Epidemiological Characteristics and Genotypic Features of Rotavirus and Norovirus in Jining City, 2021–2022

Diarrhea, often caused by viruses like rotavirus (RV) and norovirus (NV), is a global health concern. This study focuses on RV and NV in Jining City from 2021 to 2022. Between 2021 and 2022, a total of 1052 diarrhea samples were collected. Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR was used to detect RV-A, NV GI, and NV GII. For RV-A-positive samples, VP7 and VP4 genes were sequenced for genotype analysis, followed by the construction of evolutionary trees. Likewise, for NV-GII-positive samples, VP1 and RdRp genes were sequenced for genotypic analysis, and evolutionary trees were subsequently constructed. Between 2021 and 2022, Jining City showed varying detection ratios: RV-A alone (excluding co-infection of RV-A and NV GII) at 7.03%, NV GI at 0.10%, NV GII alone (excluding co-infection of RV-A and NV GII) at 5.42%, and co-infection of RV-A and NV GII at 1.14%. The highest RV-A ratios were shown in children ≤1 year and 2–5 years. Jining, Jinxiang County, and Liangshan County had notably high RV-A ratios at 24.37% (excluding co-infection of RV-A and NV GII) and 18.33% (excluding co-infection of RV-A and NV GII), respectively. Jining, Qufu, and Weishan had no RV-A positives. Weishan showed the highest NV GII ratios at 35.48% (excluding co-infection of RV-A and NV GII). Genotype analysis showed that, in 2021, G9P[8] and G2P[4] were dominant at 94.44% and 5.56%, respectively. In 2022, G8P[8], G9P[8], and G1P[8] were prominent at 75.86%, 13.79%, and 10.35%, respectively. In 2021, GII.3[P12], GII.4[P16], and GII.4[P31] constituted 71.42%, 14.29%, and 14.29%, respectively. In 2022, GII.3[P12] and GII.4[P16] accounted for 55.00% and 45.00%, respectively. RV-A and NV showed varying patterns for different time frames, age groups, and regions within Jining. Genotypic shifts were also observed in prevalent RV-A and NV GII strains in Jining City from 2021 to 2022. Ongoing monitoring of RV-A and NV is recommended for effective prevention and control.


Introduction
Diarrheal disease is one of the most common illnesses in humans, affecting approximately 6 billion people globally each year and resulting in 1.3 million deaths [1,2].It constitutes a significant global public health challenge [3,4].The vast majority of cases of diarrhea are caused by viruses, with rotavirus (RV) and norovirus (NV) being the primary pathogens responsible for human viral gastroenteritis [5][6][7].
RV is one of the leading causative agents of diarrhea, particularly in children under the age of 5, especially in developing countries.It is a major cause of diarrhea-related deaths, accounting for approximately 450,000 deaths annually [8][9][10].RV-A is the predominant strain of rotavirus infecting humans.Based on the VP7 and VP4 gene sequences of RV-A, Viruses 2024, 16, 925 2 of 15 it is classified into G and P genotypes.The globally prevalent genotypes include G1P [8], G2P [4], G3P [8], G4P [8], G8P [8], G9P [8], and G12P [8], among others.Different genotypes of RV-A may predominate in different regions and time periods [9,10].Rotavirus vaccines are effective in preventing rotavirus infections.In Jining, there are two types of rotavirus vaccines in use: the pentavalent rotavirus vaccine (RotaTeq) (G1, G2, G3, G4, P [8]) produced by Merck, Rahway, NJ, USA, which has been used since 2018 [11,12], and the live attenuated rotavirus vaccine (LLR strain, Rotavin-M1) (G10P [15]) produced by the Lanzhou Institute of Biological Products, Lanzhou China, which has been used since 2001 [13,14].Although rotavirus vaccines are not included in the routine immunization program in Jining, and parents need to pay for the vaccination themselves, there is a relatively high willingness among parents to vaccinate their children, with approximately 30% of infants and young children receiving the rotavirus vaccine.
NV causes approximately 680 million cases of diarrhea and leads to around 200,000 deaths annually [15].Based on the VP1 and RdRp gene sequences of NV, it is classified into G and P genotypes.Among them, GI and GII are the primary genotypes infecting humans [16][17][18].
Global risks of viral gastroenteritis infection and outbreak persist.In China, incidents of viral gastroenteritis remain at a significant level.The characteristics of viral gastroenteritis vary across regions and time periods.Jining City, with a population of approximately 8.3 million, holds a unique position at the confluence of the Yellow River and the Grand Canal, boasting the largest freshwater lake in northern China, Weishan Lake.In this study, specimens collected from January 2021 to December 2022 in Jining City were tested and genotyped for RV-A, NV GI, and NV GII.The aim is to explore the epidemiological and genotypic features of RV and NV during this period, providing a basis for formulating prevention and control policies.

