Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53.

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2'-5'-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.

Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model.

BACKGROUND: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk' regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. METHODS: JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. FINDINGS: NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 µg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 µg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. CONCLUSIONS: Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.

Molecular characterization of enterohemorrhagic E. coli O157 isolated from animal fecal and food samples in Eastern China.

OBJECTIVE: To elucidate the extent of food contamination by enterohemorrhagic Escherichia coli (EHEC) O157 in Eastern China. METHODS: A total of 1100 food and animal fecal samples were screened for EHEC O157. Then, molecular characterization of each isolate was determined. RESULTS: EHEC O157 was isolated as follows: pig feces, 4% (20/500); cattle feces, 3.3% (2/60); chicken feces, 1.43% (2/140); pork, 2.14% (3/140), milk, 1.67% (1/60); and chicken meat, 1.67% (1/60). The stx1, stx2, eae, and hlyA genes were present in 26.7% (8/30), 40% (12/30), 63.3% (19/30), and 50% (15/30) of the O157 isolates, respectively. Molecular typing showed that strains from fecal and food samples were clustered into the same molecular typing group. Furthermore, the isolates from pork and pig feces possessed the same characterization as the clinical strains ATCC35150 and ATCC43889. Biofilm formation assays showed that 53.3% of the EHEC O157 isolates could produce biofilm. However, composite analyses showed that biofilm formation of EHEC O157 was independent of genetic background. CONCLUSIONS: Animal feces, especially from pigs, serve as reservoirs for food contamination by EHEC O157. Thus, it is important to control contamination by EHEC O157 on farms and in abattoirs to reduce the incidence of foodborne infections in humans.

Stabilization of p53 in influenza A virus-infected cells is associated with compromised MDM2-mediated ubiquitination of p53.

Influenza A virus (IAV) induces apoptosis of infected cells. In response to IAV infection, p53, a tumor suppressor involved in regulating apoptosis and host antiviral defense, accumulates and becomes activated. This study was undertaken to examine the mechanism of p53 accumulation in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells results from protein stabilization, which was associated with compromised Mdm2-mediated ubiquitination of p53. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of polyubiquitinated p53 were not detectable in the presence of the proteasome inhibitor MG132 and were less sensitive to proteasome-mediated degradation. IAV infection did not affect the abundance of Mdm2, a major ubiquitin E3 ligase responsible for regulating p53 ubiquitination and degradation, but weakened the interaction between p53 and Mdm2. Viral nucleoprotein (NP) was able to increase the transcriptional activity and stability of p53. Furthermore, NP was found to associate with p53 and to impair the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, demonstrating its role in inhibiting Mdm2-mediated p53 ubiquitination and degradation.

Identification of human guanylate-binding protein 1 gene (hGBP1) as a direct transcriptional target gene of p53.

Human guanylate-binding protein 1 (hGBP1) plays an important role in antitumor and antiviral immune responses. Here, we show that tumor suppressor p53 positively regulated hGBP1 transcription via binding to the p53 response element (p53RE) present in the hGBP1 promoter region. p53 activation by 5-fluorouracil significantly increased hGBP1 expression in wild-type p53 cells, but not in p53-null cells. Knockdown of p53 expression remarkably impaired hGBP1 expression induced by 5-fluorouracil, type I interferon treatment, or influenza A virus infection. Among three deductive p53REs present in the hGBP1 promoter region, two p53REs were found to be transactivated by p53.

Detection and new genetic environment of the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in methicillin-resistant Staphylococcus aureus of swine origin.

OBJECTIVES: To investigate the genetic basis of pleuromutilin resistance in porcine methicillin-resistant Staphylococcus aureus (MRSA) and to map the genetic environment of the identified plasmid-borne resistance gene. METHODS: Seventy porcine MRSA isolates, which exhibited high MICs of tiamulin, valnemulin and retapamulin, were investigated for pleuromutilin resistance genes and mutations. They were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). Plasmid DNA was extracted from the lsa(E)-positive strains and transferred to S. aureus RN4220 for selection of resistance plasmids. The plasmid-borne lsa(E) gene region was sequenced and 10 overlapping PCR assays for the analysis of the genetic environment of lsa(E) were developed. RESULTS: All 70 MRSA isolates were ST9 (MLST)-t899 (spa)-IVa (SCCmec). Sixteen isolates carried the lsa(E) gene; all others were negative for known pleuromutilin resistance mechanisms. An lsa(E)-carrying plasmid of ∼41 kb was detected in a single isolate. Sequence analysis revealed that the lsa(E) gene was located in a multiresistance gene cluster, which showed partial homology to clusters identified in MRSA, methicillin-susceptible S. aureus (MSSA) and Enterococcus faecalis. PCR analysis of the remaining isolates revealed a partly deleted multiresistance gene cluster in 6/15 isolates and solely the lsa(E) gene without the known flanking regions in 9/15 isolates. CONCLUSIONS: We identified the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in porcine MRSA isolates. The multiresistance gene cluster in which lsa(E) was located differed from the previously described ones found in human MRSA/MSSA or in E. faecalis. The location of lsa(E) on a multiresistance plasmid facilitates its persistence and dissemination.

