Ijmpv v1n1 Jan.-Jun. 2011 E-Sample

International Journal of Medical and Pharmaceutical Virology
Biannual Journal of International Association of Medical and Pharmaceutical Virologists
Volume 1 Number 1 January-June 2011
Editor-in-Chief Prof. Dr. S. Ananthan Formerly, Department of Medical Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences University of Madras, Chennai - 600 113, India Associate Editors Prof. M. Elanchezhian Prof. Radha Madhavan India India
Advisory Board Prof. S.P. Thyagarajan India Dr. Eric Walker Scotland Prof. S. Rajarajan India Prof. J. Shanmugam India Prof. P. Rajendran India Prof. Abhay Chowdhary India Dr. A. Ravi India Dr. P. Sathe India Prof. Shrikant Anant USA Dr. P. Gunasekaran India Prof. S. Kumaman India Dr. R.S. Paranjape India Dr. Udhaya Shankar USA Dr. S. Dhamodaran Singapore Board of Editors (2011) Dr. Jotna Sokhey, M.D., Ph.D. (Moscow) Prof. Shoba Broor, M.D., Ph.D. India Dr. V.K. Vijayan, M.D., Ph.D. India Dr. John Mackenzie Australia Dr. S.K. Lam Malaysia Dr. Dato. V.G. Kumaravel Malaysia Dr. A.C. Mishra India Dr. Satish Kumar USA Language Editor Prof. S. Murugan India India
Print ISSN 2231-1327 Volume 1 Number 1 January-June 2011 2011 International Association of Medical and Pharmaceutical Virologists INTERNATIONAL ASSOCIATION OF MEDICAL AND PHARMACEUTICAL VIROLOGISTS Postgraduate and Research Department of Microbiology and Biotechnology Presidency College (Autonomous), Chennai - 600 005, India WISDOM ACADEMIC PUBLISHERS No. 10/8, Dr. Nammalvar Street, Triplicane, Chennai - 600 005, India Phone: +91 44 2844 7276 E-mail: wisdomedu2003@yahoo.co.in The Editor-in-Chief and IAMPV assume no responsibility for the views expressed by the authors of material printed in the International Journal of Medical and Pharmaceutical Virology. It is a condition of publication that manuscripts submitted to the International Journal of Medical and Pharmaceutical Virology have not been published and will not be simultaneously submitted or published elsewhere. By submitting a manuscript, the authors agree that the copyright for their article is transferred to the International Association of Medical and Pharmaceutical Virologists, if and when the article is accepted for publication. The copyright covers the exclusive reprints, photographic reproductions, microform or any other reproductions of similar nature and transactions. No part of the publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, electrostatic, magnetic tape, mechanical photocopying, recording or otherwise, now known or developed in the future without prior written permission from the copyright holder. International Journal of Medical and Pharmaceutical Virology is a biannual journal published by Wisdom Academic Publishers, Chennai on behalf of the International Association of Medical and Pharmaceutical Virologists. Printed in India.
INTERNATIONAL JOURNAL OF MEDICAL AND PHARMACEUTICAL VIROLOGY Copyright 2011 International Association of Medical and Pharmaceutical Virologists, Chennai, India Vol. 1 No. 1 January-June 2011, p. iii
SRI RAMACHANDRA UNIVERSITY

(Declared under Section 3 of the UGC Act, 1956) PROF. S.P. THYAGARAJAN PhD., M.D., D.Sc., FNASC, FAMS, FIMSA, FABMS (Former Vice-Chancellor, University of Madras) PRO-CHANCELLOR (RESEARCH) Porur, Chennai - 600 116, India Phone +91-44-4592 8665 (Direct) +91-44-2476 5512 Extn. 665 Fax +91-44-2476 7008 Email sptrajansrmc@gmail.com
Message
Scientific and research journals are platforms for effective communication of validated findings which add value to the existing knowledge in a given discipline or usher in new knowledge of great societal relevance. In this context, I am pleased to learn about the launch of the International Journal of Medical and Pharmaceutical Virology (IJMPV). I am happy to note that both the International Association of Medical and Pharmaceutical Virologists and the Editorial team of IJMPV have taken up peer-reviewing of all manuscripts submitted for publication and to have credentialed for indexing purposes. I am confident that this Journal would be best utilised by the researchers across the globe, especially the young scientists in the specialities of Medical and Pharmaceutical Virology. I wish that the Journal acquires appreciable impact factor at the earliest, which would be testimony to the quality of articles published in this Journal. I fondly wish the Association and the Journal every success in their academic journey!
(S.P. Thyagarajan)
A Harvard Medical International Associated Institution
INTERNATIONAL JOURNAL OF MEDICAL AND PHARMACEUTICAL VIROLOGY Copyright 2011 International Association of Medical and Pharmaceutical Virologists, Chennai, India Vol. 1 No. 1 January-June 2011, p. v
Contents
Message From the Editor-in-Chiefs Desk Is Herbal Drug Discovery Different from Single Molecule Discovery? S.P. Thyagarajan 2. Rotavirus Vaccines and Anti-rotavirus Activity of a Few Medicinal Plants Used Against Diarrhoea S. Ananthan 3. Sero-prevalence of Ross River Virus Infection in Eight Persons Among 53 from Tamil Nadu, India Suspected of Chikungunya-like Infection S. Rajarajan, K. Sangeetha, D. Santhoshkumar, G.S. Kumar, A. Divyadarshni, G. Shanthi and R. Ravanan 4. Spread of Pandemic Influenza (H1N1 2009) Virus in Tamil Nadu, India (2009-10) Gunasekaran Palani, Kaveri Krishnasamy, Mohana Sambasivam, Kavita Arunagiri, R. Senthil Raja, Suresh B.V. Babu, Kiruba Ramesh, Senthilkumar Vijayan, Padma Priya Padmanaban, N. Saran, N. Nivas Chakravarthi, V.G. Nagaraj, K. Saravanamurali and Khaleefathullah A. Sheriff 5. A Seroepidemiological Survey of Hepatitis B and C Virus and Risk Factors in Healthy School Children in Chennai, Tamil Nadu, India E. Vikram Reddy, G. Ashok and P. Rajendran 6. Interleukin-10: A Potent Antibody Promoting Cytokine Induced by Grateloupia lithophila on Human PBMC S. Dinesh, S. Sumathi, M. Manikannan, R. Balamurugan, T. Ravindran, J. Balasubramaniam, V. Mythily and M. Elanchezhiyan 7. Seroprevalence of Human Herpes Virus Type-1 and Type-2 Among Antenatal Care Women in Chennai, India and their Co-seropositivity to Human Immunodeficiency Virus, Hepatitis B Virus, Hepatitis C Virus and Cytomegalovirus S. Thiyagarajan and S. Rajarajan 8. In Vitro and In Vivo Anti-viral Activities of an Active Fraction from the Plant Ocimum sanctum Linn. Molly Antony, Ambili S. Nair, D. Vivek, Shimitha Cyril, K.B. Rameshkumar and A. Subramoniam 9. Potential Application of Bioinformatics in Development of an Efficacious Envelope Protein Based Vaccine for Chikungunya: A Pilot Study Priya Das, Indu Purushothaman and S. Rajarajan 10. Prevalence of Japanese Encephalitis in South India Gunasekaran Palani, Kaveri Krishnasamy, Mohana Sambasivam, Kavita Arunagiri, Khaleefathullah A. Sheriff, Senthilkumar Vijayan, Padma Priya Padmanaban, Suresh B.V. Babu and Kiruba Ramesh Instructions to Authors 1.
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INTERNATIONAL JOURNAL OF MEDICAL AND PHARMACEUTICAL VIROLOGY Copyright 2011 International Association of Medical and Pharmaceutical Virologists, Chennai, India Vol. 1 No. 1 January-June 2011, p. vii
From the Editor-in-Chiefs Desk
Virology is a fascinating field for members of the scientific community who like to work in challenging circumstances in order to serve the cause of well-being of humankind. Over the years, tremendous advancements have been made in the fields of pharmaceutical sciences and medicine. Since viral infections and consequent epidemics constitute setbacks to mankinds efforts towards ensuring public health care, combating them requires first hand knowledge and access to technology. The laudable coming together of virologists from the fields of medicine and pharmaceutical science as part of the International Association of Medical and Pharmaceutical Virologists (IAMPV) marks a significant milestone as the scientific community marches towards achieving triumph in the prevention and control of precarious viral infections/ diseases. The launch of this International Journal of Medical and Pharmaceutical Virology (IJMPV) is a noble initiative by the Association towards fulfillment of its goal of playing a crucial role in promoting research and technology development in these fields. I have immense pleasure in serving the Association as the Editor-in-Chief of this prestigious journal. On behalf of the Editorial Committee of IJMPV, I earnestly request workers in the fields of medical and pharmaceutical virology to contribute the findings of their latest research works to IJMPV. PROF. DR. S. ANANTHAN
INTERNATIONAL JOURNAL OF MEDICAL AND PHARMACEUTICAL VIROLOGY Copyright 2011 International Association of Medical and Pharmaceutical Virologists, Chennai, India Vol. 1 No. 1 January-June 2011, pp. 1-2
Is Herbal Drug Discovery Different from Single Molecule Discovery?

