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1.  Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival 
Infection and Immunity  2014;82(7):2840-2850.
We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants.
PMCID: PMC4097617  PMID: 24752516
2.  Late acyltransferase genes lpxX and lpxL jointly contribute to the biological activities of Moraxella catarrhalis 
Journal of Medical Microbiology  2013;62(Pt 6):807-812.
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.
PMCID: PMC3709554  PMID: 23475908
3.  A 20-Amino-Acid Deletion in the Neuraminidase Stalk and a Five-Amino-Acid Deletion in the NS1 Protein Both Contribute to the Pathogenicity of H5N1 Avian Influenza Viruses in Mallard Ducks 
PLoS ONE  2014;9(4):e95539.
Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A−) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S−) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A−S− and three variants (A−S+ with a five-amino-acid insertion in the NS1 protein, A+S− with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A− and/or S− replicated in DEF cells were higher than that of A+S+, and the A−S− virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A−S− induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S−, the A−S+ and A−S− viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.
PMCID: PMC3990698  PMID: 24743258
4.  Novel Variants of Clade 2.3.4 Highly Pathogenic Avian Influenza A(H5N1) Viruses, China 
Emerging Infectious Diseases  2013;19(12):2021-2024.
We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009–2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.
PMCID: PMC3840869  PMID: 24274396
influenza; avian influenza virus; clade 2.3.4; China; clade; influenza virus; flu; H5; H5N1; highly pathogenic; viruses; avian; AIV; phylogeny; chickens; birds; HPAI
5.  The PA-Gene-Mediated Lethal Dissemination and Excessive Innate Immune Response Contribute to the High Virulence of H5N1 Avian Influenza Virus in Mice 
Journal of Virology  2013;87(5):2660-2672.
Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.
PMCID: PMC3571398  PMID: 23255810
6.  Genome Sequence of a Novel H10N9 Avian Influenza Virus Isolated from Chickens in a Live Poultry Market in Eastern China 
Genome Announcements  2013;1(4):e00386-13.
An H10N9 avian influenza virus (AIV) strain, A/Chicken/Jiangsu/RD5/2013, was isolated in China. The hemagglutinin (HA) and neuraminidase (NA) genes in this strain originated from H10N1 and H7N9 AIVs, respectively, and the other genes derived from H7N3 AIVs. Sequence analysis implies that the H10N9 AIV may be an NA gene donor for the human H7N9 influenza viruses.
PMCID: PMC3695435  PMID: 23814107
7.  Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China 
PLoS ONE  2012;7(9):e46183.
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.
PMCID: PMC3458027  PMID: 23049973
8.  Mutant Lipooligosaccharide-Based Conjugate Vaccine Demonstrates a Broad-Spectrum Effectiveness against Moraxella catarrhalis 
Vaccine  2011;29(25):4210-4217.
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1-4Galβ1-4Glc and human Pk antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune serum levels) of serum immunoglobulin (Ig)G against O35E LOS in rabbits with an adjuvant or without an adjuvant (an 140- or 140-fold above the preimmune serum levels). Rabbit antisera demonstrated elevated complement-mediated bactericidal activities against the wild type strain O35E. The rabbit sera elicited by O35Elgt5 dLOS-TT were further examined and showed cross bactericidal activity against all additional 19 M. catarrhalis strains and clinical isolates studied. Moreover, the rabbit sera displayed cross-reactivity not only among three serotype strains but also clinical isolates in a whole-cell enzyme-linked immunosorbent assay (ELISA), which was further confirmed under transmission electron microscopy. In conclusion, O35Elgt5 dLOS-TT may act as a vaccine against most M. catarrhalis strains and therefore can be used for further in vivo efficacy studies.
PMCID: PMC3109615  PMID: 21501641
Moraxella catarrhalis; lipooligosaccharide mutant; conjugate vaccine; cross-reactivity; conserved antigen
9.  Molecular evolution of the H6 subtype influenza a viruses from poultry in eastern China from 2002 to 2010 
Virology Journal  2011;8:470.
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.
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.
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.
PMCID: PMC3213047  PMID: 21999472
H6 influenza viruses; poultry; phylogenetic analysis; molecular evolution
10.  Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence 
Microbiology  2011;157(Pt 6):1798-1805.
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.
PMCID: PMC3167914  PMID: 21415117
11.  Novel Reassortant Highly Pathogenic Avian Influenza (H5N5) Viruses in Domestic Ducks, China 
Emerging Infectious Diseases  2011;17(6):1060-1063.
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.
