The first report and biological characterization of Avian Orthoavulavirus 16 in wild migratory waterfowl and domestic poultry in China reveal a potential threat to birds.

RESEARCH HIGHLIGHTS: First confirmation of AOAV-16 in domestic and wild birds in China.AOAV-16 are low virulent viruses for chickens.Co-circulation/co-infection of AOAV-16 and H9N2 subtype AIV enhanced pathogenicity.Different intergenic sequences and recombination events exist within AOAV-16.

Long Non-Coding RNA Analysis: Severe Pathogenicity in Chicken Embryonic Visceral Tissues Infected with Highly Virulent Newcastle Disease Virus-A Comparison to the Avirulent Vaccine Virus.

There are significant variations in pathogenicity among different virulent strains of the Newcastle disease virus (NDV). Virulent NDV typically induces severe pathological changes and high mortality rates in infected birds, while avirulent NDV usually results in asymptomatic infection. Currently, the understanding of the specific mechanisms underlying the differences in host pathological responses and symptoms caused by various virulent NDV strains remains limited. Long non-coding RNA (lncRNA) can participate in a range of biological processes and plays a crucial role in viral infection and replication. Therefore, this study employed RNA-Seq to investigate the transcriptional profiles of chicken embryos' visceral tissues (CEVTs) infected with either the virulent NA-1 strain or avirulent LaSota strain at 24 hpi and 36 hpi. Using bioinformatic methods, we obtained a total of 2532 lncRNAs, of which there were 52 and 85 differentially expressed lncRNAs at 24 hpi and 36 hpi, respectively. LncRNA analysis revealed that the severe pathological changes and symptoms induced by virulent NDV infection may be partially attributed to related target genes, regulated by differentially expressed lncRNAs such as MSTRG.1545.5, MSTRG.14601.6, MSTRG.7150.1, and MSTRG.4481.1. Taken together, these findings suggest that virulent NDV infection exploits the host's metabolic resources and exerts an influence on the host's metabolic processes, accompanied by excessive activation of the immune response. This impacts the growth and development of each system of CEVTs, breaches the blood-brain barrier, inflicts severe damage on the nervous system, and induces significant lesions. These observations may be attributed to variations in pathology. Consequently, novel insights were obtained into the intricate regulatory mechanisms governing NDV and host interactions. This will aid in unraveling the molecular mechanisms underlying both virulent and avirulent forms of NDV infection.

Geographic distribution and genetic diversity of Newcastle disease virus in pigeons from Pakistan.

RESEARCH HIGHLIGHTS: Virulent NDV genotypes were repeatedly isolated from pigeons.Evidence of epidemiological links among viruses isolated from various locations.Distinct phylogenetic branches suggest separate, simultaneous evolution of NDVs.Study information could be helpful in the development of an effective vaccine.

Analysis of Avian Orthoavulavirus 1 Detected in the Russian Federation between 2017 and 2021.

Newcastle disease virus (NDV, Avian orthoavulavirus type 1, AOAV-1) is a contagious high-impact poultry pathogen with infections detected worldwide. In the present study, 19,500 clinical samples from wild bird species and poultry collected from 28 regions of Russia between 2017 and 2021 were screened for the presence of the AOAV-1 genome. NDV RNA was detected in 15 samples from wild birds and 63 samples from poultry. All isolates were screened for a partial sequence of the fusion (F) gene that included the cleavage site. Phylogenetic analysis demonstrated that lentogenic AOAV-1 I.1.1, I.1.2.1, and II genotypes were dominant among vaccine-like viruses in the territory of the Russian Federation. A vaccine-like virus with a mutated cleavage site (112-RKQGR^L-117) was detected in turkeys. Among the virulent AOAV-1 strains, viruses of the XXI.1.1, VII.1.1, and VII.2 genotypes were identified. The cleavage site of viruses of the XXI.1.1 genotype had a 112-KRQKR^F-117 amino acid sequence. The cleavage site of viruses with VII.1.1 and VII.2 genotypes had a 112-RRQKR^F-117 amino acid sequence. The data collected by the present study demonstrate the distribution and dominance of the virulent VII.1.1 genotype in the Russian Federation between 2017 and 2021.

Surveillance and Assessment of Risk Factors for Newcastle Disease Virus from Live Bird Retail Stalls in Lahore District of Pakistan.

