Molecular Epidemiology and Evolution of Noroviruses


Chapter 3.5

Molecular Epidemiology and Evolution of Noroviruses



K.Y. Green    Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States


Abstract


Noroviruses are a major cause of acute gastroenteritis and belong to the family Caliciviridae. Replication of the positive-sense single-stranded RNA genome is carried out by an error-prone RNA-dependent RNA polymerase (RdRp) that drives adaptation and evolution. The noroviruses are remarkably diverse, with seven major phylogenetic genogroups (G) each comprised of one or more genotypes. Among the human norovirus pathogens, multiple genotypes within GI, GII, and GIV, as well as variants of these genotypes, can be detected in epidemiologic surveys, with genotype GII.4 predominating globally as the major cause of infectious diarrheal disease. The marked diversity as well as the ability of strains to undergo recombination have led to the development of a unified dual nomenclature system that defines both RNA-dependent RNA polymerase (P) and capsid protein (C) genotypes. This chapter provides a summary of norovirus epidemiology and evolution, and ongoing efforts to translate this knowledge into the development of effective vaccines.



Keywords


norovirus

calicivirus

gastroenteritis

diarrhea

viral evolution

outbreaks


1. Introduction


Noroviruses are enteric pathogens associated with acute gastroenteritis. The consequences of infection can range from asymptomatic to life-threatening (Hall et al., 2013). The virus is highly infectious and can spread easily in a population, causing outbreaks in settings where individuals are in close proximity or exposed to a common source of virus in water or food (Glass et al., 2009). The cost of outbreak management and medical visits for treatment is high; it is estimated that a vaccine of only 50% efficacy could save the US economy one billion dollars per year (Bartsch et al., 2012). Noroviruses are the leading cause of severe pediatric diarrhea following the successful implementation of rotavirus vaccines, prompting an estimated one million health care visits per year in US infants and young children (Payne et al., 2013). Vaccines to prevent norovirus disease are in clinical trials (reviewed in Ramani et al., 2014 and in Chapter 3.6), and there is an acknowledged need for antiviral drugs to treat immunocompromised patients, who are at high risk for morbidity from norovirus diarrhea (Kaufman et al., 2014).

Understanding of norovirus epidemiology and evolution is advancing rapidly with the availability of state-of-the-art diagnostic assays and new nucleotide sequencing technologies (reviewed in Vinje, 2015). Furthermore, progress has been made in the development of cell culture and animal model systems for the study of some noroviruses (reviewed in Karst et al., 2014). This chapter will provide an overview of the features of norovirus genetic diversity and adaptation responsible for the epidemiologic success of these abundant pathogens and how this diversity challenges vaccine design.

2. Classification and nomenclature of the noroviruses


2.1. Genus Norovirus in the Family Caliciviridae


Noroviruses belong to the virus family Caliciviridae, a group of small icosahedral, nonenveloped viruses that possess a polyadenylated single-stranded positive-sense RNA genome (Jiang et al., 1990). Caliciviruses are diverse in their associated disease syndromes, host specificity, and genetic characteristics. However, a striking property shared by nearly all caliciviruses is their global and ubiquitous distribution in their respective hosts. The human noroviruses are highly successful in this characteristic, with evidence for multiple infections throughout life in the majority of individuals (Jing et al., 2000Smit et al., 1997O’Ryan et al., 1998).

The current taxonomic status of the virus family Caliciviridae as defined by the International Committee on Taxonomy of Viruses (ICTV) includes five distinct genera: Norovirus, Sapovirus, Lagovirus, Nebovirus, and Vesivirus (Table 3.5.1) (Clarke et al., 2012). The current calicivirus genera are further divided into one or more species (Table 3.5.1), with each species represented by a prototype strain. The genus Norovirus is represented by Norwalk virus, which was the first norovirus discovered (Kapikian et al., 1972), and the first to be characterized at the genome level as a calicivirus (Jiang et al., 1990). Classification schemes below the species level are not addressed by the ICTV, including the genetic typing (genotyping) systems implemented by norovirus researchers to track the molecular epidemiology of norovirus strains (Kroneman et al., 2013) (see later).


Table 3.5.1


Taxonomic Structure of the Caliciviridae




























Genus Species Representative strain
Norovirus (NoV) Norwalk virus (NV) NoV/GI/Hu/US/1968/GI.P1-GI.1/Norwalk
Sapovirus (SaV) Sapporo virus (SV) SaV/GI/Hu/JP/1982/GI.1/Sapporo
Lagovirus (LaV)

Rabbit hemorrhagic disease virus (RHDV)


European brown hare syndrome virus (EBHSV)


LaV/RHDV/Ra/DE/1988/GH


LaV/EBHSV/Ha/FR/1989/GD

Vesivirus (VeV)

Vesicular exanthema of swine virus (VESV)


Feline calicivirus (FCV)


VeV/VESV/Po/US/1948/VESV-A48


VeV/FCV/Fe/US/1958/F9

Nebovirus (NeV) Newbury-1 virus (NBV) NeV/NBV/Bo/UK/1976/Newbury-1

The cryptograms in this table are organized as follows: genus/genogroup or virus species/host of origin/country of origin/year of occurrence/Pol genotype (if known)/Capsid genotype (if known)/strain name. Abbreviations for the host species are: Bo, bovine; Fe, feline; Ha, Hare; Hu, Human; Po, Porcine; Ra, Rabbit. Country abbreviations are: DE, Germany; FR, France; JP, Japan; UK, United Kingdom; US, United States. GenBank Accession numbers of representative viruses: NV, M87661; SV, U65427; RHDV, M67473, EBHSV, Z69620; VESV, AF181082; FCV, M86379; NBV, DQ013304.