Proteomic analysis of swine serum following highly virulent classical swine fever virus infection.

BACKGROUND: Classical swine fever virus (CSFV) belongs to the genus Pestivirus within the family Flaviviridae. Virulent strains of classical swine fever virus (CSFV) cause severe disease in pigs characterized by immunosuppression, thrombocytopenia and disseminated intravascular coagulation, which causes significant economic losses to the pig industry worldwide. METHODS: To reveal proteomic changes in swine serum during the acute stage of lethal CSFV infection, 5 of 10 pigs were inoculated with the virulent CSFV Shimen strain, the remainder serving as uninfected controls. A serum sample was taken at 3 days post-infection from each swine, at a stage when there were no clinical symptoms other than increased rectal temperatures (≥ 40 °C). The samples were treated to remove serum albumin and immunoglobulin (IgG), and then subjected to two-dimension differential gel electrophoresis. RESULTS: Quantitative intensity analysis revealed 17 protein spots showing at least 1.5-fold quantitative alteration in expression. Ten spots were successfully identified by MALDI-TOF MS or LTQ MS. Expression of 4 proteins was increased and 6 decreased in CSFV-infected pigs. Functions of these proteins included blood coagulation, anti-inflammatory activity and angiogenesis. CONCLUSION: These proteins with altered expression may have important implications in the pathogenesis of classical swine fever and provide a clue for identification of biomarkers for classical swine fever early diagnosis.

Characterization of nonstructural protein 3 of a neurovirulent Japanese encephalitis virus strain isolated from a pig.

BACKGROUND: Japanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5'-triphosphatase, and plays important roles in viral replication and pathogenesis. RESULTS: We characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum. CONCLUSIONS: The NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.

Icariin induces the expression of toll-like receptor 9 in ana-1 murine macrophages.

Icariin is the major pharmacologically active compound of Herba epimedii which has been used as a tonic, aphrodisiac and an antirheumatic in traditional Chinese medicine. This study analysed the effect of icariin on the expression of Toll-like receptor 9 (TLR9) which plays an important role in regulation of the innate immune response. Stimulation of Ana-1 murine macrophages with icariin induced a significant dose-dependent expression of TLR9, and its mRNA expression which increased from 3 h post-treatment was approximately five-fold that of DMSO-treated cells. Several molecules, such as myeloid differentiation factor 88, tumor necrosis factor-α and interleukin 6, which are involved in the TLR9 downstream signaling pathway, were also significantly up-regulated in response to icariin stimulation. Our findings demonstrated that icariin is able to induce the expression of TLR9.

Glycyrrhetinic acid extracted from Glycyrrhiza uralensis Fisch. induces the expression of Toll-like receptor 4 in Ana-1 murine macrophages.

Glycyrrhetinic acid (GA) is an active component of licorice root that has long been used as a herbal medicine for the treatment of peptic ulcer, hepatitis, and pulmonary and skin diseases in Asia and Europe. In this study, we analyzed the effect of GA extracted from Glycyrrhiza uralensis Fisch. on the expression of Toll-like receptors (TLRs) that play key roles in regulating the innate immune response against invading pathogens. Stimulation of Ana-1 murine macrophages with GA induced a significant dose-dependent expression of TLR-4, and its mRNA expression that increased from 3-h post-treatment was approximately fivefold over the level in the mock-treated cells. No endotoxin contamination contributed to the GA-induced TLR-4 expression, because polymyxin B treatment did not alter the upregulated expression of TLR-4 in GA-treated cells. Several molecules, such as myeloid differentiation factor 88, interferon-ß, and interleukin-6, which are involved in the TLR-4 downstream signaling pathway, were upregulated significantly in response to GA stimulation. Our findings demonstrate that GA is able to induce the expression of TLR-4 and activate its downstream signaling pathway.

Changes in the porcine peripheral blood mononuclear cell proteome induced by infection with highly virulent classical swine fever virus.

Leukopenia and immunosuppression are characteristic clinical manifestations of classical swine fever and peripheral blood mononuclear cells (PBMCs) are major targets of classical swine fever virus. To investigate proteomic expression changes in swine PBMCs during lethal CSFV infection, proteins of PBMCs from five lethally CSFV-infected pigs were resolved by two-dimensional electrophoresis followed by mass spectrometry. Quantitative intensity analysis revealed that 66 protein spots showed altered expression, 44 of which were identified as 34 unique proteins by MALDI-TOF-MS/MS. Cellular functions of these proteins included cytoskeletal, energy metabolism, protein translation and processing, antioxidative stress, heat shock and blood clotting. Western blot analysis confirmed the upregulation of annexin A1 and downregulation of cofilin. Identification of these changed levels of expression provides an understanding at the molecular level of the response of in vivo target cells to CSFV infection and of the pathogenic mechanisms of leukopenia and immunosuppression induced by the virus.

The non-structural (NS1) protein of influenza A virus associates with p53 and inhibits p53-mediated transcriptional activity and apoptosis.

