Preparation of Rotavirus Culture and Quantification assays for the ...

February 2013- April 2013, Vol. 3, No. 2, 1142-1148. e- ISSN: 2249 –1929Journal of Chemical, Biological and Physical SciencesAn International Peer Review E-3 Journal of SciencesAvailable online atwww.jcbsc.orgSection B: Biological ScienceCODEN (USA): JCBPATResearch ArticlePreparation of Rotavirus Culture and Quantification assaysfor the Vaccine Production and its role in VaccinationM. PadmavathiDepartment of Biotechnology, DVR & DR. HS MIC College of Technology,Kanchikacherla, A.P. IndiaReceived: 30 January 2013; Revised: 16 February 2013; Accepted: 18 February 2013Abstract: When a new disease arises to health threat the scientists, doctors and healthworkers recognized the need for a new way to prevent the disease. The organism or toxinthat causes damage to health or illness they pursue a number of approaches to develop avaccine. One such organism is Rotavirus which infects human health especially youngchildren and a pathogen of livestock that cause acute gastroenteritis. Millions of peoplebecome severely ill due to this pathogen. Rotavirus vaccine helps to prevent this illness.The present study describes the isolation, preparation of the Rotavirus culture andquantified by different assays and the role of Rotavirus vaccine helps to treat theinfections for saving lives of children in future.Keywords: Rotavirus, Infection, Cell culture, Plaque assay, FIA, VaccineINTRODUCTIONWhen a new disease arises to health threat the scientists, doctors and health workers recognized the needfor a new way to prevent the disease. Once they identified the organism or toxin that causes damage tohealth or illness they pursue a number of approaches to develop a vaccine. The main purpose of a vaccineis to bring about active immunity or response from a person’s immune system by marshaling B and Tcells to swing into action and creating a memory within the immune system to fight the disease. Somevaccines are combinations to protect against diseases. Scientists test and make sure that none of theantigens detracts from the immune priming effect of the others. Thus the vaccine can provide tripleprotection, the recipients are spared extra needle sticks, and the public costs are reduced. Virus vaccinesare prepared sing live virus, attenuated virus. A specific virus and the virus particle play a key role invaccination. Sing different types of quantification assays or methods one can determine the virus particle.1142 J. Chem. Bio. Phy. Sci. Sec. A, 2013, Vol.3, No.2, 1142-1148.

Preparation…M. Padmavathi.Rotaviruses belong to the family Reoviridae, which are non-enveloped, icosahedra viruses with an 11-segment double-stranded RNA genome 1 . It contains six structural proteins in three concentric layers asshown in Fig: 1. The viral genome was surrounded by the internal layer or core which contains thescaffolding protein VP2, the NA dependent NA polymerase VP1 and VP3. The intermediate layer is madeup of VP6, which is a major protein. The outer layer is made p of VP with spikes of VP4 1 . In infectedcells, six non structural proteins such as NSP1-6 are produced. The functional properties of these proteinscan be carried out by crystallographic methods.Figure 1: Rotavirus shows a virionRotavirus is transmitted by the faecal-oral route, which infects and damages the small intestine and casesgastroenteritis. In addition to its impact on human health, rotavirus also infects animals and a pathogen oflivestock 2 . It is an easily managed disease of childhood, but worldwide nearly 5 lakhs children under fiveyears of age die from this infection every year 3 and millions of people become severely ill 4 Public healthcampaigns to combat rotavirus focus on providing oral rehydration therapy for infected children andvaccination to prevent the disease 5 Slowly the infections and severity of rotavirus has decreased androtavirus vaccine to their routine childhood immunization policies 6, 7 .Rotavirus infects stomach and causes gastroenteritis which is a mild to severe disease characterized byvomiting, dehydration, diarrhea and fever 8 9, 10. Subsequent infections are typically mild or asymptomaticas the immune system provides some protection 11, 12 . Consequently symptomatic infection rates arehighest in children under two years of age and decease progressively towards 45 years of age 13 .Rotavirus is transmitted by the faecal-oral route, via contact with contaminated hands, surfaces andobjects 14 and possibly by the respiratory route 15 . The faeces of an infected person can contain more than10 trillion infectious particles per gram 16 fewer than 100 of these are required to transmit infection toanother person 17 . It is stable in the environment and survives between 9 to 19 days 18 . The multipleactivities of the virus cause diarrhea. Destruction of gut cells or enterocytes causes malabsorption. Thetoxic rotavirus protein NSP4 induces age and calcium ion-dependent chloride secretion, disrupts SGLT1transporter-mediated reabsorption of water, reduces activity of membrane disaccharidases and activatesthe calcium ion-dependent secretary reflexes of the enteric nervous system 19 . The cells secrete lactase intosmall intestine; milk intolerance due to lactase deficiency is a symptom of rotavirus infection 20 which canpersist for weeks 21 . Mild diarrhea often follows the reintroduction of milk into the child’s diet, due tobacterial fermentation of the disaccharide lactose in the gut 22 .1143 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.2, 1142-1148.

