Norovirus GII . 4 antibodies in the Portuguese population

Introduction: Norovirus GII.4 is the leading cause of outbreaks of acute and sporadic acute gastroenteritis worldwide. Information on the prevalence of norovirus in Portugal is scarce or null. Methodology: We used a GII.4 norovirus virus-like particle-based enzyme immune assay to measure the seropositivity rate of GII.4 norovirus. Results: A total of 342 (70%) out of 473 serum samples tested positive for anti-GII.4 norovirus IgG. No statistically significant differences were found between regions, sex and age groups. Conclusion: Norovirus GII.4 infections are frequent in Portugal.


Introduction
Noroviruses are the most common cause of epidemic gastroenteritis and the leading cause of foodborne outbreaks [1,2].
Noroviruses can be spread via multiple routes including fecal-oral and vomit-oral transmission [3].Person-to-person transmission is the primary mode of spreading; and together with their environmental persistence and infectiousness, outbreaks are most frequently reported in semi-closed communities such as long-term-care facilities, hospitals, schools, restaurants and cruise ships [4].
Illness due to norovirus can occur in all age groups and clinical symptoms generally include vomiting, non-bloody diarrhea, fever, abdominal cramps, nausea, chills, myalgia and headaches; however, asymptomatic infections have also been described [4,5].Symptoms are typically considered mild and self-limiting, lasting for 24 to 48 hours, with an incubation period of 24 to 48 hours [4].Medical intervention is generally not required unless rapid dehydration occurs in vulnerable populations such as infants, the elderly and immunosuppressed patients, in which case fluid therapy or additional supportive care may be needed [2,5].
Noroviruses comprise a genetically and antigenically diverse group of non-enveloped singlestranded RNA viruses classified into the family Caliciviridae, genus Norovirus.They can be grouped into at least 6 different genogroups (G) with viruses infecting humans belonging to GI, GII and GIV [5].The majority of human infections are caused by GI and GII, and these genogroups are further subdivided into 31 genotypes [5].In the last decade, genogroup II, genotype 4 (GII.4)noroviruses have emerged as the most frequent cause of outbreaks of acute nonbacterial gastroenteritis worldwide [6].The high prevalence of norovirus GII.4 has been confirmed by recent seroprevalence studies in both developed and developing countries [7][8][9].
In Portugal, information on the prevalence of norovirus infections is scarce since these viruses are not included in the diagnostic algorithm of acute gastroenteritis in routine clinical laboratories.The lack of diagnostic testing capabilities leads to underreporting and only one confirmed foodborne norovirus outbreak has been described until today [10].Hence, the aim of this study was to evaluate the seroprevalence of norovirus in the Portuguese population.

Serum samples
A total of 473 serum samples were collected in January 2012 from anonymous volunteers from Northern (n = 231), Central (n = 189) and Southern (n = 53) Portugal.Individuals enrolled in this study were healthy individuals from the University of Porto and participants of a national scientific meeting.The serum samples were from 337 females (71.2%) and 136 males (28.8%).Two hundred and forty-seven (52.2%) samples represented ages 19 to 29 years; 187 (39.5%), 30 to 39 years; 33 (7.0%), 40 to 49 years; and 6 (1.3%), 50 years and over.The study was approved by an institutional review board of the University of Porto.

Enzyme immune assay for detection of GII.4 norovirus antibodies
Sera were tested for the presence of IgG antibodies against GII.4 norovirus using an enzyme-linked immune assay (EIA) using recombinant virus-like particles (VLPs) as antigens.Briefly, norovirus GII/Hu/USA/2009/GII.4/New Orleans (GenBank accession number GU445325) VLPs were produced by cloning full-length VP1/VP2 (ORF2 and ORF3 of the genome) in a baculovirus-insect cell expression system and purified through sucrose and CsCl gradients [11].Morphology and size of the purified VLPs was confirmed by electron microscopy.
For Cut-off values were determined as previously described [12].Net absorbance (P -N) was calculated as the mean value in the antigen-coated wells (P) minus the mean value in the antigen-negative wells (N).Values with a net absorbance of 0.15 and a P/N value of > 2.0 were considered positive.A chi-square test for homogeneity of proportions (SPSS 13.0, SPSS Inc., Chicago, IL, USA) was used to evaluate differences between genders, regions, and age groups.Only values of p < 0.001 were considered statistically significant.

Results
From the 473 serum samples, 342 (70%) tested positive for anti-GII.4norovirus IgG.The distribution of IgG anti-norovirus GII.4 seropositivity of the studied population according to geographical region, sex and age is presented in Table 1.A slightly higher anti-GII.4norovirus IgG seropositivity was found in people from Southern Portugal, with 79% seropositivity, compared to 71% from both Northern and Central regions of Portugal (Table 1); however, this difference was not statistically significant.No differences were observed between the seropositivity found in women and that found in men, showing 72% and 73%, respectively.Seropositivity increased with age, reaching 79% in the group aged 40-49 years.

Discussion
GII.4 norovirus antibodies were detected in 70% of the serum samples from a Portuguese cohort.Since no information on the genetic characterization of norovirus infections in Portugal was available, we selected GII.4 New Orleans antigen as this GII.4 variant emerged in 2009 and continued to cause outbreaks in 2012 [13].Compared to data from other countries, such as France which reported a prevalence of 74.1% [14]; Italy, 91.2% [15]; and the UK, 99.5% [8], the 70% we found in the Portuguese samples was relatively low.This variance could reflect real differences in exposure levels of norovirus in different European countries, or may be the result of the fact that the VLPs used in our study were unable to detect cross-reactive antibodies against non-GII.4norovirus strains including GI [16].A higher seroprevalence could have been found if a Portuguese variant was used as antigen.
In conclusion, GII.4 norovirus infections frequently occur in Portugal, but they are underreported due to either the lack of knowledge among clinicians or the limited availability of norovirus diagnostic testing in routine clinical laboratories.To the best of our knowledge this is the first seroprevalence study of norovirus in the Portuguese population.Given the importance of norovirus as foodborne pathogen, diagnostic testing of stool samples for norovirus should be considered in addition to testing for routine bacterial enteric pathogens.

Table 1 .
Distribution of IgG anti-norovirus GII.4 seropositivity of the studied population (n = 473) according to regions of Portugal, sex and age.