Sample Collection
From January 2021 to December 2022, 3-6 outpatient medical records of diarrhea cases were collected from each of the ten counties in Jining City, China, on a monthly basis.These cases met the criteria of having diarrhea (defined as three or more bowel movements per day with a change in stool consistency, fecal leukocyte count < 15, and no observed red blood cells) or exhibited symptoms of diarrhea less than three times per day, but with altered stool consistency and vomiting as a primary symptom.Fecal samples, weighing over 5 g, were then sent to the Microbiology Laboratory of Jining City Center for Disease Control and Prevention for testing.The samples were stored in a −80 • C freezer, and nucleic acid testing was completed within 30 days.

Nucleic Acid Testing
For each sample, 0.1 g of fecal material was mixed with 0.9 mL of PBS.This mixture underwent three rounds of vigorous shaking, followed by centrifugation at 8000× g for 5 min.Subsequently, 200 µL of the resulting supernatant was collected.Nucleic acid extraction was carried out using the GeneRotex 96 automatic nucleic acid extractor (GeneRotex, Xi'an Tianlong Technology Co., Ltd., Xi'an, China) in combination with the viral nucleic acid extraction reagent (T138) from the same company.Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR testing for RV-A, NV GI, and NV GII was performed using the triple nucleic acid detection kit (Catalog No. A2593) provided by Beijing Zhuocheng Hui Sheng Biotechnology Co., Ltd., Beijing, China.The Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR reaction conditions were as follows: 50 • C for 10 min, 95 • C for 30 s, followed by 45 cycles of 95 • C for 5 s and 60 • C for 30 s.The Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR (RT-qPCR) kit from Beijing Zhuocheng Hui Sheng Biotechnology Co., Ltd.(Beijing, China) is used not only for detecting rotavirus and norovirus but also includes human ribonuclease P (RNase P), a negative control, and a positive control for quality control of nucleic acid detection.The detection results for rotavirus and norovirus are considered valid if the positive control and RNase P tests are positive, and the negative control test is negative.Additionally, the kit demonstrates a coefficient of variation of less than 5% for precision reference samples and shows no cross-reactivity with other pathogens.

Statistical Analysis
Statistical analysis was conducted using SPSS 24.0 software.Inter-group comparisons were performed using the χ 2 test, with p < 0.05 indicating statistically significant differences.

RV-A Genotype Analysis
To determine the genotypes of RV-A in Jining City from 2021 to 2022, sequencing of the VP7 and VP4 genes was conducted on RV-A-positive samples, resulting in a total of 47 sequences.According to the VP7 gene typing, also known as G genotypes, in the 47 sequences collected from Jining City between 2021 and 2022, the G1 genotype accounted for 6.38% (3/47), G2 genotype accounted for 2.13% (1/47), G8 genotype accounted for
In the VP7 gene evolutionary tree, G1, G2, G8, and G9 genotypes were located on four different evolutionary branches.In the G1 evolutionary branch, the three G1P [8] genotypes from Jining City in 2021-2022 independently formed one evolutionary cluster.One G2P [4] genotype from Jining City clustered with China's Fuzhou20-40.In the G8 evolutionary branch, the G8P [8] genotypes from Jining City were distributed in two evolutionary clusters.Among them, JN25 formed an independent evolutionary cluster, while the other 21 G8P [8] genotypes were in the same cluster as China's Fuzhou20-140, NN2785-18, and others.In the G9 evolutionary branch, JN09, JN14, and JN17 were in separate evolutionary clusters, while the other 18 G9 genotypes from Jining City were in another cluster (Figure 3).
Viruses 2024, 16, x 7 of 18 evolutionary clusters, while the other 18 G9 genotypes from Jining City were in another cluster (Figure 3).RV-A P genotype analysis was performed using the rotavirus A Genotype Determination software, with reference sequences obtained from NCBI.