PMCID: PMC3358203  PMID: 21749770
H5N5; highly pathogenic avian influenza; reassortant; domestic ducks; viruses; influenza; China; dispatch
12.  Protection against nontypeable Haemophilus influenzae challenges by mucosal vaccination with a detoxified lipooligosaccharide conjugate in two chinchilla models 
Otitis media (OM) can occur following outset of upper respiratory tract infections. Inhibition of bacterial colonization in nasopharynx (NP) by mucosal vaccination may prevent OM by reducing bacterial invasion of the middle ears (MEs). In this study, 80 chinchillas were intranasally (i.n.) immunized with a detoxified lipooligosaccharide (dLOS)-tetanus toxoid conjugate vaccine of nontypeable Haemophilus influenzae (NTHi) mixed with cholera toxin (CT) or CT alone. All vaccinated animals responded with elevated levels of mucosal and serum anti-LOS antibodies. Two weeks after the last immunization, 40 chinchillas were challenged i.n. with NTHi to evaluate NP colonization and ME infection while the rest of the animals were challenged transbullarly (T.B.) to examine the development of OM. Compared to the control group, the vaccination inhibited not only bacterial colonization in NP and transmission to MEs in the i.n. challenge group but also bacterial colonization in NP and transmission to unchallenged ears in the T.B. challenge group. Though no difference was found in the challenged ears of either group right after the T.B. challenge, an early clearance of NTHi from NP and unchallenged ears as well as less severity of OM in the unchallenged ears were observed in vaccinated animals. Current results along with our previous data indicate that mucosal vaccination is capable of inhibiting NTHi NP colonization and preventing OM occurrence in chinchillas; the i.n. challenge model is preferable for testing the mucosal vaccines while the T.B. challenge model is superior for testing the systemic vaccines.
PMCID: PMC2818210  PMID: 19782149
Mucosal immunization; Nontypeable Haemophilus influenzae; Chinchilla models; Lipooligosaccharide conjugate vaccine
13.  A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998 ▿ †  
Journal of Virology  2009;83(17):8428-8438.
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.
PMCID: PMC2738149  PMID: 19553328
14.  Identification of Two Late Acyltransferase Genes Responsible for Lipid A Biosynthesis in Moraxella catarrhalis 
The FEBS journal  2008;275(20):5201-5214.
Lipid A is a biological component of the lipooligosaccharide (LOS) of a human pathogen, Moraxella catarrhalis. No other acyltransferases except UDP-GlcNAc acyltransferase responsible for lipid A biosynthesis in M. catarrhalis have been elucidated. By informatics, two late acyltransferase genes lpxX and lpxL responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E was constructed and named as O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0) while O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX gene encoded decanoyl transferase and lpxL gene encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, the O35ElpxX was sensitive to bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (p <0.01) or 6 h (p <0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. Meanwhile, the O35ElpxL presented alike patterns as the parental strain except it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl linked secondary acyl chains into the lipid A are important for biological activities of M. catarrhalis.
PMCID: PMC2585779  PMID: 18795947
Moraxella catarrhalis; late acyltransferase; lipooligosaccharide; lpxX; lpxL
15.  Moraxella catarrhalis Bacterium without Endotoxin, a Potential Vaccine Candidate  
Infection and Immunity  2005;73(11):7569-7577.
Lipooligosaccharide (LOS) is a major surface component of Moraxella catarrhalis and a possible virulence factor in the pathogenesis of human infections caused by this organism. The presence of LOS on the bacterium is an obstacle to the development of vaccines derived from whole cells or outer membrane components of the bacterium. An lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction and characterization of an isogenic M. catarrhalis lpxA mutant in strain O35E. The resulting mutant was viable despite the complete loss of LOS. The mutant strain showed significantly decreased toxicity by the Limulus amebocyte lysate assay, reduced resistance to normal human serum, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. Importantly, the mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild-type strain. These data suggest that the null LOS mutant is attenuated and may be a potential vaccine candidate against M. catarrhalis.
PMCID: PMC1273912  PMID: 16239560
16.  Roles of 3-Deoxy-d-manno-2-Octulosonic Acid Transferase from Moraxella catarrhalis in Lipooligosaccharide Biosynthesis and Virulence  
Infection and Immunity  2005;73(7):4222-4230.
Lipooligosaccharide (LOS), a major outer membrane component of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of human infections caused by the organism. However, information about the roles of the oligosaccharide chain from LOS in bacterial infection remains limited. Here, a kdtA gene encoding 3-deoxy-d-manno-2-octulosonic acid (Kdo) transferase, which is responsible for adding Kdo residues to the lipid A portion of the LOS, was identified by transposon mutagenesis and construction of an isogenic kdtA mutant in strain O35E. The resulting O35EkdtA mutant produced only lipid A without any core oligosaccharide, and it was viable. Physicochemical and biological analysis revealed that the mutant was susceptible to hydrophobic reagents and a hydrophilic glycopeptide and was sensitive to bactericidal activity of normal human serum. Importantly, the mutant showed decreased toxicity by the Limulus amebocyte lysate assay, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. These data suggest that the oligosaccharide moiety of the LOS is important for the biological activity of the LOS and the virulence capability of the bacteria in vitro and in vivo. This study may bring new insights into novel vaccines or therapeutic interventions against M. catarrhalis infections.
PMCID: PMC1168618  PMID: 15972513

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