NS1 protein of influenza A virus is involved in regulating the apoptosis of infected cells. We found that exogenously expressed NS1 was able to associate with the tumor suppressor p53 that plays an essential role in regulating apoptosis of influenza A virus-infected cells. Exogenous expression of NS1 resulted in inhibition of p53-mediated transcriptional activity and apoptosis. The p53 inhibitory domain of NS1 was located between amino acids 144 and 188. This domain is necessary for NS1 to inhibit p53 activity, but it requires additional region(s) to cooperatively exert this inhibitory function.

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities.

BACKGROUND: Marek's disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Marek's disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction. RESULTS: We confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ∆Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ∆Meq was found to act as a negative regulator of Meq. CONCLUSIONS: The Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis.

Antigenic differentiation of classical swine fever viruses in China by monoclonal antibodies.

The phylogenetic diversity of classical swine fever virus (CSFV) in China has been extensively studied previously, with the report of the classification of Chinese CSFVs into four subgroups within two of the established genotypes, but the antigenic differences amongst Chinese CSF viruses still remain unknown. To address this issue, 21 CSFV field strains isolated in China between 1996 and 2006 were grown in cell culture and characterized in comparison with two Chinese reference strains: a virulent strain Shimen and a vaccine strain CSF lapinized virus (hog cholera lapinized virus in China, HCLV), by indirect immunofluorescence assay (IFA) with a panel of 28 monoclonal antibodies (mAbs) against four pestiviruses, CSFV, bovine viral diarrhoea virus-1 (BVDV-1), bovine viral diarrhoea virus-2 (BVDV-2) and border disease virus (BDV). All 23 CSFV strains reacted only with CSFV-specific mAbs, not with those raised against BVDV-1, BVDV-2 and BDV. Of the former mAbs, those directed against CSFV E2 protein recognized more isolates than those directed against E(rns) and NS2/3. Of nine CSFV E2-specific mAbs used, WH303 and WH302 reacted with all 23 strains, confirming their value in differentiating CSFV from other pestiviruses. Furthermore, different strains had different patterns of reactivity with CSFV-specific mAbs, and mAbs other than WH303 and WH302 did not recognize all strains. This study provides the first evidence for the existence of antigenic differences among Chinese CSFVs.

In vitro inhibition of classical swine fever virus replication by siRNAs targeting Npro and NS5B genes.

Classical swine fever (CSF) is a highly contagious disease of pigs, which causes important economic losses worldwide. In the present study, the specific effect of RNA interference on the replication of CSF virus (CSFV) was explored. Three species of small interfering RNA (siRNA), targeting different regions of CSFV Npro and NS5B genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with CSFV, the viral proliferation within the cells was examined by indirect immunofluorescence microscopy. At 72 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, while most untreated virus-infected cells were positive. Treatment with the siRNAs caused a 4-12-fold reduction in viral genome copy number as assessed by real time RT-PCR. Transfection with the siRNAs also suppressed the production of infectious virus by up to 467-fold as assessed by TCID50 assay. These results suggested that the three species of siRNAs can efficiently inhibit CSFV genome replication and infectious virus production, with the inhibition persisting for 72-84 h.

Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.

A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus.

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.

[Replicative kinetics of classical swine fever virus in PK-15 cells].

In order to understand the replication kinetics of classical swine fever virus (CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37 degrees C in 5% CO2 environment when growing to 80% confluence, the cells were infected with CSFV strain Shimen at 100 TCID50 per well. At various time post infection (p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay (IFA), RNA replication using reverse transcription real-time PCR and viral production using titration of TCID50. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining, the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However, the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID50 demonstrated that the production of live virions increased at 8h and peaked between 48 - 72 hrs p.i. without significant lose of titer.

Tumor suppressor p53 protects mice against Listeria monocytogenes infection.

Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection.

Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells.

Glycoprotein E2 of classical swine fever virus (CSFV) is a key determinant and major immunogen for viral entry and immunity, but little is known about its interaction with host proteins. In a previous study, we showed by proteomic analysis that cellular membrane protein annexin 2 (Anx2) was up-regulated in PK-15 cells following CSFV infection, but its function in CSFV replication remains unknown. In the present study we observed the interaction of Anx2 with CSFV E2 following infection of PK-15 cells by co-immunoprecipitation (Co-IP), mass spectrometry, Western blot and confocal laser scanning microscopy. The interaction between CSFV E2 and Anx2 was further confirmed in an E2-expressing PK-15 cell line, in which up-regulation of Anx2 was also observed, indicating that E2 alone can interact with, and increase, the expression of Anx2 protein. Further studies showed that siRNA-mediated knock-down and plasmid-mediated over-expression of Anx2 in PK-15 cells inhibited and increased CSFV replication and proliferation respectively. Remarkably, treatment of PK-15 cells with Anx2-specific polyclonal antibody prior to virus infection significantly inhibited CSFV multiplication, indicating that Anx2 is a cellular membrane protein likely associated with CSFV entry into cells. In conclusion, Anx2 is the novel host protein identified to interact with CSFV E2 and promote CSFV multiplication. These observations provide support for the potential use of Anx2 as a cellular target for the development of novel anti-CSFV therapies.