Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

Agricultural Sciences in China

2009, 8(6): 730-739

June 2009

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus PU Xiu-ying1, 3, LIANG Jian-ping2, 3, SHANG Ruo-feng3, WANG Xue-hong3, WANG Zuo-xin1, 3, HUA Lanying3 and LIU Yu3 1

College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, P.R.China

2

Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, P.R.China

3

Lanzhou Institute of Animal and Veterinary Pharmaceutics Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, P.R. China

Abstract To study the influence of Hypericum perforatum extract (HPE) on piglets infected with porcine respiratory and reproductive syndrome virus (PRRSV), enzyme-labeled immunosorbent assay (ELISA) and cytopathic effect (CPE) were used to determine in vitro whether HPE could induce swine pulmonary alveolar macrophages (PAMs) to secrete IFN-γ, and whether PRRSV titers in PAMs were affected by the levels of HPE-induced IFN-γ. HPE (200 mg kg-1) was administrated by oral gavage to piglets infected with the PRRSV in vivo to observe whether HPE affected the viremia, lung viral titers, and weight gain of piglets infected with PRRSV. The results showed that HPE was capable of inducing PAMs to produce IFN-γ in a dose dependent manner and HPE pretreatment was capable of significantly reducing PRRSV viral titers in PAMs (P < 0.01). Administration of HPE to the PRRSV-infected animals significantly (P < 0.05) reduced viremia over time as compared with the PRRSV-infected animals. But there was not significant decrease in lung viral titers at day 21 post-infection between the HPEtreated animals and the PRRSV-infected control piglets. There were no significant differences in weight gain over time among the HPE-treatment animals, the normal control, and the HPE control animals. The PRRSV-infected animals caused significant (P < 0.01) growth retardation as compared with the HPE controls and the normal piglets. It suggested that HPE might be an effective novel therapeutic approach to diminish the PRRSV-induced disease in swine. Key words: porcine respiratory and reproductive syndrome virus (PRRSV), porcine respiratory and reproductive syndrome (PRRS), Hypericum perforatum extract (HPE), piglet

INTRODUCTION Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine. This disease was first observed in the US in 1987 (Keffaber 1989) and in Europe in 1990 (Wensvoort et al. 1991). The etiologic agent for this disease, PRRS virus (PRRSV) is an enveloped, positive-stranded RNA virus that is a member of the family Arteriviridae, or-

dered Nidovirales. The full length genomic sequence has been determined for PRRSV isolates of both European and North American lineage (Allende et al. 1999; Meulenberg et al. 1993; Nelsen et al. 1999; Shen et al. 2000; Wootton et al. 2000). The disease is symptomatically characterized by reproductive failure in pregnant sows and severe respira tory distress in pi glets a nd growing pigs (Zimmerman et al. 2006). Infection with PRRSV also predisposes pigs to infection by bacterial patho-

Received 13 October, 2008 Accepted 1 December, 2008 PU Xiu-ying, Ph D, Tel: +86-931-2976633, E-mail: [email protected]; Correspondence LIANG Jian-ping, Professor, E-mail: [email protected]