Genotype Analysis of NV GII
To determine the genotypes of NV GII in Jining City from 2021 to 2022, we sequenced the VP1 and RdRp regions of NV-GII-positive samples, successfully obtaining 27 viral sequences.

Genotype Analysis of NV GII
To determine the genotypes of NV GII in Jining City from 2021 to 2022, we sequenced the VP1 and RdRp regions of NV-GII-positive samples, successfully obtaining 27 viral sequences.

Evolutionary Tree Analysis of NV GII Genotypes
In Jining City, the main genotypes of NV GII in 2021-2022 were GII.In the evolutionary tree based on the VP1 gene, the GII.3 and GII.4 genotypes were located on two different evolutionary branches.On the GII.3 branch, JN12 and JN17 of the GII.3[P12] genotype from Jining City formed one evolutionary cluster, while the other 14 strains of GII.3[P12] from Jining City clustered separately.On the GII.4 branch, strains from Jining City were distributed among three different evolutionary clusters.Strain JN02 of the GII.4[P31] genotype from Jining City clustered with similar strains from Tokyo, Japan.Among the 10 strains of the GII.4[P16] genotype from Jining City, JN20 was grouped in one cluster, while the other nine strains formed a separate cluster (Figure 6).In the RdRp-based evolutionary tree, GII.P12, GII.P16, and GII.P31 genotypes were located on distinct evolutionary branches.On the GII.P12 branch, strain JN12 of the GII.3[P12] genotype from Jining City formed an independent evolutionary cluster, while the other 15 strains of GII.3[P12] from Jining City clustered separately.On the GII.P16 branch, JN20 was grouped in one cluster, while the other nine strains of the GII.4[P16] genotype from Jining City formed a separate cluster.On the GII.P31 branch, strain JN02 from Jining City and similar strains from Tokyo, Japan, clustered together (Figure 7).This study suggests that NV GII of the same genotype may have different origins and evolutionary pathways.

