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.

Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo.

As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.

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.

Genome-Wide Reassortment Analysis of Influenza A H7N9 Viruses Circulating in China during 2013-2019.

Reassortment with the H9N2 virus gave rise to the zoonotic H7N9 avian influenza virus (AIV), which caused more than five outbreak waves in humans, with high mortality. The frequent exchange of genomic segments between H7N9 and H9N2 has been well-documented. However, the reassortment patterns have not been described and are not yet fully understood. Here, we used phylogenetic analyses to investigate the patterns of intersubtype and intrasubtype/intralineage reassortment across the eight viral segments. The H7N9 virus and its progeny frequently exchanged internal genes with the H9N2 virus but rarely with the other AIV subtypes. Before beginning the intrasubtype/intralineage reassortment analyses, five Yangtze River Delta (YRD A-E) and two Pearl River Delta (PRD A-B) clusters were divided according to the HA gene phylogeny. The seven reset segment genes were also nomenclatured consistently. As revealed by the tanglegram results, high intralineage reassortment rates were determined in waves 2-3 and 5. Additionally, the clusters of PB2 c05 and M c02 were the most dominant in wave 5, which could have contributed to the onset of the largest H7N9 outbreak in 2016-2017. Meanwhile, a portion of the YRD-C cluster (HP H7N9) inherited their PB2, PA, and M segments from the co-circulating YRD-E (LP H7N9) cluster during wave 5. Untanglegram results revealed that the reassortment rate between HA and NA was lower than HA with any of the other six segments. A multidimensional scaling plot revealed a robust genetic linkage between the PB2 and PA genes, indicating that they may share a co-evolutionary history. Furthermore, we observed relatively more robust positive selection pressure on HA, NA, M2, and NS1 proteins. Our findings demonstrate that frequent reassortment, particular reassorted patterns, and adaptive mutations shaped the H7N9 viral genetic diversity and evolution. Increased surveillance is required immediately to better understand the current state of the HP H7N9 AIV.

Phylogenetic analysis and pathogenicity assessment of pigeon paramyxovirus type 1 circulating in China during 2007-2019.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of Newcastle disease virus (NDV) which is mainly associated with infections of pigeons and has the potential to result in disease in chickens. In this study, we characterised 21 PPMV-1 isolates from diseased pigeons in China during 2007-2019. Phylogenetic analysis revealed that all isolates belonged to genotype VI. Among them, most isolates belonged to sub-genotype VI.2.1.1.2.2, suggesting that VI.2.1.1.2.2 has become a prevalent genotype in pigeons in China. The results showed that all PPMV-1 isolates were mesogenic in nature according to the mean death time (MDT) and intracerebral pathogenicity index (ICPI). In vitro and in vivo studies demonstrated that two genetically closely related isolates (Pi-11 and Pi-10) both of which belonged to sub-genotype VI.2.1.1.2.2 had similar replication kinetics in cells derived from pigeons, while the replication titre of Pi-11 was significantly higher than that of Pi-10 in cells derived from chickens. Pi-11 and Pi-10 could contribute to morbidity and mortality in pigeons. Remarkably, although the two viruses resulted in no apparent disease symptom in chickens, Pi-11 could cause more severe histopathological lesions and had a stronger replication ability in chickens compared to Pi-10. Moreover, chickens infected with Pi-11 had higher shedding efficiency than chickens infected with Pi-10. Additionally, several mutations within important functional regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins might be associated with different pathogenicity of the two viruses in chickens. Collectively, these results indicated that the Pi-11-like virus of pigeon origin has the potential to induce severe outbreaks in chicken flocks. These findings will help us better understand the epidemiology and evolution of PPMV-1 in China and serve as a foundation for the further investigation of the mechanism underlying the pathogenic difference of PPMV-1 isolates in chickens.

Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Biological Characterization and Evolutionary Dynamics of Pigeon Paramyxovirus Type 1 in China.

