Introduction: Avian pathogenic Escherichia coli (APEC) causes serious colibacillosis and significant economic losses. Data on profiles of virulence factors and antibiotic resistances among APEC strains are crucial to the control of infection. In this study, strains were isolated from eastern China, and the prevalence of virulence factors and distribution of antibiotic resistance were determined. Material and Methods: APEC strains were isolated and characterised by PCR for O serogroups, virulence factor genes, antibiotic resistance, and phylogenetic groups. Results: O78 was the most prevalent serogroup and type A was the most frequent phylogenetic group. The fimH, feoB, and iron genes were the most prevalent among the isolates. All isolates were multiresistant, and all strains were resistant to ampicillin and tetracycline, which are widely used in the poultry industry in China. Conclusion: This study provided important data on the presence of virulence genes and antibiotic resistance profiles of APEC from poultry farms in eastern China.

Interleukin-24 (IL-24) is a promising agent for cancer immunotherapy that induces apoptosis of tumor cells and enhances T cell activation and function. In order to improve the antitumor activity induced by Newcastle disease virus (NDV)-modified tumor vaccine, we generated a recombinant NDV expressing IL-24 using reverse genetics. Irradiated tumor cells infected with LX/IL-24 showed stable IL-24 expression. The cytotoxicity assay showed that LX/IL-24-infected murine melanoma cells significantly enhanced the antitumor immune response in vitro. Then, the antitumor effects of virus-infected tumor cells were examined in the murine tumor models. LX/IL-24-infected tumor cells exhibited strong antitumor effects both in prophylaxis and therapeutic models. LX/IL-24-infected tumor cells increased infiltration of CD4(+) T cells and CD8(+) T cells in tumor sites, and the antitumor activity of the tumor vaccine modified with LX/IL-24 was dependent on CD8+ T cells. Taken together, our data well illustrates that LX/IL-24-modified tumor cells are a promising agent for cancer immunotherapy.

Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) contribute to antigen capture, uptake, presentation and activation of immune responses. We recently developed a new and lymph node (LN) targeting adjuvant (D-CpG) by chemical conjugation type B CpG DNA with FDA-approved dextran polymer for lymph node imaging. To elucidate the possible antitumor mechanisms of this adjuvant, prophylaxis and therapeutic models of melanoma were used in this study. Our results showed that D-CpG was an efficient adjuvant of protein-based tumor vaccine in both prophylaxis and therapeutic models. It enhanced the tumor-specific Th1 and CTL responses. It also facilitated the tumor infiltration of the T cells and promoted IFNγ and TNFα production of both CD4+ and CD8+ T cells. In therapeutic model, D-CpG included tumor vaccine decreased the percentage of CD11b+Gr1low+ MDSCs in spleen and inhibited their infiltration in tumor microenvironments. Administration of the D-CpG included vaccine significantly inhibited lung metastasis of the tumor through similar mechanisms. In conclusion, D-CpG used as tumor vaccine adjuvant can enhance both Th1 and CTL responses and inhibit CD11b+Gr1low MDSCs, which may have general applicability to the development of vaccines against tumors.

Interleukin 15 ( IL 15) and IL 7 are two cytokines essential for T cell development and homeostasis. In order to improve the antitumor activity by Newcastle disease virus ( NDV )‐modified tumor vaccine, we generated a recombinant NDV co‐expressing IL 15 and IL 7 ( LX / IL (15+7)) through incorporation of a 2A self‐processing peptide into IL 15 and IL 7 using reverse genetics. B16 cells infected with LX / IL (15+7) expressed both IL 15 and IL 7 stably. The cytotoxicity assay showed that murine melanoma cells modified with LX / IL (15+7) could significantly enhance the antitumor immune response in vitro. Then, the antitumor effects of tumor vaccine modified with recombinant virus were tested in the murine tumor models. We observed strong antitumor responses induced by LX / IL (15+7)‐modified tumor cells both in prophylaxis and therapeutic models. Although the tumor‐infiltrating CD 4 + T cells and CD 8 + T cells were both increased, the antitumor activity of the tumor vaccine modified with LX / IL (15+7) was dependent on CD 8 + T cells. Taken together, our data strongly indicated that tumor vaccine modified with NDV strain LX / IL (15+7) is a promising agent for cancer immunotherapy.