2.2. Norovirus Genogroups and Genotypes


Human noroviruses are genetically diverse, and early studies noted the presence of two major genetic groups, now called genogroups (G) (Ando et al., 1994Wang et al., 1994). The norovirus RNA genome (approx. 7.5–7.6 kb in size) is organized into three major open reading frames (ORFs 1–3). The length of the RNA genome can vary among strains, and the nucleotide boundaries of the ORFs are shown for representative GI (Norwalk virus) and GII (Lordsdale virus) human norovirus strains in Fig. 3.5.1A.

image

Figure 3.5.1 Norovirus genome organization, proteins, and diagnostic primers.
(A) Genome organization and nucleotide boundaries of the ORFs of representative human norovirus strains Genogroup I Norwalk virus (M87661) (top) and Genogroup II Lordsdale virus (X86557) (bottom). (B) Proteins encoded by the norovirus genome. ORF1 encodes the nonstructural proteins of the virus, NS1-NS7. ORF2 encodes the major capsid protein, VP1, and ORF3 encodes the minor capsid protein, VP2. (C) Genomic regions targeted for the norovirus dual genotyping system involve RNA-dependent RNA polymerase (P) and capsid (C) sequences. The regions of interest are amplified in a reverse transcription-polymerase chain reaction (RT-PCR) assay, sequenced, and then interrogated against a sequence database using the norovirus typing tool (Kroneman et al., 2013). References for representative primer sets are: (Anderson et al., 2001Jiang et al., 1999bKojima et al., 2002Newman and Leon, 2015Vinje and Koopmans, 1996Vinje et al., 2004). A widely-used real time RT-qPCR assay for norovirus detection is based on the primers of Kageyama et al. (Kageyama et al., 2003). Many additional primer pairs have been developed, but periodic optimization may be needed to encompass ever-evolving norovirus strains (Kong et al., 2015).

2.2.1. ORF1


The norovirus ORF1 encodes the nonstructural proteins (designated NS1 through NS7) that are cleaved from the newly-synthesized polyprotein precursor by a virus-encoded protease (NS6) (reviewed in Sosnovtsev, 2010Thorne and Goodfellow, 2014) (Fig. 3.5.1B). The nonstructural proteins play a major role in viral replication, and enzymatic activity has been identified for the NS3 (NTPase), NS6 (protease) and NS7 (RNA-dependent RNA polymerase) proteins. Nonstructural proteins NS1-2 (or Nterm) and NS4 (p22 or “3A-like”) contain hydrophobic domains and interact with host cell membranes to establish the sites of viral replication (Hyde et al., 2009). The VPg protein (NS5) is covalently linked to the 5′-end of the viral RNA genome and plays an essential role in translation and RNA replication. The RNA-dependent RNA polymerase (abbreviated as RdRp, NS7, or Pol), mapping to the C-terminal end of ORF1, is the enzyme involved in viral RNA replication and one of the most conserved proteins.

2.2.2. ORF2


The structural proteins of the virion, VP1, and VP2, are encoded by ORFs 2 and 3, respectively. The VP1 (approximately 58 kDa) is the most abundant protein in the virion, and 180 copies of VP1 form the icosahedral capsid of the virus (Prasad et al., 1996). Expression of the norovirus VP1 by recombinant baculovirus yields virus-like particles (VLPs) that resemble native virions, and VLPs have become an essential tool in norovirus research and vaccine development (Jiang et al., 1992). The VP1 is organized into two major domains, designated Shell (S) and Protruding (P) (Prasad et al., 1996). In dimeric form with another VP1 protein, the P domain forms an arch-like structure in which two arms (the P1 subdomains) present a highly variable P2 subdomain to the surface of the virion (Prasad et al., 1996). Structural studies have demonstrated that both antibodies and histo-blood group antigen (HBGA) carbohydrates (proposed attachment factors in the gut) bind within the norovirus P2 domain, confirming its importance in host interactions and immunity (Cao et al., 2007Bu et al., 2008Katpally et al., 2008). Furthermore, because multiple antigenic epitopes and HBGA binding sites overlap (Parra et al., 2012a), the measurement of antibodies that block HBGA binding to virus particles has been taken as a surrogate marker of virus neutralization and been accepted widely as a correlate of protection from norovirus infection and disease (Bok et al., 2011Reeck et al., 2010).

