Clinical, Epidemiologic and Genotypic Characteristics of Norovirus Gastroenteritis in Lebanon Nada M. Melhem, PhD American University of Beirut Faculty of Health Sciences Center for Infectious Diseases Research Faculty of Medicine December 7, 2015 Melhem, 2015

Introduction Norovirus (NoV) is responsible for at least 50% of all gastroenteritis outbreaks worldwide (Lamata et al., 2013). Data from the Centers for Disease Control and prevention (CDC) suggest that NoV is the leading cause of acute gastroenteritis across all age groups seeking medical care in emergency departments, outpatient clinics and the community (Hall et al., 2011). NoV-gastroenteritis accounts for 10-15 % of severe cases in children less than 5 years old (Patel et al., 2009). 9-15 % of mild-to-moderate diarrhea are due to NoV among individuals of all ages (Patel et al., 2008). Melhem, 2015

NoV: Transmission, Pathogenesis and Management Primary mode of transmission is through the fecal-oral route. The incubation period is 24-48 hours. The symptoms include: vomiting, diarrhea, nausea, abdominal cramps, malaise and low grade fever. The illness resolves in 12-72 hours. Treatment involves reversal of dehydration and electrolyte deficiency. Melhem, 2015

Genome Organization & Capsid Structure Melhem, 2015

Classification of Noroviruses and Viral Diversity (Vinjé et al. 2015) Melhem, 2015

Aim of the Study To our knowledge, there are no studies in Lebanon on NoV and its association with gastroenteritis. The aim our study is to determine the prevalence of Norovirus gastroenteritis as well as the genotypic characterization of the virus among hospitalized children less than 5 years old. Melhem, 2015

Methods Study Period: January 2011 to June 2013 Age group: ≤5 years old Sample size: 739 stool samples from hospitalized children presenting with diarrhea Collection sites and number of samples: American University of Beirut Medical Center (Capital Beirut): n=81 Hariri Governmental Hospital (Capital Beirut): n=47 Makassed Hospital (Capital Beirut): n=89 Nini Hospital (North Lebanon): n=316 Hammoud Hospital (South Lebanon): n=116 Nabatiyeh Hospital (South Lebanon): n=90 Melhem, 2015

Methods QIAamp Viral RNA Mini Kit (Qiagen, Germany) was used for viral RNA extraction. For reverse transcription polymerase chain reaction (RT-PCR) QIAGEN OneStep RT-PCR Kit was used, using genogroup-specific primers. PCR products were analyzed by gel electrophoresis and nucleotide sequencing of NoV positive samples was performed. Multiple sequence alignments were carried out using CLUSTALL or BioEdit. Phylogenetic trees were constructed using the MEGA 6 software. Melhem, 2015

Table 1. Demographic Characteristics of Study Participants Melhem, 2015

Table 2. Clinical Characteristics of NoV-associated Gastroenteritis Melhem, 2015

Incidence of Norovirus by Region Positives Total Number of Samples Incidence South 28 206 13.59% North 35 316 11.08% Beirut 20 217 9.22% Total 83 739 11.23% 11.08 9.22 13.59 Melhem, 2015

