Probiotic supplementation containing Bacillus velezensis enhances expression of immune regulatory genes against pigeon circovirus in pigeons (Columba livia).

AIMS: In this study, we aimed to isolate and evaluate the efficacy of Bacillus velezensis as a probiotic and to assess its activity towards pigeons infected with pigeon circovirus (PiCV). METHODS AND RESULTS: Bacillus velezensis, isolated from pigeon faeces, was orally administered to pigeons for 60 days. After pigeons were challenged with PiCV, the PiCV viral load and expression of indicator genes for innate immunity were detected in spleen tissue and faeces of pigeons. Bacillus velezensis significantly reduced the PiCV viral load in the faeces and spleen of pigeons 5 days post-challenge (dpc). The mRNA expression levels of treated pigeons showed that interferon-gamma (IFN-γ), myxovirus resistance 1 (Mx1), and signal transducers and activators of transcription 1 (STAT1) genes were upregulated, whereas no expression of interleukin-4 (IL-4) was detected. Moreover, toll-like receptor 2 (TLR2) and 4 (TLR4) were significantly upregulated in probiotic-treated pigeons (P < 0·05). CONCLUSIONS: This is the first report showing that probiotic supplementation can effectively enhance the T-helper type 1 immune response and decrease the PiCV viral loads in pigeons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes that the administration of a probiotic strain, B. velezensis, to pigeons can protect against PiCV infection.

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important and common DNA virus that infect pig and can cause immunosuppression and induce apoptosis in the infected cells. To escape the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, and that is why it is difficult to eradicate complex PCV2 infection by relying on vaccines and single compound. At present, there is few literature reports on the effective prevention and treatment of PCV2 infection by a combination of two or more compounds. Previously, we have demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further evaluated. Literatures have proven that Osthole has a variety of pharmacological activities, and we tested the ability of Osthole to inhibit PCV2 replication in cell culture. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and their synergistic anti-apoptotic mechanism. RESULTS: Osthole alone had an anti-PCV2 effect, and then its synergistic anti-PCV2 effect of Osthole and Matrine was better than that of Matrine or Osthole alone as demonstrated by qRT-PCR, IFA and Western blotting results. The anti-apoptotic mechanism of these two compounds by inducing the PERK pathway by PCV2 was elucidated through Annexin V-FITC/PI, JC-1 and Western blotting. Matrine and Osthole combination could inhibit the expression of Cap in Cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. Ribavirin was used as a positive control. CONCLUSIONS: The combination of Osthole and Matrine had the synergistic effect of anti-PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The combination of these two compounds also inhibited PERK apoptosis induced by PCV2 Cap protein, possibly by regulating the level of GRP78. The results formed a base for further studies on the mechanism of anti-PCV2 in vivo using Matrine and Osthole combination and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

Cepharanthine and Curcumin inhibited mitochondrial apoptosis induced by PCV2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an immunosuppressive pathogen with high prevalence rate in pig farms. It has caused serious economic losses to the global pig industry. Due to the rapid mutation of PCV2 strain and co-infection of different genotypes, vaccination could not eradicate the infection of PCV2. It is necessary to screen and develop effective new compounds and explore their anti-apoptotic mechanism. The 13 natural compounds were purchased, with a clear plant origin, chemical structure and content and specific biological activities. RESULTS: The maximum no-cytotoxic concentration (MNTC) and 50% cytotoxic concentration (CC50) of 13 tested compounds were obtained by the cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in PK-15 cells. The results of qPCR and Western blot showed that, compared with the PCV2 infected group, the expression of Cap in Paeonol (0.4 mg/mL and 0.2 mg/mL), Cepharanthine (0.003 mg/mL, 0.0015 mg/mL and 0.00075 mg/mL) and Curcumin (0.02 mg/mL, 0.001 mg/mL and 0.005 mg/mL) treated groups were significantly lowered in a dose-dependent manner. The results of Annexin V-FITC/PI, JC-1, Western blot and ROS analysis showed that the expression of cleaved caspase-3 and Bax were up-regulated Bcl-2 was down-regulated in Cepharanthine or Curcumin treated groups, while ROS and MMP value were decreased at different degrees and the apoptosis rate was reduced. In this study, Ribavirin was used as a positive control. CONCLUSIONS: Paeonol, Cepharanthine and Curcumin have significant antiviral effect. And the PCV2-induced Mitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin.