Preparation…M. Padmavathi.MATERIAL AND METHODSRotavirus preparation: Two serotypes of Bovine Human Reassortant virus namely BRV-3 and BRV-10were used for preparation. The materials used for it are T175 cell culture flask containing Vero cells,MEM, DPB, 2µg/ml Trypsin, 10%SPG, Co2 incubator.To the vero cells in 1XT175 cell culture flask was washed with MEM and DPB to remove the FBSremains and increases the virus infectivity and then MOI was added in 1:40 ratio incubated for 1 hr at37 0 C. After one hour 200µg Trypsin and MEM were added and incubated for 48-72 hrs at 37 0 C. The viruscells were harvested from the culture from the supernatant and stored for future use.Quantification assays: Quantification of rotavirus can be done by two methods:Plaque Assay: The most important procedure for measuring the virus titer-the concentration of virus inthe sample. Plaque assay is widely used approach for determining the quantity of infectious virus. Thistechnique was developed to calculate the titers of bacteriophage. Renato Dulbecco modified this assay in1952 for reliable determination of the titers of many different viruses.To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots areinoculated onto susceptible cell monolayer. After an incubation period, to allow virus to attach to cells,the monolayer were covered with a nutrient medium containing a substance, usually agar, which causesthe formation of a gel. When the plates are incubated, the original infected cells release viral progeny. Thespread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectiousparticle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes largeenough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrastbetween the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed.The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine thevirus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaquesare counted, depending on the size of the cell culture plate that is used. Statistical principles dictate thatwhen 100 plaques are counted, the sample titer will vary by plus or minus 10%. Each dilution is plated induplicate to enhance accuracy. In the example shown below, there are 17 plaques on the plate made fromthe 10 -6 dilution. The titer of the virus stock is therefore 1.7 x 10 8 PFU/ml.Fig 2: Isolation of Rotavirus by plaque assayTitre value can be measured as follows:Titre = log of reciprocal of dilution showing countable plaques + log of total no. of Plaque1144 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.2, 1142-1148.

Preparation…M. Padmavathi.Fig 3: The plate shows the number of rotavirusMethod II: Fluorescent Immune assay (FIA): The fluorescent immune assay is a more rapid variation ofthe plaque assay because it uses immunostaining techniques with fluorescently-labeled antibodies specificfor the virus antigen to measure host cell infection before an actual plaque is formed. Like the plaqueassay, host cell monolayer are infected with various dilutions of the virus sample and allowed to incubatefor a relatively brief incubation period (e.g., 24–72 hours). Plates are subsequently stained withfluorescent antibodies, and fluorescence microscopy is used to count and quantify how many cells areinfected. The FFA method yields results in less time than plaque or TCID50 assays but is more expensivein terms of required reagents and equipment. Assay time is also dependent on the size of area that the useris counting; the larger area will take more time but will provide a more accurate representation of thesample. Results are expressed as fluorescent focus forming units or FFU/mL 28 .CALCULATIONSTitre = log of lowest dilution where max positive wells are observed - log of inverse of dilutionRESULTS AND DISCUSSIONfactor + cumulative positive wells x log stepNo. of wells per dilutionThe cell count in different tissue culture flasks using Haemocytometer method was observed and theaverage cell count was calculated and the count was 15.3X10 6 .Quantification of BRV-3: Three trials of Plaque assay were carried with different dilutions of Rotavirus.Table 1:Titre values of Plaque assay of BRV-3 for different trialsTrail No.Titre ValueTrail -I 6. 875Trail –II 7.021Trail -III 7.213Fig 4: Titre values of Plaque Assay of BRV-3 for different trials1145 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.2, 1142-1148.

