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Virus Research 144 (2009) 280–284

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Genetic characterization of rotavirus subtypes in Pakistan-first report of G12genotype from Pakistan under WHO-Eastern Mediterranean region

Muhammad Masroor Alam a , Salman Akbar Malik b , Shahzad Shaukat a , Asif Naeem a , Salmaan Sharif a ,Mehar Angez a, Muhammad Suleman Rana a, Adnan Khurshid a, Sohail Zahoor Zaidi a,∗

a Department of Virology, National Institute of Health, Chak Shahzad, Park Road, Islamabad 54400, Pakistanb Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan

a r t i c l e i n f o

Article history:Received 26 January 2009Received in revised form 25 March 2009Accepted 26 March 2009Available online 5 April 2009

Keywords:PakistanRotavirusG12 genotypeG2 genotypeDiarrhea

a b s t r a c t

Rotaviruses are among the major causes of gastroenteritis and diarrhea among children in developed aswell as the developing countries. The rapidly evolving strain prevalence and circulation have resulted inthe emergence of novel strains over the period worldwide. The introduction of G12 prototype in 1987from Philippines and subsequently re-emergence among most of the Asian countries along with USA andEurope has provoked new research horizons to address the global distribution of rotavirus serotypes.These newly emerging subtypes and their sustenance among the population have posed tremendouschallenge to the development of an effectual vaccine with heterotypic protective efficacy. In Pakistan, nodata is available regarding the prevalent rotavirus serotypes; therefore, this is the first study to report theprevalence of G12 strain in Pakistan in hospitalized children with diarrhea addressing a dire need of furtherlarge-scale epidemiological surveys to resolve the underlying rotavirus isolates in both the hospitalizedand the community neonatal and child population before formulating the vaccine introduction policiesin the country’s routine immunization program.

© 2009 Elsevier B.V. All rights reserved.

Rotavirus contributes as a potential cause of neonatal as wellas childhood gastroenteritis with an estimated 705,000 deathsworldwide annually (Parashar et al., 2006). Rotaviruses belong toreoviridae family with 11 segmented double stranded RNA genometranslating into six structural and six non-structural proteins. Theouter coat proteins VP7 and VP4 resulted in the classification ofrotaviruses into subtypes designated G and P types, respectively(Estes, 2001). Recently, a Rotavirus Classification Working Group(RCWG) has introduced a comprehensive system for genotypeassignment to any analyzed rotavirus sequence based on the 11gene segments including both structural as well as non-structuralregions and thus defined 19 G genotypes and 27 different P types(Matthijnssens et al., 2008).

Group A rotaviruses have been associated with severe gastroen-teritis infections in various countries. It is well known that the majorserotypes associated with over 80% cases of gastroenteritis are G1,G2, G3, G4 and G9 in different combinations with P[8], P[4] and P[6](Santos and Hoshino, 2005). However, the changing temporal dis-tribution of rotavirus serotype circulation may account for a quitevarying epidemiological pattern of the disease. For example, themost common prevalent strains in India varied from year to year

∗ Corresponding author. Tel.: +92 51 9255082; fax: +92 51 9255147.E-mail address: zaidis@pak.emro.who.int (S.Z. Zaidi).

with the highest proportion of rotavirus G9 (G9P[11]) serotypes in1993–94 (Das et al., 1993, 1994) to G9P[8] in 2006 (Iturriza-Gomaraet al., 2004) while in a study from Delhi, India, G12 was found to bethe major cause of gastroenteritis outbreak in a neonatal nursery(Ray et al., 2007). Similarly, the surveillance network establishedat Rio de Janeiro, Brazil revealed that 57% of children with gas-troenteritis were found to be infected with G5 serotype in 1997 butturned to 0 in 1998–2003 indicating a periodic fluctuation to otherpre-dominant circulating serotypes over time (Santos et al., 2003,1998; Leite et al., 1996; Araujo et al., 2001; Gouvea et al., 1994).

Pakistan does not keep any epidemiological record regarding thelocal reservoirs of rotavirus strains. Due to lack of any surveillancenetwork, many of the outbreaks and hospitalizations go unnoticedand/or never screened for the rotaviruses or its genotypic/serotypicvariants except very few studies during the last decade. The cur-rent study reports the finding of an unusual and recently emergingrotavirus serotype G12 from Pakistan isolated from two childrenwith gastroenteritis and accentuate the need for an incessant recordof serotype prevalence in order to regulate the efficacy of vaccinebefore and during its introduction to a specific population group.

