[Show abstract][Hide abstract] ABSTRACT: Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection.
Frontiers in Microbiology 09/2015; 6:886. DOI:10.3389/fmicb.2015.00886 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial anti-viral signaling protein (MAVS) plays an important role in the type I IFN response. In this study, two feline MAVS transcripts were cloned. Both transcripts have the same open reading frame encoding 523 amino acids. The putative protein shares 76.6 % similarity with canine and exhibits similarity to human, mouse, rat, bovine and porcine, ranging from 46.1 to 65.8 %. Deletion mutant analysis indicated that the transmembrane (TM) domain is necessary for localization in the mitochondrial membrane, and both the caspase activation and recruitment domain and TM domain are indispensible for activating the IFN-β response. Additionally, Sendai virus-induced IFN-β promoter activation was significantly inhibited by siRNA targeting MAVS. Finally, miniMAVS, a second protein encoded by MAVS mRNA, was identified, which interfered with the IFN-β response via the inhibition of NF-κB activation. The identification of MAVS will promote the understanding and control of feline infectious diseases.
Immunologic Research 08/2015; DOI:10.1007/s12026-015-8682-9 · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Feline calicivirus (FCV) is a highly contagious pathogen that causes oral and upper respiratory tract disease in cats. Despite widespread vaccination, the prevalence of FCV remains high. Furthermore, a high gene mutation rate has led to the emergence of variants, and some infections are lethal. To date, there is no effective antiviral drug available for treating FCV infection. Here, we show that lithium chloride (LiCl) effectively suppresses the replication of FCV strain F9 in Crandell-Reese feline kidney (CRFK) cells. The antiviral activity of LiCl occurred primarily during the early stage of infection and in a dose-dependent manner. LiCl treatment also inhibited the cytopathic effect. LiCl treatment exhibited a strong inhibitory effect against a panel of other two reference strains and two recent FCV isolates from China. These results demonstrate that LiCl might be an effective anti-FCV drug for controlling FCV disease. Further studies are required to explore the antiviral activity of LiCl against FCV replication in vivo.
Archives of Virology 08/2015; 160(12). DOI:10.1007/s00705-015-2534-8 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Canine influenza virus A (H3N2) is a newly emerged etiological agent for respiratory infections in dogs. The mechanism of interspecies transmission from avian to canine species and the development of diseases in this new host remain to be explored. To investigate this, we conducted a differential proteomics study in 2-month-old beagles inoculated intranasally with 10(6) TCID50 of A/canine/Guangdong/01/2006 (H3N2) virus. Lung sections excised at 12 h post-inoculation (hpi), 4 days, and 7 days post-inoculation (dpi) were processed for global and quantitative analysis of differentially expressed proteins. A total of 17,796 proteins were identified at different time points. About 1.6% was differentially expressed between normal and infected samples. Of these, 23, 27, and 136 polypeptides were up-regulated, and 14, 18, and 123 polypeptides were down-regulated, at 12 hpi, 4 dpi, and 7 dpi, respectively. Vann diagram analysis indicated that 17 proteins were up-regulated and one was down-regulated at all three time points. Selected proteins were validated by real-time PCR and by Western blot. Our results show that apoptosis and cytoskeleton-associated proteins expression was suppressed, whereas interferon-induced proteins plus other innate immunity proteins were induced after the infection. Understanding of the interactions between virus and the host will provide insights into the basis of interspecies transmission, adaptation, and virus pathogenicity.
Frontiers in Microbiology 04/2015; 6:228. DOI:10.3389/fmicb.2015.00228 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here, we report the complete genome sequence of feline panleukopenia virus (FPLV) strain HRB-CS1, isolated from a dead domestic
cat showing enteric symptoms in China in 2014. The genome of HRB-CS1 was sequenced and analyzed, which will help to understand
the genetic characteristics and evolution of FPLV in China.