S.P. Thyagarajan*
Formerly, Vice Chancellor, University of Madras, Chennai - 600 005; Presently, Pro Chancellor (Research) Sri Ramachandra University, Porur, Chennai - 600 116, India
In the early days of drug discovery, therapeutic drugs were discovered out of medicinal plants. This was the case of aspirin, which was derived from the bark of the Willow tree. In the 1960s, advances in pharmacology led to elucidation of the function of cellular receptors, ion channels and enzymes. Drug discovery became more scientific and rational, resulting in useful drugs like -blockers, -blockers, calcium antagonists etc. However, development of blockbuster drugs with the potential to generate over US$ 1 billion in sales has decreased due to the R&D expenses rising from US$ 2 billion in 1980 to over US$ 40 billion in 2007. Parallelly, the number of approvals for new molecular entities dropped to 17 in 2002 and 10 in 2007. While mass screening of plants in the search for new leads or drugs is vastly expensive and inefficient, traditional knowledge offered better leads. It is estimated that over 100 new natural product based leads are in clinical development. About 60% of anti-cancer and 75% of anti-infective drugs approved from 1981-2002 could be traced to natural product origin. The three major perceived hurdles in drug development are time, money and toxicity. Hence, instead of the laboratories to clinics concept of drug discovery wading through target identification, lead identification, lead optimisation and candidate drug development through biotechnological, genomics, high throughput screening or combinational and asymmetric synthesis, the newer concept of, clinics to laboratories is gaining prominence by adopting reverse pharmacology and systems biology approaches. Since Ayurveda, Siddha and other traditional systems of medicine provide knowledge and experiential database on clinical experiences as well as observations of their actual use in patients, it is perceived that they would provide new functional leads to reduce time, money and toxicity by adopting reverse pharmacology and systems biology approaches. Reverse pharmacology has evolved as the science of integrating documented clinical experiences and experiential observations into leads by trans-disciplinary exploratory studies and further developing them into drug candidates or formulations through robust preclinical and clinical research. Systems biology provides predictive models of behaviour of diverse molecular systems to identify functional interactions, since macromolecules form complex networks and get organised into systems with properties that extend beyond individual functions. This is based on the principle that, when multiple cell types and diverse pathways contribute to the disease, a single molecule may not be effective in modulation of multiple targets. This is the basis for evolving combination therapy in modern systems of medicine in diseases like HIV/AIDS. Herbal extracts are combinatorial chemistry of nature with vast array of chemical compounds which deal with multiple targets simultaneously leading to synergistic systems effect. The mechanism of such activity can be evaluated by system biology of testing herbal drugs deploying microarrays.
*Author for Correspondence. E-mail: profspt@gmail.com
Rotavirus Vaccines and Anti-rotavirus Activity of a Few Medicinal Plants Used Against Diarrhoea
S. Ananthan*
Formerly Professor of Medical Microbiology, Department of Microbiology Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Chennai - 600 113, India
Introduction
Infant mortality rate (IMR) in India is presently estimated at 53 per 1,000 live births. Most of the studies have shown IMR decline in urban areas as compared to rural areas. Acute respiratory infections and diarrhoea are important causes of IMR in infants and children. Diarrhoeal diseases remain one of the worlds major public health problems as they are a leading cause of childhood morbidity and mortality, particularly in infants and children. Viral gastroenteritis is an intestinal infection caused by several different viruses. The main symptoms of viral gastroenteritis are watery diarrhoea and vomiting. Other symptoms are headache, fever, chills and abdominal pain. Symptoms usually appear within 4-48 hrs after exposure to the virus and last for 1-10 days. Viral gastroenteritis causing viruses damage the cells in the lining of the small intestine and produce watery diarrhoea. The resulting combination of diarrhoea and vomiting causes dehydration, i.e. loss of fluids from the body. Important salts or minerals known as electrolytes can also be lost along with the fluids. Mild dehydration can be treated by drinking liquid. The World Health Organisation (WHO) has developed a diarrhoeal solution, known as oral rehydration therapy (ORT). Severe dehydration may require intravenous fluids and even hospitalisation. Untreated severe dehydration can be life threatening. Although more than 20 different microorganisms (bacteria, parasites and viruses) cause diarrhoea, rotavirus causes 25-55% of all hospital admissions for diarrhoea. It is also the most frequent causative agent worldwide for the most severe disease in children under 5 yrs of age. Hygiene, sanitation and good nutrition are unlikely to have substantive effect in controlling the rotavirus gastroenteritis. The death toll due to the virus is much higher in the developing countries. WHO has recommended the inclusion of rotavirus vaccine in the national universal immunisation programme in India. Vaccine is considered the best means of controlling rotavirus gastroenteritis.
Viral Aetiology of Diarrhoea

Though many types of viruses can cause gastroenteritis, the most common ones are: (i) Rotavirus: It is the leading cause of severe gastroenteritis among 3- to 15-month-old children and the most common cause of diarrhoea in children under 5 yrs of age. Symptoms of rotavirus infection appear 1-2 days after exposure. Rotavirus typically causes vomiting and watery diarrhoea for 3-8 days, along with fever and abdominal pain. Group A rotavirus causes 25-65% severe infantile gastroenteritis worldwide. Acute infections with group C are quite frequent and worldwide. (ii) Enteric adenovirus: It occurs mainly in children under 2 yrs of age. Of the 49 types of adenoviruses, types 40 and 41 affect the gastrointestinal tract causing vomiting and diarrhoea. Symptoms typically appear one week after exposure. Adenovirus infections occur throughout the year. Studies confirm that they cause 2-6% of all cases.
*Author for Correspondence. E-mail: sowanand@hotmail.com
Intensive epidemiological studies over the last 30 yrs since rotavirus was first described have shown that it is the most common cause of life-threatening gastroenteritis in children. The incidence and distribution of Group A rotavirus serotypes and genotypes vary between geographical areas during a rotavirus season and from one season to another. Globally, G1, G2, G3, G4, P1A and P1B are the most common G- and P-types causing disease in humans. The application of reverse-transcription polymerase chain reaction genotyping and nucleotide sequencing has helped in the identification of strain diversity, with at least 42 P-G combinations being recognised in human infections, and has also helped in the identification of additional globally (G9) and regionally (G5, G8, P 2A) common strains, which are not covered by the vaccines that have undergone trials. Figure 1 shows the high genomic diversity of rotavirus.
Rotaviruses
Serogroups
Infects humans Causes severe life threatening infections in infants and children worldwide
Based on: Antigenic specificity of the capsid protein VP6 Electrophoretic pattern of II RNA segments
Subgroups
I and II
Based on antigenic epitopes on the proteins that form the inner capsid VP6 Based on proteins of the outer capsid and the glycoprotein VP7
G-serotypes
P-serotypes
VP7 and VP4 elicit neutralising antibodies
Based on the spike protein VP4
Genotypes
Globally G1, G2, G3, G4, P1A and P1B are most common in G- and P-serotypes causing disease in humans G9 (globally common strains) and G5, G8, P2A (regionally common strains) Fig. 1. High genomic diversity of rotavirus.
Additional types
Epidemiological and molecular studies in many countries have shown complex patterns of change from year to year in the serotypes and electropherotypes (E types) that cause diarrhoea in hospitalised children from the same geographical areas. Recent epidemiological studies in India, Bangladesh and the United States of America have shown that other Gand P-types (G5, G6, G8, G9, G10, P2A, P8) can be common and may be of emerging importance in some communities. Thus, extensive epidemiological studies on rotavirus serotypes are required to map annual changes in strains in different geographical areas. The data can be useful to select areas for vaccine trials and to serve as baselines for identification of new strains.
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Sero-prevalence of Ross River Virus Infection in Eight Persons Among 53 from Tamil Nadu, India Suspected of Chikungunya-like Infection
S. Rajarajan,1* K. Sangeetha,1 D. Santhoshkumar,1 G.S. Kumar,3 A. Divyadarshni,1 G. Shanthi1 and R. Ravanan2
1Department
of Microbiology and Biotechnology, 2Department of Statistics, Presidency College (Autonomous) Chennai - 600 005, India 3Computer Diagnostics Centre, Kallakurichi, India
Abstract Background and Objectives Owing to the clinical symptoms of tenosynovitis, fever, rashes, lymphadenopathy and polyarthritis among people living in coastal Tamil Nadu, IgM seroprevalent study was undertaken for the detection of IgM antibodies to Ross River virus (RRV) antigen and Chikungunya virus antigen. Methods Sera were collected from the infected patients and subjected to IgM Enzyme-linked Immunosorbent Assay (ELISA) for the detection of IgM antibodies to RRV antigen. The samples were also tested for the presence of IgM antibodies to Chikungunya virus (CHIKV) to rule out the cross reactivity. Pearson Chi-square test was used to determine the significance of the age in the obtained results and t-test for the paired samples. Results Out of the total 53 sera samples tested, eight were found to be positive to RRV and showed an index value 1.1 (minimal cutoff value for positivity). Also, 13 samples were equivocal with index values between 0.9 and 1.1. The remaining 32 samples were negative to RRV as the index values were below 0.9. Interestingly, five samples were positive and three samples were equivocal to Chikungunya infection. Interpretation and Conclusion Serologically, 15% of the tested samples were positive and 24% were equivocal to RRV. This finding had comparatively higher percentage than the percentage of positivity observed for CHIKV (positivity (9%) and equivocal (5%)). Statistical analyses justify the seropositivity to Ross River as highly significant and negativity to Chikungunya also as highly significant. Thus, it justifies the prevalence of RRV infection over CHIKV among 53 patients from Tamil Nadu, India during 2010. Keywords: RRV, IgM ELISA, CHIKV.
Introduction
Arthritogenic alphaviruses cause debilitating infections in human beings. The disease manifests in sudden onset of fever, rash and severe arthralgia [1]. Arthritogenic alphaviruses are globally distributed. Six of them are known to cause articular
*Author for Correspondence. E-mail: drsrajarajan@gmail.com
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manifestations [2-4]. They are Chikungunya, ONyong Nyong and Sindbis viruses from tropical Africa; the Ross River and Barmah Forest viruses from the South Pacific and Australia; and Mayaro virus from South America [5-7]. Propagation of these viruses occurs via a horizontal cycle involving mosquito vectors and vertebrate hosts, i.e. transmission from mosquito to vertebrate to mosquito. The main mosquito vectors are Aedes aegypti and Aedes albopictus for Chikungunya virus (CHIKV) and Culex annulirostris (inland areas), Ochlerotatus vigilax, Verrallina funerea and Ochlerotatus camptorhynchus (coastal regions) of Australia for Ross River virus (RRV) [8]. These viruses are transmitted from the place of origin to other regions through infected travellers. The RRV, primarily found in Australia, was introduced to Fuji by a single infected traveller and caused an explosive epidemic of polyarthritis [9]. This virus was endemic to Australia during 1979 and 1980. Major epidemics of RRV infection have been reported from several countries in the Pacific region while less serious outbreaks in other countries may have remained undiagnosed. During December 2009 to January 2010, thousands of people in Tamil Nadu, India were infected by a mysterious illness. The afflicted people showed symptoms of severe joint pain, fever, rashes and swelling of legs. Owing to the endemic feature of the virus in Australia and absence of the Australian mosquito species transmitting the virus in India, nobody correlated the symptoms of the disease to RRV. As non-resident Indians employed in Australia, students pursuing higher education and tourist travellers visit India and as the symptoms were similar to that of RRV, it was suspected that the sudden outbreak in Kallakurichi, Tamil Nadu in the post-rainy season may be due to RRV. Earlier studies have been confined to CHIKV [11-14] because other arthritogenic alphaviruses were not considered. Hence, the present study was undertaken to determine the seroprevalence of RRV in comparison to CHIKV among infected persons of Kallakurichi, Tamil Nadu.
Materials and Methods

