1940s-1960s: 75% of gastroenteritis episodes of unknown etiology

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2 1929: Winter vomiting disease first described 1940s-1960s: 75% of gastroenteritis episodes of unknown etiology 1968: Norwalk-like virus prototype (J Infect Dis, 123:307) An outbreak of gastroenteritis in a school in Norwalk, Ohio, in % (116/232) students and teachers developed gastrointestinal illness Virus was suspected as the etiological agent The filtrate was administrated orally to volunteers 1972: confirmed by IEM (Kapikian et al. J Virol 10: ) A 27-nm virus-like particle, icosahedral capsid Small Round Structure Virus (SRSV) : Electron microscopy and serology used to confirm outbreaks : Reverse transcription-polymerase chain reaction (RT-PCR) developed

3 Family Caliciviridae Lagovirus, Vesivirus, Sapovirus, Norovirus Only sapovirus and norovirus can infect humans Genome: Single-stranded, positive-sense RNA genome of ~7.7 kb. Encodes one major structural protein of 60 kda which forms viral capsid Capsid 180 molecules, folds into 90 dimers Viral Particle: nm in diameter Non-enveloped and icosahedral 50nm Norovirus, the perfect human pathogen Highly contagious Rapidly and prolifically shed Constantly evolving Evoking limited immunity Moderately virulent Allowing most of those infected to fully recover, thereby maintaining a large susceptible pool of hosts. (A. J. Hall, 2012, J Infect Dis) Norovirus Infection Infectious dose of virus particles 1 gram of faces= 100 billion norovirus particles, = 5 billion infectious doses Stable in the environment Resistant to routine disinfection methods Carriers may not be symptomatic Adapted from Strategies for Norovirus Infection Control Aboard Cruise Ships, Robert E. Wheeler, MD, 2003.

4 Three open reading frames (ORFs) ORF1 encodes the non-structural proteins that are crucial for virus replication ORF2 and ORF3 encode a major capsid protein VP1 and a minor structural protein VP2. VP1 consists of shell domain (S) and the protruding domain (P) P domain is further divided into two subdomains known as P1 and P Science 1990;250: Immunological Reviews : Nature Review 2010, 8, Genogroups >60% amino acid similarity 3 affect humans (GI, GII, GIV) Genotypes >80% amino acid similarity 9 GI genotypes, 22 GII genotypes Strains (variants, clusters) >95% amino acid similarity 11 GII.4 strains identified Norovirus Genogroups There are 7 norovirus genogroups Only 3 of these genogroups can infect humans (GI, GII, GIV) Genogroup GI: Norwalk virus, Southampton virus, Desert Shield virus Genogroup GII: Hawaii virus, Snow Mountain agent, Toronto virus (GII/3) Genogroup GIV: Ft. Lauderdale virus

5 -2 7 genogroups (GI-GVII) Associated with human infections GI: GI.1-9 GII: GII.1-22 (GII.11, GII infect porcine species) Most outbreak were caused by GII/4 worldwide GIV: GIV.1-2 (GIV.2 infects canine species) GIII: associated with bovine infections GV: associated with murine infections GVI & GVII: associated with canine infections (JCM 2015;53(2): ) GII.4 Winter Season Strain Name(s) Bristol (US95/96) Farmington Hills (2002) Hunter (2004) Laurens (Yerseke, 2006a) Minerva (Den Haag, 2006b) New Orleans Sydney New NoV strain GII.4 Sydney identified in March 2012 Caused acute gastroenteritis outbreaks in New Zealand, Japan, Western Europe, Canada, and the United States Became the predominant NoV strain implicated in outbreaks Has become the predominant strain of circulating NoV in the US Replaced the previously predominant GII.4 New Orleans strain The genogroup II, genotype 4 Noroviruses, designated GII.4, are currently responsible for 70 80% of Norovirus outbreaks worldwide.