[Show abstract][Hide abstract] ABSTRACT: Some highly pathogenic H5N1, H7N9 and H10N8 isolated from China carried six internal genes from H9N2 AIVs, and the key amino acids at 627 in PB2 of these viruses had mutated to K. To investigate the mechanism of increased pathogenicity for H9N2 AIV PB2 627K, we analyzed the difference in mouse lung proteins expression response to PB2 K627E. By iTRAQ method, we found that the mutated K627E contributed to a set of differentially expressed lung proteins, including five upregulated proteins and nine downregulated proteins at 12 h post infection; ten upregulated proteins and twenty five downregulated proteins at 72 h post infection. These proteins were chiefly involved within the cytoskeleton and motor proteins, antiviral proteins, regulation of glucocorticoids signal-associated proteins, pro- and anti-inflammatory proteins. Alteration of moesin, FKBP4, Hsp70, ezrin and sp-A may play important roles in increasing virulence and decreasing lungs antiviral response. Further, three upregulated proteins (moesin, ezrin and sp-A) caused by PB2 K627E were also confirmed in A549 cells. Moreover, overexpression of sp-A in A549 inhibited virus replication and downregulation promoted virus replication. In this study, sp-A as a potential virulence determinant associated H9N2 AIV PB2 E627K mutation was identified using comparative proteomics.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: The pathological damage inflicted by virulent AIV strains is often caused by inducing a positive feedback loop of cytokines in immune cells that cause excessive inflammation. Previous research has shown that a G protein-coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1), plays a crucial role in the development of excessive inflammation in influenza virus infection (Cell 146:861--862, 2011; Cell 146:980--991, 2011). BALB/c mice are common laboratory animals used in research of influenza virus; however the effects of influenza infections on expression patterns of S1PR1 in mice are unknown.
We investigated the expression patterns of S1PR1 in normal BALB/c mice and those infected by two distinct H9N2 AIV strains, one (A/chicken/Guangdong/V/2008,V) highly pathogenic, and the other (A/chicken/Guangdong/Ts/2004,Ts), non-pathogenic in mice, using quantitative PCR and immunohistochemistry (IHC) to detect S1PR1 mRNA and protein, respectively.
S1PR1 mRNA was ubiquitously expressed in all the tissues examined, and significant differences were seen in mRNA expression between infected Ts, V and control mice in detected tissues, heart, liver, spleen, kidney and brain. S1PR1 protein was expressed in the cytoplasm and also demonstrated quantitative changes in expression in the various tissues between mice infected with the two strains of AIV.
Our results provided the first look at differences in S1PR1 expression patterns in BALB/c mice infected by non-pathogenic and highly pathogenic H9N2 influenza viruses. This information will not only be helpful in designing experiments to better understand the role of S1PR1 in virus-host interactions but also in developing novel anti-influenza agents to minimize the mortality and morbidity associated with highly virulent strains in avian and human populations.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Cases of human infections with H5N1 avian influenza viruses have been reported all over the world with the reason of direct contact with sick or diseased poultry, which suggests the direct contact with poultry may be one of the major risk factors for human infection. OBJECTIVES: In this study, we estimated the seroprevalence of antibodies against avian influenza A (H5N1) virus in veterinarians with exposure to avians. STUDY DESIGN: From May 21, 2011 through April 22, 2012, 406 veterinarians exposure to poultry in Guangdong province were interviewed a questionnaire. A serum specimen was collected from participants to test for H5N1 antibodies by HI and NT assay. RESULTS: None of the 406 sera from occupationally exposed veterinarians was positive according to the HI test and the NT test with the H5N1 AIV. CONCLUSION: Our seroepidemiologic survey suggests that the risk of avian-to-human transmission of the H5N1 AIV is very low based on the samples that we tested. However, prevention regarding the risk of H5N1 AIV transmission is essential and should be recommended as public health measures.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 01/2013; 56(4). DOI:10.1016/j.jcv.2012.12.009 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Except severe pulmonary disease caused by influenza virus infection, an impaired immune system is also a clinic characteristic. However, the mechanism(s) of influenza virus infection-induced depletion of B cells was unknown. Here, we compared the effect of two variant virulence H9N2 virus infections on mouse B cells. Our study found that the infection with highly pathogenic virus (V) of led to depletion of spleen B cells and bone marrow (BM) early B cells, compared to lowly pathogenic virus (Ts). Moreover, high apoptosis and cell cycle arrest in spleen and BM were detected, suggesting important factors for the reduction of B cells in both organs. Further, this effect was not caused by virus replication in spleen and BM. Compared to Ts virus infection, V virus resulted in higher glucocorticoids (GCs) and lower leptin level in plasma. Intraperitoneal GCs receptor antagonist RU486 injection was sufficient to prevent the loss of spleen B cell and BM pro- and immature B cells, but similar result was not observed in leptin-treated mice. Depletion of spleen B cells and BM pro-B cells was also reversed by chemical sympathectomy mediated by the norepinephrine (NE) analog 6-hydroxydopamine (6-OHDA), but the treatment didn't affect the GCs level. This study demonstrated that depletion of B cells induced by H9N2 AIV was dependent on HPA axis and sympathetic response.