Pigeon paramyxovirus type 1 (PPMV-1) is considered as an antigenic variant of Newcastle disease virus (NDV) which has an obvious host preference for pigeons and has caused significant economic losses to the global poultry industry. The evolutionary dynamics of PPMV-1 in China, however, are poorly understood. In this study, we characterized seven PPMV-1 isolates from diseased pigeons collected in Jiangsu, Anhui, and Henan provinces during 2020. Phylogenetic analysis revealed that seven isolates belonged to sub-genotype VI.2.1.1.2.2. Biological characterization indicated that seven isolates were mesogenic based on the mean death time (69.6-91.2 h) and intracerebral pathogenicity index (1.19-1.40) and had similar growth kinetics in chicken embryos and CEFs. Furthermore, the four representative viruses (AH/01/20/Pi, JS/06/20/Pi, HN/01/20/Pi, and HN/02/20/Pi) could result in marked cytopathic effects (CPE) in CEFs and induced syncytium formation in Vero cells. Our Bayesian phylogenetic analysis showed that PPMV-1 might first emerge in East China in 1974 and East China had the highest genotypic diversity of PPMV-1. Besides, phylogeographic analysis indicated that East China and South China were probably the major epicenters of dissemination of PPMV-1 in China. Selection pressure analysis and amino acid substitutions analysis revealed that the viral replication complex (NP, P, and L proteins) was likely related with the host preference of PPMV-1. Collectively, this study uncovered the epidemiology and evolutionary dynamics of PPMV-1 circulating in China, emphasizing the importance of strengthening the monitoring of PPMV-1 in East China and South China and providing significant clues for further studies on the molecular mechanism underlying host preference of PPMV-1.

Genetic and antigenic diversity of H7N9 highly pathogenic avian influenza virus in China.

Avian influenza virus (AIV) H7N9 that emerged in 2013 in eastern China is a novel zoonotic agent mainly circulating in poultry without clinical signs but causing severe disease with high fatality in humans in more than 5 waves. Since the emergence of highly pathogenic (HP) H7N9 variants in 2016, it has induced heavy losses in the poultry industry leading to the implementation of an intensive nationwide vaccination program at the end of wave 5 (September 2017). To characterize the ongoing evolution of H7N9 AIV, we conducted analyses of H7N9 glycoprotein genes obtained from 2013 to 2019. Bayesian analyses revealed a decreasing population size of HP H7N9 variants post wave 5. Phylogenetic topologies revealed that two novel small subclades were formed and carried several fixed amino acid mutations that were along HA and NA phylogenetic trees since wave 5. Some of the mutations were located at antigenic sites or receptor binding sites. The antigenic analysis may reveal a significant antigenic drift evaluated by hemagglutinin inhibition (HI) assay and the antigenicity of H7N9 AIV might evolute in large leaps in wave 7. Molecular simulations found that the mutations (V135T, S145P, and L226Q) around the HA receptor pocket increased the affinity to α2,3-linked sialic acid (SIA) while decreased to α2,6-linked SIA. Altered affinity may suggest that HP H7N9 variations aggravate the pathogenicity to poultry but lessen the threat to public health. Selection analyses showed that the HP H7N9 AIV experienced an increasing selection pressure since wave 5, and the national implementation of vaccination might intensify the role of natural selection during the evolution waves 6 and 7. In summary, our data provide important insights about the genetic and antigenic diversity of circulating HP H7N9 viruses from 2017 to 2019. Enhanced surveillance is urgently warranted to understand the current situation of HP H7N9 AIV.

Comparative pathogenicity of two closely related Newcastle disease virus isolates from chicken and pigeon respectively.

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a highly contagious disease that has led to tremendous economic losses worldwide. Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic and host variant of NDV. However, limited in-depth studies are available concerning side-by-side comparison of pathogenicity of PPMV-1 and its phylogenetically close NDV both in chickens and pigeons. To this end, two phylogenetically closely related NDV isolates, Kuwait 256 and JS/07/04/Pi from chicken and pigeon respectively were pathotypically and genotypically characterized in this study. The results indicated that Kuwait 256 was a velogenic strain, while JS/07/04/Pi was a mesogenic strain based on the mean death time of chick embryos (MDT) and intracerebral pathogenicity index in 1-day-old chicks (ICPI). Pathogenicity tests showed that Kuwait 256 caused severe clinical signs and 100 % mortality, while JS/07/04/Pi caused no apparent disease in chickens. Interestingly, both Kuwait 256 and JS/07/04/Pi caused morbidity and mortality in pigeons. Notably, pigeons infected with JS/07/04/Pi exhibited viral shedding for longer time compared to Kuwait 256-infected pigeons. Collectively, the findings of this study suggested that PPMV-1 decreased the pathogenicity in chickens but gained a survival advantage over NDV of chicken origin after its adaptive variation in pigeons based on the previous evidence that PPMV-1 originated from chicken-origin viruses. This study laid the foundation for the elucidation of the molecularmechanism underlying difference in pathogenicity of PPMV-1 and chicken-origin NDV in chickens.