6 Incubation period: hours Symptoms usually last between hours Infection is generally self-limiting Acute-onset vomiting and/or diarrhea Watery, non-bloody stools Abdominal cramps, nausea, low-grade fever, Headache, muscle aches Fever (minority) Dehydration Most recover after hours Up to 10% seek medical attention; some require hospitalization and fluid therapy More severe illness and death possible in elderly and those with other illnesses Up to 30% may be asymptomatic Multiple routes seen in outbreaks Person to person Direct fecal-oral Ingestion of aerosolized vomitus Direct person-to-person spread Good evidence exists for transmission due to aerosolization of vomitus droplets contaminating surfaces or entering oral mucosa and swallowed Food/ water Contamination by infected food handlers Point of service or source (raspberries, oysters) Well contamination from septic tank Chlorination system breakdown Environmental/fomite contamination n Humans only known reservoir Some noroviruses present in swine, cattle, and mice, but these genogroups do not infect humans.

7 Primarily in stool, but also in vomitus Viral shedding usually begins with onset of symptoms Presymptomatic shedding may occur Unclear to what extent viral shedding 72 hours after recovery signifies continued infectivity Shedding may continue for 2 weeks after recovery Peaks 4 days after exposure viral copies/gram of feces Infectious dose: 10 to 100 viral particles A droplet of vomitus has enough viral particles to infect over 100,000 people NoV can survive 12 hours on a surface, 12 days in contaminated fabric. A study demonstrated survival for 61 days in well water. Asymptomatic Vomiting and Diarrhea Atmar 2008 EID

8 Immunity No long-term immunity, only short-term homologous immunity after infection No persistent cross-protective immunity Protection believed to be less than a year Antibodies initially in young children ages 3 to 4 Antibody prevalence > 50% by age 50 Genetic susceptibility, Some may be resistant to NoV infection Vaccine Intranasal vaccine Safe and immunogenic 47% effective against NoV gastroenteritis Bivalent GI.1/GII.4 vaccine currently being tested in human volunteers individuals are likely to be repeatedly infected throughout their lifetimes.

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16 No cell culture or animal model available Polymerase chain reaction (PCR) Gold standard: Quantitative real-time assay Conventional RT-PCR used for genetic sequencing Enzyme Immunoassays (EIA) Recent use of virus-like particles (VLP) Inadequate sensitivity (<50%) for clinical use Electron Microscopy (EM) Resource intensive Poor sensitivity Identification of virus can be best made from stool taken within 48 to 72 hours after onset Good results can be obtained on samples taken as long as 5 days after onset Virus can sometimes be found in stool samples taken 2 weeks after recovery Lack of in vitro systems for human NoV research Animal models NoV failed to induce illness of several animals: mice, guinea pig, rabbits, kittens, calves, baboons, rhesus monkeys,. Cell culture systems Human NoV failed to propagated in vitro in different cell lines: A549, AGS, Caco-2, CCD-18, CRFK, HEp- 2, MDCK, MA104, Vero, (Ester et al., J Gen Virol, 2004, 85:79-87)

17 JCM 2015; 53(2): Clinical Microbiology Reviews 2015;28(1):

18 GI Stool specimen 10% suspension Total RNA Extraction GII Conventional RT-PCR Compare virus sequences with database Cycle sequencing (1-6) (%) 662 (49) 255 (39) 167 (25) 266 (39) 695 (39) 532 (28) 517 (36) 163 (16.1) 210 (19.1) 324 (26.8) 3390 (34)

19 GII.4: 54.9% (497/905)

20 RODS

21 , 1/1 ~2/24 : 311 (1/195, 2/116) : 107 (107/311=34.4%) - 1/1-2/24GII/17: 67 (67/95=70.5%) GII /10:42014/ (2014)12GII.17 Norovirus strains surveillance

22 : % (49/84) 73.2%(30/41)68.3% (28/41) 56.1%(23/41)

23 2/12 2/13 2/14 2/15 2/16 2/17 2/18 2/19 2/20 2/21 2/22 2/23 2/24 2/25 2/26 2/27 2/28 2/29 3/1 3/2 SRTPNFC 2/20 2/21 2/22 2/23 2/24 2/25 RC~01, 03, 12, 16, 20, 26, 281~3

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25 GII O O CDC FETP 586 2/6 2/7 2/7 2/8 2/10 2/ /11 2/14 2/22 2/24 2/16 2/ /5 3/5 3/5 3/5 3/6 3/ /8 3/11 3/12 3/12 3/14 3/20 50

26 586242% (OR2.7795%CI ) (OR3.2895%CI ) GI % (RR1.5395%CI ) (RR2.3095%CI ) 5 1F(GII)(GII) B1RO (GI+GII) GI4GII6GI+GII2 1 GI (1). (2). 52

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