AbstractStimulation of tumor-specific responses in both CD4+ and CD8+ T cells has been a challenge for effective tumor vaccines. We designed a vaccine vector containing the AIDA-1 autotransporter and DNA vaccine elements, generating a murine melanoma vaccine that was delivered by the attenuated Salmonella strain SL7207. Growth of murine subcutaneous melanoma was significantly inhibited by intranasal immunization with the Salmonella tumor vaccine. The vaccine activated tumor-specific CD4+ and CD8+ T-cell responses, with increased T-cell proliferation, tumor antigen–specific Th1 cytokine production, increased percentages of tetramer positive cells, and cytotoxicity. CD4+ or CD8+ T-cell depletion resulted in the loss of antitumor activity of the Salmonella tumor vaccine, suggesting that the efficacy of the vaccine was dependent on both CD4+ and CD8+ T cells. Lung metastasis of the tumor was also inhibited by vaccine treatment. Similarly, the percentages of tumor-specific Th1 cytokine production by CD4+ and CD8+ T cells in the spleen, tumor, and bronchoalveolar lavage were increased after vaccine treatment. Tumor-specific proliferation of CD4+ and CD8+ T cells was also promoted by the vaccine. Tetramer staining and cytotoxicity assay showed enhanced tumor-specific CD8+ T-cell response after vaccine treatment. Therefore, the Salmonella tumor vaccine could activate both tumor-specific CD4+ and CD8+ T-cell responses. This vaccine strategy may be widely applicable to the development of oral or nasal vaccines against tumors. Cancer Immunol Res; 5(6); 503–14. ©2017 AACR.

In order to test and enhance the antitumor activity against mice melanoma by NDV-modified tumor vaccine, a recombinant NDV expressing IL-15 (LX/(IL-15)) was generated by reverse genetics. Then, the expression level and biological activity of IL-15 were examined. Our results showed that mice tumor cell lines infected with LX/(IL-15) expressed IL-15 at a high level, and that expressed IL-15 was biologically active. Expression kinetics demonstrated that the highest expression level of IL-15 was at 48 h post infection. The cytotoxicity assay showed that murine melanoma cells modified with LX/(IL-15) could significantly enhance the antitumor immune response in vitro. In vivo study also showed that murine melanoma cells modified with LX/(IL-15) could prevent melanoma growth in mice. Taken together, our data strongly indicated that recombinant LX/(IL-15) is a promising agent for cancer immunotherapy both for human and animal.

Autologous tumor vaccine modified with nonlytic Newcastle disease virus (ATV-NDV) is a promising vaccine for cancer immunotherapy. IL-7 plays a critical role in lymphocyte development and homeostasis. To improve the efficacy of ATV-NDV, we inserted the murine IL-7 gene into the genome of nonlytic NDV strain LX using reverse genetic system. The insertion of the IL-7 gene neither affected the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Then we tested the antitumor effects of the autologous tumor vaccine modified with LX/(IL-7) in the murine tumor models. We showed that tumor cells modified with LX/IL-7 induced a strong antitumor activity both in prophylaxis and therapeutic models. The IFN-γ production and the cytotoxicity of tumor-specific CD8(+) T cells were significantly enhanced after immunization with tumor cells modified with LX/(IL-7) in both models. Although the tumor-infiltrating CD4(+) T cells and CD8(+) T cells were both increased and their IFN-γ productions also were upregulated, the antitumor activity of the tumor vaccine modified with LX/(IL-7) was dependent on CD8(+) T cells. Our results demonstrated that the autologous tumor vaccine modified with NDV strain LX/(IL-7) could promote the antitumor immune responses mediated by CD8(+) T cells and significantly improve the efficacy of the ATV-NDV.