Figure 1. Norovirus and Seasonal Distribution Melhem, 2015

Figure 2. Phylogenetic Analysis of NoV VP1 Capsid Gene LBM073/2011 GII LBH065/2011 GII LBM123/2011 GII LBM122/2011 GII LBM104/2011 GII LBN159/2011 GII LBH048/2011 GII LBH075/2011 GII LBH062/2011 GII LBH077/2011 GII LBN120/2011 GII LBN111/2011 GII LBH009/2011 GII LBN047/2011 GII LBN086/2011 GII LBH089/2011 GII LBH049/2011 GII LBN131/2011 GII JB-15/KOR/2008 GII.4 Apeldoorn 2008 LBR034/2011 GII LBN095/2011 GII LBN142/2011 GII LBN185/2011 GII LBM097/2011 GII LBH066/2011 GII LBN154/2011 GII LBM109/2011 GII Orange/NSW001P/2008/AUS GII.4 New Orleans 2009 Farmington Hills/2002/USA GII.4 2002 Guangzhou/NVgz01/CHN GII.4 Asia 2003 Hunter504D/04O/AU GII.4 Hunter 2004 Kenepuru/NZ327/2006/NZL GII.4 2006a Osaka1/2007/JP GII.4 Osaka 2007 Yerseke38/2006/NL GII.4 Shellharbour-NSW696T/2006/AUS GII.4 2006b LBA147/2012 GII Sydney/NSW0514/2012/AUS GII.4 Sydney 2012 LBA163/2013 GII LBN516/2013 GII LBNG112/2012 GII LBR040/2011 GII LBN365/2012 GII LBN330/2012 GII LBN323/2012 GII 2012/FRA GII.4 variant Sydney LBA113/2012 GII LBN384/2012 GII LBN478/2012 GII LBN382/2012 GII LBN393/2012 GII LBN328/2012 GII LBH208/2012 GII LBNG175/2013 GII LBN458/2012 GII LBH054/2011 GII LBH068/2011 GII LBH185/2012 GII LBH203/2012 GII LBN259/2011 GII LBR081/2012 GII LBN339/2012 GII LBN374/2012 GII LBA126/2012 GII LBH186/2012 GII Sutherland/NSW505G/2007/AUS GII.4 Cairo 2007 Maizuru8915/2008/JPN GII.6 LBA057/2011 GII LBN360/2012 GII CBNU1/2006/KOR GII.3 LBNG107/2012 GII LBA125/2012 GII LBNG098/2012 GII NSW743L/2008/AUS GII.7 HCMC-VNM30241/2009/VNM GII.9 LBN480/2012 GII LBN052/2011 GII LBH046/2011 GII LBR082/2012 GII LBM093/2011 GII LBA038/2011 GII LBH028/2011 GII LBNG147/2012 GII Ascension208/2010/USA GII.1 LBM101/2011 GII HS207/2010/USA GII.12 Vaals87/2005/NL GII.2 LBN329/2012 GII LBN358/2012 GII LBM102/2011 GII 10N4555/2010/NP GII.13 LBH091/2011 GII Salisbury150/2011/USA GII.21 LBH221/2012 GII LBNG129/2012 GII LBN387/2012 GII LBN373/2012 GII LBN366/2012 GII LBH098/ GI LBH112/2011 GI LBN187/2011 GI LBN040/2011 GI 141/2009/BFA GI.3 JKPG 881/SWE/2007 GI.3 2008890321/2008/US GI.8 1643/2008/US GI.4 LBN343/2012 GI Leuven/2003/BEL GI.2 CS-841/2001/USA GI StromstadP7-587/2007/Sweden GI.1 74 70 88 92 98 97 91 96 75 94 99 93 100 0.2 GII.4 68% (52/76) GII.3 (5/76, 6.6%) GII.9 (1/76, 1.3%) GII.6 (7/76, 9.2%) GII.13 (3/76, 3.95) GII.1 (1/76, 1.3%) GII.2 (1/76, 1.3%) GII.21 (5/76, 6.6%) GII.3 GII.9 GII.6 GII.1 GII.2 GII.13 GII.21 GI.3 80% (4/5) GI.4 Melhem, 2015

