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Enterovirus 71 (EV71) is a 7411 bp positive stranded RNA virus belongs to the Enterovirus genus in the family of Picornaviridae. Since its first characterization in 1969 in California, USA, several outbreaks have been report in United States, Australia, Japan, Hong Kong, and Taiwan. Meningitis, brainstem encephalitis, and acute flaccid paralysis have been reported to result from these outbreaks. However, it is not until late 1990s that EV71 had reached global attention when symptoms such as mucocutaneous manifestation of Hand, Foot and Mouth Disease (HFMD), herpangina, and cardiopulmonary complications, which were not originally relates with the viral infection, have been observed. Children with brainstem encephalitis resulted from EV71 infection can rapidly develop cardiopulmonary compromise, often result in death.
During the past 14 years, EV71 outbreaks often correlate with a sharp rise in HFMD cases. In Malaysia, HFMD outbreaks were first recorded in 1997, with case count of 5999 and 42 deaths. Since then, periodic outbreaks are subsequently reported in Malaysia, with majority of the outbreaks happen in Sarawak. Closures of daily childcare and pre-school education centre and proper hygiene and sanitation in daily operations have been recommended as a measurement to break the circle of transmission of EV71, but its effectiveness in controlling the spread of the virus remains unclear.
EV71 spreads through direct contacts with blisters and other surfaces contaminated by nose and throat discharge of infected person, while fecal-oral route is reported to be the main mode of transmission of the virus. Not everyone infected by the virus shows the symptoms of the infection. Symptoms of disease mostly observed in children of younger than 6 years old, when their immune system is not yet fully developed. Therefore, an adult may harbor the virus without knowing it and spread the virus especially easy when improper hygiene in food preparation and handling of contaminated objects is practiced. As a result, outbreaks almost always occur in daycare and preschool education centers, where the children are crowded and sanitation is hard to be controlled.
To date, no effective vaccine is available, and attempts to treat EV71 infection with existing antiviral compounds were largely unsuccessful, either due to low efficiency or high degree of side effects. Viewing that the cost of vaccines or remedy can be overwhelming to developing countries during disease outbreaks, an effective, cheap, and readily available alternative is urgently needed.
Medicinal plants are promising candidates that fits the requirements. Before the evident-based medicine became the standard in modern society, herbs were used as remedy in prevention and treatment of diseases for thousands of years, while many of them are still in used today. Medicinal plants harbour wide ranges of compounds that their value as effective control against disease is not fully studied. In this project, attempts to screen and isolate antiEV71 compound from two medicinal herbs, Pueraria lobata and Glycyrrhiza glabra, will be performed.
Sheng Ma Ge Gen Tang (SMGGT; å‡éº»è‘›æ ¹æ±¤) has been used as traditional prescription to treat measles of children for thousands of years in China. Several viral activities have been reported to be inhibited by different ingredients in SMGGT. In a particular report, SMGGT have been found to inhibit EV71 infection in vitro. As one of the main ingredients in SMGGT, water extract of kudzu (è‘›æ ¹, Pueraria lobata) is reported to have antiEV71 property. However, the bioactive compound that exhibits this property is still to be determined.
Another potential herb that contains promising antiviral compound is licorice (ç”˜è‰, Glycyrrhiza uralensis). Liquorice extract is used as often as food flavoring additives and also in traditional medicine. Several findings on the medicinal and pharmacological uses of licorice extract have been previously described, possessing functions such as anti immunity regulation, anti-tumor and anti viral. In particular, its use as antiviral compound have been studied and shown to be effective against Human Immunodeficiency Virus, Hepatitis B Virus, and Herpes Simplex Virus, mainly pointing to glycyrrhizin as the bioactive component. Nevertheless, licorice extract as a whole, its use and effectiveness against EV71 infection have yet to be evaluated, and shall be determined in this project.
In short, this project aims to screen for antiEV71 compound from two medicinal herbs, namely P. lobata and G. glabra. Crude water extract of the herbs shall be prepared and concentrated and purified using chromatography. Effectiveness of the pure extract shall be tested against EV71 culture, and structure of the bioactive compound will be determined.