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60272-2

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

gens as well as other viral pathogens (Benfield et al. 1992) and PRRSV is a key etiologic agent of the economically important porcine respiratory disease complex (PRDC). The most consistent pathological lesions caused by PRRSV during acute infection are interstitial pneumonia and mild lymphocytic encephalitis (Halbur et al. 1995; Plagemann 1996; Rossow et al. 1995, 1996). Tissue macrophages and monocytes are the major target cells during both acute and persistent infection (Molitor et al. 1997), especially, pulmonary alveolar macrophages (PAMs) are one of the primary target cells of PRRSV infection (Collins et al. 1992). In vitro studies have revealed that PRRSV is lethal to PAMs within 48 h post-infection (Oleksiewicz and Nielsen 1999). Producers often vaccinate swine against PRRSV with modified-live attenuated strains or killed virus vaccines. However, current vaccines do not provide satisfactory protection, possibly due to strain variation and inadequate stimulation of the immune system. A protective immune response is possible since it has been demonstrated that previous exposure can provide protection when pigs are challenged with a homologous strain of PRRSV (Lager et al. 1999). However, protective immunity has never been consistently demonstrated for challenge with heterologous strains (Lager et al. 1999; Mengeling et al. 2003). Several studies have evaluated the immune response to PRRSV infection in swine. Although PRRSV induces both humoral and cell-mediated immune responses, the virus continues to replicate for months after initial infection in some animals (Allende et al. 2000). The inability of swine to resolve a PRRSV infection may be linked to the low levels of interferongamma (IFN-γ) expression (Suradhat et al. 2003). IFN-γ is a type II interferon mainly produced by activated natural killer (NK) and T cells. IFN-γ secreted from NK and T cells enhances major histocompatibilty complex (MHC) classes I and II expression, nitrous oxide production, and superanion oxide radical formation from antigen presenting cells (APCs). In addition to its effects on APCs, IFN-γ plays a role in the development of T helper 1 (Th1 cells, from naive T helper cells), generation of antigen specific memory T cells, and production of an antiviral state in uninfected cells and tissues. In vivo studies have revealed a weak IFN-

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γ response in pigs infected with PRRSV (Meier et al. 2003). It has been suggested that PRRSV is capable of suppressing IFN-γ synthesis and consequently its effect on the immune system during viral infection (Rowland et al. 2001). Interestingly, treatment of PAMs with recombinant porcine IFN-γ blocks PRRSV replication (Bautista and Molitor 1999). The persistence of PRRSV infection is likely related to the inability of pigs to generate an effective immune response against this pathogen. The plant Hypericum perforatum belongs to the genus Guttiferae which contains about 400 species, found in Europe, West Asia, North Africa, and North America. It is used in herbal medicine externally for treatment of skin wounds, eczema, burns, and internally for diseases of the alimentary tract and others (Lavagna et al. 2001; Nicolaeva 1977). Many studies have shown that H. perforatum extract (HPE) has antiviral activity (Andersen et al. 1991; Serkedjieva et al. 1990) and can also take considerable action against cytomegalovirus (CMV) and human immunodeficiency virus (HIV) (Barnard et al. 1992; Hudson et al. 1991; Sandstrom 1989; Schinazi et al. 1990). The antiviral action of HPE has been studied on lipid enveloped and non-enveloped DNA and RNA viruses (Lavie et al. 1995; Lenard 1993; Tang et al. 1990) and also, clinical studies have been conducted in HIV infected patients (Bone 1994; Thiers 1989). Besides the above-mentioned activities, HPE has been reported to possess marked antimicrobial (Li and Jiang 2007), antiretroviral activity (Lavie et al. 1989), and immune augmented function (Evstifeeva and Sibiryak 1996). However, scientific information is not available for the report of anti-PRRSV activity of HPE in vitro and in vivo. Therefore, the purpose of this study was to determine whether HPE treatment enhances the resistance of piglets to PRRSV. It showed that freshly isolated PAMs are capable of producing IFN-γ in response to HPE. HPE pretreatment of PAMs significantly reduced PRRSV titers in vitro. in vivo administration of HPE significantly decreased total viremia, and prevented PRRSV-infected animals from significant growth retardation. Taken together all data, it indicates that treatment with HPE is an effective novel therapeutic approach to diminish PRRSVinduced disease in swine.

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MATERIALS AND METHODS

Viral challenge

Extract

Piglets were challenged with an intranasal inoculation of PRRSV (isolated by Shanghai Institute of Animal and Veterinary Sciences, Chinese Academy of Agricultural Sciences) at a concentration of 2 × 10-6.1 TCID50 per animal. The inoculation device was a teat cannula on a 3-mL syringe administering a 2-mL dose of PRRSV suspension. All test piglets were challenged the PRRSV in the P3 Animal Laboratory of Chengdu Medicine Machinery Company, China Animal Husbandry Industry Co., Ltd. Animals of each group were completely isolated from the other animals.