Antiviral Effect of Epigallocatechin Gallate via Impairing Porcine Circovirus Type 2 Attachment to Host Cell Receptor.

The green tea catechin epigallocatechin gallate (EGCG) exhibits antiviral activity against various viruses. Whether EGCG also inhibits the infectivity of circovirus remains unclear. In this study, we demonstrated the antiviral effect of EGCG on porcine circovirus type 2 (PCV2). EGCG targets PCV2 virions directly and blocks the attachment of virions to host cells. The microscale thermophoresis assay showed EGCG could interact with PCV2 capsid protein in vitro with considerable affinity (Kd = 98.03 ± 4.76 µM), thereby interfering with the binding of the capsid to the cell surface receptor heparan sulfate. The molecular docking analysis of capsid-EGCG interaction identified the key amino acids which formed the binding pocket accommodating EGCG. Amino acids ARG51, ASP70, ARG73 and ASP78 of capsid were found to be critical for maintaining the binding, and the arginine residues were also essential for the electrostatic interaction with heparan sulfate. The rescued mutant viruses also confirm the importance of the key amino acids of the capsid to the antiviral effect of EGCG. Our findings suggest that catechins could act as anti-infective agents against circovirus invasion, as well as provide the basic information for the development and synthesis of structure-based anti-circovirus drugs.

Molecular detection of Hsp90 inhibitor suppressing PCV2 replication in host cells.

Porcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause devastating disease manifestations in pig populations with major economic implications. Our previous research found that Hsp90 is required for PCV2 production in PK-15 and 3D4/31 cells. The aim of this study was to evaluate the effect of Hsp90 inhibitor regulating PCV2 replication and to explore its underlying mechanism. In PK-15 and 3D4/31 cells treated with 17-AAG after viral adsorption, replication of PCV2 was attenuated as assessed by quantitating the expression of viral protein. Following NF-κB activation it was observed that 24hpi with PCV2 was significantly inhibited in the presence of 17-AAG. The expression of Hsp90 associated client proteins in PCV2-infected cells were also reduced in the presence of 17-AAG. However, treatment with MG-132 failed to rescue 17-AAG mediated reduction of PCV2 production in host cells. Thus, Hsp90 regulates PCV2 by modulating cellular signaling proteins. These results highlight the importance of cellular proteins during PCV2 infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.

A radical form of nitric oxide inhibits porcine circovirus type 2 replication in vitro.

BACKGROUND: Porcine circovirus type 2 (PCV2) is the causal agent of postweaning multisystemic wasting syndrome (PMWS), causing large economical losses of the global swine industry. Nitric oxide (NO), as an important signaling molecule, has antiviral activity on some viruses. To date, there is little information on the role of NO during PCV2 infection. RESULTS: We used indirect fluorescence assay (IFA), TCID50, real-time RT-qPCR and western blot assay to reveal the role of NO in restricting PCV2 replication. PCV2 replication was inhibited by a form of NO, NO•, whereas PCV2 was not susceptible to another form of NO, NO+. CONCLUSION: Our findings indicate that the form of NO• has a potential role in the fight against PCV2 infection.

Ivermectin treatment inhibits the replication of Porcine circovirus 2 (PCV2) in vitro and mitigates the impact of viral infection in piglets.

Porcine circovirus 2 (PCV2) capsid protein (Cap) has a nuclear localization signal (NLS) and can enter the nucleus. In this study, ivermectin, a small-molecule nuclear import inhibitor of proteins was used to determine the role of nuclear localization of Cap on PCV2 replication. Observation by fluorescence microscopy of the intracellular localization of Cap and Cap NLS in cells cultured with ivermectin (50 µg/mL) determined that Cap and Cap NLS were located in the cytoplasm; in contrast, for cells cultured without ivermectin, they accumulated in the cell nucleus. Ivermectin treatment also reduced nuclear transport of Cap derived from PCV2 infection as well as PCV2 replication in PK-15 cells. In addition, lower levels of PCV2 in tissues and sera of piglets treated with ivermectin were detected by qPCR. These results established for the first time that ivermectin has potent antiviral activity towards PCV2 both in vitro and vivo.