2.2.3. ORF3 (and ORF4)


The ORF3 encodes a small, basic protein, VP2, considered a minor structural protein because of its low abundance in virions (Sosnovtsev and Green, 2000). The VP2 plays a role in particle stability and infectivity (Bertolotti-Ciarlet et al., 2003Sosnovtsev et al., 2005), and it interacts directly with a highly conserved region of the inner shell domain in VP1 (Vongpunsawad et al., 2013). The norovirus VP2 may exert host effects: a murine norovirus VP2 was recently shown to dampen the maturation of antigen presenting cells (Zhu et al., 2013).

An ORF4 encoding a host regulatory protein (designated virulence factor-1) has been described for murine norovirus (McFadden et al., 2011), a member of the genus that replicates efficiently in cell culture (Wobus et al., 2004), but an ORF4 (or functional homolog) has not yet been identified in human strains.

2.2.4. Genotyping Methods


Diagnostic RT-PCR primers for the analysis of human noroviruses are targeted to conserved regions of the genome, and references describing representative primer sets for the detection and genotyping of noroviruses are diagrammed in Fig. 3.5.1C. The RNA-dependent RNA polymerase and capsid encoding regions are important targets for virus genotyping (see later).

2.2.5. Genotyping System Based on the Viral Capsid


Comparative phylogenetic analysis of the complete capsid VP1-encoding gene (ORF2) has led to the division of noroviruses into seven genogroups (G) designated with Roman numerals as GI through GVII (Vinje, 2015Kroneman et al., 2013) (Fig. 3.5.2). The genogroups are sufficiently divergent to require the use of different diagnostic primer sets for identification (Fig. 3.5.1C). The genogroups are further subdivided into capsid (C) genotypes that are numbered sequentially as they are identified (Table 3.5.2). The two genogroups with the majority of human pathogens, GI and GII, currently contain 9 and 22 capsid genotypes, respectively. The reference strain for a genotype, by convention, is the first full-length capsid sequence entered into a public sequence database. Each capsid genotype is further segregated into “clusters” or “variants,” and this diversity is especially noteworthy for genotype GII.4 because of its major role in epidemic disease (reviewed in White, 2014). Eight pandemic variants of norovirus GII.4 have emerged since 1995, when molecular diagnostic assays began to gain widespread use (Kroneman et al., 2013). The GII.4 variants and their GenBank accession numbers are listed in the footnote of Table 3.5.2. The variants are named according to one of the early sites where the emergence of a new pandemic strain was first recognized.

image

Figure 3.5.2 Phylogenetic relationships among the noroviruses.
There are presently seven major genogroups within the genus Norovirus, with human pathogens found in GI, GII, and GIV. Porcine norovirus strains have been found also in GII, and canine and feline strains have been detected in GIV. Strains belonging to certain genogroups have not been detected in humans thus far and include GIII (bovine), GV (murine), and GVII (canine). Phylogenetic analyses were carried out with MEGA v6 using Neighbor-Joining as the algorithm for reconstruction and amino acid sequences from the entire capsid protein VP1. Scale bar represents the number of amino acids substitutions per site. (Source: Analysis and image courtesy of Gabriel I. Parra.)


Table 3.5.2


Norovirus Genogroups and Genotypes as Determined by Capsid (C) Gene Relatedness












































































































































































Reference virus Genogroup. C genotype GenBank accession number
GI/Hu/US/1968/GI.1/Norwalk GI.1 M87661
GI/Hu/UK/1991/GI.2/Southampton GI.2 L07418
GI/Hu/SA/1990/GI.3/DesertShield 395 GI.3 U04469
GI/Hu/JP/1987/GI.4/Chiba 407 GI.4 AB042808
GI/Hu/GB/1989/GI.5/Musgrove GI.5 AJ277614
GI/Hu/DE/1997/GI.6/BS5(Hesse3) GI.6 AF093797
GI/Hu/GB/1994/GI.7/Winchester GI.7 AJ277609
GI/Hu/US/2001/GI.8/Boxer GI.8 AF538679
GI/Hu/CA/2004/GI.9/Vancouver730 GI.9 HQ637267
GII/Hu/US/1971/GII.1/Hawaii GII.1 U07611
GII/Hu/GB/1994/GII.2/Melksham GII.2 X81879
GII/Hu/CA/1991/GII.3/Toronto 24 GII.3 U02030
GII/Hu/GB/1993/GII.4/Bristola GII.4 X76716
GII/Hu/GB/1990/GII.5/Hillingdon GII.5 AJ277607
GII/Hu/GB/1990/GII.6/Seacroft GII.6 AJ277620
GII/Hu/GB/1990/GII.7/Leeds GII.7 AJ277608
GII/Hu/NL/1998/GII.8/Amsterdam GII.8 AF195848
GII/Hu/US/1996/GII.9/VA97207 GII.9 AY038599
GII/Hu/DE/2000/GII.10/Erfurt546 GII.10 AF427118
GII/Po/JP/1997/GII.11/Sw918 GII.11 AB074893
GII/Hu/GB/1990/GII.12/Wortley GII.12 AJ277618
GII/Hu/US/1998/GII.13/Fayetteville GII.13 AY113106
GII/Hu/US/1999/GII.14/M7 GII.14 AY130761
GII/Hu/US/1999/GII.15/J23 GII.15 AY130762
GII/Hu/US/1999/GII.16/Tiffin GII.16 AY502010
GII/Hu/US/2002/GII.17/CS-E1 GII.17 AY502009
GII/Po/US/2003/GII.18/OH-QW101 GII.18 AY823304
GII/Po/US/2003/GII.19/OH-QW170 GII.19 AY823306
GII/Hu/DE/2002/GII.20/Luckenwalde591 GII.20 EU373815
GII/Hu/IR/2003/GII.21/IF1998 GII.21 AY675554
GII/Hu/JP/2003/GII.22/Yuri GII.22 AB083780
GIII/Bo/De/1980/GIII.1/Jena GIII.1 AJ011099
GIII/Bo/GB/1976/GIII.2/Newbury-2 GIII.2 AF097917
GIII/Ov/NZ/2007/GIII.3/Norsewood30 GIII.3 EU193658
GIV.1/Hu/NL/1998/GIV.1/Alphatron 98-2 GIV.1 AF195847
GIV.2/Fe/IT/2006/GIV.2/Pistoia 387 GIV.2 EF450827
GV/Mu/US/2002/GV.1/MNV-1 GV.1 AY228235
GV/Rn/HK/2011/GV.2/HKU_CT2 GV.2 JX486101
GVI/Ca/IT/2007/Bari 91 GVI.1 FJ875027
GVI/Ca/PT/2007/Viseu GVI.2 GQ443611
GVII/Ca/HK/2007/GVII/026F GVII FJ692500

