| Bovine viral diarrhea(BVD), with the characteristics of diarrhea, acute/chronic mucosal inflammation, reproductive disorders, persistent infection and immune tolerance, is one of the important infectious diseases caused by bovine viral diarrhea virus(BVDV). BVDV is a positive single-strand RNA virus, belongs to the genus Pestivirus in the family Flaviviridae with the other two members of classical swine fever virus(CSFV) and border disease virus(BDV). It is reported that BVDV have a broad host tropism, apart from cattle, sheep, pig, deer, camel and many other animals can be infected and caused the related diseases, which made it difficult to eliminate from the natural environment and finally caused the huge threat to the entire livestock industry. Pregnant cows infected by BVDV often result in immune tolerance and persistent infection(PI). Persistent infected cattles did not develop detectable antibodies specificly against the virus after vaccination that lead to BVDV persistently present in the body and shed to the ambient and infect other animals, which made them become the most important source of infection. The present control measures are difficult to eradicate BVDV from the body or the herds. Therefore, a new strategy is needed for controlling BVD.Heme oxygenase-1(HO-1), an important anti-oxidative stress protein in the body, possesses numerous biological functions including anti-oxidant, anti-inflammatory, and anti-apoptotic. Recent studies have shown that HO-1 also has significant antiviral properties. HO-1 expression was supressed following infection of hepatitis B virus(HBV), hepatitis C virus(HCV) and porcine reproductive and respiratory syndrome virus(PRRSV), up-regulation of HO-1 expression could inhibit their replication in vitro or in vivo. However, on the contrary, infection of classical swine fever virus(CSFV) to the cells could increase HO-1 expression, and down-regulation of HO-1 expression could inhibit its proliferation. These findings suggest that HO-1 plays a different roles for different virus infection. Does HO-1 have regulation effect on BVDV infection and if has, what is the role? In this study, we conducted a serial experimentations to answer these questions. The results from this study were as follows: 1. BVDV infection decreases the expression level of HO-1The expression of BVDV, HO-2 and two antioxidant response element(AER)-driven genes including HO-1 and NADPH quinone oxidoreductase 1(NQO1) was examined at different times after BVDV infected MDBK cells. The result showed that both HO-1 and NQO1 expression were decreased remarkably in BVDV infected cells, the reduction of HO-1 expression was progressive and time dependent. But there is no significant change in expression of HO-2 both in mRNA or protein level. The results suggest that BVDV infection significantly decreasethe abundance of HO-1 in MDBK cells and the reduction of HO-1 is related to antioxidant response, and BVDV infection has no effect on HO-2 expression. 2. Upregulation of HO-1 expression represses BVDV replicationTo investigate whether HO-1 could affect BVDV replication, a classical inducer of HO-1, CoPP, was incubated with MDBK and the effect of HO-1 expression on BVDV replication was examined. The result showed that HO-1 but not HO-2 was substantially induced by CoPP and cell viability was no significantly affected. BVDV RNA and NS5 B protein as well as virus titration were decreased in a CoPP concentration-dependent manner. Treatment of MDBK cells pre- or post-infection with CoPP significantly reduced BVDV repilication. The results demonstrate that CoPP-induced HO-1 up-regulation could inhibit BVDV replication without affection of the cell viability.To determine whether inhibition of BVDV replication by CoPP is HO-1 dependent, a recombinant adenovirus that expression of HO-1 was constructed and used to inoculate MDBK cells before BVDV infection. The results showed that HO-1 was remarkablely increased after cells incubated with recombinated adenovirus, but the abundence of BVDV RNA and NS5 B protein as well as virus titration was significantly decreased. The results suggest that specific up-regulation of HO-1could inhibit BVDV replication. 3. Decreased basal levels of HO-1 promotes BVDV replicationSince up-regulation of HO-1 by CoPP or recombinant adenovirus could suppress BVDV infection, we wanted to know whether reduction of HO-1 expression could affect BVDV infection in cells. siRNA was used to specifically decrease basal level of HO-1 in MDBK cells, and the effect on BVDV replication was determined. The result showed that the expression of HO-1 was considerably reduced after transduction of siRNA to the cells, but BVDV RNA and virus titers in the cell supernant were markedly increasd. It is suggested that decreased basal levels of HO-1 could promotes BVDV replication. 4. CoPP-mediated attenuation of BVDV infection is HO-1 dependentTo further confirm the inhibition of BVDV replication by CoPP is HO-1 dependent, siRNAs transducted MDBK cells were infection of BVDV followed by treated with CoPP, BVDV NS5 B protein and virus titer were determined. The results indicated that HO-1 knocked-down by siRNA partially reversed the inhibitory effect of CoPP treatment on BVDV replication, which suggesting that CoPP-mediated inhibition of BVDV replication is HO-1 dependent. 5. The effect of HO-1 inhibiting BVDV replication is related to HO-1 byproductsTo further understand the mechanism of the effect of HO-1 expression on BVDV inhibition, three chemical products, biliverdin, carbon monoxide-releasing molecules 2(CORM-2) and ferric chloride(FeCl3), which mimic the natural HO-1 byproducts of biliverdin, carbon monoxide(CO) and iron(Fe2+), were used to treatment of BVDV infected cells. The results showed that the infection of BVDV in cells treated with biliverdin or CORM-2, but not FeCl3, was significantly decreased. The results indicated that biliverdin and CO was responsible for the HO-1 mediated inhibition of BVDV replication.In summary, the study demonstrated that BVDV infection decreases the expression level of HO-1, CoPP-induction or adenovirus-mediated HO-1 expression effectively inhibits BVDV infection and HO-1 byproducts biliverdin and CO were responsible for the effect. These results provide useful information for further understanding BVDV pathogenesis and pave the way as well as provide a target for future development of new strategy to prevent BVDV infection. |
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