H. perforatum extract was extracted, isolated, and purified by Lanzhou Institute of Animal and Veterinary Pharmaceutics Sciences, Chinese Academy of Agricultural Sciences, China. Hypericin and pseudohypericin are considered the main active ingredients in this extract. The reversed-phase high performance liquid chromatography (RP-HPLC) was used to detect the content of hypericin in HPE. Hypericin was separated by Symmetry C18 column (4.6 mm × 250 mm, 5 µL) with methanol-a cetonitile-1.0% sodium dihydrogen phosphatc (170:10:10) as mobile phases. The detecting wave length was 588 nm, the flow rate was 1.0 mL min-1, the column temperature was room temperature and the injection volume was 20 µL. In this condition, the content of hypericin was 4.73% within H. perforatum extract (Yu et al. 2000).

Virus Virus (VJX0612 PRRSV variation virus) was provided by Prof. Yuan Shishan from Shanghai Institute of Animal and Veterinary Sciences, Chinese Academy of Agricultural Sciences, China. TCID50 was 10-6.1 0.1 mL-1.

Animals Piglets weighing approximately 5-6 kg were purchased from Jingyuan County, Gansu Province, China. To insure that PRRSV-free animals were used in these experiments, serum samples were collected prior to experimentation and tested for the presence of viremia and antibody titers to PRRSV using an ELISA Kit (IEDXX, USA). All piglets tested negative for PRRSV-specific antibodies and PRRSV viral titers. These piglets were not vaccinated with any modified live PRRSV vaccines. Animals with clinical signs of disease or abnormalities were not used in the study. The piglets were tagged, allowed free access to water, and fed a non-medicated ration. The pigs were acclimated for at least 2 d prior to experimentation.

In vivo experimental design (14 d study) 30 viral challenged piglets were randomly divided into HPE treatment group and PRRSV-infected control group. After viral challenged 72 h, each piglet in the treatment group was given HPE prepared into concentration of 200 mg kg-1 using 10 mL distilled water by mouth daily for 3 d. Piglets of the PRRSV-infected control group were given phosphate buffer saline (PBS) daily by mouth on the same days. Five animals from both groups were killed according to the international act of animal welfare and other relevant guidelines on days 3, 7, and 14 after treatment and bronchoalveolar lavages of the lungs were performed to isolate BAL cells.

In vivo experimental design (28 d study) 10 viral challenged piglets were randomly divided into HPE treatment group and PRRSV-infected control group. After viral challenged 72 h, each piglet in the treatment group was given HPE prepared into concentration of 200 mg kg-1 using 10 mL distilled water by mouth daily for 3 d. In addition, 10 PRRSV-free piglets were used for HPE control animals and normal control animals, respectively. There were 5 piglets each group. The piglets of the HPE control group were given only HPE 200 mg kg-1 daily by mouth on the same days. The piglets of normal and PRRSV-infected control groups were treated with PBS on the same days. The animals in all groups were analyzed for weight changes on days 7, 14, 21, and 28 of the study. Blood serum samples

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Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

for viral titer analyses were collected on days 3, 7, 14, and 21 of the study. All groups were killed on day 28 according to the international act of animal welfare and other relevant guidelines. On a single experiment, bronchoalveolar lavages for each of the animals in every group were performed. The lavage fluids and BAL cells were collected for lung viral titer determinations.