Note: According to classification system of the online norovirus typing tool at www.rivm.nl/mpf/norovirus/typingtool (Kroneman et al., 2011)


The cryptogram is organized as follows: Genogroup/host species of origin /country of origin/year of occurrence/Capsid genotype/strain name


Host species abbreviations are: Bo, bovine; Ca, canine, Fe, feline; Hu, human; Mu, murine; Ov, ovine; Po, porcine; Rn, rat


Country abbreviations are: CA, Canada; DE, Germany; IR, Iraq; IT, Italy; JP, Japan; NL, Netherlands; NZ, New Zealand; PT, Portugal; SA, Saudi Arabia; UK, United Kingdom; US, United States


a The pandemic GII.4 variants and their GenBank accession numbers are: US95_96 (AJ004864), Farmington_Hills_2002 (AY485642), Asia_2003 (AB220921), Hunter_2004 (AY883096), Yerseke_2006a (EF126963), Den Haag_2006b (EF126965), NewOrleans_2009 (GU445325), and Sydney_2012 (JX459908) (Kroneman et al., 2013).


2.2.6. Genotyping System Based on the Viral RNA-Dependent RNA Polymerase


The RdRp (Pol) gene can vary among strains of the same capsid genotype (Hardy et al., 1997Jiang et al., 1999a), which has led to the development of a polymerase (P) genotyping system based on phylogenetic analysis of the 3′-terminal 1300 nucleotides of the norovirus ORF1 (that encodes a large part of the RdRp protein) (Kroneman et al., 2013). In an effort to unify the disparate nomenclature in the literature, the known P genotypes were recently assigned number or letter designations (Kroneman et al., 2013). The P genotyping system currently focuses on GI and GII noroviruses, each containing 14 and 27 unique P types, respectively (Table 3.5.3). A number of reference viruses share the same P and C genotyping designations (Table 3.5.3), but recombination events (Jiang et al., 1999a) can lead to varying combinations of ORF1 and ORF2. Some C genotypes are found in combination with two or more P genotypes, as illustrated by the GII.4 Bristol (GII/Hu/GB/1993/GII.P4-GII.4/Bristol), Sakai (GII/Hu/JP/2005/GII.P12-GII.4/Sakai/04-179, and OC07138 (GII/Hu/JP/2007/GII.Pe-GII.4/OC07138) noroviruses (Table 3.5.3).


Table 3.5.3


Norovirus Genogroups and Genotypes as Determined by Polymerase (P) Gene Relatedness












































































































































