Chinese medicinal plants
Medicinal herbs were used extensively in the past. The use of plant as cure can be traced back to prehistoric era. There are evidence that Neanderthals (present day Iraq) living 60000 years ago uses plants such as hollyhock (Cowan, 1999). Medicinal herbs were not only used in wide ranges traditionally from antidotes of deadly snake venom to common cold, but also thought to strengthen immunity and very much used as flavoring in food and beverages. Plants contain wide variety of secondary metabolites, many of which the possibility to be used in antiviral agent still remains unknown.
Many works has been done to study the potential use of herb extracts in treatments of different illness or viral infection. Hot water extract and dimethylsulfoxide of Viola yedoensis were reported to inhibit HIV growth in vitro (Chang and Yeung, 1988; Ngan et al., 1988). Ma et al. (2002) successfully isolated and purified the bioactive component in Sophora flavescens and Scutellaria baicalensis, which were found to have inhibitory effect on respiratory syncytial virus.
However, some study shows some medicinal herbs to have adverse effect on viral infection. In one randomized trial conducted by Liu et al. (2003), the result suggest no firm evidence to support medicinal herbs in treating hepatitis C virus infection. In fact, some herbs were found to have adverse effect on the infection.
In particular to EV71 infection, several researches have been carried out to test the possibility of traditional herbs to be used as antiviral agent, some of them reported to be a success. Wu et al. (2007) studied the anti EV71 property of Salvia miltiorrhiza (Dan Shen). He reported water extract of S. miltiorrhiza at room temperature possesses antiviral capability by preventing EV71 entry into the cell. However, hot water extract of S. miltiorrhiza does not seem to exhibit the same antiviral capability, leading to the suggestion that the active component might be phenolics and heat sensitive. Also, S. miltiorrhiza extract shows different degree of antiviral effect in 4 different cell lines, namely MRC-5, RD and Vero cell lines, while HeLa cell lines shows only partial inhibition of the cytopathic effect. The authors suggest that the extract may exhibits cell-type specific or selectivity inhibition.
Research done on ge gen
Sheng Ma Ge Gen Tang (SMGGT; å‡éº»è‘›æ ¹æ±¤) was used traditionally to treat measles of children in China. Huang (1995) have reported that SMGGT are capable to inhibit measles virus infection in vitro. Although measles virus and EV71 belongs to different family in taxonomy, they are both single stranded RNA virus and can cause acute encephalitis in children. Active compounds in SMGGT also was shown to inhibits several viruses in vitro, namely varicella-zoster virus (Baba and Shigeta, 1987), SARS-associated coronavirus (Cinatl et al., 2003), Hepatitis A virus (Crance et al., 1994), Hepatitis B virus (Sata et al., 1996), flaviviruses (Crance et al., 2003), human immunodeficiency virus (Ito et al., 1999), Epstein-Barr virus (Lin, 2003), and influenza virus (Utsunomiya et al, 1997). As one of the main ingredients of SMGGT, water extracts of Ge Gen was expected to possess wide spectrum antiviral properties.
Su et al. (2008) had reported antiviral properties of water extract of P. lobata. IC50 of as low as 0.2 - 0.3μg/ml can be effective; with CC50 of more than 5000 μg/ml was recorded. Its considerably lower effective dosage as compared to other more expensive counterpart such as S. miltiorrhiza (IC50 of 724μg/ml) and H. cordata, ( IC50 of 125.9μl/ml) suggest that it is more effective alternatives in terms of cost and antiviral activity during the outbreaks of the virus. Time-course assay had revealed that this extract is most effective in preventing viral entries rather than pre- or post-infection. Its high CC50 also suggest it is safe for daily consumptions. In the same paper, the authors also pointed out that the possible limitation of this extract in crossing blood brain barrier to prevent brain encephalitis due to its hydrophilic nature. Correlation of EV71 encephalitis with peak HFMD outbreak has been observed and it is proposed that in the event of EV71 infection is controlled, the possibility in developing encephalitis caused by EV71 can also be reduced.
Research done on gan cao
Licorice, collectively referring to Glycyrrhiza spp., have been used and included in manuscripts of the Chinese, Greece, and India. Chinese licorice, G. uralensis, is dominant in the Asia region, and widely used in Chinese traditional medicine in treatment of respiratory tract infections and hepatitis.
