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Epidemiological and Genetic Characterization of Norovirus Outbreaks That Occurred in Catalonia, Spain, 2017–2019

Molecular characterization of human norovirus (HuNoV) genotypes enhances the understanding of viral features and illustrates distinctive evolutionary patterns. The aim of our study was to describe the prevalence of the genetic diversity and the epidemiology of the genotypes involved in HuNoV outbreaks in Catalonia (Spain) between 2017 and 2019. A total of 100 HuNoV outbreaks were notified with the predominance of GII (70%), followed by GI (27%) and mixed GI/GII (3%). Seasonality was observed for GII outbreaks only. The most prevalent genotypes identified were GII.4[P31] Sydney 2012, GII.4[P16] Sydney 2012 and GII.2[P16]. As compared to person-to-person (P/P) transmitted outbreaks, foodborne outbreaks showed significantly higher attack rates and lower duration. The average attack rate was higher in youth hostel/campgrounds compared to nursing homes. Only genotypes GI.4[P4], GII.2[P16], GII.4[P16], GII.4[P31] and GII.17[P17] were consistently detected every year, and only abundance of GII.2[P16] showed a negative trend over time. GII.4 Sydney 2012 outbreaks were significantly associated to nursing homes, while GII.2[P16] and GI.3[P3] were most frequently identified in youth hostel/campgrounds. The average attack rate was significantly higher when comparing GII.2[P16] vs. GI.4[P4], GII.2[P16] vs. GII.4[P31] Sydney 2012, and GII.6[P7] vs. GII.4[P31] Sydney 2012. No correlations were found between genotype and outbreak duration or age of affected individuals.


Introduction
Enteric viruses are the most common cause of acute gastroenteritis. Specifically, human noroviruses (HuNoV) are recognized as the most prevalent agent, causing approximately 17-18% of the total diarrheal diseases and 200,000 annual deaths [1,2]. They also represent

Sample Collection
Human stool samples were collected from affected individuals during HuNoV outbreaks reported in Catalonia (Spain) through January 2017 to December 2019 in closed and semi-closed settings such as nursing homes, youth/campgrounds, long-term care facilities, schools and other institutions. All epidemiological data were collected by the different Epidemiologic Surveillance Units belonging to the Public Health Agency of Catalonia (ASPCAT). This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the University of Barcelona (IRB00003099).

HuNoV RTqPCR and Genotyping Assays
The presence of HuNoV in stool was assessed by real-time RTqPCR at the Microbiology Laboratoy at Hospital Universitari Vall d'Hebron and the Agència de Salut Pública de Barcelona (ASPB) [28]. Viral RNA was extracted from a 10% stool suspension using the NucliSENS ® easyMAG ® system (BioMérieux, Marcy-L'Etoile, France), and the presence of HuNoV was assessed by RTqPCR according to ISO 15216-2:2019 [29].
A semi-nested RT-PCR at the Enteric Virus Laboratory (University of Barcelona) targeting ORF1 and ORF2 genes, including RdRp and VP1, was used for genotyping [8], considering 2-4 positive specimens randomly selected from each outbreak. RT-PCR products were purified and sequenced on an ABI Prism 3700 automatic sequencer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Genotypes were assigned for RdRp and VP1 using the Norovirus Typing Tool (version 2.0) [30]. Phylogenetic analysis was performed using the neighbour-joining method (distance calculation by the Kimura-2parameter correction; pairwise deletion) implemented in the MEGA7 program [31], and results were validated by 1000 bootstrap replicates.

Statistical Analysis
Chi-square test was used to compare categorical variables by OpenEpi website. Comparisons between means were performed using ANOVA analysis by the Good Calculators website. In addition, p-values < 0.05 were considered statistically significant.