Preparation…M. Padmavathi.Method II: Fluorescent Immuno assay (FIA): Three trials of FIA were carried out. For each trial singleplate was taken with different dilutions of rotavirus were added in different columns of microtitre plate.One was taken as control. The titre values for the three trials of FIA for BRV-10 were calculated from theobservations and tabulated as follows:Table 2: Titre values of Plaque assay of BRV-3 for different trialsTrail No.Trail –I 7.567Trail –II 7.431Trail –III 7.428Titre ValueFig 5: Titre value of Plaque assay of BRV-3 for different trialsQuantification of BRV- 10: Three trials of Plaque assay were carried with different dilutions ofRotavirus. Three plaque values were tabulated as follows:Table 3: Titre values of Plaque assay of BRV-10 for different trialsTrail No.Titre ValueTrail -I 6. 657Trail –II 7.239Trail -III 7.125Fig 6: Titre value of Plaque assay of BRV-10 for different trialsMethod II: Fluorescent Immunoassay (FIA): Three trials of FIA were carried out. For each trial singleplate was taken with different dilutions of rotavirus were added in different columns of microtitre plate.One was taken as control. The titre values for the three trials of FIA for BRV- 10 were calculated from theobservations and tabulated as follows:1146 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.2, 1142-1148.

Preparation…M. Padmavathi.Table 4: Titre value of FIA of BRV-10 for different trialsTrail No.Titre ValueTrail -I 7.637Trail –II 7.525Trail -III 7.526Fig 7: Titre values of FIA of BRV-10 for different trialsBy comparing in both the serotypes of Rotavirus taken i.e., BV-3, BRV-10 the titre values obtained usingFIA are greater than the titre values obtained using Plaque assay. From the results it can be concluded thatFIA is more accurate and specific method than Plaque assay for quantification of rotavirus. The titrevalues obtained by both the quantification methods lies in between 7.0 and 7.5 which means titre valuesare considered to be good and achieved.Role in vaccination: Rotavirus vaccines are licensed in more than 100 countries, but only 30 countieshave introduced routine rotavirus vaccination 23 . The health burden of rotavirus gastroenteritis rapidly anddramatically reduced despite lower coverage levels compared to other routine infant immunizations 24 .Asia found that the vaccines dramatically reduced severe disease among infants in developing countrieswhere the majority of rotavirus deaths occur 25 . In September 2013, the vaccine will be offered to allchildren in the UK, aged between two and three months, and it is expected to halve the cases of severeinfection and reduce the number of children admitted to hospital because of the infection by 70 percent 26 .The Rotavirus Vaccine Program and the Accelerating Vaccine Introduction initiative, these groups arepartnering with research institutions and governments to reduce child morbidity and mortality fromdiarrheal disease by making a vaccine against rotavirus available for use in developing countries 27 .CONCLUSIONThe rotavirus vaccine field has recovered and gone on to make great progress with the development of thetwo recently licensed vaccines. The development of any future rotavirus vaccine and improvement of thecurrently licensed vaccines is limited knowledge of the mechanisms of rotavirus pathogenesis and thebasis for protection against rotavirus-associated gastroenteritis in humans. Finally, developed on the basisof the observation that neonatal rotavirus infections are generally asymptomatic and can protect againstsubsequent severe rotavirus gastroenteritis, two rotavirus vaccines derived from human neonatalrotaviruses are under study.REFERENCES1. M.K. Estes A.Z. Kapikian, D.M. EdsKnipe, In Fields Virology 917–1974 (LippincottWilliams & Wilkins/Wolters Kluwer, Philadelphia, 2006.2. H.F. Clark, P.A. Offit, S.A. Plotkin, P.M. Heaton, The new pentavalent rotavirus vaccinecomposed of bovine (strain WC3)-human rotavirus reassortants.Pediatr. Infect. Dis.J.,2006, 25, 577–583.3. R.J. Glass, et al. Rotavirus vaccines: current prospects and future challenges.Lancet,2006, 368, 323–332.1147 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.2, 1142-1148.

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