While most of the research on G. uralensis was concentrated on glycyrrhizin as a single bioactive compound, Sun and Pan (2005) reported G. uralensis saponins increase specific antibody binding strength and cellular response in ovalbumin of mice, with low haemolytic effect on the mice itself. However, the author suggest further work have to be done before the saponins can be fully developed as immunological adjuvant.
Ethanol extract of G. uralensis was also suggested to induce cell apoptosis and arrest G1 cell cycle in MCF-7 human cancer cell line (Jo et al., 2005). Although the use of phytoestrogens against hormone dependent cancers or as natural alternatives to hormone replacement therapy remains controversial, as mentioned by the author, but nevertheless the data supported the possible uses of G. uralensis in cancer treatment.
Water soluble polysaccharides were isolated in another study conducted by Wan and Cheng (2009). The author reported an increased production of acid phosphatase, adenosine triphosphatase, acid alpha-naphthyl acetate esterase and succinate dehydrogenase and recorded macrophages activity is induced by the polysaccharaide. This suggest that Glycyrrhiza polysaccharide is partly responsible for the beneficial therapeutic effects for G. uralensis.
Glycyrrhizin is one of the more well studied constituents in ethnobotanicals. It is a major bioactive component in G. uralensis, and is widely used as a natural sweetener in foods and tobacco. Estimated consumption of glycyrrhizin is at 0.027-3.3mg/kg/day in the US, with recommended usage of 0.015-0.225mg/kg/day (Isbrucker and Burdock, 2006). Glycyrrhzinates can inhibit 11beta-hydroxsteroid dehydrogenase, which responsible for the inactivation of cortisol. As a result, mineralocorticoid-like symptoms, characterized by hypertension, hypokalemic alkolosis (low plasma potassium), and low rennin and aldosterone secretion are observed in both human and animals, but were reversible upon reduced consumption of foods containing glycyrrhizin (Isbrucker and Burdock, 2006).
Yoshida et al. (2010) reported that mice with burn injury treated with glycyrrhizin shows presence of antimicrobial peptides on injury area in contrast to untreated burnt injury. Immature myeloid cells, which indicate inability of the mice to produce antimicrobial peptides needed to prevent infection, were found presence in untreated mice but not in treated mice. The authors suggested the use of glycyrrhizin as a treatment of Psenomonas aerugenosa infection in burn patient, which is a major cause of death among patient with burn injuries.
As a treatment to chronic hepatitis B, glycyrrhizin has been used in Japan intravenously and improves liver function, occasionally were linked to complete recovery from hepatitis (Sato et al., 1996). Glycyrrhizin were suggested to act by modifying intracellular transport and suppresses sialylation (distribution of sialic acid in glycan) of Hepatitis B virus (HBV) surface antigen when it attain a concentration of 31.8μg glycyrrhizin /g cell in the liver. Therefore, when taken intravenously directed to the liver, glycyrrhizin might bind to hepatocytes at which the expression of HBV-related antigen can be modified and sialylation of HBV antigen can be suppressed (Sato et al, 1996).
HFMD can be caused by several viruses in enterovirus group, including polioviruses, coxsackieviruses, echoviruses, and enteroviruses, often the clinical features are indistinguishable. The most common etiological agent bring Coxsackievirus A16, which results in a mild illness with most patient recover within two weeks of first symptom appears. However, more severe form of HFMD can be result from EV71 infection, which can lead to encephalitis and acute flaccid paralysis (WHO, 2007).
Symptoms of HFMD include mild fever and often soar throats, which followed by sores or ulcers in the mouth, and development of rashes or blisters on the surface of hands and feet within 24 to 48 hours after fever. Symptoms of HFMD usually disappear within 10 days of viral infection without any medical intervention (Chan and Sazaly, 2004). However, HFMD caused by EV71 infection is far more likely to lead to complication and death especially in young children of age less than 5 years old. Early symptoms of EV71 infection shows similarity in HFMD, but can progress to headache, irritability and acute limb weakness. Its ability to infect Central Nervous System also can cause neurological complications including brainstem encephalitis, acute flaccid paralysis, pulmonary adema, and myocarditis, which are the symptoms not observed in HFMD cases caused by other viruses.
