The virulence of NDV NA-1 strain regulated by the 3' leader or 5' trailer sequences.

The 3' and 5' terminal regions of Newcastle disease virus (NDV) genome are cis-acting regulatory elements involved in replication, transcription, and packaging of genomic and anti-genomic viral RNA. There are 6 different nucleotides (nts) at the 3' and 34 different nts at the 5' end of genome in the velogenic NA-1 strain and lentogenic LaSota strain, sharing 90.00% and 70.18% identity, respectively. We investigated the roles of 3' and 5' terminus in the NA-1 strain in viral replication, virulence and pathogenicity. Three NA-1 strain-based recombinant viruses (rNA-L, rNA-T, and rNA-LT) were generated using reverse genetics by either replacing the 3' leader or 5' trailer sequence of NA-1 strain or both with the corresponding sequences of the LaSota strain. Viral replication kinetics and pathogenicity of rNA-L and rNA-T were indistinguishable to that of the parental NA-1 strain, demonstrating that individual replacement or 3' or 5' terminal sequences had little influence. However, the synchronal replacement of both 3' and 5' terminal sequences resulted in decreased viral plaque size, reduced virulence and weaker pathogenicity in 2-week-old chickens. Therefore, our results suggest that the 3' and 5' terminal sequences of NDV genome could only influence the viral virulence when worked collaboratively, while separate replacement would not alter its biological characteristics.

Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus.

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.