Sample Collection A questionnaire was used to elicit clinically relevant information on symptoms such as fever, rashes, myalgia, polyarthritis, headache, lymphadenopathy, onset of symptoms etc., from the patients reporting to Computer Diagnostics Centre, Kallakurichi, Villupuram District, Tamil Nadu. Patients with acute infections within five days from the onset of symptoms were chosen for the study. About 5 ml of blood was collected from each patient and centrifuged at 1500 g for 15 min at 4C. The serum was separated and stored at 80C for screening. The patients were selected based on clinical symptoms related to that of Chikungunya and diagnosed by a clinician, Dr. G.S. Kumar, Computer Diagnostics Centre, Kallakurichi. A written consent was taken from the patients. The sample size was decided based on World Health Organisation (WHO) guidelines that mandate a minimum of 50 samples to do a significant finding. The present study neither involved any control nor subjected the patients to an invasive study. The study was part of routine diagnostic work involving the serum of patients reporting to the Centre for Chikungunya illness, with an additional screening for another arboborne virus RRV. The study protocol was approved by the Institutes Ethics Committee. IgM ELISA IgM ELISA was performed for the 53 samples to detect IgM antibodies to RRV antigen in conformity to the Instruction Manual supplied by Panbio kit (Batch No. 09110, EV RVO1M). Parallely, the samples were tested for the presence of IgM antibodies to CHIKV in conformity with the Instruction Manual supplied by National Institute of Virology, Pune (in-house kit). Statistical Analysis Pearson Chi-square test was applied to determine the significance of seropositivity for the age group below 45 years for RRV and above 60 years for CHIKV. The t-test was used for dual samples of patients negative to CHIKV and positive to RRV, as also those equivocal to RRV and negative to CHIKV.
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Spread of Pandemic Influenza (H1N1 2009) Virus in Tamil Nadu, India (2009-10)
Gunasekaran Palani, Kaveri Krishnasamy,* Mohana Sambasivam, Kavita Arunagiri, R. Senthil Raja, Suresh B.V. Babu, Kiruba Ramesh, Senthilkumar Vijayan, Padma Priya Padmanaban, N. Saran, N. Nivas Chakravarthi, V.G. Nagaraj, K. Saravanamurali and Khaleefathullah A. Sheriff
Department of Virology, King Institute of Preventive Medicine and Research (KIPMR), Guindy, Chennai - 600 032, India
Abstract Background and Objectives Influenza virus is a common human pathogen that has caused serious respiratory illness, pandemics and death over the past century. The emergence of a novel strains of influenza virus A (pH1N1) in April 2009 focused attention on influenza surveillance capabilities worldwide. Materials and Methods Testing for pH1N1 was initiated in the Department of Virology, King Institute of Preventive Medicine and Research, Chennai in May 2009. Out of the total 10,086 samples screened by Real Time RT-PCR, 1,623 (16.09%) were found to be positive for pH1N1. Results Of the 1,623 positive cases, 560 (34.5%) were pediatric cases and 1,063 (65.5%) were adult cases. Among the pediatric age group, the 5-12 yrs age group was maximum affected and among adults the 19-30 yrs age group was maximum affected. The pH1N1 positive cases reported from June 2009 peaked in September 2009 and continued actively till January 2010. Thereafter a decline was witnessed. Interpretation and Conclusions From this influenza outbreak it was learnt that such an emergency can only be dealt with by a well established public health and surveillance programme, which allows appropriate response. It is very important, therefore, to consider actions concerning influenza A H1N1 containment by following the recommendations of public health officials. Keywords: Pandemic H1N1, Influenza, rRT-PCR.
Introduction
The first case of H1N12009, reported on March 18, 2009 by the Federal District of Mexico [1], marked the beginning of a pandemic involving almost all the countries worldwide. By September 20, 2009 human infections caused by H1N1 virus had been identified in 191 countries [1]. On June 11, 2009 the World Health Organisation (WHO) raised its pandemic alert to the highest-level phase 6, indicating widespread community transmission. The successful zoonotic transfer of Influenza A virus, containing gene segments of avian, swine and human origin, to humans along with consistent human-to-human transmission on each of the worlds continents fulfilled each of the current criteria for a pandemic strain [2]. A higher rate of transmission was observed among A H1N1viruses compared
*Author for Correspondence. E-mail: virology@dataone.in
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to seasonal influenza viruses [3]. The Influenza A H1N1 infection, primarily seen among young and previously healthy adults, suggested that these groups were most vulnerable to infection [4]. While some fatal cases have been attributed to secondary bacterial infections or exacerbation of other health conditions, as was commonly seen in fatal cases of seasonal influenza in the elderly, an unusual feature of Influenza A (H1N1) 2009 infection was severe viral pneumonitis, leading to acute respiratory distress syndrome, prolonged stay in intensive care units (ICUs) and extended use of mechanical ventilation [5, 6]. Pandemic H1N12009 has several distinctive epidemiologic features. The distribution of cases is similar across multiple geographic areas. The distribution of cases by age group is markedly different from that of seasonal influenza, with more cases in school children and fewer cases in older adults; it was the former group which had the highest mortality [7]. The first case of pH1N12009 was identified in India on May 16, 2009 in Hyderabad with subsequent cases observed in many other states [8]. Influenza A H1N1 2009 virus was identified in Coimbatore, Tamil Nadu, India on June 1, 2009 [9]. Initially, people returning after travel from abroad were found to be affected. However, later on the common public was also infected, thus, indicating community transmission. The circulation of seasonal and pandemic influenza A H1N1 strains makes the flu season more challenging. The present study is a retrospective analysis of H1N1 2009 during the pandemic period and its impact on the population of Tamil Nadu.