Live bird markets (LBMs) in Asian countries are considered hubs for the spread of several poultry viruses. In Pakistan, there is a lack of uniformity in practices used in LBMs, which leads to the spread of poultry diseases. A cross-sectional survey was conducted in June-October 2017 to determine the circulation of Newcastle disease virus (NDV) in chickens being sold in live bird retail stalls (LBRSs) and to identify potential risk factors associated with estimated prevalence. A total of 189 stalls (n = 1134 birds) distributed in eight administrative towns of Lahore were visited. A pool of six oropharyngeal swabs was collected from each stall and tested by real-time reverse transcriptase PCR for the presence of NDV. Forty-two out of 189 swabs were found positive with an overall prevalence of 22.22% (95% confidence interval [Cl]: 16.88%-28.67%). Data for 11 potential risk factors acquired through questionnaires were analyzed by survey-weighted logistic regression and prevalence odds ratios (ORs) for associated risk factors were calculated. A final multivariable model identified three risk factors for NDV prevalence in LBRSs, including trading other poultry breeds alongside broilers (OR = 2.41; 95% confidence interval [CI] = 1.5-6.1), purchasing birds from mixed sources (OR = 3.12; 95% CI = 1.4-11.9), and number of birds sold per day (OR = 6.32; 95% CI = 1.9-23.5). Additionally, 24 selected samples were sequenced and phylogenetic analysis of the complete fusion gene (1662 bp) revealed that all isolates belonged to Subgenotype VII.2. This study provides important information on the epidemiology of NDV in Pakistan and highlights the importance of implementing surveillance and biosecurity practices in LBRSs.

Vigilancia y evaluación de factores de riesgo para el virus de la enfermedad de Newcastle en puestos de venta al menudeo de aves vivas en el distrito de Lahore en Pakistán. Los mercados de aves vivas (LBM, por sus siglas en inglés) en los países asiáticos se consideran centros de propagación de varios virus aviares. En Pakistán, existe una falta de uniformidad en las prácticas utilizadas en los mercados de aves vivas, lo que conduce a la propagación de enfermedades avícolas. Se realizó una encuesta transversal de junio a octubre del 2017 para determinar la circulación del virus de la enfermedad de Newcastle (NDV) en pollos que se venden en puestos minoristas de aves vivas y para identificar posibles factores de riesgo asociados con la prevalencia estimada. Se visitó un total de 189 puestos (n = 1134 aves) distribuidos en ocho ciudades administrativas de Lahore. Se recolectó un grupo de seis hisopos orofaríngeos de cada puesto y se analizó mediante transcripción reversa y PCR en tiempo real para detectar la presencia del virus de Newcastle. Cuarenta y dos de los 189 hisopos resultaron positivos con una prevalencia general del 22.22 % (intervalo de confianza [IC] del 95 % = 16.88­28.67). Los datos para 11 factores de riesgo potenciales adquiridos a través de cuestionarios se analizaron mediante regresión logística ponderada por encuesta y se calcularon las razones de probabilidad (OR) de prevalencia para los factores de riesgo asociados. Un modelo multivariable final identificó tres factores de riesgo para la prevalencia del virus de Newcastle en puestos minoristas de aves vivas, incluido el comercio de otras razas de aves de corral junto con pollos de engorde (OR = 2.41; IC del 95 % = 1.5­6.1), la compra de aves de fuentes mixtas (OR = 3.12; IC del 95 % = 1.4 ­11.9), y número de aves vendidas por día (OR = 6.32; IC 95% = 1.9­23.5). Además, se secuenciaron 24 muestras seleccionadas y el análisis filogenético del gene de fusión completo (1662 pb) reveló que todos los aislamientos pertenecían al subgenotipo VII.2. Este estudio brinda información importante sobre la epidemiología del virus de Newcastle en Pakistán y destaca la importancia de implementar prácticas de vigilancia y bioseguridad en los en puestos minoristas de aves vivas.

Intense Innate Immune Responses and Severe Metabolic Disorders in Chicken Embryonic Visceral Tissues Caused by Infection with Highly Virulent Newcastle Disease Virus Compared to the Avirulent Virus: A Bioinformatics Analysis.

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host's innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host-cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host's uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo.

Novel avian orthoavulavirus 13 in wild migratory waterfowl: biological and genetic considerations.