Taurine attenuates OTA-promoted PCV2 replication through blocking ROS-dependent autophagy via inhibiting AMPK/mTOR signaling pathway.

Previous research found that ochratoxin A (OTA) could promote PCV2 replication by inducing autophagy. The aim of this study is to evaluate the effect of dietary amino acid derivative taurine on OTA-promoted PCV2 replication and explore the underlying mechanism. The results showed that taurine could inhibit OTA-promoted PCV2 replication in PK-15 cells. The effect of taurine could be mediated by its ability to attenuate ROS level and block OTA-promoted autophagy. Indeed, induction of autophagy by rapamycin could suppress the inhibitory effect of taurine on OTA-promoted PCV2 replication. Furthermore, taurine supplementation inhibited 5'AMP-activated protein kinase (AMPK) and activated mammalian target of rapamycin (mTOR). Activation of AMPK by acadesine (AICAR) could suppress the effect of taurine. In conclusion, taurine treatment suppresses autophagy by regulating the ROS/AMPK/mTOR signaling axis, thereby inhibiting OTA-promoted PCV2 replication. These findings provide the rationale for the use of taurine as an intervention against PCV2 infection.

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

Porcine circovirus type 2 inhibits inter-ß expression by targeting Karyopherin alpha-3 in PK-15 cells.

Interferon (IFN)-mediated antiviral response is an important part of host defense. Previous studies reported that porcine circovirus type 2 (PCV2) inhibits interferon production, but the mechanism is still poorly understood. In this study, PCV2 suppresses IFN-ß and IRF3 promoters and mRNA level of IFN-ß induced by ISD or Poly(I:C), but has no effect on the activation of AP-1 and NF-κB. Furthermore, PCV2 decreases the mRNA level of IFN-ß and IFN-ß promoter activity driven by STING, TBK1, IRF3, and IRF3/5D, and causes a reduction in the protein level of nuclear p-IRF3. In addition, PCV2 interrupts the interaction of KPNA3, rather than KPNA4, with p-IRF3. Overexpression of KPNA3 restores IFN-ß promoter activity. These results indicate that PCV2 disrupts the interaction of KPNA3 with p-IRF3 and blocks p-IRF3 translocation to the nucleus, thereby inhibiting IFN-ß induction in PK-15 cells.

The level of decoy epitope in PCV2 vaccine affects the neutralizing activity of sera in the immunized animals.

Viral pathogens have evolved a wide range of tactics to evade host immune responses and thus propagate effectively. One efficient tactic is to divert host immune responses toward an immunodominant decoy epitope and to induce non-neutralizing antibodies toward this epitope. Therefore, it is expected that the amount of decoy epitope in a subunit vaccine can affect the level of neutralizing antibody in an immunized animal. In this study, we tested this hypothesis by generating an antibody specific to the decoy epitope on the capsid protein of porcine circovirus type 2 (PCV2). Using this antibody, we found that two commercial vaccines contained statistically different amounts of the decoy epitope. The vaccine with lower levels of decoy epitope induced a significantly higher level of neutralizing antibody after immunization. This antibody can be used as an analytical tool to monitor the quality of a vaccine from batch to batch.

Genetic and immunogenicity analysis of porcine circovirus type 2 strains isolated in central China.

Porcine circovirus type 2 (PCV2) is an economically important pathogen in domestic pigs and wild boars all around the world. To understand the molecular epidemiology of PCV2 strains circulating in central China and to provide a potential vaccine candidate strain, we analyzed the genetic variations of 46 PCV2 isolates circulating from 2009 to 2016 in Henan Province (24 detected in the field from 2009-2013 and 22 from 2013-2016) and evaluated the efficacy of an isolate as a vaccine candidate strain in a mouse model. We found that PCV2b was the predominant genotype and PCV2b-1C was the main subtype. The PCV2 isolate DF-1, which had a virus titer of 106.5 TCID50/mL and a stable genome, was selected and used to immunize Kunming mice. Enzyme-linked immunosorbent assay (ELISA), immunoperoxidase monolayer assay (IPMA), and virus neutralization test (VNT) results indicated that the DF-1 vaccine candidate strain could elicit a level of specific antibodies and neutralizing antibodies similar to those induced by a commercial vaccine. Polymerase chain reaction (PCR) detection of virus in vaccinated mice after challenge revealed that DF-1 vaccination was effective in clearing the virus in different tissues. Hence, the PCV2 isolate DF-1, a circulating subtype of PCV2b-1C, might be used as a potential vaccine candidate strain.