PLoS ONE 12/2012; 7(12):e51029. DOI:10.1371/journal.pone.0051029 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza A viruses occasionally cause large epidemics and kill thousands every year. While little is known about the mechanism of cell fusion in diseases, especially influenza virus infected and protection from Amaryllidaceae Alkaloids. The two compounds lyconne (AA1) and haemanthamme (AA3) were obtained from bulbs ofL. radiate, exhibited anti-influenza activity after influenza virus entry cells. The two compounds were investigated for in vitro displaying different levels of resistance to pro-apoptotic stimuli. Seven influenza viruses were used, A/CK/GD/178/04 (H 5N, 178), A/DK/GD/212/04 (H 5N l5 212), A/swine/GD/166/06 (H 3N 2,166), A/CK/HN/170/03 (H 1N 1, 170), A/Puerto Rico/8/34 (H 1N 1, PR8), A/Ck/GD/400/07 (H 9N 2, 400), A/Ck/GD/228/04 (H 9N2, 228), the two compounds exhibited potency in the single-digit micromolar range. The studies also showed that AA1 and A A3 exerted their in vitro anti-influenza activity through cytostatic rather than cytotoxic effects. Many viruses interact with the host cells to change their own growth which they favor the speed. The cells infected with virus, growth of MDCK cells was slowed down by arresting cell cycle at Gl/S phase. With the compound treated, the growth cycle was decreased in S phase. With H 5N 1, influenza virus treated, the cytoskeleton of cells was changed while with the compound treated the protection of cytoskeleton was obviously protected. The data showed differences between drug treated cells and virus infected cells, provided a basis to further explore cell fusion and anti-influenza mechanism of the two compounds.
Journal of Animal and Veterinary Advances 12/2012; 11(14):2485-2492. DOI:10.3923/javaa.2012.2485.2492 · 0.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Few drugs are currently licensed to treat influenza A infection, and new therapies are needed, especially for highly pathogenic strains. Traditional medicinal plants, such as Lycoris radiata, are a potential source of new antiviral agents. Objective To test 15 Amaryllidaceae alkaloids isolated from the bulbs of L. radiata in vitro for antiviral activities against influenza virus type A, A/Chicken/GuangDong/178/2004 (H5N1, 178). Methods Antiviral activities of the compounds were tested in time-of-addition assays, hemagglutination inhibition (HI) assays, neuraminidase (NA) activity assays, and viral entry inhibition assays using H5N1-HIV pseudoviruses. Effects of the compounds on localization and activity of the viral ribonucleoprotein (RNP) were determined by immunofluorescence and an RNP minigenome assay, respectively. Results Among the alkaloids, lycorine (AA1), hippeastrine (AA2), hemanthamine (AA3) and 11-hydroxy vittatine (AA4) exhibited antiviral activities, with EC90 values of 0.52, 82.07, 4.15, and 13.45 mu m, respectively. These compounds did not affect the function of the outer membrane proteins or the viral entry process and viral RNP activity. As AA1 and AA3 exhibited stronger antiviral activities, they were further analyzed. Intracellular nucleoprotein (NP) localization showed that AA1 and AA3 inhibited the RNP complex in the nucleus at an early stage of a single-round and multi-round of replication. Conclusion Four Amaryllidaceae alkaloids were first determined that could exert anti-influenza activities after virus entry into cells. Furthermore, AA1 and AA3 could inhibit nuclear-to-cytoplasmic export of the RNP complex of virus replication. Thus, these compounds may be developed further as anti-influenza drug candidates.
Influenza and Other Respiratory Viruses 11/2012; 7(6). DOI:10.1111/irv.12035 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a potential threat to both human and avian health. We report a complete genome sequence of a novel reassortant H3N2 avian influenza virus. Phylogenetic analysis showed that HA and NA showed the highest sequence homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2). However, the internal genes had the highest sequence homologies with those of H6 and H7 subtypes. The data provide further evidence of the existence of a natural reassortant H3N2 strain in southern China.
Journal of Virology 09/2012; 86(17):9553-4. DOI:10.1128/JVI.01523-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.
PLoS ONE 06/2012; 7(6):e40118. DOI:10.1371/journal.pone.0040118 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB(2) residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that V(K627) and rTs(E627K) infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rV(K627E) and Ts(E627). Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by V(K627) and rTs(E627K) infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung. These data demonstrated that V(K627) and rTs(E627K) infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV.
PLoS ONE 06/2012; 7(6):e38233. DOI:10.1371/journal.pone.0038233 · 3.23 Impact Factor