Avian orthoavulavirus 13 (AOAV-13), formerly known as Avian paramyxovirus 13 (APMV-13), is found scatteredly in wild birds around the world. Although four complete genome sequences of AOAV-13 had been identified since the first discovery in Japan in 2003, the information available on the genetic variation and biological characteristics of AOAV-13 is still limited. In the present study, we isolated six AOAV-13 strains from fecal samples of wild migratory waterfowls during annual (2014-2018) viral surveillance of wild bird populations from wetland and domestic poultry of live bird markets (LBMs) in China. The phylogenetic analyses based on the HN and F genes showed that they had very close relationship and the molecular clock estimations showed a low evolutionary rate of AOAV-13. However, Bean goose/Hubei/V97-1/2015 is 1953 nt in size (ORF, 1, 776 nt), which is a unique size and longer than other reported AOAV-13 strains. Additionally, four repeats of conserved sequences "AAAAAT" was presented in the 5'-end trailer region of Swan goose/Hubei/VI49-1/2016, which is unprecedented in the AOAV-13. These findings highlight the importance of continuous monitoring the specific species of APMVs.

Genomic and biological characteristics of Avian Orthoavulavirus-1 strains isolated from multiple wild birds and backyard chickens in Pakistan.

Circulation of the dominant sub-genotype VII.2 of Avian Orthoavulavirus-1 (AOAV-1) is affecting multiple poultry and non-poultry avian species and causing significant economic losses to the poultry industry worldwide. In countries where ND is endemic, continuous monitoring and characterization of field strains are necessary. In this study, genetic characteristics of eleven AOAV-1 strains were analyzed isolated from wild birds including parakeets (n = 3), lovebird parrot (n = 1), pheasant (n = 1), peacock (n = 1), and backyard chickens (n = 5) during 2015-2016. Genetic characterization (genome size [15,192 nucleotides], the presence of typical cleavage site [112-RRQKRF-117]) and biological assessment (HA log 27 to 29 and intracerebral pathogenicity index [ICPI] value ranging from 1.50 to 1.86) showed virulent AOAV-1. Phylogenetic analysis showed that the studied isolates belonged to sub-genotype VII.2 and genetically very closely related (> 98.9%) to viruses repeatedly isolated (2011-2018) from commercial poultry. These findings provide evidence for the existence of epidemiological links between poultry and wild bird species in the region where the disease is prevalent. The deduced amino acid analysis revealed several substitutions in critical domains of fusion and hemagglutinin-neuraminidase genes. The pathogenesis and transmission potential of wild bird-origin AOAV-1 strain (AW-Pht/2015) was evaluated in 21-day-old chickens that showed the strain was highly virulent causing clinical signs and killed all chickens. High viral loads were detected in different organs of the infected chickens correlating with the severity of lesions developed. The continuous monitoring of AOAV-1 isolates in different species of birds will improve our knowledge of the evolution of these viruses, thereby preventing possible panzootic.

Dominant subtype switch in avian influenza viruses during 2016-2019 in China.

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014-2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.

Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge.

Newcastle disease virus (NDV) and H9N2 subtype Avian influenza virus (AIV) are two notorious avian respiratory pathogens that cause great losses in the poultry industry. Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses, while an attenuated live vaccine bears the risk of mutation. Dendritic cell (DC) targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens. In this study, DC-binding peptide (DCpep)-decorated chimeric virus-like particles (cVLPs), containing NDV haemagglutinin-neuraminidase (HN) and AIV haemagglutinin (HA), were developed as a DC-targeting mucosal vaccine candidate. DCpep-decorated cVLPs activated DCs in vitro, and induced potent immune stimulation in chickens, with enhanced secretory immunoglobulin A (sIgA) secretion and splenic T cell differentiation. 40 µg cVLPs can provide full protection against the challenge with homologous, heterologous NDV strains, and AIV H9N2. In addition, DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly, as indicated by robust sIgA secretion and a reduced virus shedding period. Taken together, this chimeric VLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

Evaluation of the safety and protection efficacy of an attenuated genotype vii newcastle disease virus strain as a candidate vaccine.