Epidemiological Features of HuNoV Outbreaks and Cases
During the study period, a total of 100 HuNoV outbreaks were reported to the Public Health authorities. The total number of outbreaks increased every year, resulting in 27 outbreaks in 2017, 34 in 2018 and 39 in 2019. GII was the predominant genogroup, being involved in 70 outbreaks, followed by GI in 27 and mixed genogroups GI and GII in 3. Monthly distribution of total outbreaks also exhibited a pronounced seasonality with a higher occurrence during cold months (October-March) rather than warm months (April-September) ( Figure 1). Information about the outbreak size was reported for 83 of them. Eleven outbreaks affected >50 cases (six in 2017, one in 2018 and three in 2019) and one >250 cases (2017). The main epidemiological features of the studied outbreaks are summarized in Tables 1 and 2. Average attack rates were significantly higher in foodborne outbreaks as compared to person-to-person (P/P) transmitted outbreaks (45.76% vs. 29.71%, p = 0.0106), but duration of outbreak was significantly lower (3.20 vs. 8.93 days, p = 0.001). Regarding outbreaks occurring at different settings, differences in mode of transmission were observed between hotels and nursing homes (p < 0.003), hotels and schools (p = 0.049), and between youth hostel/campgrounds compared with nursing homes (p < 0.001) and schools (p = 0.0037). Similarly, nursing home outbreaks occurred with a significant higher frequency during cold months, as compared to outbreaks occurring in youth hostel/campgrounds (p < 0.001). The average attack rate was significantly higher in youth hostel/campgrounds, as compared to nursing homes (49.22% vs. 28.62%, p = 0.013). In addition, a kindergarten/preschool setting showed a significantly longer duration when comparing it with youth hostel/campgrounds (p = 0.0059) and hotels (p = 0.0077). No significant association was observed between genogroup and mode of transmission. Modes of transmission for GI outbreaks were 77.8% P/P and 22.2% foodborne; for GII outbreaks were 72.9% P/P, 25.7% foodborne and 1.4% waterborne; and for mixed GI/GII outbreaks were 66.7% P/P and 33.3% foodborne.
Symptom information was collected for 533 HuNoV cases (Table 3). Diarrhea presented at a significantly higher frequency in patients older than 65, as compared to patients younger than 15 and to 16-65 age group (p < 0.001), and in patients from 16-65 age group compared to patients younger than 15 (p < 0.001). Vomiting and fever were significantly less frequent in patients older than 65 years as compared to patients younger than 15 or 16-65 age group (p < 0.001). Fever was very rare in patients older than 65.
comparing it with youth hostel/campgrounds (p = 0.0059) and hotels (p = 0.0077). No significant association was observed between genogroup and mode of transmission. Modes of transmission for GI outbreaks were 77.8% P/P and 22.2% foodborne; for GII outbreaks were 72.9% P/P, 25.7% foodborne and 1.4% waterborne; and for mixed GI/GII outbreaks were 66.7% P/P and 33.3% foodborne.       (Table S1). The number and evolution of sequence identification per trimester over the study period are illustrated in Figure 2.
No strong correlations were observed when analysing epidemiological features according to genotype (  21.24%, p = 0.047). No other genotype correlations were found with either the duration of the outbreaks or with the mean age of affected individuals. Although not statistically significant, GI outbreaks were less influenced by season as compared to GII outbreaks (p = 0.058).

Phylogenetic Analysis
Phylogenetic analysis confirmed genotype assignment performed using a Norovirus Typing Tool (version 2.0) (Figure 3). Information of 77 of the 93 genotyped outbreaks is shown in the phylogenetic trees.