Initially, samples were collected from clinically suspected individuals referred by Madras International Airport, Chennai for screening. Later, from June 2009 onwards, samples were collected from patients referred to KIPMR by the physicians, from clinically suspected out- and in-patients of both government and private hospitals. At the time of sample collection, informed consent from all individuals was obtained by using a standard questionnaire. Laboratory request forms were completed with all necessary demographic and case details for all cases to be tested. The strength of the present study includes its prospective design test setting and use of Real Time RT-PCR assays to detect pandemic (H1N1) 2009. Nasopharyngeal (NP) secretions were collected from all suspected cases of Influenza A H1N1 who showed acute febrile respiratory illness with onset within seven days of contact with positive cases. The NP swabs were inserted into 3 ml of universal transport medium (Himedia). A total of 10,086 samples were analysed with the pH1N1 Real Time RT-PCR assay. All samples were subjected to RNA Extraction using viral RNA kit (Qiagen) and Real Time RT-PCR was performed as per Centers for Disease Control and Prevention, USA protocol, wherein four sets of primers and probes are used. Each sample was subjected to primers against Pan A, Swine A, Swine H1 and RNAse P. Ambion super script one-step RT-PCR kit was used for performing Real Time RT-PCR. The reaction mix concentration included a PCR reaction (one-step RT-PCR kit, Ambion) volume of 25 l for the matrix assay that contained 5 l of RNA template, 2 reaction buffer 12.5 l, 0.5 l of four different target assays (probe and primer), 6 l nuclease free water and 25 of 1 l enzyme mix. Thermal cycling was done on Real Time PCR systems (ABI 7500 and Roche lightcycler 480) instrument under different cycling conditions: 30 min at 50C, 2 min at 95C followed by 45 cycles of 15 s at 95C, and 30 s at 55C. The CDC supplied all primers and probes. The positive and negative controls were supplied by CDC, which was run with each test. The primers details are as follows: Primers and probes Sequence (5 3) 1. InfA F GAC CRA TCC TGT CAC CTC TGA C 2. INFA R AGG GCA TTY TGG ACA AAK CGT CTA 3. InfA P TGC AGT CCT CGC TCA CTG GGC ACG 4. SW InfA F GCA CGG TCA GCA CTT ATY CTR AG 5. SW InfA R GTG RGC TGG GTT TTC ATT TGG TC 6. SW InfA P CYA CTG CAA GCC CAT ACA CAC AAG CAG GCA 7. SW H1 FGTG CTA TAA ACA CCA GCC TYC CA 8. SW H1 R CGG GAT ATT CCT TAA TCC TGT RGC 9. SW H1 P CA GAA TAT ACA TCC RGT CAC AAT TGG ARA A 10. RnaseP F AGA TTT GGA CCT GCG AGC G 11. RnaseP Reverse GAG CGG CTG TCT CCA CAA GT 12. RnaseP Probe TTC TGA CCT GAA GGC TCT GCG CG INTERNATIONAL JOURNAL
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A Seroepidemiological Survey of Hepatitis B and C Virus and Risk Factors in Healthy School Children in Chennai, Tamil Nadu, India
E. Vikram Reddy,1 G. Ashok1 and P. Rajendran2*
1Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Chennai - 600 113, India 2Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur Chennai - 600 116, India
Abstract Background/Objective Available literature on the epidemiological pattern of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in healthy school children of India is scanty. In view of the paucity of data, the present study looks at the epidemiological pattern of HBV and HCV infection as well as the associated risk factors of acquiring HBV/HCV in healthy school children from the state of Tamil Nadu in South India. Methods In the study, 856 serum samples from apparently healthy children aged 3-18 yrs, studying in three schools in and around Chennai, Tamil Nadu were analysed for hepatitis B surface antigen (HBsAg). Hepatitis B virus e antigen (HBeAg) screening was done only for HBsAg positive samples. Out of the 856 samples only 88 randomly selected samples were further screened for antibody to HBsAg (anti-HBs) and antibody to hepatitis B core antigen (anti-HBc IgG). Antibody to HCV (anti-HCV) was screened for 840 serum samples by ELISA. Samples were collected from children aged 3-5 yrs by finger prick method and by venepuncture from those in higher age groups. The collected serum was separated and stored at 20C prior to testing. Informed consent was obtained from either the children or their parents prior to the collection of blood samples. Children with history of HBV vaccination were excluded from the study. Results Out of the 856 serum samples from apparently healthy school children, 16 (1.86%) samples tested positive for HBsAg. Out of these 16 HBsAg positive samples, 6 (37.5%) samples were positive for anti-HBeAg, suggesting increased risk of chronicity. However, anti-HBs and anti-HBc IgG were screened in only 88 randomly selected serum samples. Out of these 88 serum samples, 6 (6.28%) samples were positive for anti-HBs and 4 (4.55%) samples were positive for anti-HBc IgG. Out of 840 samples screened, 8 (0.95%) samples were positive for HCV (anti-HCV). Conclusion Low prevalence of HBV infection in urban school children of Chennai was observed, perhaps due to increased health awareness among the urban population. The 0.95% anti-HCV positivity among healthy school children is probably the first reporting in the country. This finding suggests that HCV infection also occurs among the younger age group in urban school population. Keywords: HBV, HCV, Chennai, School children, Seroepidemiology. *Author for Correspondence. E-mail: rajendranparam@hotmail.com
A Seroepidemiological Survey of Hepatitis B and C Virus and Risk Factors in Healthy School Children
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Introduction
Chronic viral hepatitis is a major important public health problem in the world, especially in India. Chronic hepatitis B (HBV) virus and hepatitis C virus (HCV) infection are important causes for chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. Asymptomatic carriers of both these viruses serve as reservoir of infection to the uninfected population. Although vaccines are currently available against HBV, mass vaccination strategies in population make it mandatory to study the epidemiological pattern of HBV infection in various population. A few studies have documented HBV infection among healthy school children in India [1-4]. However, studies on the prevalence pattern of HBV and HCV in healthy school children in Tamil Nadu are extremely rare. Hence, the present study was undertaken.

Out of the 856 samples collected, 741 were venous blood samples from children above 4 yrs age and 115 dried blood spots collected by finger prick from children aged 2-4 yrs studying in three different city schools in and around Chennai by simple random sampling procedure. The finger prick blood samples were collected in Whatmans filter paper discs and stored in serum vials at 4C, after drying at room temperature, with code numbers until processed. Before testing 200 l of PBS was added and the discs were soaked for 10 min at room temperature and centrifuged at 2000 rpm for 10 min. The elute was collected and tested for HBV/HCV markers. The serum separated from blood samples was stored at 20C until it was tested. The following commercial micro ELISA kits were used for testing HBV and HCV markers in the above samples: (i) (ii) (iii) (iv) Hepatitis B surface antigen (HBsAg) by Hepanostika II (Organon Teknika, Belgium). Antibody to HBsAg (anti-HBs) by Hepanostika anti-HBs (Organon Teknika, Belgium). Antibody to hepatitis B core antigen (anti-HBc IgG) (Sanofi Pasteur Kit, France). Antibody to HCV (anti-HCV) by UBI-anti-HCV-III Gen. (UBI Biologicals, USA).
Results
The overall HBsAg positivity among healthy school children was 1.86% (95% CI 1.11-2.95) (Table 1). Surprisingly, 37.5% of the HBsAg positive (6 out of 16 children) samples were Table 1. Overall prevalence of chronic HBV (HBsAg) carriage in replicative carriers as shown by their HBeAg positivity. school children of Chennai The 88 randomly selected samples were tested for antiHBs and anti-HBc IgG. The seropositivity rate was 3.3% HBV markers No. of children No. of positive 95% CI (4 out of 121) pre-school children, 1.4% (8 out of 549) tested (%) primary school children, and 2.13% (4 out of 187) juniormiddle school students. The prevalence was 1.5% in HBsAg 1.11-2.95 856 16 (1.86) Anti-HBs 88 2.81-12.84 6 (6.82) children less than 10 yrs of age and 2.01% in those above Anti-HBc IgG 88 4 (4.55) 1.46-10.6 10 yrs of age (p > 0.05). The prevalence of HBV markers HBeAg 16 6 (37.5) Nil did not show any significance between males and females (Table 2). Anti-HCV positivity (Table 3) among healthy school children of Chennai was 0.95% (85% CI 0.44-1.80). The HCV prevalence was slightly higher in males (1.32%) than in females (0.64%).
Table 2. Prevalence of chronic HBV infection (HBsAg carriage) in school children HBV markers HBsAg Anti-HBs Anti-HBc IgG Sex Male Female Male Female Male Female No. of children tested 381 475 49 39 49 39 No. of positives (%) 10 6 4 2 2 2 (2.62) (1.25) (8.61) (5.12) (4.08) (5.12) P value > 0.05 > 0.05 > 0.05
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Interleukin-10: A Potent Antibody Promoting Cytokine Induced by Grateloupia lithophila on Human PBMC
S. Dinesh,1 S. Sumathi,1 M. Manikannan,1 R. Balamurugan,1 T. Ravindran,2 J. Balasubramaniam,3 V. Mythily3 and M. Elanchezhiyan1*
1Virology and Immunology Division, Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences University of Madras, Chennai - 600 113, India 2Department of Humanities and Social Services, Sri Venkateswara College of Engineering, Sriperumbudur - 602 105, India 3Rotary Central T.T.K. V.H.S. Blood Bank, Voluntary Health Services, Chennai - 600 113, India
Abstract Purpose Inducing required cytokine niche is an important paradigm for the development of better antiviral defence mechanisms. The present study was aimed at determining the in vitro cytokine stimulation property of extracts of Grateloupia lithophila (G. lithophila), a red marine algae on human peripheral blood mononuclear cell (PBMC). Methods Human PBMCs were isolated and stimulated with aqueous and ethanolic extracts of G. lithophila. Culture supernatants were collected at different time points and tested for Th-1 cytokine (IFN) and Th-2 cytokine (IL-10) using ELISA. Results Aqueous extracts of G. lithophila stimulated remarkable levels of IL-10 on human PBMC. Time dependent increase of IL-10 was noticed among 200 g/ml ethanolic extracts. However, there was no correlation between the varying concentrations of the extract and amount of IL-10 produced. Though there was a detectable amount of IFN-, the levels were much lower, unlike IL-10. Conclusion G. lithophila is a strong stimulator of IL-10, which is a potent Th-2 cytokine produced by CD4 cells. The IL-10 governs antibody production (humoral immunity), which is one of the strongest arms of adaptive immunity. The uses and limitations of G. lithophila as a possible immunotherapy agent against viral disease have been discussed. Keywords: Cytokine, Immunotherapy, PBMCs, Th1, Th2, Grateloupia lithophila.
Introduction
Microorganisms, especially viruses, are constantly on the lookout for opportunities to trick and exploit the immune system and cause infections. As a countermeasure, the immune system is loaded with various defence strategies. To maintain homeostasis, the immune system remains in defence mode to handle the billions of antigens that it faces every second. However, the system is also equipped with precision-guided immune cells that can adopt an offensive mode whenever faced with danger signals [1] or deceived by employment of immune evasive properties [2-9] by the viruses. Thus, deployment of defensive vs. offensive vs. anergy responses of the immune system determines the health status
*Author for Correspondence. E-mail: emanickan@yahoo.com
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of an individual. In this continuing battle a decisive role is played by cytokines, which do a plethora of activities (e.g. antiviral). By default, the immune system responds to viral infections by using a set of cytokines that are beneficial to the host as they coordinate with host immune cells and eliminate viruses efficiently. To counter this viruses have evolved mechanisms that can alter the otherwise protective cytokine phenotype into a non-protective or virus promoting phenotype. In this context, intervention strategies to help the host regain production of protective cytokines would be a novel approach that assists the immune system in its fight against infectious diseases. Immunotherapy represents a novel approach by boosting the immune components that would have a direct impact on antimicrobial immunity [10]. Immune modulation and immunotherapy has become the most attractive area of research following the discovery and understanding of several cytokines as well as their central role during disease prognosis. Using immunotherapy protocols, attempts have been made to prevent the damages caused by pro-inflammatory and inflammatory cytokines [11]. These cytokines mediate and regulate the cell-cell communication between the lymphocytes and other host immune cells in the course of an immune response [12]. Inhibition strategies adapted by infectious agents against the protective cytokine response of the host leads to severity of the disease either by up-regulating the antagonistic cytokine response or by inducing mechanisms that lead to the depletion of the protective cytokine response [13]. Cytokines secreted by T helper (Th) cells against infectious agents are important for the outcome of many diseases. The Th cell response can be divided into Th-1 or Th-2 based on the pattern of cytokine secretion. Th-1 cells produce gamma interferon (IFN-) [14], interleukin-2 (IL-2), and tumour necrosis factor- (TNF-), whereas Th-2 cells produce IL4, IL-10 etc. [15, 16]. T-cells that produce cytokine pattern distinct from the well-defined Th-1/Th-2 sets have been described as Th-0 [17, 18], Th-17 [19, 20] etc. Th-1 cells normally promote cell mediated immune (CMI) response cells and produce IFN- and IL-2 while Th-2 cells promote humoral immunity and produce IL-4, IL-5 and IL-10. Marine organisms have been found to have several bioactive compounds [21-25]. Medicinal values of several seaweeds have been explored. They have been found to contain antibacterial, antifungal and antifouling activities [26]. In addition, seaweeds are used in food, agar, cosmetics, medicines, minerals and fertilisers [27]. Among seaweeds the genus Grateloupia has been found to have antiviral activity against HIV [28]. Other species such as G. indica, G. filicina have been documented for their biological activities [29]. However, reports on their role against other viruses and immune modulation are scanty. For the present evaluatory study, G. lithophila was chosen for its T cell cytokine stimulation potential. To test this, extracts of G. lithophila were prepared and tested for IFN- (Th-1 cytokine) and IL-10 (Th-2 cytokine). The results have been presented and discussed in the context of antiviral cytokine therapy.