Reference Virus Genogroup. P Genotype GenBank Accession Number
GI/Hu/US/1968/GI.P1-GI.1/Norwalk GI.P1 M87661
GI/Hu/GB/1991/GI.P2-GI.2/Southampton GI.P2 L07418
GI/Hu/US/1998/GI.P3-GI.3/VA98115 GI.P3 AY038598
GI/Hu/JP/1987/GI.P4-GI.4/Chiba407 GI.P4 AB042808
GI/Hu/SE/2005/GI.P5-unknown/07_1 GI.P5 EU007765
GI/Hu/DE/1997/GI.P6-GI.6/BS5(Hesse) GI.P6 AF093797
GI/Hu/SE/2008/GI.P7-GI.7/Lilla Edet GI.P7 JN603251
GI/Hu/US/2008/GI.P8-GI.8/890321 GI.P8 GU299761
GI/Hu/FR/2004/GI.P9-GI.9/Chatellerault709 GI.P9 EF529737
GI/Hu/SA/1990/GI.Pa-GI.3/DesertShield GI.Pa U04469
GI/Hu/JP/2002/GI.Pb-GI.6/WUG1 GI.Pb AB081723
GI/Hu/JP/2000/GI.Pc-GI.5/SzUG1 GI.Pc AB039774
GI/Hu/FR/2003/GI.Pd-GI.3/Vesoul576 GI.Pd EF529738
GI/Hu/JP/1979/GI.Pf-GI.3/Otofuke GI.Pf AB187514
GII/Hu/US/1971/GII.P1-GII.1/Hawaii GII.P1 U07611
GII/Hu/GB/1994/GII.P2-GII.2/Melksham GII.P2 X81879
GII/Hu/CA/1991/GII.P3-GII.3/Toronto GII.P3 U02030
GII/Hu/GB/1993/GII.P4-GII.4/Bristol GII.P4 X76716
GII/Hu/HU/1999/GII.P5-GII.5/MOH GII.P5 AF397156
GII/Hu/JP/2002/GII.P6-GII.6/Saitama U16 GII.P6 AB039778
GII/Hu/JP/2002/GII.P7-GII.6/Saitama U4 GII.P7 AB039777
GII/Hu/JP/2002/GII.P8-GII.8/Saitama U25 GII.P8 AB039780
GII/Po/US/1997/GII.P11-GII.11/Sw918 GII.P11 AB074893
GII/Hu/JP/2005/GII.P12-GII.4/Sakai/04-179 GII.P12 AB220922
GII/Hu/FR/2004/GII.P13-GII.17/Briancon870 GII.P13 EF529741
GII/Hu/JP/2006/GII.P15-GII.15/Hiroshima66 GII.P15 AB360387
GII/Hu/DE/2000/GII.P16-GII.16/Neustrelitz260 GII.P16 AY772730
GII/Po/US/2003/GII.P18-GII.18/OH-QW101 GII.P18 AY823304
GII/Hu/DE/2005/GII.P20-GII.20/Leverkusen267 GII.P20 EU424333
GII/Hu/FR/2004/GII.P21-GII.2/Pont de Roide673 GII.P21 AY682549
GII/Hu/JP/2003/GII.P22-GII.5/Hokkaido133 GII.P22 AB212306
GII/Hu/JP/2004/GII.Pa-GII.3/SN2000JA GII.Pa AB190457
GII/Hu/US/1976/GII.Pc-GII.2/SnowMountain GII.Pc AY134748
GII/Hu/JP/2007/GII.Pe-GII.4/OC07138 GII.Pe AB434770
GII/Hu/FR/1999/GII.Pf-GII.5/S63 GII.Pf AY682550
GII/Hu/AU/1983/GII.Pg-GII.13/Goulburn Valley GII.Pg DQ379714
GII/Hu/JP/1997/GII.Ph-GII.2/OC97007 GII.Ph AB089882
GII/Hu/GR/1997/GII.Pj-GII.2/E3 GII.Pj AY682552
GII/Hu/JP/1996/GII.Pk-Unknown/OC96065 GII.Pk AF315813
GII/Hu/IN/2006/GII.Pm-GII.12/PunePC24 GII.Pm EU921353
GII/Hu/CN/2007/GII.Pn-GII.22/Beijing53931 GII.Pn GQ856469

Note: According to classification system of the online norovirus typing tool at www.rivm.nl/mpf/norovirus/typingtool (Kroneman et al., 2011)


Cryptogram is organized as follows: Genogroup/host species of origin/country of origin/year of occurrence/Pol (P) genotype-Capsid (C) genotype/strain name


Host species abbreviations: Hu, human; Po, porcine


Country abbreviations are: AU, Australia; CA, Canada; CN, China; DE, Germany; GB, Great Britain; GR, Greece; FR, France; HU, Hungary; IN, India; IT, Italy; JP, Japan; SA, Saudi Arabia; SE, Sweden; UK, United Kingdom; US, United States


Bold lettering represents P genotypes with matched C genotype numbering


2.2.7. Norovirus Online Genotyping Tool


An online typing tool has been developed for identification of norovirus genogroup, genotype (P or C), and GII.4 variant cluster (Kroneman et al., 2011Verhoef et al., 2011) (www.rivm.nl/mpf/norovirus/typingtool). Full-length norovirus genomic or partial nucleotide sequences can be submitted and quickly matched to a database of known reference strains for genotype assignment. New genotype and variant designations are assigned by an international norovirus working group (reviewed in Kroneman et al., 2013). The development of a unified nomenclature system should facilitate surveillance efforts to track the emergence and spread of epidemic norovirus strains. Data sharing networks such as the international NoroNet group (Siebenga et al., 2009) and CaliciNet in the United States (Vega et al., 2011) provide platforms that allow communication of molecular epidemiological findings in real time.

3. Mechanisms for the generation of norovirus diversity


Noroviruses are diverse and constantly changing. A number of mechanisms have been identified that allow the generation of diversity and the emergence of new norovirus strains (White, 2014Bull and White, 2011). Genetic drift, transmission bottlenecks, and recombination are among the factors driving norovirus evolution, and are briefly described later.

