Study On Activity Of The Qiqing Baidu Granules Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome（PRRS）is a kind of infectious disease which seriously endangers the development of the pig industry.Its main clinical features are reproductive disorders of sows and respiratory diseases of piglets.In 1996,Porcine Reproductive and Respiratory Syndrome virus（PRRSV）was firstly isolated from infected pigs in China.Since then,PRRS has been widespread in the country,and has caused huge economic losses to the pig industry.At present,the main prevention and control measures against PRRSV are vaccine immunization.However,there are some limitations,such as immune failure,the virulence of the attenuated vaccine return to strong and other uncertain factors.Therefore,finding new prevention and control programs against PRRS has become a research hot spot in recent years.Traditional Chinese medicines（TCM）contain natural compounds with rich structure and various activities,and thus become an important resource for searching effective antiviral medicines.According to relevant data,there are as many as 189 kinds of TCM with antiviral effects.Among them,TCM monomers are the focus of study,such as Radix isatidatis,Astragalus and Honeysuckle.By contrast,there are few studies on the screening and efficacy of compound TCM against PRRSV,and the antiviral mechanism and optimal dosage of the medicine are not very clear.Therefore,this study intends to take Qiqingbaidu Granules as the breakthrough point to carry out in-depth research,as a way to systematically evaluate the anti-PRRSV activity of Qiqingbaidu Granules through in vitro and in vivo tests,and preliminary explore its mechanism of action.The main findings are as follows:（1）The anti-PRRSV effect of Qiqingbaidu Granules in vitro.The CCK-8 method and fluorescence microscope were used to observe and screen the dosing time and concentration of drugs;RT-q PCR and TCID₅₀were conducted to analyze the effect of drugs on the proliferation of PRRSV.The results showed that compared with the virus control group,the mRNA expression of PRRSV decreased by 68%in the prevention mode,53%in the treatment mode and 89%in the direct virucidal mode.The TCID₅₀of virus was 10^-3.25per 100μL,10^-4.36per 100μL and 10^-2.36per 100μL respectively,significantly lower than the virus control group(10^-5.15per 100μL).These results indicated that Qiqingbaidu Granules could inhibit the proliferation of PRRSV-GFP in Marc-145 cells.（2）The anti-PRRSV effect of Qiqingbaidu Granules in vivo.Group A was given medicine three days before the infection and Group B was given medicine and infection at the same time.Group C and D were infection and non-infection control.Medicine was mixed in the basal diet（10 g per pig one day）and supplied from the start time points to the end of the experiment.The rectal temperature of each piglet was recorded each day.On the 14th day post infection（PI）,one piglet in each group was randomly selected and euthanized to observe the lesions caused by viral infection.RT-qPCR analysis of PRRSV viral copies of the blood and ELISA analysis humoral immune response on day 1,3,5,7,14,21,28,and 35 PI.The results showed that rectal temperature of the piglets in Group A and Group B were raised up to 40℃at 6th PI and returned to normal on the 11th and 12th days PI.While,high rectal temperature（>40℃）of the piglets in Group C lasted to the end of the experiments.The lung lesions in piglets from Group A and B were lighter with less alveolar structure damage,inflammatory cell invasion and less exudate in the bronchus.However,the lesions in lung from Group C were serious.The alveolar structures were missing and the bronchus in the lung were filled with lots of exudates.Serious inflammatory cell invasion was also observed around the bronchus.The viremia of Group A was highly significantly lower than Group C at 3,5,7,and 14 days PI of PRRSV,（P<0.01）.And Group B was highly significantly lower than Group C at 3,5 days PI,（P<0.01）,significantly lower at 7 and 14 days PI,（P<0.05）.Antibodies against PRRSV in Group A and B reached the peak at 21 days PI,and the antibodies of Group C reached the peak at 28 days PI.This indicated that the Qiqingbaidu Granules significantly reduce the lesions caused by PRRSV and also the viral loads in bloods and lungs.The drug brought forward the peak value of PRRSV-specific antibody and reduced the duration of viremia.（3）The effect of Qiqingbaidu Granules on the expression of immune factors in peripheral blood mononuclear cells（PBMCs）from piglets.Medicine was mixed in the basal diet continuously for 7 days（10 g per pig one day）and collected PBMCs on the0,1,3,5,and 7 days.RT-qPCR was conducted to analyze of the expression of drug on IFN-α,IFN-γand TNF-αmRNA in PBMCs.These results showed that the drug had no significant effect on the expression of IFN-α,IFN-γ,and TNF-αmRNA.This indicated that indirect antiviral effect of Qiqingbaidu Granules was not obvious,and the anti-PRRSV effect was mainly through the direct antiviral pathway.In summary,it is proved that Qiqingbaidu Granules have anti-PRRSV effects in vitro and in vivo.It has significant preventive and direct virucidal effect on PRRSV.Animal experiments showed that Qiqingbaidu Granules could effectively alleviate the clinical symptoms of PRRS,and reduce the invasion of PRRSV to the organs of piglets,providing theoretical basis for the clinical prevention and control of PRRSV.