Figure 3. Phylogenetic Analysis of NoV VP1: GII.4 LBM073/2011 GII LBH065/2011 GII LBM123/2011 GII LBM122/2011 GII LBM104/2011 GII LBN159/2011 GII LBH048/2011 GII LBH075/2011 GII LBH062/2011 GII LBH077/2011 GII LBN120/2011 GII LBN111/2011 GII LBH009/2011 GII LBN047/2011 GII LBN086/2011 GII LBH089/2011 GII LBH049/2011 GII LBN131/2011 GII JB-15/KOR/2008 GII.4 Apeldoorn 2008 LBR034/2011 GII LBN095/2011 GII LBN142/2011 GII LBN185/2011 GII LBM097/2011 GII LBH066/2011 GII LBN154/2011 GII LBM109/2011 GII Orange/NSW001P/2008/AUS GII.4 New Orleans 2009 Farmington Hills/2002/USA GII.4 2002 Guangzhou/NVgz01/CHN GII.4 Asia 2003 Hunter504D/04O/AU GII.4 Hunter 2004 Kenepuru/NZ327/2006/NZL GII.4 2006a Osaka1/2007/JP GII.4 Osaka 2007 Yerseke38/2006/NL GII.4 Shellharbour-NSW696T/2006/AUS GII.4 2006b LBA147/2012 GII Sydney/NSW0514/2012/AUS GII.4 Sydney 2012 LBA163/2013 GII LBN516/2013 GII LBNG112/2012 GII LBR040/2011 GII LBN365/2012 GII LBN330/2012 GII LBN323/2012 GII 2012/FRA GII.4 variant Sydney LBA113/2012 GII LBN384/2012 GII LBN478/2012 GII LBN382/2012 GII LBN393/2012 GII LBN328/2012 GII LBH208/2012 GII LBNG175/2013 GII LBN458/2012 GII LBH054/2011 GII LBH068/2011 GII LBH185/2012 GII LBH203/2012 GII LBN259/2011 GII LBR081/2012 GII LBN339/2012 GII LBN374/2012 GII LBA126/2012 GII LBH186/2012 GII 0.005 Melhem, 2015

Conclusions: NoV in Lebanon This is the first study assessing NoV infections in Lebanon. Our results are compatible with globally reported ones where GII.4 contributes to the majority of viral gastroenteritis outbreaks ( Bull and White, 2011; Guo et al., 2009; Siebenga et al., 2007). We report JB-15/KOR/2008 GII.4 Apeldoorn 2008-like variant strain circulating in 2011 among children less than 5 years. Between 2012 and 2013, the Apeldoorn variant was replaced by a variant sharing homology with Sydney/ NSW0514/2012/AUS GII.4 Sydney 2012 and the Sydney 2012/FRA GII.4. Melhem, 2015

Conclusions: NoV in Lebanon NoV GII.6 incidence was the second predominant cause of gastroenteritis among hospitalized children in Lebanon, similar to what has been reported in several countries including Brazil, Japan, South Africa and Finland (Ferreira et al., 2012). NoV GII.3 ranked third along with NoV GII.21 among our study participants; after GII.4 and GII.6, while it ranked second in other countries. Reports show that NoV cases peak during the cold months in parts of Europe and North America with sporadic cases detected all year round as well as outbreaks during the summer time (Mounts et al., 2000; Rohayem, 2009; Thongprachum et al., 2015; Verhoef et al., 2008; Ahmed et al. 2013) . Our data support a peak incidence in July; the seasonal pattern of NoV in Lebanon should be further investigated. our data supports a peak incidence in July Melhem, 2015

NoV in the Middle East and North Africa (MENA) Region In the MENA region, NoV was detected in stool samples of 6-30% of hospitalized children under 5 years. Only few studies further reported on the geno-subgroups. 3 reported GII.3 being more prevalent (Romani et al., 2012; Leshem et al., 2015; Muhsen et al., 2013; Kaplan et al., 2011). Clear lack of data on the genetic relatedness and subtyping in the MENA . Melhem, 2015

Conclusions Variants of the NoV GII.4 lineage have been associated with 62 to 80% of NoV outbreaks worldwide (Donaldson et al., 2010; Siebenga et al., 2009). NoV remains an understudied causative agent of acute gastroenteritis and not included in the Global Burden of Diseases (GBD) among the infectious agents causing gastroenteritis and diarrhea. Thus there’s a lack of clinical diagnosis and reporting of NoV as an important cause of disease and further studies are needed to support intervention strategies. Melhem, 2015

Acknowledgments Faculty of Health Sciences Khalil Kreidieh Rana Charide Nour Rahal Center for Infectious Diseases Research, Faculty of Medicine Ghassan Dbaibo and team (Amjad Haidar) Hassan Zaraket Melhem, 2015