Autologous tumor cell modified with non-lytic Newcastle Disease Virus (ATV/NDV) is a promising vaccine for cancer immunotherapy. IL-7 plays a critical role in lymphocyte development and homeostasis. In order to improve the efficacy of ATV/NDV, we inserted IL-7 gene into the genome of non-lytic NDV strain LX using reverse genetic system. The insertion of the IL-7 gene neither affected the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Then we tested the anti-tumor effects of the autologous tumor vaccine modified with LX/(IL-7) in the melanoma model. We showed that B16-F10 cells modified with LX/IL-7 induced a strong anti-tumor activity against melanoma both in prophylaxis and therapeutic models. The IFN-γ production and the cytotoxicity of tumor-specific CD8+ T cells were significantly enhanced after immunization with B16-F10 cells modified with LX/(IL-7) in both models. Although the tumor infiltrating CD4+ T cells and CD8+ T cells were both increased and their IFN-γ productions were also up-regulated, the anti-tumor activity of the tumor vaccine modified with LX/(IL-7) was dependent on CD8+ T cells. Our results demonstrated that the autologous tumor vaccine modified with NDV/(IL-7) could promote the anti-tumor immune responses mediated by CD8+ T cells and significantly improve the efficacy of the ATV/NDV.

ABSTRACT In this study, the complete genomic sequence of a novel reassortant H9N2 avian influenza virus (AIV) from domestic ducks in eastern China was reported. Phylogenetic analysis showed that seven of the eight genes were all highly homologous to the chicken-origin H9N2 viruses, whereas the PB2 gene was homologous to the human-origin H1N1 virus, which suggested that domestic ducks might play a key role in the genetic reassortment and evolution of H9N2 AIVs in eastern China.

In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007–2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

Background The 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. Results In 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. Conclusions There 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.

Background: Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF) chicken eggs with Influenza virus (AIV) and Newcastle disease virus (NDV) demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for AIV and NDV to specifically detect the viral genomes in mixed infections.Results: Data from this survey showed that when different doses of NDV (Lasota or F48E8) and AIV (F98 or H5N1) were simultaneously inoculated into embryonating chicken eggs (ECE), interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus.Conclusion: AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Background Six nucleotide (nt) insertion in the 5'-noncoding region (NCR) of the nucleoprotein (NP) gene of Newcaslte disease virus (NDV) is considered to be a genetic marker for recent genotypes of NDV, which emerged after 1960. However, F48-like NDVs from China, identified a 6-nt insert in the NP gene, have been previously classified into genotype III or genotype IX. Results In order to clarify their phylogenetic position and explore the origin of NDVs with the 6-nt insert and its significance in NDV evolution, we determined the entire genome sequences of five F48-like viruses isolated in China between 1946 and 2002 by RT-PCR amplification of overlapping fragments of full-length genome and rapid amplification of cDNA ends. All the five NDV isolates shared the same genome size of 15,192-nt with the recent genotype V-VIII viruses whereas they had the highest homology with early genotype III and IV isolates. Conclusions The unique characteristic of the genome size and phylogenetic position of F48-like viruses warrants placing them in a separate geno-group, genotype IX. Results in this study also suggest that genotype IX viruses most likely originate from a genotype III virus by insertion of a 6-nt motif in the 5'-NCR of the NP gene which had occurred as early as in 1940 s, and might be the common origin of genotype V-VIII viruses.

