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. 2016 Feb;88(2):216-23.
doi: 10.1002/jmv.24329. Epub 2015 Jul 20.

Genetic characterization of norovirus strains in hospitalized children from Pakistan

Amna Alam  1 , Sohail A Qureshi  2 , Jan Vinjé  3 , Anita Zaidi  1   4
Affiliations

Genetic characterization of norovirus strains in hospitalized children from Pakistan

Amna Alam et al. J Med Virol. 2016 Feb.

Abstract

Norovirus is one of the most common causes of acute gastroenteritis among children in developing countries. No data on the prevalence and genetic variability of norovirus are available for Pakistan, where early childhood mortality due to acute gastroenteritis is common. We tested 255 fecal specimens from children under 5 years of age hospitalized between April 2006 and March 2008 with severe acute gastroenteritis in five hospitals in the four largest cities in Pakistan for norovirus by real-time RT-PCR. Positive samples were further genotyped by conventional RT-PCR targeting the 5'-end of the capsid gene followed by sequencing of the positive PCR products. Overall, 41 (16.1%) samples tested positive for norovirus with an equal frequency in rotavirus-positive and rotavirus-negative samples. Nine (22%) samples were genogroup (G)I positive, 30 (73%) GII positive and two (5%) samples contained a mixture of GI and GII viruses. Sequence analyses demonstrated co-circulation of 14 norovirus genotypes including four GI genotypes (GI.3, GI.5, GI.7, GI.8) and 10 GII genotypes (GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.9, GII.13, GII.16, and GII.21). The most prevalent genotypes were GI.7 and GII.4 both causing 12.2% of the infections. This report confirms the presence of multiple norovirus genotypes in hospitalized children with acute gastroenteritis in Pakistan and a lack of clear predominance of GII.4 viruses.

Keywords: Pakistan; acute gastroenteritis; co-infection; genogroup I and II; norovirus.

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Figures

Fig. 1
Fig. 1
Seasonality of norovirus. Seasonal distribution of norovirus infections in hospitalized children with acute gastroenteritis, Pakistan from April 2006 to March 2008. Bars represent number of specimens tested each month and the line diagram shows number of norovirus positive specimens.
Fig. 2
Fig. 2
Genotyping of norovirus GI strains. Phylogenetic analysis of GI norovirus strains detected in Pakistan from April 2006 to March 2008. The phylogenetic tree was constructed by the maximum likelihood method with 100 bootstrap replicates using MEGA5.05. Sequences of the following GI norovirus reference strains were included in the tree: GI.1_Gothenburg_SWE04 (EU085529), GI.1_Norwalk_USA68 (M87661), GI.2_Constellation59_USA99 (AF435807), GI.2_Southampton_GBR91 (L07418), GI.3A_Beijing55042_CHN07 (GQ856473), GI.3A_DesertShield_USA90 (U04469), GI.3B_Potsdam_DEU00 (AF439267), GI.3B_Stavanger_NOR95 (AF145709), GI.3_Beijing 54108_CHN07 (GQ856470), GI.3_Otofuke_JPN79 (AB187514), GI.3_Akabane991130_JPN99 (EF547396), GI.3_Beijing54114_C HN07 (GQ856471), GI.4_Chiba407_JPN87 (AB042808), GI.4_-Koblenz433_DEU00 (AF394960), GI.5_AppalachicolaBay318_USA95 (AF414406), GI.5_Musgrove_GRB89 (AJ277614), GI.5_SzU G1_JPN99 (AB039774), GI.6_Beijing53997_CHN07 (GQ856463), GI.6_Sindlesham_GBR95 (AJ277615), GI.6_Hesse_DEU97 (AF0937970), GI.6_VA497_USA99 (AF538678), GI.7_Winchester_GBR94 (AJ277609), GI.7_Providence191_USA1 (JN899243), GI.7_Chiba030100_JPN03 (AJ844469), GI.7_IF2036_IRQ03 (AY675555), GI.8_1837_USA08 (GU299761), GI.8_Boxer_USA01 (AF538679), and GI.9_Vancouver730_CAN04 (HQ637267). Norovirus strains (in bold) detected in this study are indicated with unique ID’s representing the city and hospital origin (PKPM, Mercy Hospital Peshwar; PKLC, Children’s Hospital Lahore; PKRR, Rawalpindi General Hospital; PKKK, Kharadar General Hospital; and PKKN, National Institute of Child Health Karachi).
Fig. 3
Fig. 3
Genotyping of norovirus GII strains. Phylogenetic analysis of GII norovirus strains detected in Pakistan from April 2006 to March 2008. Sequences of the following GII norovirus reference strains were included in the tree: GII.1_Hawaii_USA71 (U07611), GII.1_Dillingen391_DEU01 (AF425767), GII.1_Ascension208_USA10 (JN797508), GII.2_Melksham_GBR94 (X81879), GII.2_SnowMountain_USA76 (AY134748), GII.2_Vaals_NLD05 (AB281090), GII.3_Toronto_CAN91 (U02030), GII.3_Milwaukee009_USA10 (JN565063), GII.3_Arg320_ARG99 (AF190817), GII.3_Herzberg385_DEU01 (AF539439), GII.3_Rotterdam_NLD06 (AB385626), GII.4_Bristol_GBR93 (X76716), GII.4_Chiba040974_JPN04 (AB294782), GII.4_Farmington Hills_USA02 (AY502023), GII.4_Henry_USA00 (FJ411170), GII.4_Hunter_AUS04 (DQ078794), GII.4_DenHaag_NL06 [2006b] (EF126965), GII.4_NewOrleans [NO1805a_USA09] (GU445325), GII.4_Osaka_JPN07 [Riviera] (AB434770), GII.4_Richmond_USA94 (EU078406), GII.4_Yerseke_NLD06 [2006a] (EF126963), GII.4 Sydney_AUS12 (JX459908), GII.5_Hillingdon_GBR90 (AJ277607), GII.5_Hokkaido133_JPN03 (AB212306), GII.6_Shizuoka_JPN08 (HM633213), GII.6_Miami292_USA94 (AF414410), GII.6_Seacroft_GBR90 (AJ277620) GII.7_Gwynedd_USA94 (AF414409), GII.7_Leeds_GBR90 (AJ277608), GII.8_Amsterdam_NLD98 (AF195848), GII.8_SaitamaU25_JPN00 (AB039780), GII.9_Goulburn ValleyG5175_AUS83 (DQ379715), GII.9_VA97207_USA97 (AY038599), GII.10_Erfurt546_DEU00 (AF427118), GII.10_Vietnam026_VNM02 (AF504671), GII.12_NSW199U_AUS08 (GQ845370), GII.12_Wortley_GBR90 (AJ277618), GII.13_Fayetteville_USA98 (AY113106), GII.13_VA173_USA10 (JN899242), GII.14_M7_USA99 (AY130761), GII.14_Maizuru8533_JPN08 (GU017903), GII.16_Hiram_USA00 (AY502006), GII.16_Tiffin_USA99 (AY502010), GII.17_CSE1_USA02 (AY502009), GII.17_Katrina-17_USA05 (DQ438972), GII.20_Leverkusen267_DEU05 (EU424333), GII.20_Luckenwalde591_DEU02 (EU373815), GII.21_IF1998_IRQ03 (AY675554) and GII.21_Salisbury150_USA11(JN899245), GII.22_Yuri_JPN02 (AB083780), GII.22_Beijing53931_CN07 (GQ856469). The phylogenetic tree was constructed by maximum likelihood method with 100 bootstrap replicates using MEGA5.05.norovirus. The key of the Pakistani strains is the same as in Figure 2.
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