Stools samples were referred to the Serology Laboratories,Department of Virology, National Institute of Health, Islamabadfrom a local hospital for the detection of rotavirus. Three stoolsamples were kindly provided by Serology Laboratories uponrequest to determine the rotavirus serotypes. Stool samples were

0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.virusres.2009.03.015

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Fig. 1. Dendogram based on VP7 gene of G2 strain isolated from Pakistan in 2008. The tree robustness has been tested by 500 replicates using neighbor-joining methodwith Kimura-2 parameter. The values less than 70 have not been shown The Genebank sequences have been retrieved from NCBI databank. The box represents the isolateidentified during this study while the arrowhead represents G2 isolate reported from Pakistan in 1995.

re-suspended in phosphate buffered saline. RNA from the clear stoolsupernatants was extracted using QiaAmp viral RNA minikit andstored at −20 ◦C till further testing.

PCR was performed to amplify VP7 gene of rotavirus followingthe protocol as described earlier (Gouvea et al., 1990). The amplifiedproduct was purified using Qiagen kit (Qiaquick PCR purificationkit) and genetic sequences were generated through automatedGenetic Analyzer (Applied Biosystems 3130). The sequence datawas analyzed by performing BLAST comparison of VP7 gene withthe rotavirus genome sequences downloaded from NCBI Genebank.Multiple alignments were performed using Clustal W (Higgins et al.,1994). The phylogenetic distances and tree were reconstructed by

Table 1Epidemiological data of the cases infected with diarrhea and enrolled for the subjectstudy.

Address Year Rotavirus(serotype)

Disease Age(months)

Sex

Rawalpindi 2008 G12 Watery diarrhea 24 MaleRawalpindi 2008 G12 Watery diarrhea 14 FemaleRawalpindi 2008 G2 Watery diarrhea 02 Male

MEGA 4.0 (Tamura et al., 2007) using neighbor-joining method tostature out the possible genetic co-relations between the circulat-ing serotypes in Pakistan and those in the other countries.

For the three ELISA positive rotavirus samples, VP7 was success-fully amplified and directly sequenced to determine G subtypes. Thedetailed epidemiological data of all three isolates are presented inTable 1. The phylogenetic analysis performed through the MEGA4.0 revealed one isolate as G2 closely matched to the Bangladeshstrains (Fig. 1) while the other two isolates shown the contigu-ous homology with the recently emerging rotavirus strain G12 asreported from the other regions. The closest nucleotide sequencematches were found with G12 isolates reported from South Koreaand Slovenia with 98% homology score (Fig. 2). Globally, rotavirusserotypes G1, G2, G3, G4 and G9 in different combinations withP serotype have been documented accounting considerable highdisease burden in populations. According to World Health Organi-zation survey report conducted in 1991 among the five developingcountries including Pakistan, rotavirus accounts for 20% cases ofgastroenteritis with the highest infection rate among 6–11-month-old children. Also, WHO estimated that diarrheal diseases causeabout 286,000 deaths annually in Eastern Mediterranean region,while rotavirus may account for 30–40% of hospitalizations and

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Fig. 2. Dendogram based on VP7 gene of G12 strain isolated from Pakistan in 2008. The tree robustness has been tested by 500 replicates using neighbor-joining methodwith Kimura-2 parameter. The values less than 75 have not been shown. The Genebank sequences have been retrieved from NCBI databank. The box represents the isolateidentified during this study.

deaths due to diarrhea among children less than 5 years of ageor 6% of all childhood deaths worldwide. Similarly, World HealthOrganization revealed 31.7% cases positive for rotavirus from Pak-istan during April, 2006; however, the rate was lowest among theother countries of Eastern Mediterranean region like Iran (53.6%),Oman (60.5%) and Tunisia (40.5%) (Rotavirus Surveillance News, vol.1, Issue 3, June 2006).

Very few hospital-based studies focused on the prevalence ofrotavirus have been conducted in Pakistan (Table 2) but none

has included the indication of prevalent serotypes in the countryexcept two studies. One was conducted in 1995 with the find-ings of serotype G2 (Genebank Accession number—D50126) asendemic serotype in Pakistan. The second study conducted during1990–1997 has reported serotypes G1 and G4 as the pre-dominantrotavirus subtypes in Pakistan. However, the studies were carriedout about a decade ago and had reported the same serotypes whichare commonly associated with 95% cases of gastroenteritis in theworld.

Table 2A review of Rotavirus prevalence studies conducted in Pakistan (1988–2006).

Authors Year Rotavirus prevalence Rotavirus serotype Ref.

