Circulation of Human Norovirus in Ekaterinburg, 2020-2023: A Pilot Hospital-Based Surveillance Study

1. Introduction

Human norovirus (HuNoV) is a prevalent cause of acute gastroenteritis (AGE), associated with approximately 18% of cases [1] with significant mortality [2] and economic burden [3] worldwide. In addition, based on notification data, norovirus infection (NovI) is a common cause of acute gastroenteritis in Sverdlovsk Oblast with annual incidence rate up to 147 cases per 100,000 inhabitants with yearly seasonal peak from November to April [4].

The HuNoV genome consists of a positive-sense single-stranded RNA and organized in three open reading frame (from ORF1 to ORF3), with specific importance of ORF1 coding the RNA-dependent RNA polymerase (RdRp) and ORF2 coding the major capsid protein (VP1). Based on VP1 structure, HuNoV are classified into seven genogroups, with at least three (I, II, IV) are known for causing disease in human. HuNoV GII genogroup is significantly more prevalent. As it has been proposed previously, HuNoV are classified using a dual typing system by sequencing RdRp and VP1 [5]. Among all HuNoV types, the GII.4 strain is overrepresented and has been associated with the most of the outbreaks and sporadic cases globally over the least two decades. Nevertheless, high prevalence of GII.17, another common genotype, was also observed [6].

In contrast to rotavirus, another common etiological agent of AGE, norovirus vaccine is unavailable due to a high genetic diversity of the virus, a poor knowledge of immunity characteristics and the lack of high-performance cultivation technique [7]. Therefore, ongoing surveillance for the circulation of is HuNoV strongly recommended to estimate diseases’ burden and track its genetic diversity [8].

The aim of this study is to estimate the prevalence of HuNoV in patients with AGE in Ekaterinburg, Russian Federation, overall and by specific genotypes, to further consider a sentinel surveillance program in the studied area.

2. Materials and Methods

2.1. Sample Collection

The study was approved by the Ethical Board of the Ural State Medical University (protocol N26, date of approval 10 May 2020). All clinical samples were collected after obtaining written informed consent from the children’s legal guardian. Additional personal information, allowing participant identification, were not provided.

From November 2020 to April 2023 (three consecutive epidemic seasons), we collected fecal samples from all pediatric patients with AGE, admitted to the Infectious disease unit of the main pediatric hospital in Ekaterinburg, Sverdlovsk Oblast, Russian Federation – Municipal Clinical Hospital No 15. The city studied is the Capital of the corresponding region with total population approximately 1.5 millions including 122,000 children under the age of six – the main affected cohort for NovI. The samples were collected during routine clinical testing for patients with gastroenteritis. 10% fecal suspensions were prepared in PBS and stored at –20℃.

2.2. Nucleic Acid Extraction and Sample Screening for HuNoV

Nucleic acid from fecal suspensions were extracted by an alcohol precipitation technique using “RIBO-prep” nucleic acid extraction kit followed by reverse transcription using “REVERTA-L” kit (both purchased from FBIS CRIE of Rospotrebnadzor, Moscow, Russia). Later, samples were screened for HuNoV using “Amplisens Norovirus GI/GII” qPCR assay (FBIS CRIE of Rospotrebnadzor, Moscow, Russia) according to manufacturer’s instructions. Amplification and detection were performed on a StepOne Plus Real Time PCR System (ThermoFisher Scientific, Foster City, CA, USA).

2.3. Sanger Sequencing and HuNoV Typing

Positive samples with detected HuNoV strains were genotyped as it has been described previously using the Sanger sequencing technique [9]. Briefly, we amplified a part of HuNoV genome corresponding to ORF 1/2 junction, that make it possible to type both RdRp and VP1 genes. We used MON432 or MON431 primers (for GI and GII genogroups, respectively) targeting RdRp [10] and GISKR or GIISKR for VP1 [11] (this set has been described previously by Chhabra et al. [9]). As it will be described below, there were no samples positive for HuNoV GI genogroup in qPCR, therefore, sequencing reactions for this target were not performed. In addition, to study genetic diversity of most significant strains we used sequence analysis of highly variable P2 domain as it has been described previously [12]. All these reactions were performed using Evrogen 5x ScreenMix Ready Reaction Mix (Evrogen JSC, Moscow, Russia) following manufacturer’s protocol on a Veriti Thermal Cycler (Life Technologies, Carlsbad, CA, USA).

PCR products were separated on a 1% agarose gel by electrophoresis. Bands with length of approximately 570bp (for the ORF1/2 junction) and 900bp (for the P2 domain) were extracted and purified using CleanUp Standard Kit (Evrogen JSC, Moscow, Russia). Further, target genome fragment amplification was followed by chain termination through the incorporation of dideoxynucleotides utilizing the BrilliantDye Terminator v3.1 (NimaGen, Nijmegen, the Netherlands) Cycle Sequencing Kit for the sequencing reaction. Sequencing products were purified using the iX-Pure DyeTerminator Cleanup Kit (NimaGen). These later were sequenced on a ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were assigned to specific genotypes using the Norovirus Genotyping Tool (RIVM, The Netherlands). For phylogenetic analysis, we used the BEAST software (version 1.10.4.) and the FigTree software (version 1.4.4) to visualize results.

Table 1. HuNoV strains included to phylogenetic analysis in this study.