EV71 was first described in 1969 in USA. During the first two decades of its discovery, sporadic outbreaks of EV71 were reported in various locations throughout the world including United States, Japan, Hong Kong, and Taiwan, where the first reported outbreak outside USA in Melbourne, Australia, in 1972 (Kennett et al., 1974). Symptoms of infection included brain meningitis, bulbar, acute paralysis, but were not found to be associated with mucocutaneous of HFMD or herpangina or cardiopulmonary complications.
Second global epidemic of EV71 infection occurs in early 1998 with a huge change in epidemiological pattern. Outbreaks have been reported in countries that were not previously shown sign of infection such as Malaysia, Singapore, Korea, and India. Death associated with HFMD outbreaks has been reported in Taiwan and Malaysia during 1997 to 1998 (Chen et al., 2007) with a mortality rate of up to 19%, of which 91% of such cases occurs to children under age of 5, with highest fatality in 7-12 months old children (Abzug, 2009). Symptoms of the viral infection characterized not only by neurological manifestation but also including HFMD and herpangina. Cardiopulmonary compromise, which were not observed in previous EV71 infection have also been developed among some children, with high mortality rate. Apparently most EV71 infections are mild. However, serious infection can result in brainstem encephalitis and acute paralysis has become common and have caused high death toll among young children (Abzug, 2009).
The most contagious stage of EV71 infected person is during the first week of infection. It spreads through fecal-oral route, respiratory droplets and direct contact with objects contaminated by vesicular fluids from infected person. The virus can be continuously shredded in feces for up to one month after infection. Standard precaution was recommended to avoid contact with the virus and the disease itself. Good hygiene practice such as proper hand washing with soap, cleaning infected surface with soap and then chlorine bleach, avoid close contact with persons with HFMD, and not sharing personal items such as cup and spoons and properly washing them after use are suggested steps in avoiding the disease.
Enterovirus 71 (EV71) is a Human enterovirus A species and belongs to the family of Picornaviridae under the genus Enterovirus. Its genetic core consists of 7.4-7.5 kb of positive strand of RNA and icosahedral non-enveloped capsid proteins composed of 60 protomers, structured by four different structural viral proteins (VP), each numbered 1-4 (VP 1-VP 4). It is only capable of infecting humans and thus does not have animal reservoir.
First attempt to determine variations of EV71 strains was done by Hagiwara et al. (1984), whereby its capsid polypeptide and RNAse T1 sequences of virus isolated from Japan, Taiwan, Bulgaria, Hungary, and USA. However, the author failed to identify specific molecular markers of the virus. Several years later, Brown et al. (1995) identified three subgroup of EV71, namely A, B, and C, based on the comparison of complete VP1 sequence of 113 isolates of EV71 in different countries. The author reported similarity in gene sequence in over 92% within the same EV71 subgroup, while 78 to 83% similarity as compared between each subgroup, suggesting that the virus undergone high degree of mutation since its discovery.
The VP1 gene has attracted much attention due to it being the most informative region in the study of evolutionary relationship of this virus. Its product, the VP1 protein, in its tertiary form, is highly conserved and identified to be the source of virulence determinants for several enterovirus. A deep cleft at the junction of VP and VP2 on the virion surface was proposed to function as attachment site of the cellular receptor. Furthemore, a polymorphism at the 170 amino acid in VP1 from alanine to valine was discovered through comparative analysis of EV71 with uncomplicated HFMD cases. This change has thought to increase hydrophobicity of the region and change the overall protein conformation of the protein, leading to the suggestion that this change is responsible for the increased neurovirulence in certain EV71 strain.
The 5' UTR of EV71 contain a group of conserved secondary structure element which is called the internal ribosome entry site (IRES). The IRES regulates enterovirus replication through the control of cap-independent translation of the polyprotein. Within the 5' UTR of EV71, IRES-like stem loop structure has been identified (Abubakar et al., 1999). A single nucleotide change within the poliovirus IRES resulted in a large alteration of the neurovirulence of the virus (Evans et al., 1985). Although, the EV71 IRES may play an important role in neurovirulence its actual role remains unclear because nucleotide variation linked to neurovirulence in the EV71 IRES has not been identified to date.
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