Newcastle disease (ND) is a serious avian infectious disease, causing severe economic loss worldwide. Its prevention depends on comprehensive vaccination scheme against Newcastle disease virus (NDV). However, current vaccine strains are of different genotypes with prevalent circulating strains (genotype VII), with significant genetic distance. Our team previously generated a genotype matched attenuated NDV strain (rmNA-1). In this study, its safety and immunization efficacy were evaluated. Its lentogenic characteristic was stable for 25 generations in embryonated chicken eggs and for six generations in SPF chickens. Overdosed administration did not cause any clinical signs or pathogenic changes in chickens. As to its immunization effect, rmNA-1 stimulated a comparable serum NDV specific antibody level to a LaSota (genotype II) strain based commercial vaccine, and provided full protection against virulent genotype VII strain challenge, with significantly reduced virus shedding period.

Emergence of a Novel Ehrlichia minasensis Strain, Harboring the Major Immunogenic Glycoprotein trp36 with Unique Tandem Repeat and C-Terminal Region Sequences, in Haemaphysalis hystricis Ticks Removed from Free-Ranging Sheep in Hainan Province, China.

Ehrlichia minasensis, a recently described Ehrlichia species that is the most closely related to, but clearly distinct from, Ehrlichia canis, has been circulating in not only bovines, cervids, and dogs but also several tick species from Canada, Brazil, France, Pakistan, Ethiopia, and Israel. However, there are no reports of E. minasensis in China. The purpose of this study was to explore whether E. minasensis is present naturally in ticks in China. Through PCR targeting of the genus-conserved dsb gene, E. minasensis DNA was detected in Haemaphysalis hystricis ticks removed from free-ranging sheep in Hainan Province, South China in 2017. The partial sequence of the dsb, 16S rRNA, and groEL genes demonstrated that the Hainan strain shared 99% identity with the dsb gene of E. minasensis strain UFMG-EV (GenBank: JX629808), with the 16S rRNA of E. minasensis isolate E-2650 (MH500005) and with the groEL gene of E. minasensis strain UFMG-EV (JX629806), respectively. Moreover, sequence analysis of the major immunogenic tandem repeat protein (trp36) revealed that the Hainan strain harbored a unique tandem repeat sequence (APEAAPVSAPEAAPVSAPVS) and a C-terminal region that differed from those of other known E. minasensis strains. Additionally, phylogenetic analysis based on the entire amino acid sequence of trp36 revealed that the Hainan strain was closely related to a recently described E. minasensis strain from Brazil, of which the sister clade contained different strains of E. canis. The discovery of this novel Hainan strain in H. hystricis ticks represents the first known natural presence of E. minasensis in South China, highlighting the need for its constant surveillance.

Cellular MicroRNA Expression Profile of Chicken Macrophages Infected with Newcastle Disease Virus Vaccine Strain LaSota.

Vaccines with live, low-virulence Newcastle disease virus (NDV) strains are still the most accepted prevention and control strategies for combating Newcastle disease (ND), a major viral disease that hampers the development of the poultry industry worldwide. However, the mechanism underlying vaccine-mediated innate cell immune responses remains unclear. Here, a high-throughput Illumina sequencing approach was employed to determine cellular miRNA expression profiles in chicken macrophages infected with the LaSota virus, a widely used vaccine strain for mass vaccination programs against ND in poultry. Compared to the control group, 112 and 115 differentially expressed (DE) miRNAs were identified at 24 hpi (hours post inoculation) and 48 hpi, respectively. Meanwhile, 174 DE miRNAs were identified between 24 hpi and 48 hpi. Furthermore, 12 upregulated and 6 downregulated DE miRNAs were observed in common at 24 and 48 hpi compared with 0 hpi. In addition, target prediction and functional analysis of these DE miRNAs revealed significant enrichment for several signaling pathways, especially in the immune-related genes and pathways, such as the RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. Our findings not only lay the foundations for further investigating the roles and regulatory mechanisms of miRNA in vaccine-mediated innate cellular immune responses, but also extend new insights into the interactions between the host and NDV infection.

The Emergence of Avian Orthoavulavirus 13 in Wild Migratory Waterfowl in China Revealed the Existence of Diversified Trailer Region Sequences and HN Gene Lengths within this Serotype.

Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, "AAAAAT", in the 5'-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds.

Newcastle disease virus-like particles containing the Brucella BCSP31 protein induce dendritic cell activation and protect mice against virulent Brucella challenge.