Selenizing astragalus polysaccharide attenuates PCV2 replication promotion caused by oxidative stress through autophagy inhibition via PI3K/AKT activation.

Our previous studies have shown that oxidative stress could promote the porcine circovirus type 2 (PCV2) replication, and astragalus polysaccharide (APS)/selenium could suppress PCV2 replication. However, whether selenizing astragalus polysaccharide (sAPS) provides protection against oxidative stress-induced PCV2 replication promotion and the mechanism involved remain unclear. The present study aimed to explore the mechanism of the PCV2 replication promotion induced by oxidative stress and a novel pharmacotherapeutic approach involving the regulation of autophagy of sAPS. Our results showed that H2O2 promoted PCV2 replication via enhancing autophagy by using 3-methyladenine (3-MA) and autophagy-related gene 5 (ATG5) knockdown. Sodium selenite, APS, the mixture of sodium selenite and APS, and sAPS significantly inhibited H2O2-induced PCV2 replication promotion, respectively. Among these, sAPS exerted maximal inhibitory effect. sAPS could also significantly inhibit autophagy activated by H2O2 and increase the Akt and mTOR phosphorylation. Moreover, LY294002, the specific phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor, significantly alleviated the effects of sAPS on autophagy and PCV2 replication. Taken together, we conclude that H2O2 promotes PCV2 replication by inducing autophagy and sAPS attenuates the PCV2 replication promotion through autophagy inhibition via PI3K/AKT activation.

Immunomodulatory and antioxidant effects of total flavonoids of Spatholobus suberectus Dunn on PCV2 infected mice.

Oxidative stress plays an important role in the pathogenesis of virus infection and antioxidants are becoming promising candidates as therapeutic agents. This study is designed to investigate the effect of total flavonoids of Spatholobus suberectus Dunn (TFSD) on oxidative stress in mice induced by porcine circovirus type 2 (PCV2) infection. The PCV2 infection leads to significant decrease in thymus and spleen indices, elevation of xanthine oxidase (XOD) and myeloperoxidase (MPO) activities, reduction in GSH level and GSH to GSSG ratio and decline of superoxide dismutase (SOD) activity, indicating the formation of immunosuppression and oxidative stress. TFSD treatment recovered the alteration of viscera index, antioxidant content and activities of oxidative-associated enzymes to a level similar to control. Our findings suggested that PCV2 induced immunosuppression and oxidative stress in mice and TFSD might be able to protect animals from virus infection via regulation of immune function and inhibition of oxidative stress.

The Hsp90 inhibitor 17-DMAG decreases infection of porcine circovirus type 2 in mice.

Although several factors affecting porcine circovirus type 2 (PCV2) infection have been reported, their precise roles are far from clear. The aim of this study was to determine whether 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could significantly affect PCV2 infection and immune responses in BALB/c mice. Intraperitoneal injection of 17-DMAG significantly reduced viral loads in the blood and tissues of mice infected with PCV2, compared with control groups. The 17-DMAG treatment decreased serum interleukin (IL)-10 and tumor necrosis factor(TNF)-α levels, but it did not have a significant effect on the IL-1ß level. These data demonstrate that 17-DMAG is highly effective in suppressing PCV2 replication in BALB/c mice, indicating that it has potential value as an antiviral drug against PCV2 infection.

Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. Recently, we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo, but the underlying mechanism needed further investigation. The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. Our studies also showed that OTA induced autophagy in PK-15 cells, as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, autophagy-related protein 5 (ATG5), and Beclin-1 and the accumulation of green fluorescent protein (GFP)-LC3 dots. OTA induced complete autophagic flux, which was detected by monitoring p62 degradation and LC3-II turnover using immunoblotting. Inhibition of autophagy by 3-methylademine (3-MA) and chloroquine (CQ) significantly attenuated OTA-induced PCV2 replication promotion. The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA. Further studies showed that N-acetyl-L-cysteine (NAC), an ROS scavenger could block autophagy induced by OTA, indicating that ROS may be involved in the regulation of OTA-induced autophagy. Furthermore, we observed significant increases in OTA concentrations in lung, spleen, kidney, liver and inguinal lymph nodes (ILN) and bronchial lymph nodes (BLN) of pigs fed 75 and 150 µg/kg OTA compared with controls in vivo. Administration of 75 µg/kg OTA significantly increased PCV2 replication and autophagy in the lung, spleen, kidney and BLN of pigs. Taken together, it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro.

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 µg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

HMGCR inhibits the early stage of PCV2 infection, while PKC enhances the infection at the late stage.

Porcine circovirus type 2 (PCV2) is the smallest DNA virus, which causes porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD). Due the small size of viral genomic DNA, PCV2 replication predominantly relies on the host factors. In this study, effects of PKC and HMGCR on PCV2 infection were evaluated using real time PCR and western blot. We found that PKC and HMGCR participated in different stages of PCV2 infection. HMGCR works on the early stage of the infection to inhibit the virus infection, while PKC enhances the infection at the late stage. Furthermore, PKC enhances PCV2 replication by activating JNK1/2 and inactivating HMGCR via regulating phosphorylation of these two proteins, while HMGCR can suppress phosphorylation of JNK1/2. The results in the present study will provide new sights in the pathogenesis of PCV2 infection, as well as interactions between host factors during PCV2 infection.

Hsp90 inhibitor reduces porcine circovirus 2 replication in the porcine monocytic line 3D4/31.

Porcine circovirus 2 (PCV2) is an important pathogen of swine, which causes porcine circovirus disease and porcine circovirus-associated diseases (PCVD/PCVAD). However, no effective countermeasures exist to combat this virus infection so far. Recently, heat shock protein 90 (Hsp90) was found to be an important host factor for the replication of multiple viruses and the inhibition of Hsp90 showed significant antiviral effects. Inhibition of Hsp90 by treatment of porcine monocytic line 3D4/31 with geldanamycin (GA), a specific inhibitor of Hsp90, caused a 70 % decrease in viral Cap protein expression. Further, individual knockdown targeting Hsp90α or Hsp90ß with siRNAs resulted in down to 20-25 % of decrease in viral replication, and inhibited the PCV2 titer by approximately 12- and 15-fold, respectively. In addition, we investigated alteration of several cytokine production in PCV2-infected cells following treatment with GA. Then, we found that GA could decrease IL-1ß, IL-6, and IL-12p40 mRNA levels, respectively, by 30, 40, and 40 % in PCV2-infected cells. Our results shed light on the possibility of developing potential therapeutics targeting Hsp90 against PCV2 infection.

Heat shock protein 90 is essential for replication of porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated disease (PCVAD). However, the mechanism of PCV2 replication has not been understood completely. Heat shock protein 90 (Hsp90) plays an important role in viral genome replication, viral genes expression, and viral particle packaging. In this study, we firstly found that inhibition of Hsp90 by pretreatment of host cells with 17-AAG, a specific inhibitor of Hsp90, or blocking Hsp90α/Hsp90ß with siRNA, resulted in significantly reduced viral replication in PK-15 cells. But inhibition of Hsp90 by 17-AAG did not affect PCV2 entry into the host cells. Meanwhile, over-expression of Hsp90α/Hsp90ß enhanced PCV2 genome replication and virion production. In addition, Hsp90ß was enriched in the nuclear zone in the cells infected with PCV2. But it did not interact with the viral Cap/Rep proteins. It suggested that Hsp90 is required for PCV2 production in PK-15 cells culture. It should be helpful for further evaluating the mechanism of replication and pathogenesis of PCV2 and developing novel antiviral therapies.