Algae Collection In December 2007 G. lithophila Boergesen was collected from Kanyakumari coast, India. The impurities were removed by rinsing in sterile distilled water and authenticated at the Department of Botany, University of Madras, Chennai. The algae was shade dried, powdered and stored at room temperature until use. Preparation of Extracts of G. lithophila Boergesen Aqueous extract was prepared by adding 50 gm of powder in 500 ml of sterile distilled water and mixed thoroughly. After overnight soaking the supernatant was collected and filtered with Whatman filter paper (no. 1) to remove debris. Then the filtrate was syringe filtered, lyophilised and stored at 4C. Ethanolic extract was prepared by adding 50 gm of algal powder in 500 ml of solvent and then filtered using Whatman filter paper (no. 1). The filtrate was allowed to evaporate for about 2-3 days. The dried filtrate was collected, weighed and re-suspended in appropriate solvent just prior to use. The processed ethanolic extract was stored at 4C until use. Isolation of Human PBMCs Blood obtained from healthy donors (Blood Bank, Voluntary Healthy Services, Chennai) was used for the study. Separation of blood cells was performed using Histopaque (Sigma Aldrich Chemicals, USA) and peripheral blood
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Seroprevalence of Human Herpes Virus Type-1 and Type-2 Among Antenatal Care Women in Chennai, India and their Co-seropositivity to Human Immunodeficiency Virus, Hepatitis B Virus, Hepatitis C Virus and Cytomegalovirus
S. Thiyagarajan1 and S. Rajarajan2*
1Department 2Department
of Microbiology, Asan Memorial College of Arts and Science, Chennai - 600 100, India of Microbiology and Biotechnology, Presidency College (Autonomous), Chennai - 600 005, India
Abstract: The human herpes viruses type-1 (HHV-1) and type-2 (HHV-2) are considered to be significant sexually transmitted infection (STI) viruses owing to their ability to establish latent infection in susceptible persons and to predispose to other viral infections including HIV due to the ulceration they cause on the skin or genitalia. The genital infection caused by these viruses in pregnant women leads to complication both in the mother and the neonate on skin, eye, mouth and central nervous system (CNS). In the present study 850 pregnant women reporting to two Government hospitals in Chennai for antenatal care (ANC) were screened for their seropositivity to immunoglobulin M (IgM) and immunoglobulin G (IgG) of HHV-1 and HHV-2 and their co-seropositivity to IgG of other STI viruses. The serological screening was performed by Enzyme-linked Immunosorbent Assay (ELISA) using standard serodiagnostic kits. The seroprevalence to HHV-1 and HHV-2 was 65.64 and 14.35%, respectively. The individual positivity to IgM and IgG was 8.47 and 47.41%, respectively. Combined positivity was 9.76%, respectively, to both IgM and IgG antibodies to HHV-1. For HHV-2 these were 6.82, 7.29 and 0.24%, respectively. The percentage of positivity to IgM was found to be statistically significant. The co-seroprevalence to other STI viruses among HHV-1 and HHV-2 seropositive individuals, respectively, was 0.36 and 0% (HIV), 6.99 and 8.20% (hepatitis B virus (HBV)), 1.43 and 3.28% (hepatitis C virus (HCV)), 50.72 and 62.30% (cytomegalovirus (CMV/HHV-5)). The symptoms of HHV-1 or HHV-2 were comparatively more prevalent among the individuals who showed combined seropositivity to IgM and IgG antibodies and were statistically significant. Keywords: ANC women, HHV-1, HHV-2, Seroprevalence, Positivity to IgM, Positivity to IgG, Combined positivity to IgM and IgG.
Introduction
Many agents of sexually transmitted infections (STIs), particularly of viral origin, have been proved to be potentially harmful to pregnant women. They affect both the mother and the fetus, thus, leading to complications. Genital herpes has been recognised as one of the most prevalent STIs globally. Its prevalence over the years has been increasing significantly [1-3]. Approximately, 70% of newly acquired human herpes virus-1 (HHV-1) or HHV-2 infections among pregnant women are asymptomatic or unrecognised [4, 5]. The remaining 30% of women with new infections have clinical presentations that range from minimal lesions and mild discomfort to widespread genital lesions. Globally about 1.6 million new cases of genital herpes occur annually [6], 22% of pregnant women are seropositive to HHV-2 and more than 2% of women acquire genital herpes during pregnancy [7]. Neonatal HHV-1 or HHV-2 causes disseminated or central nervous system (CNS) disease in approximately 50% of the cases. Up to 30% of these infants will die, and up to 40% of survivors will have neurologic damage, despite anti-
*Author for Correspondence. E-mail: drsrajarajan@gmail.com
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viral therapy. The HHV-1 or HHV-2 mediated CNS disease is characterised by seizures, lethargy, irritability, tremors, poor feeding, temperature instability and bulging fontanelle. The remaining 25% of cases have disseminated infection involving multi-organ systems and may result in death from sever coagulopathy, liver dysfunction and pulmonary failure [8]. The social and economic costs of long-term care of infants with sequelae from neonatal herpes are substantial [9]. Therefore, prevention of neonatal herpes infection remains critically important in decreasing the sequelae from this disease. Several reports on the seroprevalence of HHV-1 and HHV-2 among antenatal care (ANC) women worldwide have been published. Ades et al. [10] studied the seroprevalence of HHV-1 immunoglobulin G (IgG) among pregnant women in West London for two years and reported that HHV-1 seroprevalence was in the range of 60.0-80.0% in white, Asian and UK born black women. Ibrahim et al. [11] reported the seroprevalence of HHV-1 among pregnant women from different cities in Syria; the seroprevalences of HHV-1 IgM and HHV-1 IgG, respectively, in pregnant women was 5.45 and 100%. Alanen et al. [12] observed the seroprevalence of IgM and IgG antibodies to HHV-2 and among pregnant women in South-Western Finland; the seroprevalence of HHV-2 was found to be 9.3%. The incidences of symptomatic cases among HHV-2 seropositive pregnant women were 8.0% (orofacial HSV) and 43.5% (genital HSV). Chen et al. [13] reported 10.8% of seroprevalence of HHV-2 IgG in women attending an antenatal clinic of Fujian Provincial Maternal and Child Hospital in Fuzhou, China. There is a paucity of data on the co-seroprevalence of other STI viruses among HHV-1 and HHV-2 seropositive ANC women of India. Also, few data are available on the co-seroprevalence of HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) among the HHV-2 seropositive antenatal care women. Data for the same among HHV-1 seropositive ANC women is not available. Msuya et al. [14] reported 61.4% seroprevalence of HIV and 37.5% seroprevalence of HBV (HBsAg) among HHV-2 IgG seropositive pregnant women attending primary health care clinics in the urban district of Moshi, Tanzania. Kibur et al. [15] reported 41.8% seroprevalence of HBV and 2.8% seroprevalence of HCV among HHV2 IgG seropositive pregnant women consulting antenatal clinics in Estonia. The data on the seroprevalence of HHV-1 and HHV-2 among ANC women in India are not just scanty but either IgG based or carried out with combined antigen kits for HHV-1 and HHV-2. Therefore, the present study has been carried out with specific antibody detection kits to observe the seroprevalence of HHV-1 and HHV-2 and to estimate the IgM seropositive ANC women, who are considered to be one of the STI susceptible groups and study their co-seropositivity to other STI viruses such as human immunodeficiency virus (HIV), HBV, HCV and cytomegalovirus (CMV).