Khan et al. 1988 9.6% Not detected Khan et al. (1988)Mubashir et al. 1990 8.2% Not detected Mubashir et al. (1990)Wen et al. 1995 – G2 Wen et al. (1995)Agboatwalla et al. 1995 12.3% – 1990 Not detected Agboatwalla et al. (1995)

24.4% – 199113.0% – 1992

Shah et al. 1999 29% Not detected Shah et al. (1999)Nishio et al. 2001 37% G1 and G4 Nishio et al. (2001)Khan et al. 2006 38% Not detected Khan et al. (2006)

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According to a study conducted by WHO on 4936 rotavirus-positive specimen from all the six WHO regions, 50% and 60%rotavirus infections from the Eastern Mediterranean and Africanregions, respectively, remained uncharacterized for G or P serotype(Rotavirus Surveillance Worldwide, 2001–2008. MMWR, 2008).The G12 rotavirus isolates described in this study resulted in theclosest homology scores with previously reported strains fromSouth Korea (strain CAU214-2006) and Slovenia (strain SI264-2006)indicating a continued circulation of same lineage over a signif-icant period of time. The phylogenetic relationship revealed thatthe isolates recovered, from Pakistani children harboring rotavirusinfection; correspond to the lineage prevalent across the Euro-pean countries like Slovenia as well as from East Asian regions likeSouth Korea despite its closest geographical neighbors like Indiaand Bangladesh indicating co-circulation stretched across a vastgeographical zone right from Saudi Arabia to South Korea includingPakistan during the same time frame because most of these coun-tries have reported G12 presence in 2006. Albeit, the G2 rotavirussubtype isolated in this study depicts quite a different lineage thanthe previously isolated strain (Accession number—D50126) of sameserotype from the country that was reported about 13 years ago.Although this G2 strain sequence correspond well with those cir-culating in Bangladesh but with significant sequence divergencesymbolizing a long time introduction, circulation and establish-ment among the local population of Pakistan.

The prototype strain of G12 (L26-G12P[4]) was first reported in1987 in Philippines and then re-emerged after about a decade inThailand (1998), United States of America (1999) and other Asiancountries like India (1999), Bangladesh (2000), Japan (2003), Korea(2002), UK (2002) and Belgium (2003) (Rahman et al., 2007). Theprobable origin of G12 progenitor has not yet been confirmedand proposed to be swine rotavirus strain G12P[7] (strain-RU172)recently isolated from India (Ghosh et al., 2006) or introducedthrough the re-assortment of segmented rotavirus genome. It hasbeen recognized that the common rotavirus serotypes in variousareas also correlate with the prevalent serotypes in animals in thoseareas indicating the zoonotic importance of rotavirus infections, forinstance, detection of rotavirus serotype G10 in India, G8 in Africaand G5 in Brazil (G10 has been found in cattle; G5 and G9 in horsesand pigs and G9 strains in lambs (Kelkar and Ayachit, 2000; Adahet al., 2001; Cunliffe et al., 2001; Fischer et al., 2003; Gouvea andSantos, 1999; Desselberger et al., 2001; Fischer et al., 2005). Thereis need of further broad-range continued surveillance across vari-ous regions especially those where human populations are in closecontact with the pets or farm animals like Pakistan as majority ofthe country’s population is living in rural areas and keeping animalsfor land farming and the source of income.

To our knowledge, this is the first report presented on the find-ings of G12 rotavirus genotype in the Eastern Mediterranean region.However, the study has few limitations like undersized samplesize as well as lack of comprehensive laboratory analysis (due toresource limitations) to explicitly reveal the true genetic as wellas epidemiological status of rotavirus disease burden in the coun-try. Pakistan has an annual birth rate of approximately five millionchildren; the rotavirus vaccine would be greatly helpful to pro-tect its newborns from the serious and often fatal form of diarrhea(http://www.app.com). Pakistan would receive $60 million worthof Rotavirus vaccines from the Global Alliance for Vaccine Immu-nization (GAVI) for its Expanded Program of Immunization (EPI)to check rotavirus associated diarrheal infections. Pakistan, willhopefully, introduce the rotavirus vaccine in its routine immu-nization program in 2014 which will be fully scaled up in 2017resulting in about 129,923 deaths averted by rotavirus vaccineintroduction (Accelerating the introduction of rotavirus vaccineinto GAVI-eligible countries. Investment Case for GAVI Secretariat,2006). Although this report about the rotavirus strains in Pakistan

does not depict a true picture of the overall prevalent genotypesin the country but keeps significance to attract attention of healthpolicy advisors for a thorough and large-scale epidemiological sur-vey before implementing immunization activities in the country. Itis therefore suggested that a wide-range epidemiological data maylikely to be generated about the disease burden in the Pakistanipopulation along with the pre-dominant rotavirus genotypes to for-mulate enhanced and time-honored policies resulting in effectualchildhood survival efforts.

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