Isolation, purification, and culture of bronchoalveolar lavage (BAL) cells, and porcine alveolar macrophages (PAMs) Porcine BAL cells were collected aseptically according to a modified bronchoalveolar lavage protocol (Girard et al. 2001). After being killed, lungs were lavaged with 500 mL of PBS supplemented with 1% penicillinstreptomycin-amphotericin (the General Pharmaceutical Factory of Harbin Pharmaceutical Group, Harbin, China). The BAL cells were centrifuged at 3 000 × g for 10 min at 4°C and resuspended in RPMI 1640 (Sigma, USA) culture medium supplemented with 2 mmol L-1 glutamine, 24 mmol L-1 NaHCO3 (Jianghai Biological Engineering Co., Ltd., Beijing, China), 1% penicillin-streptomycin-amphotericin, and 10% fetal bovine serum (FBS; Sijiqing Co., Hangzhou, China). Ficoll-Paque (Sigma, USA) density gradient was used according to manufacturer’s recommendations in order to remove dead cells, red blood cells, and granulocytes from BAL cells. PAMs were purified based on a method used in previous study (Zou et al. 2003). The BAL cells and PAMs were incubated at a concentration of 2 × 106 cell mL-1 at 37°C in 5% CO2/95% humidified air for 2 h. The cultured cells were then stimulated with either varying concentrations of HPE (25, 50, 100, and 200 mg L-1), and 5 mg mL-1 of phytohemagglutinin (PHA) from Phaseolus vulgaris (Sigma, USA).

Viral titer determination PRRSV titers were determined (Yin and Lin 1998). Mark-145 cells grown in 96 well plates in complete medium, Dulbecco’s Modified Eagle Medium (DMEM, Sigma, USA) supplemented with 2 mmol L-1 glutamine, 1% penicillin-streptomycin-amphotericin, and 10% FBS) for 2 d. PRRSV were diluted vary concentration (10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9) using FBS-

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free DMEM. 50 µL PRRSV was respectively overlaid on Mark-145 cells for 2 h at 37°C in a 5% CO2 incubator. Eight wells were set each concentration. Cells were washed three times with PBS and then cultured in the presence of complete medium until CPE were not observed by inverted microscope. The viral TCID50 was determined by measuring CPE according to ReedMuench method (Yin and Liu 1997).

Cytokine analysis Cell-free supernatant fluids were collected at 3, 6, 12, 24, 48, and 72 h after adding HPE, and cytokine levels of IFN-γ were determined by using enzyme-labeled immunosorbent assay (ELISA) according to the manufacturer’s instructions (Adlitteram Diagnostic Laboratories, USA).

Statistical analysis Data were analyzed by applying SPSS 13.0 for Windows statistics software. t-test was used to analyze data shown in Figs.1, 2, 4, and 5. Variance used to analyze data in Fig.6 (P < 0.05) were considered to be significant.

RESULTS In vitro effects of HPE on alveolar macrophages Since BAL cells in normal piglets are a collection of pulmonary alveolar macrophages (90-99% of total normal immune lung cell population) and pulmonary lymphocytes (1-10% of total normal immune lung cell population) (Ganter and Hensel 1997). We wanted to determine whether freshly isolated BAL cells from piglets were capable of producing IFN-γ in response to HPE. The BAL cells and purified PAMs were stimulated with HPE. The IFN-γ protein was detected in the supernatants of HPE-stimulated PAMs and BAL cells (Fig.1). As shown in Fig.1, the purified HPE-treated PAMs produced significantly (P< 0.01) higher amounts of IFN-γ protein (458 ± 2.36) pg mL-1 as compared with the HPE-treated BAL cells (305 ± 5.49) pg mL-1. Since PAMs were the sole producers of IFN-γ in re-

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sponse to HPE and they comprise the majority of immune cells in the lungs.

HPE dose response BAL cells were isolated from piglets and cultured with increasing concentrations of HPE. HPE induced IFN-γ in a dose-dependent manner (Fig.2). As little as 25 mg L-1 of HPE was sufficient to induce IFN-γ secretion (P < 0.01, as compared with the medium-incubated BAL cells). No significant differences were observed in the induction of IFN-γ protein levels between 25 and 200 mg L-1 of HPE. Thus we chose to use 100 mg L-1 of HPE, a concentration 4 times that of the minimal dose needed to induce IFN-γ, in all subsequent in vitro experiments.

