Aerobactin genes are known to be present in virulent strains and absent from avirulent strains, but contributions of iucC and iucA, which are involved in aerobactin synthesis, to the pathogenicity of avian pathogenic Escherichia coli (APEC) have not been clarified. In this study, effects of double mutants (iucA/iutA or iucC/iutA) compared to those of single mutants (iucA, iucC or iutA) of aerobactin genes on the virulence of APEC strain E058 were examined both in vitro (aerobactin production, ingestion into HD-11 cells, survival in chicken serum) and in vivo (competitive growth against parental strain, colonization and persistence). In competitive co-infection assays, compared to the E058 parental strain, the E058ΔiucA mutant was significantly reduced in the liver, kidney, spleen (all P<0.01), heart and lung (both P<0.001). The E058ΔiutA mutant also was significantly reduced in the liver, lung, kidney (all P<0.01), heart and spleen (both P<0.001). The E058ΔiucC mutant was significantly attenuated in the heart and kidney (both P<0.05) and showed a remarkable reduction in the liver, spleen and lung (P<0.01); meanwhile, both E058ΔiucAΔiutA and E058ΔiucCΔiutA double mutants were sharply reduced as well (P<0.001). In colonization and persistence assays, compared with E058, recovered colonies of E058ΔiucA were significantly reduced from the lung, liver, spleen and kidney (P<0.01) and significantly reduced in the heart (P<0.001). E058ΔiutA was significantly reduced from the heart, lung, liver, spleen and kidney (P<0.01). E058ΔiucC, E058ΔiucAΔiutA and E058ΔiucCΔiutA were significantly decreased in all organs tested (P<0.001). These results suggest that iutA, iucA and iucC play important roles in the pathogenicity of APEC E058.

Here, the whole genome sequences of two Newcastle disease viruses (NDV) of genotype VIII, which were isolated from west China in the 1980s, were determined and characterized phylogenetically. This is the first report with respect to the complete genomic information of genotype VIII NDV strains.

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

Background

Genotype VIId Newcastle disease virus (NDV) isolates induce more severe damage to lymphoid tissues, especially to the spleen, when compared to virulent viruses of other genotypes. However, the biological basis of the unusual pathological changes remains largely unknown.

Methods

Virus replication, cytokine gene expression profile and cell death response in chicken splenocytes infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV strain F48E8 and genotype IV NDV strain Herts/33 were evaluated. Statistical significance of differences between experimental groups was determined using the Independent-Samples T test.

Results

JS5/05 and JS3/05 caused hyperinduction of type I interferons (IFNs) (IFN-α and -β) during detection period compared to F48E8 and Herts/33. JS5/05 increased expression level of IFN-γ gene at 6 h post-inoculation (pi) and JS3/05 initiated sustained activation of IFN-γ within 24 h pi, whereas transcriptional levels of IFN-γ remained unchanged at any of the time points during infection of F48E8 and Herts/33. In addition, compared to F48E8 and Herts/33, JS3/05 and JS5/05 significantly increased the amount of free nucleosomal DNA in splenocytes at 6 and 24 h pi respectively. Annexin-V and Proidium iodid (PI) double staining of infected cells showed that cell death induced by JS3/05 and JS5/05 was characterized by marked necrosis compared to F48E8 and Herts/33 at 24 h pi. These results indicate that genotype VIId NDV strains JS3/05 and JS5/05 elicited stronger innate immune and cell death responses in chicken splenocytes than F48E8 and Herts/33. JS5/05 replicated at a significantly higher efficiency in splenocytes than F48E8 and Herts/33. Early excessive cell death induced by JS3/05 infection partially impaired virus replication.

Conclusions

Viral dysregulaiton of host response may be relevant to the severe pathological manifestation in the spleen following genotype VIId NDV infection.

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

Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) are the two main subsets of extraintestinal pathogenic E. coli (ExPEC). Both types have multiple iron acquisition systems, including heme and siderophores. Although iron transport systems involved in the pathogenesis of APEC or UPEC have been documented individually in corresponding animal models, the contribution of these systems during simultaneous APEC and UPEC infection is not well described. To determine the contribution of each individual iron acquisition system to the virulence of APEC and UPEC, isogenic mutants affecting iron uptake in APEC E058 and UPEC U17 were constructed and compared in a chicken challenge model.