Study Population In all, 850 pregnant women who reported for ANC to Government Hospital, Chrompet and Government Maternity and Child Care Hospital, Egmore in Chennai, India were enrolled in the study on voluntary basis. The seroscreening lasted for four years from October 2004 to September 2008. The enrolled volunteers were interviewed using a questionnaire to obtain information on the sociological and demographical details pertaining to the study of interest. The relevant sociological factors included name, sex, age, religion, residential area, marital status, family type, educational qualification, occupation and nature of job. The demographic factors included clinical symptoms observed (by the Medical Officer), type of first sexual contact, sexual relationship, practice of condom use, practice of injection drug abuse, duration of injection drug abuse, habit of injection needle sharing and other habits (smoking, alcoholism, oral substitution drug abuse). For comparative study, 64 healthy women belonging to the same age group were inducted in the study and screened using similar procedures. Serological Assay In the study 2 ml of blood sample was collected at the hospital from each enrolled volunteer. The samples were transferred to the laboratory under cold chain. All the collected blood samples were processed at the Laboratory of Postgraduate and Research Department of Microbiology and Biotechnology, Presidency College (Autonomous), Chennai, India. Each specimen was then subjected to centrifugation to separate the serum samples. The serum samples of each volunteer were then marked with a unique code for identification.
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IgM, 6.82 IgM and IgG, 0.24 IgG, 7.29 IgM, 6.25 IgM and IgG, 1.56 IgG, 6.25 Negative, 85.94 Fig. 5. Seroprevalence (%) of HHV-2 among control group.
Negative, 85.65 Fig. 4. Seroprevalence (%) of HHV-2 among ANC women.
seropositivity to IgM and IgG of HHV-2 (P = 0.257) (Table 4; Fig. 7) half of the individuals (50.0%) were symptomatic. Earlier workers have observed that IgM antibodies, after their production, may persist upto one year [17, 18] and that they may be detected during the recurrent phase of infections [19]. Hence, HHV-1 or HHV-2 seropositive individuals who show combined seropositvity to IgM and IgG could be considered as patients with active symptomatic infection. This finding can be considered as significant because it is new to the literature. Thus, screening of IgM antibodies and identifying the individuals who are seropositive to both IgM and IgG antibodies would help not only in seroprevalence studies but also in monitoring the status of the infection and its response to drugs.
100 90 80 70 60 50 40 30 20 10 0 ANC Control 100 90 80 70 60 50 40 30 20 10 0
ANC Control
50.00 33.87 50.00
20.48
IgM
2.78
IgM + IgG
5.45 9.37
IgG
IgM
13.79
IgM + IgG
IgG
Fig. 6. Prevalence (%) of symptomatic cases among HHV-1 seropositive ANC and control.
Fig. 7. Prevalence (%) of symptomatic cases among HHV-2 seropositive ANC and control.
On the co-seroprevalence of other STI viruses among the HHV-1 and HHV-2 seropositive antenatal group (Figs. 8 and 9; Tables 5 and 6), HIV co-positivity was found to be associated significantly with HHV-1 (0.36%; P = 0.019). Ironically, it was not associated with HHV-2. The immunodeficiency caused by HIV in ANC group, especially among those who are already infected with HHV-1, could be considered at potential risk. Although HHV-1 is comparatively less
100 90 80 70 60 50 40 30 20 10 0 ANC
50.72 6.99 55.55
Control
4.44
0.36
HIV
HBV
1.43
HCV
HHV-5
HHV-2
Fig. 8. Co-positivity (%) of viral STI among HHV-1 seropositive ANC and control group.
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6.66
In Vitro and In Vivo Anti-viral Activities of an Active Fraction from the Plant Ocimum sanctum Linn.
Molly Antony,1 Ambili S. Nair,1 D. Vivek,2 Shimitha Cyril,1 K.B. Rameshkumar2 and A. Subramoniam2*
1Department of Microbiology, Sree Chitra Thirunal Institute of Medical Sciences and Technology Thiruvananthapuram - 695 029, India 2Phytochemistry and Phytopharmacology Division, Tropical Botanic Garden and Research Institute, Palode Thiruvananthapuram - 695 562, India
Abstract: Anti-viral property of a traditional medicinal plant, Ocimum sanctum has been studied using cell culture system and in vivo animal models. Alcohol extract of O. sanctum showed activities against Coxsackie B virus (CBV), measles virus and herpes simplex virus (HSV) in Vero cells. An anti-viral butanol fraction (active fraction) was isolated from the alcohol extract. This fraction showed anti-hepatitis B viral activity also, in cell line, HepG2.2.15. The fraction exhibited remarkable in vivo anti-CBV activity. At a dose of 250 mg/kg (per oral; twice daily) it protected all the virus challenged suckling mice whereas all the untreated virus challenged control pups died due to infection. The fraction also showed some level of efficacy against ducklings challenged with duck hepatitis virus (DHV). This active fraction was found to be devoid of any conspicuous sub-acute toxicity in mice. The HPTLC profile of the fraction was also determined. One of the active compounds in the butanol fraction was identified as ursolic acid. Thus, O. sanctum is an attractive plant for anti-viral drug development against CBV and other viruses. Keywords: Ocimum sanctum, Coxsackie virus, Hepatitis B virus, Anti-viral activity.
Introduction
Even today, infectious diseases remain one of the major causes of deaths worldwide [1, 2]. These diseases, in particular viral diseases, are leading to unpredictable epidemics and pose difficult challenges to public health [1]. Coxsackie viruses cause myocarditis. This virus infection is a serious medical problem, especially in newborns [3, 4]. There is no effective drug to combat Coxsackie viral infection. Nucleoside analogues and other drugs are available to treat most of the viral diseases, but these drugs have undesired side effects [5]. Further, appearance of resistance against these drugs has been reported [6]. Hence, there is an urgent need to develop new, more effective and safe anti-viral agents. One of the sources of anti-viral agents is traditional medicinal plants. A well-known traditional medicinal plant being used to treat several ailments, including bacterial and viral fevers, is Ocimum sanctum Linn. [7]. It is considered as an adaptogenic plant with several pharmacological properties [8-19]. In traditional medicine, this plant is used to treat viral diseases in many parts of India [7]. The in vitro anti-viral properties of a related species, O. basilicum has been reported [20]. The present study was undertaken to determine in vitro anti-viral activity, if any, of the plant against Coxsackie B virus (CBV), hepatitis B virus (HBV), measles virus and herpes simplex viruses (HSV) and in vivo efficacy against CBV in sucking mice and duck hepatitis virus (DHV) in ducklings.
*Author for Correspondence. E-mail: asubramoniam@yahoo.com
In Vitro and In Vivo Anti-viral Activities of an Active Fraction from the Plant Ocimum sanctum Linn.
1000 900 400 300 200
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100 400 300 200 100 A
0 0.09
0.12
0.32
0.52
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Fig. 1. TLC separation of butanol fraction (AF) from the alcohol extract of O. sanctum. Silica gel 60 F254 was used with the solvent system, chloroform-methanol (9.5:0.5), visualisation by spraying with anisaldehydesulphuric acid reagent. A: Butanol fraction showing resolved bands; band with Rf value of 0.44 corresponds to ursolic acid. B: Authentic ursolic acid (1 g in 1 l).
Fig. 2. HPTLC profile of O. sanctum butanol fraction (AF). Butanol fraction showing 15 peaks; peak with Rf value of 0.44 corresponds to ursolic acid. B: Authentic ursolic acid. HPTLC was done using CAMAG HPTLC system, equipped with the sample applicator Linomat V, scanner III and integration software CATS 4.04. The TLC was done on pre-coated aluminium plates of silica gel GF254 (Merck). Details of chromatography are as given in Fig. 1.
In Vitro Anti-hepatitis B (HB) Activity The AF showed anti-HB activity in HepG 2.2.15 cell line (Table 3). The fraction in a concentration dependent manner inhibited the production of HB surface antigen (HBsAg) in HepG 2.2.15 cell line. The concentration required for effective inhibition was 190 g/ml. The disappearance of HBsAg from the cell line treated with the AF was also time dependent (Fig. 3). Lamivudine was used as a positive control. The AF was found to be superior to Lamivudine. The efficacy of the AF was markedly evident from day one onwards (Fig. 3), whereas in the case of the standard drug marked effect was observed on day 12 and thereafter.
Table 3. Anti-HB viral activity of O. sanctum AF in HepG 2.215 cell line Virus Cell line Concentration of AF required for anti-HB activity (g/ml)* 190 14 Concentration of AF required for exhibiting cytotoxicity to Vero cells (g/ml) 2666 62
HB
HepG 2.215 (HB producing cell line)
Values are mean S.D. of three separate determinations. Cytotoxicity was detected using XTT assay. *Concentration of the AF required to lower the level of HBsAg below 2 ng/ml in 48 hrs. In the control cells, the concentration of antigen was around 15 ng/ml.
In Vivo Anti-Coxsackie Viral Activity in Suckling Mice The AF, when administered twice daily starting from the day of viral challenge, at a dose of 250 or 500 mg/kg body weight (per oral; daily), protected all the viral challenged pups. Lower dose (125 mg/kg) was not very effective (Fig. 4). The AF treatment did not influence body weight and behaviour of the pups. In each group, the fraction treated pups, which survived the viral challenge, also showed normal body weight (on day 8) and behaviour. In every aspect, they looked INTERNATIONAL JOURNAL
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25 20
HBsAg (ng/ml)
Control O. sanctum Lamivudine
15 10 5 0
4 7 Time (days)
12
17
Fig. 3. Time dependent decline of HB surface antigen (HBsAg) under the influence of O. sanctum AF in HBV producing cell line (HepG 2.215). Values are average of three determinations. Cytotoxic concentration to Vero cells, when tested using XTT assay, was 2666 62 g/ml. 7 6
Number of surviving pups
5 4 3 2 1 0 1 2 4 8 Days after virus challenge 20
Normal control Virus control Virus + AF (125 mg/kg) Virus + AF (250 mg/kg) Virus + AF (500 mg/kg) AF only (250 mg/kg) AF only (500 mg/kg)
Fig 4. Protection of CBV challenged suckling mice with butanol fraction (AF) of alcohol extract from O. sanctum leaf. Six 2or 3-day-old pups were used in each group. Indicated doses of AF (from alcohol extract) were administered orally, daily, twice a day, for eight days starting from the day of intra-peritoneal (100% mortality titre) virus challenge.
