Yantao Wu

Virulence of H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene

More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263–277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID50 or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD50 in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.

A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has been circulating in poultry in eastern China since 1998.

ABSTRACT Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.

Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication

Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005–2008

Seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons (Columba livia), geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. The results showed genetic diversity of these viruses: three class I viruses and one genotype I and 12 genotype II viruses of class II circulating in chickens were avirulent; four genotype VIb viruses isolated from pigeons were moderately virulent; and two genotype III viruses and 57 genotype VIId viruses were highly virulent. The three class I viruses were further classified as genotypes 2 and 3. The very high F protein sequence identity of one genotype I virus with strain Queensland V4 and 12 genotype II viruses with strain La Sota indicated that these viruses originated from the two vaccine strains. Two genotype III viruses shared greater than 99% sequence identity with the moderately virulent vaccine strain Mukteswar but exhibited significantly higher virulence, suggesting that they evolved from the vaccine virus and that the Mukteswar vaccine should be banned in China. Fifty-seven of the 63 virulent NDVs in this study belonged to genotype VIId, indicating its predominance in Eastern China. Genotype VIId viruses could be further classified into two subgroups. Four of the five NDVs isolated from pigeons belonged to genotype VIb, indicating its host-specific preference. Both the genotype VIb and VIId NDVs showed low amino acid similarity to the vaccine strains currently used in China, implying the urgent need to develop better vaccines against the most prevalent NDVs in China.

Avian reovirus triggers autophagy in primary chicken fibroblast cells and Vero cells to promote virus production

Avian reovirus (ARV) is an important cause of disease in poultry. Although ARV is known to induce apoptosis in infected cells, the interaction between ARV and its target cells requires further elucidation. In this report, we show that the ARV isolate strain GX/2010/1 induces autophagy in both Vero and primary chicken embryonic fibroblast (CEF) cells based on the appearance of an increased number of double-membrane vesicles, the presence of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) dot formation, and the elevated production of LC3II. We further demonstrate that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction by ARV infection. Moreover, treatment of ARV-infected cells with the autophagy inducer rapamycin increased viral yields, while inhibition of the autophagosomal pathway using chloroquine led to a decrease in virus production. Altogether, our studies strongly suggest that autophagy may play a critical role in determining viral yield during ARV infection.

Identification of a variable epitope on the Newcastle disease virus hemagglutinin-neuraminidase protein.

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin-neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345-353) on the HN protein.

Apoptin Enhances the Oncolytic Properties of Newcastle Disease Virus

Objective: Naturally occurring strains of Newcastle disease virus (NDV) have demonstrated the potential to kill cancer cells in both preclinical and clinical studies. Previous studies showed that apoptin, the VP3 protein of chicken infectious anemia virus, is a p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in transformed cells. In this study, we tested the hypothesis that apoptin enhances NDV-mediated tumor cell death. Methods: Reverse genetics was used to engineer an oncolytic NDV strain, FMW, to express apoptin. The antitumor effects of the recombinant virus (rFMW/AP) were also evaluated in the tumor cell lines and tumor-bearing mice. Results: Compared to the parental strain FMW, rFMW/AP was more potent in killing A459 and SMMC7721 tumor cells. Recombinant NDV also exhibited higher efficacy in suppressing tumor growth in mice bearing A549-induced tumors. Furthermore, rFMW/AP did not display apparent toxic effects in either normal cells or control mice. Conclusion: Our results suggest that the recombinant NDV expressing apoptin is a promising novel antitumor agent.

Prevalence and diversity of enterotoxin genes with genetic background of Staphylococcus aureus isolates from different origins in China.

Staphylococcal enterotoxins (SE) induce toxin-mediated diseases, such as food poisoning. In the present study, 568 isolates from different sources were tested for the prevalence of 18 SE genes and performed spa typing. In addition, we characterized the relationships between the distribution of SE genes and molecular clones based on multilocus sequence typing (MLST), spa and staphylococcal cassette chromosome mec (SCCmec) typing in selected 250 isolates. Approximately 54.40% of the isolates from different sources harbored one or more SE genes forming 120 distinct gene profiles. Seven genes, sea, seb, seg, seo, sem, seq, and sel were more frequently detected. The distributions of the SE genes among the isolates from human, animals, and foodborne origins were highly different with isolates from environments (P<0.01). The classic SE genes in both foodborne and human origin isolates were significantly higher than that in animal origin isolates (P<0.01), whereas the prevalence of genes of egc cluster and the other genes was similar in human, animal, and foodborne origin isolates (P>0.05). We identified two important gene clusters, sea-sek-seq, which is closely related to hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA)-III, and the egc cluster, which accounts for nearly half of all genes. Approximately 71% isolates could be typed by spa, yielding 103 spa types, of which 18 spa types were primary types. In clonal complex (CC) 239, an important Asian HA-MRSA-III clone from humans, nearly all isolates harbored complete or partial sea-sek-seq cluster; the main spa types were t030 and t037. In CC630, an important new community-associated (CA) MRSA-V CC in China, only sporadic SE genes, three main spa types, t4549, t2196, and t377 were observed. The egc cluster coexisting with other genes was present in isolates of CC5, CC9, CC1281, CC1301, CC30 and sequence type (ST) 25, but completely absent in isolates of CC239, CC59, CC7, and CC88. The results illustrate the genetic clonal diversity and the identity of S. aureus isolates from different sources with respect to SE genes and highlight a correlation between SE genes or gene clusters and CCs, spa, and MRSA clones. The foodborne and human origin isolates were the main potential causes of classic staphylococcal foodborne poisonings, whereas isolates harboring novel genes were new potential hazards to food safety.

Toll-like receptor 3 inhibits Newcastle disease virus replication through activation of pro-inflammatory cytokines and the type-1 interferon pathway.

Newcastle disease virus (NDV) is an avian paramyxovirus that can selectively replicate in and destroy human tumor cells. In this report, we demonstrate that NDV infection in HeLa cells leads to the activation of the pattern recognition Toll-like receptor 3 (TLR3). Overexpression of TLR3 enhanced the activity of the IFN-β promoter and the transcription factor NF-kappa B (NF-κB), thereby decreasing viral protein synthesis and the virus titer. In addition, the reduction of endogenous TLR3 by small interfering RNA (siRNA) increased NDV replication. Similar anti-NDV effects were observed in DF-1 chicken fibroblast cells with the exogenous expression of chicken TLR3 (cTLR3). Immunofluorescence staining of HeLa cells indicated that the dsRNA generated during NDV replication colocalized with TLR3 in punctate subcellular structures. Altogether, our results strongly suggest that TLR3 actively participates in the recognition of the innate pro-inflammatory response after NDV infection and leads to the consequent antiviral cytokine/interferon secretion.

Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

BackgroundThe goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial.ResultsIn this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33.ConclusionsThere is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.