Results

Salmochelin-defective mutants E058ΔiroD and U17ΔiroD showed significantly decreased pathogenicity compared to the wild-type strains. Aerobactin defective mutants E058ΔiucD and U17ΔiucD demonstrated reduced colonization in several internal organs, whereas the heme defective mutants E058ΔchuT and U17ΔchuT colonized internal organs to the same extent as their wild-type strains. The triple mutant ΔchuTΔiroDΔiucD in both E058 and U17 showed decreased pathogenicity compared to each of the single mutants. The histopathological lesions in visceral organs of birds challenged with the wild-type strains were more severe than those from birds challenged with ΔiroD, ΔiucD or the triple mutants. Conversely, chickens inoculated with the ΔchuT mutants had lesions comparable to those in chickens inoculated with the wild-type strains. However, no significant differences were observed between the mutants and the wild-type strains in resistance to serum, cellular invasion and intracellular survival in HD-11, and growth in iron-rich or iron-restricted medium.

Conclusions

Results indicated that APEC and UPEC utilize similar iron acquisition mechanisms in chickens. Both salmochelin and aerobactin systems appeared to be important in APEC and UPEC virulence, while salmochelin contributed more to the virulence. Heme bounded by ChuT in the periplasm appeared to be redundant in this model, indicating that other periplasmic binding proteins likely contributed to the observed no phenotype for the heme uptake mutant. No differences were observed between the mutants and their wild-type parents in other phenotypic traits, suggesting that other virulence mechanisms compensate for the effect of the mutations.

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

The virulent class I Newcastle disease virus (NDV) variant 9a5b was generated from a nonvirulent NDV isolate Goose/Alaska/415/91 via nine consecutive passages in the chicken air sac, followed by five passages in the chick brain. The evolutionary mechanism of virulence in the class I NDV isolate is not fully understood. To elucidate this evolutionary mechanism, a reverse genetics manipulation specific for class I NDV is indispensable.

Results

A full-length cDNA clone of 9a5b and the helper plasmids pCI-NP, pCI-P, and pCI-L were constructed from segments of cDNA. After these plasmids were co-transfected into BSR T7/5 cells, infectious viral particles were obtained. The rescued viruses were genetically and biologically identical to the parental strain and showed similar pathogenicity in chickens.

Conclusion

A stable recovery method for class I NDV was established. Reverse genetics of the class I NDV variant 9a5b allowed for the generation of genetically altered and virulent NDV, and can be used as a foundation for research on the evolution of virulence in class I NDV isolates.

Background

In many countries, the predominant field isolates of infectious bronchitis virus (IBV) have been classified as QX-like strains since 1996. However, no commercial vaccines that are specific for this type of IBV are currently available. Therefore, there is an urgent need to develop novel vaccines that prevent QX-like IBV infection.

Results

A recombinant Marek’s disease virus (MDV), rMDV-S1, that expresses the S1 subunit of the spike (S) protein from the QX-like infectious bronchitis virus (IBV) was constructed by inserting the IBV S1 gene into the genome of the CVI988/Rispens strain of MDV. Specific pathogen-free (SPF) chickens that were vaccinated with rMDV-S1 were protected when challenged with the QX-like IBV. They were observed to have mild clinical signs of disease, a short virus-shedding period and low mortality. Additionally, the rMDV-S1 conferred full protection to chickens against virulent MDV, as did the CVI988/Rispens strain.

Conclusions

Our results demonstrate that rMDV-S1 is an effective and promising recombinant vaccine for the prevention of QX-like IBV infection.

The recombination rate in Newcastle disease virus (NDV) was as high as 10% in RDP analysis with full-length NDV genome sequences available in GenBank. We found that two NDV strains, China/Guangxi09/2003 and NDV/03/018, previously reported as recombinants, failed to show any evidence of recombination upon complete genome resequencing. Furthermore, we were able to reproduce artificial recombination by amplification of the M gene in a mixed sample of strains LaSota and ZJ1. It appears that the recombination of NDV is not as common as has been reported. NDV sequences in GenBank should be analyzed with caution during bioinformatic analyses for natural recombination events.

Background

The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.

Methodology/Principal Findings

Mice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.

Conclusion/Significance

Our findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.

Background

Although extensive data demonstrates that the majority of H6 duck isolates belonged to a single H6N2 virus lineage with a single gene constellation in southern China from 2000 to 2005, the prevalence of H6N2 virus in poultry in Eastern China is largely unknown.