like normal pups. Average body weight of one-day-old pup was 1.9 0.2 g and the weight on day 8 was 5.8 0.5 g. The body weight of the AF treated pups (drug control) on day 8 was 5.7 0.6 g and that of viral challenged and AF treated (250 mg/kg) pups, which survived the challenge, was 5.5 0.5 g. The mice which survived the viral challenge looked normal in every way and reached adulthood with normal growth and body weight. The viral control pups, however, died with hind limb paralysis within three or four days. In vivo Anti-DHV Activity in Ducklings The AF at a dose of 250 mg/kg (per oral; daily) protected 50% of the viral challenged ducklings. Figure 5 shows that lower dose (125 mg/kg) was not very effective. The herbal drug treatment (drug control) did not influence body weight and behaviour of the ducklings. The drug treated ducklings which survived the viral challenge also showed normal body weight and behaviour. In every aspect, they looked like normal control ducklings. The body weight of one-day-old duckling was 35.1 3.2 g (mean S.D.) and weight on day 17 was 50.8 3.4 g. The body weight was not influenced INTERNATIONAL JOURNAL MEDICAL PHARMACEUTICAL VIROLOGY Vol. 1 No. 1 Jan.-Jun. 2011
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Table 5. Effect of short term (15 days) O. sanctum AF (butanol fraction from alcohol extract) administration on the hematological and serum bio-chemical parameters of adult male mice Parameter Control 250 mg/kg Haemoglobulin (mg/100 ml) WBC (106/mm3) Peritoneal macrophages (106/mouse) Urea (mg/dL) Glucose (mg/dL) GPT (IU/L) GOT (IU/L) AP (KA unit) Protein (g/dL) Cholesterol (mg/dL) 12.4 9.1 9.4 26.1 117.2 46.2 84.2 25.0 6.2 126.2 1.6 0.2 0.4 2.3 6.6 3.5 5.5 2.9 0.9 5.7 13.3 9.9 10.3 22.2 102.1 44.1 88.1 29.0 7.6 116.2 1.4 0.3 0.5 2.4 6.4* 2.3 4.5 3.4 1.1 6.4 AF treated 500 mg/kg 16 11.6 11.4 19.0 81.4 40.4 83.5 24.0 8.0 128.2 1.7* 0.3* 0.4* 2.1* 5.4** 3.3 5.0 3.1 0.8* 4.7
Values are mean S.D.; values marked with *are significantly different from control values; **P < 0.01; *P < 0.05 (Students t test).
levels were significantly decreased in a concentration dependent manner. Urea levels were slightly decreased by the high dose (500 mg/kg) but not by low dose (250 mg/kg) of AF. Serum protein content increased by the treatment (Table 5). These changes are normally considered as beneficial effects. In preliminary studies, it was found that the alcohol extract of this plant, even at a high dose of 2 g/kg body weight, did not cause any mortality. The herbal drug is also used in ethnomedicine without any known side effects. So attempts were not made to determine LD50. Interestingly, the plant (butanol fraction of alcohol extract) did not show any conspicuous toxicity except a decrease in spleen weight in the short term toxicity evaluation. At the optimum dose it protected all the mice challenged with the virus. It should be noted that while in vitro screening of plants have been carried out in India [26, 27], follow up in vivo studies are rare. The butanol fraction may be used as a phytomedicine whereas compounds ursolic acid may be developed as pure chemical entity anti-viral medicines. It is relatively inexpensive and less time consuming to develop phytomedicines for viral diseases [28, 29]. The ursolic acid isolated was found to be only 96% pure. The butanol AF is likely to contain other active molecules. Further studies are required to identify them. The presence of alkaloid in Ocimum sp. has been suggested by other investigators [30]. In the present study, plants grown in the Institute garden were used. Plants collected from the wild could, possibly, differ in their efficacy depending on soil conditions and nutrition. Besides, association of other organisms, including microbial pathogens could, possibly, affect the medicinal efficacy. O. sanctum is a herb which can be easily cultivated. Thus, availability of raw materials for drug development can be ensured. Ongoing studies at the authors laboratory include identification and chemical characterisation of all the principles and mechanism of action of the active principles. Although O. sanctum is known to have immuno-stimulatory property [10], in vivo protection of the viral challenged mice and duck may not be solely due to the immune stimulation because, in the present study, the anti-viral activity was observed in the cell lines under in vitro conditions. The mechanism of action of the drug remains to be studied. Interestingly, in the present study, therapeutically promising in vivo and in vitro anti-CBV activity of O. sanctum (AF) was shown. Besides, the AF showed activities against HB, measles and HSV in vitro in cultured cells. It was shown that ethanol extract of O. sanctum inhibits poliovirus replication in Vero cells [31]. Further, it has been reported that O. basilicum and ursolic acid isolated from it also exhibited in vitro anti-viral activity against HSV, ADV, CVB and EV71 [20]. Interestingly, anti-viral activities against phylogenically different viruses were also shown by O. sanctum. A common major mechanism of action is likely to exist. It may not be due to stimulation of interferon production because the anti-viral activity is seen in Vero cells, which are not known to produce interferon. INTERNATIONAL JOURNAL
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Potential Application of Bioinformatics in Development of an Efficacious Envelope Protein Based Vaccine for Chikungunya: A Pilot Study
Priya Das,1* Indu Purushothaman2 and S. Rajarajan3
of Biotechnology and Bioinformatics, Mar Augusthinose College, Ramapuram, Kottayam - 686 576, India Infrastructure Facility Center, 3Postgraduate and Research Department of Microbiology and Biotechnology University of Madras, Presidency College (Autonomous), Chennai - 600 005, India
2Bioinformatics 1Department
Abstract: The present study used bioinformatics to develop a potential efficacious envelope protein based vaccine for Chikungunya, a re-emergent infection that affected and immobilised millions of people from Asia and Africa in 2006. The amino acid sequence of a highly immunogenic envelope protein, E2 was taken for comparative analyses from the different strains of Chikungunya virus (CHIKV). Emboss-Align and ClustalW2 were the bioinformatics tools utilised for this study. A comparative analysis of E2 was done among 29 Asian strains and 12 African strains of CHIKV. The comparison among Asian strains revealed a close alignment score ranging from 90 to 99% with an exception of 89% for ABN04200. Similarly, the comparison among African strains revealed an alignment score ranging from 95 to 97%. Among the 29 Asian strains, 15 strains showed 99% similarity. Among the 12 African strains, three strains showed 97% similarity. Among the 18 (15 + 3) strains, the ones of recent incidence were taken for the next level analysis. Pairwise comparison among six Asian strains ACA81773, ABP88822, ACD93610, ABN04194, ABN04192 and ABN04190 with three African strains BAH97933, ABX38965 and ABU93705 revealed an alignment score of 100%. Hence, it is suggested to use E2 protein of recent CHIKV Asian or African strains from the year 2007 for production of subunit vaccine. Keywords: CHIKV, E2 protein, Envelope protein, Subunit vaccine, Bioinformatics.
Introduction
Epidemiology Chikungunya virus (CHIKV) is an alphavirus transmitted through Aedes aegypti belonging to family Togaviridae. The Tanzania strain was isolated by Rose for the first time from an outbreak in 1952-53 [1]. CHIKV is an important human pathogen that causes symptoms like fever, headache, rash, nausea, vomiting, myalgia, arthralgia and, occasionally, neurological manifestations such as acute limb weakness. It is also associated with a fatal haemorrhagic condition. CHIKV is geographically distributed from Africa to Southeast Asia [2]. Chikungunya originated in Africa [3] and has been maintained in the sylvatic cycle involving wild primates and forest dwelling mosquitoes such as Aedes furcifer, Aedes luteocephalus, or Aedes Taylori [4, 5]. It was subsequently introduced in Asia where it gets transmitted from human to human mainly by Aedes aegypti and, to a lesser extent, by Aedes Albopictus through urban transmission cycle [5]. Since 1963, when CHIKV was isolated in Calcutta (now Kolkata), there have been several reports of CHIKV infection in different parts of India [6]. In Nagpur, the incidence in certain wards was as high as 40-70%. Thereafter, it was considered that CHIKV had disappeared from India and Southeast Asia [7, 8]. In 2006, there was a large outbreak of CHIKV infection that affected 1.39 million people in India [9].
*Author for Correspondence. E-mail: priyadas001@gmail.com
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Serological surveys of CHIKV reported cases support this view [10]. The age of patients ranged from 16 to 59 yrs with the majority in the 30-39 yrs age group (29.4%) and the 40-49 yrs age group (31.4%). Of the total, 76.5% were females and Indians formed the majority (72.5%), followed by Malays (25.5%) and Chinese (2%) [11]. Vaccine Studies The CHIKV vaccines prepared by Tween 80 and ether inactivation of virus grown in green monkey kidney cell cultures were as immunogenic as comparable Formalin-inactivated vaccines. Both types of vaccines stimulated hemagglutinationinhibiting, complement-fixing and neutralising antibody, and afforded protection to mice against live virus challenge [12]. A comparative study was made of Formalin-inactivated Chikungunya vaccines prepared from the virus propagated in African green monkey kidney monolayers and concentrated chick embryo suspension cultures. The vaccine prepared in the chick embryo suspension cultures was significantly more protective to mice against a live homologous virus challenge. It also stimulated the production of 4-5 times more circulating antibodies than the vaccine prepared with virus grown in African green monkey kidney monolayer cultures [13]. The sensitivities of the CHIKV E1 and E2 envelope proteins for anti-CHIKV positive serum samples were 77.5 and 90%, respectively. The sensitivity of CHIKV E2 envelope protein was higher than that of CHIKV E1 envelope protein. This difference between CHIKV E1 and E2 envelope proteins might be explained by the fact that the former lies almost buried in the virus structure, which may give it less chance of exposure to the host immune system [9]. Phase II, randomised, double-blind, placebo-controlled, safety and immunogenicity study of a serially passaged, plaque-purified live Chikungunya vaccine was conducted with 73 healthy adult volunteers. The live Chikungunya vaccine was safe. It produced well-tolerated side effects such as transient arthralgia in five (8%) of the 59 volunteers, and was highly immunogenic. It is, therefore, a promising vaccine for use in alphavirus naive individuals [14].