3.1. Genetic Drift


The RdRp of the Caliciviridae, like that of other positive strand RNA viruses, does not have an editing function, resulting in a comparably higher error rate per genome per replication cycle than DNA viruses (Duffy et al., 2008). The subsequent nucleotide mutations can lead to fixed substitutions at the viral population level, a key component in adaptation. Genetic drift may best be illustrated by the evolutionary patterns of the predominant GII.4 noroviruses that show the emergence of a new pandemic variant every few years (Fig. 3.5.3). The genetic changes of an emerging variant are often clustered in epitopes in the surface-exposed P2 region of the capsid, suggesting a role for herd immunity and selective antibody pressure (antigenic drift) in its evolution (reviewed in Debbink et al., 2012). In immunocompromised individuals, the population of RNA molecules is even more diverse, presumably because of the absence of selective pressure to drive escape from antibodies and other immune responses (Bull et al., 2012).

image

Figure 3.5.3 Mechanisms by which GII.4 noroviruses evolve to become pandemic.
Evidence for two distinct mechanisms in the emergence of pandemic GII.4 noroviruses has been documented in molecular epidemiologic studies: antigenic drift and recombination (White, 2014). The VP1 capsid protein is organized into domains (Shell and Protruding) and subdomains (P1 and P2) (Prasad et al., 1999). A short N-terminal region (N) precedes the highly conserved S domain. The P2 domain, highly variable among strains, bears key antigenic and host carbohydrate ligand binding sites and is a major site of host selective immune pressure (Debbink et al., 2012). The asterisk represents a GII.4 P2 domain that has sustained amino acid substitutions that escape herd immunity. Proposed recombination breakpoints (BP) in GII.4 recombinants are indicated, with BPs identified at the ORF1/ORF2 junction, within ORF2, and near the ORF2/ORF3 junction (Eden et al., 2013). Examples of pandemic GII.4 variants and the mechanism by which they may have emerged are shown.

One study compared the replication and mutation rates of circulating norovirus strains that varied in global prevalence to assess whether the RdRp error rate might influence strain success. Three pandemic GII.4 strains, a nonpandemic recombinant GII.4/GII.10 strain, a prevalent GII.b/GII.3 virus, and the infrequently detected GII.7 strain were evaluated (Bull et al., 2010). Evidence was found for a higher error rate (decreased fidelity) for the pandemic GII.4 RdRp, leading to the suggestion that a more error-prone polymerase might facilitate the rapid evolution of GII.4 viruses by introducing a higher rate of adaptive substitutions, especially in the VP1 (Bull et al., 2010).

Estimates of the evolutionary rates of two prevalent norovirus genotypes, GII.3 and GII.4, were determined by comparative phylogenetic analysis of viruses occurring over a span of approximately 40 years (Boon et al., 2011Bok et al., 2009). The GII.3 and GII.4 norovirus genotypes were calculated to evolve at similar rates of 4.16 × 10(−3) and 4.3 × 10(−3) nucleotide substitutions/site/year (strict clock), respectively, even though differences in their evolutionary patterns within the VP1 sequence were noted (Boon et al., 2011Bok et al., 2009). This evolutionary rate is comparable to that of other positive strand RNA viruses with rates of within one order of magnitude of 1 × 10(−3) nucleotide substitutions/site/year (Duffy et al., 2008). These data suggest that noroviruses, although diverse and subject to genetic drift, are not equipped with a replication machinery that drives them to evolve at higher rates than most other positive strand RNA viruses.

3.2. Transmission Bottlenecks


Deep sequencing of clinical specimens has shown that norovirus strains exist as a mixed population of genetically closely related RNA variants in the host, with a predominant RNA sequence that can comprise >60% of the total population (Bull et al., 2012). However, transmission of this mixed population into a new host may create a fitness bottleneck, and the emergence of a minor population can give rise to new population diversity (Bull et al., 2012). The highly infectious nature of norovirus and its efficient spread by person-to-person transmission (Koopmans, 2008) creates ample opportunity for the emergence of new variants via this mechanism. A recent study of the norovirus genomic RNA population in an immunocompromised host showed evidence for the presence of novel GII.4 variants with the potential for transmission into a susceptible population (Vega et al., 2014a). The impact of transmission bottlenecks in norovirus epidemiology will require additional viral metagenomics studies. (See Chapter 5.1.)