Results

Epidemiology revealed that H6N2 viruses were the most frequently detected influenza subtypes in live bird markets from 2002 to 2008 in Eastern China, but from 2009 onwards, they were replaced with novel H6N6 viruses. We phylogenetically and antigenically analyzed 42 H6 viruses isolated mainly in domestic ducks from 2002 to 2010 in Eastern China. Surprisingly, none of these isolates grouped with the previously described H6N2 viruses which belonged to a single H6N2 virus lineage with a single gene constellation in domestic ducks in southern China from 2000 to 2005. Two distinct hemagglutinin lineages were identified and they all underwent frequent reassortment with multiple virus subtypes from the natural gene pool, but few reassortants were persistent or prevalent.

Conclusions

Five subtypes of H6 influenza viruses (H6N1, H6N2, H6N5, H6N6 and H6N8) cocirculated in Eastern China, which form a significant part of the natural influenza virus reservoir in domestic ducks, and significant viral reassortment is still ongoing in this species.

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.

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

In China, domestic ducks and wild birds often share the same water, in which influenza viruses replicate preferentially. Isolation of 2 novel reassortant highly pathogenic avian influenza (H5N5) viruses from apparently healthy domestic ducks highlights the role of these ducks as reassortment vessels. Such new subtypes of influenza viruses may pose a pandemic threat.

Background

To investigate the relationship between the selective pressure and the sequence variation of the hemagglutinin-neuraminidase (HN) protein, we performed the positive selection analysis by estimating the ratio of non-synonymous to synonymous substitutions with 132 complete HN gene sequences of Newcastle disease viruses (NDVs) isolated in China.

Results

The PAML software applying a maximum likelihood method was used for the analysis and three sites (residues 266, 347 and 540) in the HN protein were identified as being under positive selection. Codon 347 was located exactly in a recognized antigenic determinant (residues 345-353) and codon 266 in a predicted linear B-cell epitope. Substitutions at codon 540 contributed to the N-linked glycosylation potential of residue 538. To further evaluate the effect of positively selected sites on the vaccine efficacy, we constructed two recombinant fowlpox viruses rFPV-JS6HN and rFPV-LaSHN, expressing the HN proteins from a genotype VII field isolate Go/JS6/05 (with A266, K347 and A540) and vaccine strain La Sota (with V266, E347 and T540), respectively. Two groups of SPF chickens, 18 each, were vaccinated with the two recombinant fowlpox viruses and challenged by Go/JS6/05 at 3 weeks post-immunization. The results showed that rFPV-JS6HN could elicit more effective immunity against the prevalent virus infection than rFPV-LaSHN in terms of reducing virus shedding.

Conclusions

The analysis of positively selected codons and their effect on the vaccine efficacy indicated that the selective pressure on the HN protein can induce antigenic variation, and new vaccine to control the current ND epidemics should be developed.

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.

H5N1 highly pathogenic avian influenza virus (HPAIV) has posed a great threat not only for the poultry industry but also for human health. However, an effective vaccine to provide a full spectrum of protection is lacking in the poultry field. In the current study, a novel prime-boost vaccination strategy against H5N1 HPAIV was developed: chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella enterica serovar Typhimurium, and boosting with a killed vaccine followed. Chickens in the combined vaccination group but not in single vaccination and control groups were completely protected against disease following H5N1 HPAIV intranasal challenge, with no clinical signs and virus shedding. Chickens in the prime-boost group also generated significantly higher serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA titers against avian influenza virus (AIV) and higher host immune cellular responses than those from other groups before challenge. These results demonstrated that the prime-boost vaccination strategy provides an effective way to prevent and control H5N1 highly pathogenic avian influenza virus.

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.

Background

Germline mutations in the tumor suppressor PTEN predispose human beings to breast cancer, and genetic and epigenetic alterations of PTEN are also detected in sporadic human breast cancer. Germline Pten mutations in mice lead to the development of a variety of tumors, but mammary carcinomas are infrequently found, especially in mice under the age of six months.

Results

To better understand the role of PTEN in breast tumor development, we have crossed Pten heterozygous mice to MMTV-Wnt-1 transgenic mice that routinely develop ductal carcinomas in the mammary gland. Female Wnt-1 transgenics heterozygous for Pten developed mammary tumors earlier than Wnt-1 transgenics that were wild type for Pten. In most tumors arising in Pten heterozygotes, the Pten wild-type allele was lost, suggesting that cells lacking Pten function have a growth advantage over cells retaining a wild type allele. Tumors with LOH contained high levels of activated AKT/PKB, a downstream target of the PTEN/PI3K pathway.

Conclusions

An animal model has been developed in which the absence of Pten collaborates with Wnt-1 to induce ductal carcinoma in the mammary gland. This animal model may be useful for testing therapies specific for tumors deregulated in the PTEN/PI3K/AKT pathway.