Fig. 1 IFN-γ produced by the HPE-treated porcine alveolar macrophages. BAL cells and PAM were stimulated with HPE (200 mg L-1 ) for 24 h. The supernatant fluids from treated cells were analyzed for IFN-γ content by ELISA. Samples were performed in triplicate. The same as the below. *P < 0.01 as compared with HPE-treated BAL cells.

Kinetics of IFN-γ production in response to HPE BAL cells were isolated and stimulated with HPE for 3, 6, 12, 24, 48, and 72 h. As early as 3 h post-treatment, IFN-γ protein was detected in the supernatants of the HPE-treated BAL cells (Fig.3). The levels of HPE-induced IFN-γ protein expression peaked at 24 h and were almost sustained as long as 72 h post-stimulation, the last time point sampled.

Effects of HPE on PRRSV titers in vitro BAL cells were isolated from uninfected piglets and either pretreated with HPE or left untreated prior to PRRSV infection. The BAL cells pretreated with HPE exhibited a significantly (P < 0.05) lower log10TCID50 (2.24 ±0.16) than the PRRSV-infected control BAL cells (3.17 ± 0.20) (Fig.4).

Fig. 2 Dose response of HPE-induced IFN-γ in BAL cells. BAL cells were stimulated with increasing concentrations of HPE for 24 h. ELISA was used to analyze the supernatant fluids from stimulated cells for IFN-γ expression. *P < 0.01 as compared with mediumincubated BAL cells.

Effects of HPE treatment on resistance to PRRSV infection Experiments were conducted to investigate whether HPE enhances resistance to PRRSV infection. Administration of HPE to PRRSV-infected animals significantly (P < 0.01) reduced viremia over time as compared with PRRSV-infected animals (Fig.5). Additionally, analysis of lung viral titers collected from

Fig. 3 Kinetics of IFN-γ protein expression induced by HPE in BAL cells. BAL cells were stimulated with HPE (100 mg mL-1 ) for 3, 6, 12, 24, 48, and 72 h. ELISA was used to analyze the supernatant fluids from stimulated cells for IFN-γ protein expression.

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Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

BAL fluids of infected animals at day 21 post-infection revealed a slight but not significant decrease in HPEtreatment animals as compared with PRRSV-infected control piglets. In order to determine the effect of HPE treatment on weight gain in infected animals, the animals were weighed on days 7, 14, 21, and 28 post-infection. There were no significant differences in weight gain over time between the normal control and the HPE control group. More importantly, no significant differences in weight gain overtime were observed among HPE-treatment animals, the normal animals and the HPE control animals. However, the animals infected with PRRSV caused significant (P < 0.01) growth retardation as compared with those of HPE controls (Fig.6).

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Fig. 6 Effect of HPE treatment on weight gain in PRRSV-infected animals over time. The weight was recorded at days 3, 7, 14, 21, and 28 post-infection from all 5 animals in each group, normal control, HPE control, HPE treatment group, and PRRSV-infected group. *P < 0.01 as compared with PRRSV-infected control animals.

DISCUSSIION

Fig. 4 Effect of HPE pretreatment on PRRSV titers in vitro. BAL cells were cultured with medium alone or pretreated with HPE for 48 h prior to infection with medium or infection with PRRSV. Supernatant fluids were collected and analyzed at 24 h post infection. *P < 0.05 as compared with PRRSV-infected control BAL cells.

Fig. 5 Analysis of viremia over time. Serum samples were collected from all five animals at days 3, 7, 14, and 21 post-infection in each of the following groups: PRRSV-infected control group and HPE treatment group animals. *P < 0.05 as compared with PRRSVinfected control animals.