Brucellosis is a widespread zoonosis that poses a substantial threat to human and animal public health due to the absence of a sufficiently safe and efficient vaccine. Virus-like particles (VLPs) have been developed as novel vaccine candidates and suitable carrier platforms for the delivery of exogenous proteins. Herein, we constructed chimeric virus-like particles (cVLPs) assembled by a Newcastle disease virus (NDV) M protein and glycosylphosphatidylinositol-anchored Brucella BCSP31 protein (GPI-BCSP31). cVLPs-GPI-BCSP31 were highly efficient in murine dendritic cell (DC) activation, both in vitro and in vivo. Moreover, they elicited strong specific humoural immune responses detected through ELISA assay with inactivated Brucella and recombinant BCSP31 protein and by elevated cellular immune responses indicated by intracellular IFN-γ and IL-4 levels in CD3+CD4+ T and CD3+CD8+ T cells. Importantly, cVLPs-GPI-BCSP31 conferred protection against virulent Brucella melitensis strain 16 M challenge, comparable to the efficacy of Brucella vaccine strain M5. In summary, this study provides a new strategy for the development of a safe and effective vaccine candidate against virulent Brucella and further extends the application of NDV VLP-based vaccine platforms.

Detection of viral components in exosomes derived from NDV-infected DF-1 cells and their promoting ability in virus replication.

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection.

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.

A genotype VII Newcastle disease virus-like particles confer full protection with reduced virus load and decreased virus shedding.

Newcastle disease (ND) is one of the most severe avian infectious disease inflicting a great loss on poultry industry worldwide. The control of ND relies on proper vaccination strategies. The vaccine strains of Newcastle disease virus (NDV) mainly belong to genotype I, II or III, which cannot fully prohibit virus shedding against the prevalent genotype VII virulent strain attack. To develop a safe, genotype matched vaccine candidate, we employed a bac-to-bac expression system and constructed a genotype VII NDV strain based virus-like particles (NDV VLPs). It was constructed with NDV M protein as the skeleton, and protective antigen F and HN proteins displayed on the surface. The NDV VLPs exhibited a similar appearance to the live NDV particles, but with denser F and HN proteins displayed on the surface. The immunization assay indicated that NDV VLPs stimulated a longer protection period, less tissue virus loading and shorter virus shedding period than the commercialized LaSota-formulated vaccine when challenged with genotype VII NDV strain. These results proposed the potential role of NDV VLPs as an alternative to current live genotype unmatched vaccine for the control and eliminate NDV in the avian flocks.

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.

Biological and phylogenetic characterization of a novel hemagglutination-negative avian avulavirus 6 isolated from wild waterfowl in China.

Up to now only nine whole genome sequences of avian avulavirus 6 (AAvV-6) had been documented in the world since the first discovery of AAvV-6 (AAvV-6/duck/HongKong/18/199/77) at a domestic duck in 1977 from Hong Kong of China. Very limited information is known about the regularities of transmission, genetic and biological characteristics of AAvV-6 because of the lower isolation rate and mild losses for poultry industry. To better further explore the relationships among above factors, an AAvV-6 epidemiological surveillance of domestic poultry and wild birds in six provinces of China suspected of sites of inter-species transmission and being intercontinental flyways during the year 2013-2017 was conducted. Therefore, 9,872 faecal samples from wild birds and 1,642 cloacal and tracheal swab samples from clinically healthy poultry of live bird market (LBM) were collected respectively. However, only one novel hemagglutination-negative AAvV-6 isolate (AAvV-6/mallard/Hubei/2015) was isolated from a fresh faecal sample obtained from mallard at a wetland of Hubei province. Sequencing and phylogenetic analyses of this AAvV-6 isolate (AAvV-6/mallard/Hubei/2015) indicated that this isolate grouping to genotype I were epidemiological intercontinentally linked with viruses from the wild birds in Europe and America. Meanwhile, at least two genotypes (I and II) are existed within serotype AAvV-6. In additional, this novel hemagglutination-negative AAvV-6 isolate in chicken embryos restored its hemagglutination when pre-treated with trypsin. These findings, together with data from other AAvV-6, suggest potential epidemiological intercontinental spreads among AAvV-6 transmission by wild migratory birds, and reveal potential threats to wild birds and domestic poultry worldwide.