Discussion
The harm caused by viral infection to the human body is significant [23].This study presents a detailed two-year systematic investigation of the infection ratios and genotypic characteristics of RV-A and NV in diarrheal cases in Jining City, Northern China.The findings reveal that RV-A and NV collectively accounted for 13.69% of diarrheal cases, which is lower than the positivity ratios reported in the Southwest region of China from 2018 to 2020 [24].Specifically, the RV-A positivity rate in Jining City was 7.03%, a figure similar to the 7.22% reported in Beijing, Northern China, from 2018 to 2022 [25].This rate is, however, lower than the 15.63% positivity rate observed in Wuhan, Central China, from 2019 to 2022 [26].This contrasts with broader studies which indicate more consistent detection ratios across different age groups, illustrating the variability in RV-A epidemiology across different settings.However, it should be noted that the RV-A detection kit used in this study cannot distinguish between vaccine strains and wild strains, which may affect the detection results.In Jining City, the positivity rate for NV is 5.51%, predominantly attributed to NV GII, while the positivity rate for NV GI is only 0.10%.This distribution aligns with the prevalent results of NV genotypes in China.The positivity rate for NV GII in Jining City is lower compared to the Southwest region of China from 2018 to 2020 but is in line with findings from Wuhan, China [24,25,27].
In China, RV-A and NV show distinct seasonal patterns, with RV-A more prevalent from December to May, and NV exhibiting a higher incidence from October to May [24].This study in Jining City found that RV-A had the highest detection ratios from January to April, while NV peaked from December to May.
These trends were consistent with established patterns in Northern China.There were significant differences in detection ratios among counties in Jining City.For example, Jinxiang County had a high RV-A detection rate of 24.37%, whereas Weishan and Qufu counties showed no RV-A detection.Weishan County stood out with a remarkably high NV GII detection rate of 35.48%, while other counties had ratios below 6.14%.This emphasizes how distinct county environments impact RV-A and NV GII prevalence.Notably, Weishan County hosts one of Northern China's largest freshwater lakes, Weishan Lake, which is a major source of freshwater products in China.It produces over ten types of shellfish and various freshwater fish species.There may be norovirus contamination in these freshwater products, and improper consumption of these products is one of the significant causes of norovirus infections, likely influencing the distribution of RV-A and NV [28,29].Additionally, a significant number of diarrheal cases involved mixed RV-A and NV GII infections [24,30].This study identified a substantial number of such cases, and their distribution mirrors patterns observed for norovirus and rotavirus in terms of age, month, and county.
RV-A can be classified into different G and P genotypes based on its VP7 and VP4 gene sequences.In Asia, the predominant genotypes of RV-A have shown variation over the years [10,25].From 2009 to 2013, G1P [8] and G2P [4] were prevalent.This shifted to G8P [8] in 2014-2015 [10].Between 2016 and 2020, the main genotypes became G9P [8] and G3P [8], and post-2021, they further transitioned to G9P [8] and G8P [8] [10,25,31-33].This study successfully identified the G and P genotypes of 47 RV-A viruses, and the results were in agreement with the phylogenetic tree analysis.In 2021, G9P [8] was predominant, cocirculating with G2P [4].In 2022, G8P [8] emerged as the predominant genotype, alongside G9P [8] and G1P [8].This indicates that although G9P [8] and G8P [8] have been predominant in Asia in recent years, other genotypes still coexist, in line with findings in Beijing and Wuhan, China [25,26].The G8P [8] genotype of RV-A began to emerge in China in 2020-2021, and by 2022, it became the prevailing genotype, accounting for a high proportion of 75.86%.This mirrors results in Beijing and Wuhan, suggesting that G8P [8] is currently the dominant genotype in China.In the phylogenetic trees based on VP7 and VP4 genes, the G8P [8] genotype RV-A in Jining City is closely related to strains from Fuzhou, a city in southern China, in 2020.This suggests that the circulating G8P [8] genotype RV-A in China may have a common or closely related origin.Moreover, RV-A vaccines have proven effective in preventing RV-A infections [34,35].In Jining, China, two types of rotavirus vaccines are used: the RotaTeq rotavirus vaccine, which contains the G1, G2, and P [8] genotypes, and the LLR strain vaccine [11,12].The use of rotavirus vaccines in infants and young children in Jining may provide immunological protection and reduce infections, particularly from genotypes G1, G2, G3, G4, and P [8].Additionally, considering the complex diversity of rotavirus genotypes, both domestic and imported rotavirus vaccines should consider multivalent vaccines and include genotypes G8, G9, and P [8] to promote better protection against G8P [8] and G9P [8] genotype rotaviruses.
Based on the VP1 and RdRp gene sequences of NV, it can be classified into different G and P genotypes.Since 2015, the predominant genotypes in China have been GII.It should be noted that in September 2022, Jining City experienced an outbreak of the novel coronavirus.Due to strict local COVID-19 control measures, the collection of diarrheal specimens in some counties was affected, leading to a lower sample volume during this period.As a result, the final analysis results may have potential biases.
In summary, RV-A and NV GII are the primary pathogens causing diarrheal cases in Jining City, with a higher incidence in the winter and spring seasons.The prevalence of RV-A and NV GII varies among different counties.From 2021 to 2022, the predominant genotype of RV-A shifted from G9P [8] to G8P [8], while the dominant genotypes of NV GII remained unchanged over these two years.It is crucial to strengthen long-term monitoring and genotype analysis of RV-A and NV in various regions of China, providing valuable data for the development of new vaccine strains.Additionally, tailored vaccination strategies should be devised for different counties and age groups to more effectively mitigate the risk of RV-A and NV infections.

Figure 1 .
Figure 1.The epidemiological characteristics of norovirus and rotavirus.(A) Positive rates of norovirus and rotavirus in 2021 and 2022.(B) Infection-positive rates of norovirus and rotavirus in different age groups.(C) Infection-positive rates of norovirus and rotavirus in different genders.(D) Infection-positive rates of norovirus and rotavirus in different months.(E) Infection-positive rates of norovirus and rotavirus in different counties."■" RV-A, "■" NVGI, "■" NVGII, "■" RV-A + NV GII.