Discussion
A total of 100 HuNoV outbreaks were reported during the three-year study, affecting 2677 individuals. In addition, 70% of outbreaks were caused by GII as the leading genogroup, and GII.4 was present in~40% of them. When compared to a similar study performed in Catalonia during 2010-2012, which identified GII.4 in a higher proportion of outbreaks (66/103) [28], we observed an overall lower proportion of GII.  (Table S1). These differences could be due to an emerging trend of genotypes other than GII.4, to a lower persistence in the environment or to an absence of immunity against particular genotypes [32].
Despite this lower abundance, GII.4 genotypes were still predominant during the study period. GII.4[P31] Sydney 2012 was the most predominant genotype in Thailand [33] and China [34] during the same period, and along with GII.4[P16] Sydney 2012 were the most identified genotypes in the study, as happened in Germany in 2018 [16]. After GII.4, GII.2[P16] was identified in more than 10% of the outbreaks. This genotype was also the most frequently isolated genotype from sewage in Valencia, Spain in 2016-2017 [35].
We identified GII. 17[P17] in all three years, mostly in 2018, without observing an increasing trend. Globally, it has been reported that GII.17[P17] reached a peak in 2014/2015, but its incidence began to decline after that [8,39]. A remarkable proportion of GI. 3[P3] outbreaks was observed in 2019, being not previously identified in 2017 or 2018, although it did not represent a significant increase in the number of affected individuals that year, as in other genotypes.
Almost one half of the outbreaks occurred in nursing homes, as elderly residents are a highly vulnerable group to the infection who could also experience more severe symptoms. A longer illness duration and extended episodes of excretion have been associated with aged patients, increasing the probability of transmission [40][41][42]. Nevertheless, in our report, a longer duration of outbreaks was significantly associated with kindergarten/preschool setting. Young children are more likely to infect other people possibly because they have wider spheres of activity, lower levels of hygiene and a higher susceptibility to agents due to insufficient acquired immunity [43,44].
Among transmission modes, P/P was identified in 74% of outbreaks as the most common route to acquire HuNoV infection [8,45] followed by foodborne in 24% of them, similar to what has been observed elsewhere [20,46,47]. However, a significantly higher attack rate was observed in foodborne outbreaks, since this mode of transmission is capable of occurring in larger outbreaks affecting a major number of individuals rather than P/P transmission, probably by the ingestion of higher infections doses and the easier identification of individuals exposed to the contaminated source [48,49]. Thus, youth hostel/campgrounds had a significant higher average attack rate over nursing homes because of its mostly identified foodborne transmission origin. Apart from particular food contamination, poor hygienic practices during food preparation can be related to food contamination and the source of foodborne transmission, which could result in a faster virus expansion to consumers [50].
Considered jointly, GII.6[P7] and GII.2[P16] had a higher average attack rate as compared to GII.4[P31] Sydney 2012, probably due to lack of immunity against these less frequent genotypes. In addition, these genotypes were mostly identified in foodborne outbreaks occurring in youth hostel/campgrounds, while GII.4[P31] Sydney 2012 outbreaks occurred predominantly in nursing homes with P/P transmission. An association between GII, particularly GII.4, to P/P transmission and to nursing homes/older patients during cold months has also been reported by previous studies [9,28,51,52]. In our study,~75% of all three GII.4 Sydney 2012 outbreaks occurred in nursing homes, >85% of the outbreaks occurring during the cold season peak, and~90% of them were interpersonal outbreaks. Alternatively, in other settings, GI and non-GII.4 are more frequent [53]. In the study, we did observe a significant association of GII.2[P16] and GI.3[P3] with outbreaks occurring in youth hostels or campgrounds and affecting children and young adults. Finally, 68% of the HuNoV outbreaks were reported during cold months (October-March), as people are more frequently clustered indoors, which enhances P/P transmission, and a marked seasonality was especially observed for GII outbreaks only. A lower seasonality observed for GI has also been reported by Matthews et al. [49].
A study of amino acid sequences of partial ORF1 and ORF2 among all typed sequences has been performed to describe novel substitutions. Four amino acid changes have been described in GI.P1, GI.P11, GII.P16 and GII.P31 RdRp sequences compared to those uploaded to GenBank. Point mutations in non-structural proteins could lead to novel properties with better fitness potential in different norovirus genotypes [54].

Conclusions
Overall, this study shows a great diversity of HuNoV detected as cause of gastroenteritis outbreaks during the three-year period, with 21 different genotypes circulating in the community. Only genotypes GI. 4 3[P3] occurred more frequently in youth hostels or campgrounds. Seasonality was strongly observed for GII outbreaks only. Surveillance of HuNoV strains circulating in the community is important for a better understanding of factors driving virus evolution and to provide information for vaccine development.