H. perforatum (HP) is a plant that has been used as a medicinal herb since ancient times. Its extract contains several active compounds including hypericin, hyperforin, and fla vonoids (Zou e t al. 2004). Hypericin, the presumed active moiety within HP, exhibits potent pharmacological effects that include light-dependent antiretroviral activity against HIV infection (Miskovsky 2002), inhibition of protein kinase C (Takahashi et al. 1989) and inhibition of the activation of the transcription factor nuclear factor kappa B (NF-κB) (Bork et al. 1999). After the acute phase of PRRSV infection, which is typically characterized by viremia and clinical disease, many pigs fully recovered yet carry a low-level viral infection for an extended period of time. PRRS is one of the most challenging subjects of research in veterinary viral immunology, and the immune response against PRRSV still is poorly understood. Although scientists have long recognized that IFN-γ is mainly produced by NK and T cells, recent publications have suggested that myeloid cells, such as macrophages, may be capable of producing IFN-γ in response to multiple combinations of stimuli (Munder et al. 1998; Fenton et al. 1997; Robinson et al. 1985). In this study, freshly purified PAMs but not freshly isolated porcine pulmonary T cells was showed to produce IFN-γ in response to HPE for the first time. As little as 25 mg L-1 of HPE was sufficient to induce IFN-γ

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protein expression from BAL cells. Additionally, IFN-γ protein expression within 3 h of HPE stimulation has been detected. Taken together the kinetics data, it suggests that freshly isolated BAL cells are able to readily secrete IFN-γ in response to HPE. This IFN-γ production could be the result of a direct event and not mediated by additional factors such as cytokines or cell-tocell interactions. Pre-treatment of PAMs with IFN-γ has been shown to inhibit viral replication and to lower the numbers of macrophages that support PRRSV replication (Bautista and Molitor 1999). Also, Mark-145 cells pre-treated with IFN-γ prior to PRRSV challenge resulted in fewer infected cells, fewer viral particles, and lower levels of viral RNA genome in individual cells (Rowland et al. 2001). The ability of PAMs, the target cell of PRRSV, to respond to HPE by secreting IFN-γ suggests that these cells may be capable of blocking PRRSV replication after HPE stimulation. Therefore, we wanted to determine whether HPE treatment was capable of suppressing PRRSV replication in PAMs. The results showed that pretreatment with HPE 48 h prior to viral challenge significantly diminished the TCID50 of PRRSV infection in the supernatants of infected PAMs. In this set of experiments, we also treated cell cultures for 24 h prior to and at the time of viral challenge. HPE treatment had no effect on viral titers when co-administered with the viral challenge. Additionally, cultures pre-treated 24 h prior to viral challenge showed slightly lower titers but not significantly different from those seen in infected controls (data were not shown). It is likely that the lower levels of PRRSV titers seen after 48-h pre-treatment with HPE are mediated by the antiviral properties of IFN-γ. However, the mechanisms involved in preventing PRRSV replication in HPE-stimulated PAMs will need to be further explored. Since HPE treatment was capable of suppressing PRRSV titers in vitro, we wanted to determine whether HPE treatment enhanced resistance to PRRSV as defined by reduction in viral burden and improvement of growth performance in infected piglets. The result showed that HPE treatment of PRRSV-infected animals significantly reduced viremia over time. A previous report has indicated that PRRSV viremia peaks between 7 and 9 days post-infection (Labarque et al. 2000).

PU Xiu-ying et al.