3.3. Recombination


During calicivirus replication, two major positive-sense RNA species are generated, with one corresponding to the full-length genome (approx. 7.6 kb in size) and the other to a 3′-end coterminal subgenomic region (approx. 2.6 kb in size) (Neill and Mengeling, 1988). The abundant subgenomic RNA serves as a template for translation of the VP1 and VP2 capsid proteins. There is evidence that during coinfection of a cell with two caliciviruses, the two genomic RNAs replicate in proximity so that recombination events can readily occur (Abente et al., 2013), likely due to template switching by the RdRp (Bull et al., 2005). A recent in vitro study found that after coinfection of cells with two parental feline calicivirus strains, recombination was detected as early as 6 h after infection at a rate of 6.8 × 10(−6) single direction recombinant genomes generated per parental virus genome (Symes et al., 2015). Although the recombination rate is not known for the human noroviruses, epidemiological studies suggest that recombination is not a rare event (Lu et al., 2015). A hotspot for recombination in the noroviruses exists at the junction between ORF1 and ORF2 as a breakpoint (BP) for template switching, yielding new combinations of VP1 and polymerase that may confer adaptive advantages to the new strain (Bull et al., 2005Mans et al., 2014Shen et al., 2012) (Fig. 3.5.3). For example, the “GII.Pb” polymerase has been detected globally for nearly a decade and has been associated with at least five different GII capsid proteins (GII.1, GII.2, GII.3, GII.4, and GII.13) (Lim et al., 2013Reuter et al., 2005). The GII.4 noroviruses may have additional recombination BPs within ORF2 and between ORF2 and 3, leading to new combinations of VP1 and VP2 (Kamel et al., 2009Eden et al., 2013). Evidence for additional BPs in ORF1 was found in the murine norovirus (MNV) VPg, RdRp, and protease genes following coinfection of mice with two distinct MNV strains (Zhang et al., 2015). A common feature of the MNV BP sites was high sequence identity between the two parental strains, strongly suggesting that any highly conserved region may, in theory, be a site for template switching and recombination.

Tracking the molecular epidemiology of the Pol gene (ORF1) in the natural history of norovirus infection may give insight into the factors driving recombination and why different capsid and RdRp combinations occur in nature (Hasing et al., 2014). Recombination occurs predominantly among strains of the same genogroup (intragenogroup), although rare intergenogroup recombinants have been reported (Nayak et al., 2008Nataraju et al., 2011). There may be specific structural constraints for capsid and polymerase interactions as evidence has been reported for a direct interaction between the two proteins during RNA replication (Subba-Reddy et al., 2012).

4. Molecular epidemiology and transmission


4.1. Distribution of Norovirus Genotypes


Norovirus genotypes vary among geographical locations, seasons, and years. Outbreaks occur more often in the winter months of temperate climates (Ahmed et al., 2013). A link has been proposed between higher particle stability and low absolute humidity (below 0.007 kg/kg air), which could explain, in part, the increase of norovirus transmission and illness during cold months when these conditions predominate (Colas de la Noue et al., 2014). The marked diversity and unpredictable pattern of norovirus genotypes is reflected in epidemiologic surveys conducted across the globe. The diversity is especially striking in environmental studies, reflecting the ubiquitous distribution of these viruses (Fernandez et al., 2012van den Berg et al., 2005). Two consistently observed features in most clinical surveys are the predominance of GII compared to GI viruses, and the higher prevalence of the GII.4 genotype (Matthews et al., 2012Vega et al., 2014b). In contrast, studies of environmental samples, waterborne outbreaks, or shellfish may find a predominance of GI strains (Matthews et al., 2012Lowther et al., 2012). The molecular basis for this variation in genotypic distribution is not yet known, but the emergence of a pathogenic strain likely involves properties of the virus (eg, physicochemical features, replicative fitness) and the susceptible host (eg, immune status, HBGA profile).

Most human norovirus capsid proteins interact with HBGA carbohydrate ligands displayed on intestinal tissue (Marionneau et al., 2002), and there is a diverse array of HBGA molecules and other glycan-containing molecules such as galactosylceramide in the gut that could potentially serve as attachment factors (Bally et al., 2012). The precise mechanism of viral entry into host cells is not known. The HBGA phenotype of an individual, as well as the presentation of these HBGA antigens on intestinal epithelial cells (ie, secretor status) is determined by inherited alleles that affect norovirus binding to host cells and thus, susceptibility to infection (reviewed in Le Pendu, 2004 and in Chapter 3.3). Ancestry-based differences in HBGA genetics can influence norovirus susceptibility in different populations (Currier et al., 2015). Norovirus strains vary in their recognition of HBGA carbohydrates, and these interactions have been studied extensively at the structural level with recombinant norovirus P domains and synthetic saccharides (Cao et al., 2007Huang et al., 2003). The GI and GII noroviruses exhibit sequence differences in their HBGA binding interfaces, although an overall structural similarity in the HBGA recognition sites of the P2 domain has remained conserved during evolution of the two distinct phylogenetic lineages (Tan et al., 2009). An association between susceptibility to GII.4 noroviruses and the presence of a functional FUT2 enzyme has been noted in epidemiologic surveys, suggesting that the higher prevalence of secretors (80%) versus nonsecretors (20%) in many populations insures a large pool of susceptible individuals for this predominant genotype (Currier et al., 2015Frenck et al., 2012). The specific amino acid sequences for HBGA binding are often highly conserved in VP1 proteins of the same genotype or cluster, but subtle changes in sequence can affect the binding of a norovirus strain to a specific HBGA ligand that consequently might influence host cell interactions (Tan et al., 2003de Rougemont et al., 2011).