Gga-miR-19b-3p Inhibits Newcastle Disease Virus Replication by Suppressing Inflammatory Response via Targeting RNF11 and ZMYND11.

Newcastle disease (ND), an acute and highly contagious avian disease caused by virulent Newcastle disease virus (NDV), often results in severe economic losses worldwide every year. Although it is clear that microRNAs (miRNAs) are implicated in modulating innate immune response to invading microbial pathogens, their role in host defense against NDV infection remains largely unknown. Our prior study indicates that gga-miR-19b-3p is up-regulated in NDV-infected DF-1 cells (a chicken embryo fibroblast cell line) and functions to suppress NDV replication. Here we report that overexpression of gga-miR-19b-3p promoted the production of NDV-induced inflammatory cytokines and suppressed NDV replication, whereas inhibition of endogenous gga-miR-19b-3p expression had an opposite effect. Dual-luciferase and gene expression array analyses revealed that gga-miR-19b-3p directly targets the mRNAs of ring finger protein 11 (RNF11) and zinc-finger protein, MYND-type containing 11 (ZMYND11), two negative regulators of nuclear factor kappa B (NF-κB) signaling, in DF-1 cells. RNF11 and ZMYND11 silencing by small interfering RNA (siRNA) induced NF-κB activity and inflammatory cytokine production, and suppressed NDV replication; whereas ectopic expression of these two proteins exhibited an opposite effect. Our study provides evidence that gga-miR-19b-3p activates NF-κB signaling by targeting RNF11 and ZMYND11, and that enhanced inflammatory cytokine production is likely responsible for the suppression of NDV replication.

Genetic analysis and biological characteristics of different internal gene origin H5N6 reassortment avian influenza virus in China in 2016.

Clade 2.3.4.4 of H5N6 subtype Avian Influenza Viruses (AIVs) has become dominant clade in South-East Asia. So far, a total of 16 cases of human infection, including 6 deaths, have been confirmed since 2014. In this study, we systematically investigated the genetic evolution and biological characteristics of these viruses. We first carried out phylogenetic and statistical analysis of all H5N6 viruses that were downloaded from Influenza Research Database, GISAID and isolates from our lab. We found that H5N6 AIVs continued to reassort with other AIVs subtypes since 2014. Among these H5N6 reassortments, four main gene types were identified: A (internal genes of H5N1-origin), B (PB2 of H6-origin, and others of H5N1-origin), C (internal genes of H9-origin) and D (PB2 of H6-origin and PB1of H3-origin, and others of H5N1). In addition, after several years of evolution, gene type D is currently the dominant gene type. To systematically compare the genetic and evolutionary characteristics and pathogenicity of these viruses, four H5N6 AIVs of different gene types were selected for further analysis. S4, XZ6, GD1602 and YZ587 virus represented gene type A, B, C and D, respectively. Their NA genes were all originated from H6 and their whole genome showed a high similarity with human isolates. All these isolates could both bind with SA-α2,3 Gal and SA-α2,6 Gal receptors. Pathogenicity test showed that these viruses were highly pathogenic in chickens, while YZ587 showed the lowest virulence. Moreover, XZ6 and S4 viruses were highly pathogenic in ducks and moderately pathogenic in mice, while GD1602 and YZ587 viruses were no-pathogenic in these animals. Interestingly, GD1602 and YZ587-like viruses were responsible for 4 and 2 human infection cases in 2016, respectively. Therefore, our study showed that the YZ587 virus which has mixed internal genes, showed lower virulence in avian species and mammals compared to other genotype viruses. Overall, our findings suggest that the H5N6 avian influenza virus is undergoing constantly evolving and reassortment. Thus, our study highlights the necessary of continued surveillance of the H5N6 AIVs in birds and paying close attention to the spread of these novel reassortment viruses.