Interestingly, the reduction in viremia in our studies was most obvious at days 3 and 6 post-infection suggesting that HPE treatment was effective in suppressing peak PRRSV titers in vivo. In vitro data from Bautista and Molitor (1999) and Rowland et al. (2001) indicate that pre-treatment with IFN-γ prior to PRRSV challenge results in fewer numbers of infected cells, fewer viral particles, and lower levels of viral PRRSV RNA genome in individual cells. The reduction of viral titers in serum of HPE-treatment animals is significant difference as compare with PRRSV-infected animals (days 3-6 post-infection). But beyond day 7 postinfection, there were no differences in viremia or PRRSV viral titers between PRRSV control animals and HPE treatment animals. Furthermore, in this experiment, a slight but not significant difference in viremia observed at day 14 post-infection supporting the conjecture that HPE might only be suppressing peak viral titers but not affecting low level viral loads. It is possible that lung viral titers are simply reflective of differences in blood viral burden and not of local events. Conversely, HPE treatment of PRRSV infected animals might be inducing local production of cytokines, or cellular responses that led to the control of peak levels of viral titers but did not affect low levels of viral burden. Weight gain in these animals was measured to determine whether HPE treatment was capable of preventing PRRSV-induced growth retardation. In the studies, an initial separation in piglet’s weight between PRRSVinfected group and HPE-treatment animals on day 7 and this weight difference continued increasing were observed throughout the duration of the experiment. The data showed that HPE treatment prevented significant growth retardation in infected animals overtime as compared with PRRSV-infected controls. On average, by the end of the experiment, each untreated infectedanimal weighed (3.6 kg) less than their uninfected counterparts. HPE-treatment animals weighed (2.9 kg) more than their untreated counterparts. A correlation between increases in log titers of PRRSV viremia with decreases in pig weight gain and feed intake has been established (Greiner et al. 2000). The improved weight gain overtime in HPE-treatment animals as compared with PRRSV-infected controls is possibly associated with the lower levels of viremia observed in these animals.

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Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

Besides, the use of HPE as a novel plant sources agent for PRRS offers the a dva nta ges of ea sy production, low cost, extensive availability, adequate water solubility and minimal side effects. It was demonstrated that HPE can be used as an effective treatment to increase resistance of pigs to PRRSV infection. The use of HPE may provide novel therapeutic approaches for the treatment of porcine viral diseases.

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anthraquinones, anthrones and anthraquinones derivatives against human cytomegalovirus. Antiviral Research, 17, 6377. Bautista E M, Molitor T W. 1999. IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages. Archives Virology, 144, 1191-1200. Benfield D A, Nelson E, Collins J E, Harris L, Goyal S M, Robinson D, Christianson W T, Morrison R B, Gorcyca D, Chladek D. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus isolate ATCC VR-2332.

CONCLUSION

Journal of Veterinary Diagnostic Investigation, 4, 127-133. Bone K. 1994. The herbal treatment of viral infections. European

In this study, HPE affected the PRRSV titers by the levels of induced IFN-γ in vitro; and affected the viremia, lung viral titers and weight gain of piglets infected with PRRSV in vivo. Collectively, all data suggested that treatment with HPE was effective in decreasing the severity of PRRSV infection while increasing the growth performance of infected animals. These results laid the foundation for developing an effective therapeutic agent for PRRS. It also suggested HPE might be an effective novel therapeutic approach to diminish PRRSV-induced disease in swine.

Journal of Herbal Medicine, 1, 23-28. Bork P M, Bacher S, Schmitz M L, Kaspers U, Heinrich M. 1999. Hypericin as a non-antioxidant inhibitor of NF-kappa B. Planta Medica, 65, 297-300. Collins J E, Benfield D A, Christianson W T, Harris L, Hennings J C, Shaw D P, Goyal S M, McCullough S, Morrison R, Joo H, et al. 1992. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. Journal of Veterinary Diagnostic Investigation, 4, 117126. Evstifeeva V A, Sibiryak S V. 1996. Immunotropic properties of biologically active products obtained from Jonh’s wort

Acknowledgements This work was supported by One Hundred Person Project of the Chinese Academy of Sciences (Renjiaozi [2008] 287) and the Special Fund to Aid Basic Scientific Research of State Level Research Institutes for Public Welfare, China (BRF070402).

Hypericum perforatum. Eksperimental’Naya i Klinicheskaya Farmakologiya, 59, 51-54. Fenton M J, Vermeulen M W, Kim S, Burdick M, Strieter R M, Kornfeld H. 1997. Induction of gamma interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infection and Immunity, 65, 5149-5156. Ganter M, Hensel A. 1997. Cellular variables in bronchoalveolar lavage fluids (BALF) in selected healthy pigs. Research in

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