Databases National Center for Biotechnology Information (NCBI) and International Committee on Taxonomy of Viruses database (ICTVdB). Softwares Emboss-Align and ClustalW2. Morphology Virions consist of a nucleocapsid and an envelope. The virus capsid is tightly enveloped by a detergent sensitive lipoprotein. Virions are spherical to pleomorphic and measure 70 nm in diameter. Surface projections are distinctive glycoprotein spikes composed of two virus proteins forming heterodimers evenly covering the surface. The capsid/ nucleocapsid is round and exhibits icosahedral symmetry (T = 4). The nucleocapsid is isometric and has a diameter of 40 nm. Capsids appear round. Genome The CHIKV is an envelope, positive-strand RNA virus. The genome (Fig. 1) is not segmented and contains a single molecule of linear positive-sense, single-stranded RNA. The complete genome is 11826 nucleotides long. In RNA the
26S 5' cap nsP1 nsP2 Helicase, proteinase nsP3 RNA synthesis nsP4 RNA polymerase C E3 E2 6K E1 Poly|A|-3*
() Strand RNA synthesis capping of RNA
Capsid Fig. 1. CHIKV genome.
Envelope glycoproteins
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Prevalence of Japanese Encephalitis in South India

Gunasekaran Palani, Kaveri Krishnasamy,* Mohana Sambasivam, Kavita Arunagiri, Khaleefathullah A. Sheriff, Senthilkumar Vijayan, Padma Priya Padmanaban, Suresh B.V. Babu and Kiruba Ramesh
Department of Virology, King Institute of Preventive Medicine and Research (KIPMR), Guindy, Chennai - 600 032, India
Abstract: The prevalence of Japanese Encephalitis (JE) among Acute Encephalitis Syndrome (AES) cases in Tamil Nadu, South India during 2007 and 2008 was investigated. A total of 412 cases were reported, of which 13 were JE positive. Out of the positives 84.6% belonged to the paediatric age group < 12 yrs. Keywords: AES, JE, South India.
Introduction
Japanese Encephalitis (JE), also known as the plague of the orient is a significant public health problem in India [1]. An increasing number of JE cases have been reported in the country [2]. In South India, JE virus (JEV) is prevalent in certain regions where the topography is conducive to its spread [3]. Vast stretches of rice fields and plenty of irrigation provide ideal breeding conditions for mosquitoes that transmit the virus from pigs to humans. Since JEV is the most common cause of childhood viral encephalitis in the world, vaccination against this virus plays a crucial role in its eradication and control [4, 5]. The present study reports a two-year (2007-08) retrospective analysis of JE in Acute Encephalitis Syndrome (AES) cases referred to the Department of Virology, KIPMR, Chennai.
Study
In the present study, samples of suspected AES cases were referred from Government General Hospital, Institute of Child Health, Stanley Medical College and Hospital, Chennai, other Government hospitals and private institutions. Since they are tertiary referral centres, the cases were referred from various districts of South India. Serum and cerebrospinal fluid (CSF) samples were collected and sent in cold chain, under sterile conditions. Serum was subjected to JE-DENGUE IgM COMBO ELISA, Australia and CSF JEV-CheX IgM ELISA (XCyton Diagnostics Pvt. Ltd., Bangalore, India). Out of the total of 412 cases referred during 2007-08, 13 were found to be positive. As per standard diagnostic criteria defined by World Health Organisation (WHO), serum IgM positive cases, CSF IgM positive cases and both serum and CSF IgM positives were considered as confirmed JEV cases. The total number of cases in 2007 was 186 with 6 (3.22%) positives; in 2008 out of 226 cases 7 (3.09%) were positive. Out of the 13 cases, 8 were serum positive, 3 were positive for CSF, while 2 were serum and CSF positive for IgM (Table 1). For the clinically suspected and serology negative cases, repeat serum and CSF samples were requested. Out of the 13 cases, 10 were males and 3 were females. There was no difference in the genderwise seropositivity in 2007 as all cases were males, whereas in 2008 the difference was statistically insignificant. Among the positive cases, 11 belonged to the pediatric age group and 2 were adults. Among the paediatric positive cases, 6 cases fell in the 1-5 yrs age group, followed by 5 cases in the 5-12 yrs age group.
*Author for Correspondence. E-mail: kaveri_raj1967@yahoo.com
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Ijmpv v1n1 Jan.-Jun. 2011 E-Sample

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International Journal of Medical and Pharmaceutical Virology

Biannual Journal of International Association of Medical and Pharmaceutical Virologists

Volume 1 Number 1 January-June 2011

SRI RAMACHANDRA UNIVERSITY

A Harvard Medical International Associated Institution

From the Editor-in-Chiefs Desk

Is Herbal Drug Discovery Different from Single Molecule Discovery?

*Author for Correspondence. E-mail: profspt@gmail.com

Viral Aetiology of Diarrhoea

VP7 and VP4 elicit neutralising antibodies

Based on the spike protein VP4

Vol. 1 No. 1 Jan.-Jun. 2011

9. 10. 11. 12. 13.

27. 28. 29. 30.

Vol. 1 No. 1 Jan.-Jun. 2011

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

*Author for Correspondence. E-mail: drsrajarajan@gmail.com

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

Negative, 85.65 Fig. 4. Seroprevalence (%) of HHV-2 among ANC women.

Vol. 1 No. 1 Jan.-Jun. 2011

*Author for Correspondence. E-mail: asubramoniam@yahoo.com

Track 1, ID: OCM-01

100 400 300 200 100 A

HepG 2.215 (HB producing cell line)

Vol. 1 No. 1 Jan.-Jun. 2011

Control O. sanctum Lamivudine

5 4 3 2 1 0 1 2 4 8 Days after virus challenge 20

Vol. 1 No. 1 Jan.-Jun. 2011

Materials and Methods

() Strand RNA synthesis capping of RNA

Capsid Fig. 1. CHIKV genome.

Vol. 1 No. 1 Jan.-Jun. 2011

Prevalence of Japanese Encephalitis in South India

*Author for Correspondence. E-mail: kaveri_raj1967@yahoo.com

Materials and Methods

Vol. 1 No. 1 Jan.-Jun. 2011

Figures and Tables

Vol. 1 No. 1 Jan.-Jun. 2011

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