4.2. Diversity and Norovirus Vaccine Development


Serologic surveys have shown evidence for the acquisition of both GI and GII norovirus-specific antibodies early in life in most populations, although regional differences in rates of antibody acquisition and specificity have been reported (Jiang et al., 2000Menon et al., 2013Gray et al., 1993). Susceptibility to norovirus disease spans all age groups, and this lifetime susceptibility enables the occurrence of sharp gastroenteritis outbreaks in a wide range of settings involving otherwise healthy individuals. Severe, life-threatening norovirus illness occurs most often in the young and old, suggesting that both, immunological immaturity in the young and immunosenescence in the elderly, hamper virus clearance. This is consistent with the link between immunocompromised patients and chronic norovirus infection, in which impaired immune function affects virus clearance (Vega et al., 2014a, reviewed in Bok and Green, 2012).

A key goal in norovirus vaccine development is the formulation of a vaccine that will provide broad protection against the myriad of circulating genotypes. Early cross-challenge studies had shown that Norwalk virus (Genogroup I) and Hawaii virus (Genogroup II) were serotypically distinct (Wyatt et al., 1974), consistent with a proposed minimal need for representative strains from these two genogroups in a vaccine (Bok et al., 2011Malm et al., 2015). Presently, a bivalent norovirus VLP-based candidate is under investigation containing both GI and GII components (Treanor et al., 2014), with the GII component consisting of an engineered consensus GII.4 VLP (Parra et al., 2012b). Efficacy data will be important to assess whether additional antigenic components are needed. Immunity to noroviruses is complex, and infection with a virus of one genotype may not induce immunity to a second genotype in young individuals (Parra and Green, 2014). This observation is reflected in natural history studies as well, in which a high rate of sequential reinfection occurs in children with noroviruses belonging to different genotypes within the same genogroup (Ayukekbong et al., 2014Saito et al., 2014).

Efforts are underway to identify cross-reactive antigenic sites that are shared among the diverse genotypes (Kitamoto et al., 2002Parker et al., 2005Crawford et al., 2015Parra et al., 2013). An understanding of heterotypic immunity will be important in the development of broadly-protective vaccines or immunotherapy (see Chapter 3.6).

4.3. Transmission and Site of Replication


Noroviruses are transmitted by the fecal–oral route, and shed in the feces. It is estimated that approximately 14% of norovirus outbreaks are foodborne (Verhoef et al., 2015). The virus is apparently most infectious in the early phase of the acute illness, when the symptoms of vomiting and/or diarrhea are most severe and norovirus shedding is at its peak (Zelner et al., 2013). The infectious dose has been estimated in volunteer studies. In one such study, aggregation of Norwalk virus particles was detected and the ID50 dose of disaggregated virus was estimated to be approximately between 18 and 1015 genome equivalents (Teunis et al., 2008). In a second dose-response study of Norwalk virus, a higher ID50 dose of 1320–2800 genome equivalents was reported, with dose depending on secretor and blood group status (Atmar et al., 2014). Norovirus is shed to similar levels (as measured by RT-qPCR) in symptomatic and asymptomatic individuals, making it problematic to use virus genome copy numbers as a marker of disease (Teunis et al., 2014).

Viral replication is thought to occur predominantly in the small intestine, and studies in adult volunteers (Agus et al., 1973) and a calf model (Otto et al., 2011) show a striking pathology of blunted villi. Extra-intestinal sites of replication have not been confirmed in humans, but murine norovirus can be detected in multiple organs in the immunocompromised mouse host (Karst et al., 2003). A recent study found no evidence of viremia in healthy adults challenged with norovirus, suggesting local replication in the gut (Newman et al., 2015). Norovirus RNA has been detected in the serum of immunocompromised children (Frange et al., 2012), but the presence of the virus in organs outside the gut has not been confirmed. Norovirus strains may vary in their pathogenicity, as has been noted for the GII.4 noroviruses (Desai et al., 2012), but there is presently no marker to track or predict the virulence of circulating viruses. Identification of the mechanisms of norovirus pathogenesis among all members of this diverse genus will be an important area of future research.

5. Summary and future directions


Noroviruses are major enteric pathogens, and efforts are underway to develop vaccines and antiviral drugs to control morbidity and mortality. Molecular epidemiologic studies will continue to play a key role in the development of effective strategies. The implementation of a recently developed unified genotyping nomenclature system will facilitate the tracking (and possible prediction) of emerging pandemic strains and inform vaccine design. Investigations will undoubtedly continue to develop tractable and efficient cell culture systems as well as “humanized” animal models to elucidate the mechanisms of norovirus pathogenesis and immunity. These model systems, in concert with clinical investigations and vaccine efficacy trials, will be empowered by ground-breaking new technologies to understand norovirus evolution and immunity at the molecular level. Elucidation of the complex interactions between virus and host that result in acute and chronic norovirus infection will undoubtedly remain a compelling area of enteric virus research.

Acknowledgments


This work was supported by the Division of Intramural Research (DIR), NIAID, NIH, Bethesda, Maryland. The author would like to thank Gabriel I. Parra, LID, NIAID, for data analysis and critical review. In addition, the author thanks Jordan Johnson, Stanislav V. Sosnovtsev, Eric Levenson, and Allison Behrle of LID, NIAID, for reviewing the chapter and providing constructive comments.


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Apr 25, 2018 | Posted by in MICROBIOLOGY | Comments Off on Molecular Epidemiology and Evolution of Noroviruses
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