OBJECTIVE:The purpose of this research is trying to uncover the homology between two velogenic genotype III Newcastle disease virus (NDV) isolates with NDV strain Mukteswar, which was commonly used as vaccine in China.METHODS:The full-length genome of NDV isolates, JS/7/05/Ch and JS/9/05/Go, were determined by RT-PCR and then analyzed.RESULTS:The full-length genome of 2 genotype III velogenic NDV isolates shared 99.7% nucleotide identity with that of Mukteswar. The results of alignment of 6 viral genes showed that JS/7/05/Ch and JS/9/05/Go shared nucleotide and amino acid identities of 99.6% - 99.9% and 98.8% - 99.8% with that of Mukteswar, respectively. Furthermore, the IVPI score of JS/7/ 05/Ch and JS/9/05/Go was 2.18 and 1.33, remarkablely higher than that of Mukteswar, while the 3 NDV strains shared the consensus cleavage site of virulent NDV strains (112RRQRRF117). Virulence of NDV is mainly determined by the amino acid sequence of the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of lentogenic strains. However, 3 NDV strains with same F protein cleavage site showed different virulence. The entire genomic sequence of JS/7/05/Ch, JS/9/05/Go and Mukteswar was further analyzed. Three strains shared some gene-start signal, gene-end signal, intergenic region and six highly identical viral genes. Most amino acid differences among JS/7/05/Ch, JS/9/05/Go and Mukteswar were found in the predicted HN and L protein, and the predicted NP, P, V, W, M and even F protein had few amino acid differences.CONCLUSION:First, it is concluded that field isolates JS/7/05/ Ch and JS/9/05/Go are derived from Mukteswar and more attention must be paid to the virulence enhancement of vaccine strains. Second, it is proposed that the differences in the amino acid sequence of HN and L protein may give rise to the significant virulence differences between two NDV isolates and Mukteswar.

OBJECTIVE:The purpose of this research is to establish a simple rapid amplification of cDNA ends (RACE) strategy for direct mapping of the 3' end and 5' end of the genomic RNA of Newcastle disease virus (NDV), and to analyze the leader and trailer sequence of NDV strains belonging to different genotypes.METHODS:Classic RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) was specifically modified for mapping both ends of the NDV genome. 3'-RACE was carried out by genomic RNA ligation with 5' end phosphated adaptor CL+, and the 5' end was obtained by first strand cDNA with adaptor CL+.RESULTS:A modified RLM-RACE strategy was established in this paper, which proved simple, low-cost, repetitive and could be specifically used to map genome ends of NDV. By using this method, the leader and trailer sequence of 5 NDV strains, termed JS/5/05/Go, JS/07/04/Pi, JS/07/16/Pi, JS/7/05/Ch and JS/9/05/Go, belonging to genotype III, VI and VII was determined, respectively.CONCLUSION:The initial 8nt at the 3' and 5' ends of the genome of genotype I-VI NDV strains were complementary, whereas, the complementary sequences of strain JS/5/05/Go were up to 9 nt due to a mutation from T to C at the 9th nt in the 5' end. The 3' end of NDV genomic and anti-genomic RNA was predicted to form a potential hairpin structure. The U-->C(T-->C) mutation was located in the circle part of the hairpin in the 5' end of anti-genomic RNA, and had no visible influence on the formation of RNA secondary structure. However, the sequence of the circle part of the hairpin was changed from 3'-UUUC-5' to 3'-UCUC-5', more similar to the 3'-UCUUA-5' in the hairpin of genomic RNA.

新城疫（ND）和禽流感（AI）是严重危害养禽业的重要病毒性疫病[1],在给养禽业造成巨大经济损失的同时,在公共卫生上有其重要性。目前,在我国主要通过广泛疫苗的免疫接种来控制ND与AI的发生与流行[2]。但是,在临床上由于疫苗使用不当或滥用疫苗,造成免疫失败的现象屡有发生。因此,制定合理的免疫程序对预防上述疫病的暴发具有重要作用。

Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain isolated from an outbreak in the goose, seven pairs of primers were designed to amplify cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of NDV ZJI strain. The pNDV/ZJI with three helper plasmids, pCI-NP, pCI-P and pCI-L, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, infectious NDV ZJI strain was successfully rescued. The recombinant plasmid pNDV/ZJIFM was generated by converting the multi-basic amino acid sequence of the F0 protein cleavage region in pNDV/ZJI to the non-basic amino acid sequence characteristic of avirulent NDV strain. After cotransfection of the resultant plasmid and the three helper plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIFM, was generated. The mean death time (MDT) of NDV/ZJIFM was more than 120h and the intrancerebral pathogenicity index (ICPI) was 0.16, indicating that the rescued virus was highly attenuated. This attenuated genotype VIId NDV of goose origin could be a desirable vaccine in controlling the current epidemic of ND.