Figure 1 .
Figure 1.The epidemiological characteristics of norovirus and rotavirus.(A) Positive rates of norovirus and rotavirus in 2021 and 2022.(B) Infection-positive rates of norovirus and rotavirus in different age groups.(C) Infection-positive rates of norovirus and rotavirus in different genders.(D) Infection-positive rates of norovirus and rotavirus in different months.(E) Infection-positive rates of norovirus and rotavirus in different counties."■" RV-A, "■" NVGI, "■" NVGII, "■" RV-A + NV GII.

Figure 3 .
Figure 3. Phylogenetic trees based on VP7 nucleotide sequences of RV-A.An evolutionary tree was constructed using 47 rotavirus VP7 gene sequences from Jining City and 19 rotavirus VP7 gene sequences downloaded from NCBI from recent years, using the maximum-likelihood method with

Figure 3 .
Figure 3. Phylogenetic trees based on VP7 nucleotide sequences of RV-A.An evolutionary tree was constructed using 47 rotavirus VP7 gene sequences from Jining City and 19 rotavirus VP7 gene sequences downloaded from NCBI from recent years, using the maximum-likelihood method with 1000 bootstrap replicates in the MEGA 7.0.14software.The RV-A G genotype analysis was conducted using the rotavirus A Genotype Determination software, with reference sequences sourced from NCBI."■" represents RV-A in 2021, "■" represents RV-A in 2022.

Figure 4 .
Figure 4. Phylogenetic trees based on VP4 nucleotide sequences of RV-A.An evolutionary tree was constructed using 47 rotavirus VP4 gene sequences from Jining City and 19 rotavirus VP4 gene sequences downloaded from NCBI from recent years using the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0.14software."■" represents RV-A in 2021, and "■" represents RV-A in 2022.RV-A P genotype analysis was performed using the rotavirus A Genotype Determination software, with reference sequences obtained from NCBI.

Figure 4 .
Figure 4. Phylogenetic trees based on VP4 nucleotide sequences of RV-A.An evolutionary tree was constructed using 47 rotavirus VP4 gene sequences from Jining City and 19 rotavirus VP4 gene sequences downloaded from NCBI from recent years using the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0.14software."■" represents RV-A in 2021, and "■" represents RV-A in 2022.RV-A P genotype analysis was performed using the rotavirus A Genotype Determination software, with reference sequences obtained from NCBI.

Viruses 2024, 16 , x 12 of 18 Figure 6 .
Figure 6.Phylogenetic trees based on VP1 nucleotide sequences of NV GII.An evolutionary tree was constructed using 27 NV GII VP1 gene sequences from Jining City and 13 NV GII VP1 sequences

Figure 6 .
Figure 6.Phylogenetic trees based on VP1 nucleotide sequences of NV GII.An evolutionary tree was constructed using 27 NV GII VP1 gene sequences from Jining City and 13 NV GII VP1 sequences downloaded from NCBI from recent years using the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0.14software.NV GII G genotype analysis was conducted using the norovirus Typing Tool Version 2.0 software, with reference sequences sourced from NCBI."■" represents NV GII in 2021, and "■" represents NV GII in 2022.

Figure 7 .
Figure 7. Phylogenetic trees based on RdRp nucleotide sequences of NV GII.An evolutionary tree was constructed using 27 NV GII RdRp gene sequences from Jining City and 13 NV GII RdRp sequences downloaded from NCBI from recent years using the maximum-likelihood method with 1000 bootstrap replicates in the MEGA 7.0.14software.NV GII P genotype analysis was performed

Figure 7 .
Figure 7. Phylogenetic trees based on RdRp nucleotide sequences of NV GII.An evolutionary tree was constructed using 27 NV GII RdRp gene sequences from Jining City and 13 NV GII RdRp sequences downloaded from NCBI from recent years using the maximum-likelihood method with 1000 bootstrap replicates in the MEGA 7.0.14software.NV GII P genotype analysis was performed using the norovirus Typing Tool Version 2.0 software, with reference sequences obtained from NCBI."■" represents NV GII in 2021, and "■" represents NV GII in 2022.

Table 1 .
Distribution characteristics of rotavirus and norovirus in outpatient diarrhea-positive cases in Jining City, 2021-2022.