Sample ID	GenBank Accession number	Year of sample collection	Age of patient, years
GII.4
474_20	PZ251255	2020	4
916_21	PZ251256	2021	3
917_21	PZ251257	2021	3
926_21	PZ251258	2021	4
934_21	PZ251259	2021	3
1380_21	PZ251252	2021	3
159_22	PZ251253	2022	4
942_22	PZ251260	2022	3
3051_22	PZ251254	2022	3
GII.17
3066_22	PZ251267	2022	3
3450_22	PZ251266	2022	3
3454_22	PZ251265	2022	3
109_23	PZ251268	2023	3

Table 1. HuNoV strains included to phylogenetic analysis in this study.

Sample ID	GenBank Accession number	Year of sample collection	Age of patient, years
GII.4
474_20	PZ251255	2020	4
916_21	PZ251256	2021	3
917_21	PZ251257	2021	3
926_21	PZ251258	2021	4
934_21	PZ251259	2021	3
1380_21	PZ251252	2021	3
159_22	PZ251253	2022	4
942_22	PZ251260	2022	3
3051_22	PZ251254	2022	3
GII.17
3066_22	PZ251267	2022	3
3450_22	PZ251266	2022	3
3454_22	PZ251265	2022	3
109_23	PZ251268	2023	3

3. Results

3.1. Incidence of Norovirus Infection Based on Notification Data

In the Russian Federation, NovI is a notifiable disease since 2009. Over the studied period, the total number of cases in our study area was 5883 (373.67 per 100,000 inhabitants), and crude incidence rate was for 29% more than average for the Sverdlovsk Oblast (289.76). The disease was most common in children under the age of six (76.0%), including infants in first year of life (18.8%). The seasonality was typical for a temperate climate with the highest number of cases were observed from October/November to March/April (excluding 2020-2021, when seasonal peak was vague, Figure 1) [13].

3.2. Norovirus Prevalence and Genotype Distribution

Over the studied period, a total of 495 patients were enrolled, and 68 of them tested positive for HuNoV genome (prevalence 13.7%, 95% CI 10.8 – 17.1). The median age of the included patients was 3 years (IQR 3-4), with a male to female ratio 1.03 to 1. No lethal cases were reported during the study period.

Seasonality of sampling and positive for HuNoV cases was typical for NoVI with an increase during cold months (Figure 2).

A total of 61 HuNoV samples were successfully genotyped (both for VP1 and RdRp), while 7 samples did not amplify by PCR and thus were not available for sequencing. The most prevalent genotype was GII.4 (44%, 27/61), followed by GII.17 (23%, 14/61) and GII.3 (16%, 10/61). Other rare genotypes were also detected: GII.2, GII.6, GII.8, GII.14 (Figure 3). Surprisingly, in 2022 the number of GII.4 and GII.17 cases were equal (n=11). No GI genotypes were detected in the study.

First, among the GII.4 strains, the most prevalent RdRp type was recombinant GII.4[P16] (81%, 22/27), followed by GII.4[P4] (11%, 3/27) and GII.4[P31] (7%, 2/27). Second, all detected GII.17 strains were GII.17[P17]. Finally, most of GII.3 isolates (9/10) were GII.3[P12].

Phylogenetic analysis of GII.4 strains demonstrates its genetic diversity with at least six independent clusters. These clusters correspond to previously detected isolates from Russia as well as the Asia-Pacific Region and Europe (Figure 4). In contrast, GII.17 strains were genetically similar with those circulated in Russia (Figure 5).

4. Discussion

In defiance of intensive studying of NovI epidemiology in European Russia [14,15] and Western Siberia [16,17], this is the first hospital-based surveillance study for HuNoV in Ekaterinburg, Russia Federation, central city of the Sverdlovsk oblast. In this study, we estimated the prevalence of HuNoV in hospitalized AGE cases in study area. Despite limited number of positive cases, our estimation seems lower than previous studies including a meta-analysis by Farahmand et al. [1], but similar to previous results from Novosibirsk (Eastern Siberia) [16,17] and another Russian cities (Moscow, St. Petersburg, Chelyabinsk, Nizhniy Novgorod, Tyumen, Khabarovsk, Makhachkala, and Yakutsk) [14].

Despite high incidence of rotavirus infection, another common diarrheal pathogen, available rotavirus vaccines are not mandatory in the national vaccine schedule in Russia, therefore vaccination is almost absent in the Sverdlovsk region. Therefore, a large proportion of diarrhea cases associated with rotaviruses may be due to the absence of rotavirus vaccination in the studied area [18].

The observed predominance of GII.4 genotype is consistent with main body of literature, including Russia [19], but in contrast to previous studies [20,21,22], the proportion of GII.17 was surprisingly high. As opposed to our previous study, we did not detect wide proportion of HuNoV GI, possibly because of a different study design (voluntary collecting of specimens previously) [19] and the non-inclusion of other communities in the Sverdlovsk region.

The main limitation of the study is the small number of enrolled, therefore we plan to include additional hospitals for further analysis. In addition, the study was conducted only in hospital, not in out-patient facility, that may introduce selection bias in terms of disease severity.

Over the studied period, the incidence of NovI in Ekaterinburg was higher than average in the region, that may be explained by more largescale diagnostics and a large proportion of children due to ongoing urbanization. Therefore, we consider continuing the study as a sentinel surveillance network in Ekaterinburg as the capital of the region to enroll additional patient groups and to perform longitudinal analysis of circulated genotypes.

Beyond regional implications, these data reaffirm that integrated hospital-based surveillance is vital for capturing the true epidemiological scale of norovirus in Russia and guiding future vaccination or prevention strategies.