Inhibitory Effects Of Nitric Oxide On Porcine Circovirus Type2Replication In Vitro And In Vivo

Porcine circoviruses (PCVs) are belonging to the family Circoviridae which can be divided into two major genotypes: nonpathogenic PCV type1(PCV1) and pathogenic PCV type2(PCV2). Post-weaning multisystemic wasting syndrome (PMWS) is identified by the presence of porcine circovirus type2(PCV2) as the primary causative agent, in addition, many other diseases, often referred to as porcine circovirus diseases (PCVD) or porcine circovirus associated diseases (PCVAD), are also induced by PCV2, including porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproductive failure, granulomatous enteritis, exudative epidermitis, necrotizing lymphadenitis and congenital tremor (CT), etc. Horizontal and vertical transmission are all determined during PCV2infection. PCV2can be shed through various ways like feces, urine, milk, serum, semen and so on, which is often co-infected with other pathogens, including bacteria, viruses and parasites, etc. So it is a great challenge to prevent and control PCV2infection. To date, pathogenesis of PCV2is still unclear to us, and there is no specific remedy against PCV2infection. We mainly depend on vaccines to reduce outbreaks of PCVAD. However, the protective efficiency of vaccines is sometimes not so satisfied because of various influencing factors. NO, as a signaling molecule, has complex and diverse functions in physiological and pathophysiological phenomena, exerting microbistatic/microbicidal effects on a broad spectrum of pathogens, including bacteria, parasites, fungi and helminths, etc. NO can also inhibit replication and infection of many DNA and RNA viruses. Few studies were performed between NO and PCV2replication. We confirmed the inhibitory effects of NO on PCV2replication by in vitro and in vivo studies and underlying mechanisms of these effects were also explored, by which we assessed partial pathogenesis of PCV2, moreover, all the results provided new insights into prevention and control of PCVAD.1. Inhibitory effects of nitric oxide on porcine circovirus type2replication via NF-κB pathwayThis study was conducted to investigate the effects of NO on PCV2replication in Dulac cells. S-nitroso-acetylpenicillamine (SNAP) was used as an exogenous nitric oxide donor, while acetylpenicillamine (NAP), a structure analog but not a NO donor, was used as a durg control. After incubation with the drugs for6h, the cells were inoculated with PCV2in the presence of the drugs for additional72h. We evaluated effects of NO on PCV2replication by measuring NO production of cell culture supernatant, percentages of PCV2-infected cells, virus titers and viral DNA copies. Moreover, The pNF-KB-luc reporter gene constructs were transfected into Dulac cells, Activities of NF-κB-dependent luciferase of cell extracts were measured to explore whether NO affected PCV2replication through NF-κB pathway. The results indicated that SNAP showed a dose dependent effect on NO production. PCV2replication was inhibited by SNAP in a dose-dependent manner. Furthermore, whether Dulac cells were infected with PCV2or not, the NF-κB activity was suppressed by NO in a dose dependent manner. In conclusion, inhibited PCV2replication mediated by exogenous NO in vitro is associated with down-regulation of NF-κB pathway.2. NO inhibits PCV2replication in vitro mediated by NO*In the present study, NO were generated from SNP or SNP in the presence of ascorbate. SNP-ascorbate (Vc) generates mainly NO*in culture medium while NO+is the major product of SNP alone. PCV2-infected Dulac cells were treated with SNP or SNP+Vc. Data obtained from our study demonstrated that both of the donors exerted dose dependent effects on NO release. Virus titers from SNP+Vc-treated group were significantly lower than that from infected control (P<0.01). In addition, PCV2-infected cells and viral DNA copies from the samples treated with SNP+Vc also decreased significantly in relation to those from infected control (P<0.05). However, almost no antiviral activities of SNP or Vc alone were indicated during PCV2infection. In conclusion, inhibition of NO on PCV2replication was mediated by NO’, independent on NO+3. Comparison of effects of exogenous and endogenous NO on PCV2replication in vitroIn this study, nitrosoglutathione (GSNO) was used as an exogenous NO donor, while L-arginine was used as the precursor of endogenous NO. Citrulline, a metabolic product of L-arginine, was used as the drug control. The effects of the drugs on PCV2replication were investigated by measuring kinetics of NO production, PCV2-infected cells, virus titers and viral DNA copies. Results were shown as follows:when compared with non-treated group, NO increased significantly in GSNO-treated group (P<0.01), reaching a plateau at around72, in addition, GSNO exerted a dose dependent effect on NO production. However, L-arginine and citrulline did not generate NO. In comparison with infected control, no significant alteration of PCV2-infected cell, virus titers and viral DNA copies was observed in L-arginine/citrulline treated group (P>0.05). Virus titers from GSNO-treated group were significantly lower than that from infected control (P<0.05), while PCV2-infected cells and viral DNA copies from GSNO-treated group decreased significantly in relation to those from infected control (P<0.01). In conclusion, PCV2replication was inhibited by exogenous NO generated from GSNO, moreover, L-arginine exerted no effects on PCV2replication in Dulac cells because it did not generate endogenous NO.4. Effects of hemoglobin on inhibition of PCV2replication induced by NO in vitroInhibitory effects of exogenous NO generated from GSNO on PCV2replication in vitro was confirmed in our previous study of chapter3. In the current study, Dulac cells were treated with GSNO and hemoglobin (Hb), which was used as a NO scavenger. This study was conducted to investigate whether anti-PCV2activities of exogenous NO were affected by the NO scavenger. The results obtained revealed that NO levels were lower from the groups treated with higher concentrations of Hb, manifesting in a dose dependent manner. In addition, NO from the groups treated with GSNO plus Hb all decreased to some degree when compared with that from the group treated with GSNO alone. In comparison with the group treated with GSNO alone, increased observations were indicated in PCV2-infected cells, virus titers and viral DNA copies from the groups treated with GSNO plus Hb, and Hb exerted a dose dependent effect on these parameters as noted above. furthermore, the parameters from the group treated with GSNO plus20μM Hb showed no significant difference in relation to those from the infected control group (P>0.05). In summary, Hb effectively scavenged NO generated from GSNO, moreover, the inhibitory effects of NO on replication in vitro could be blocked by Hb in a dose dependent manner, which provided another evidence that PCV2replication was inhibited by NO in vitro.5. Suppressed PCV2replication induced by exogenous NO in BALB/c mice In the present study, BALB/c mice were artificially inoculated with PCV2, meanwhile, intraperitoneal injection was also performed with an exogenous NO donor, GSNO. We evaluated the effects of NO on PCV2-infected mice by measuring average daily weight gains, indexes of immune organs, PCV2-positive tissue and serum samples, PCV2DNA copies of serum samples as well as PCV2-specific antibodies. The data were indicated as follows: Increased average daily weight gains and indexes of immune organs were observed, while decreased tendency was shown in PCV2-positive tissue and serum samples and PCV2DNA copies, in addition, whether mice treated with GSNO or not, PCV2-specific antibodies, detected by indirect fluorescence assay, could not be recorded. Taken together, PCV2replication in BALB/c mice was markedly inhibited by exogenous NO generated from GSNO, although no alteration of PCV2-specific antibodies was induced by NO.