Genetic Diversity of Bovine Group A Rotavirus Strains Circulating in Korean Calves during 2014 and 2018

Simple Summary Morbidity and mortality rates due to bovine group A rotavirus (BoRVA) infections are high, resulting in direct and indirect economic losses to the beef and dairy industries. RVA strains, which are antigenically heterogeneous, are classified into multiple G and P types; these two types are defined by the two outer capsid proteins VP7 and VP4, respectively. A clear understanding of the various VP7 and VP4 type-specificities is required to ascertain whether it is necessary to construct polyvalent RVA vaccines for calves. In the past, BoRVA G8P[7] was the predominant type in Korean calves; however, we consistently identified G6P[5] as the main cause of diarrhea in young calves from 2014 to the present day. Abstract The purpose of this study was to investigate annual changes in BoRVA strains by examining the VP4 and VP7 genes of rotaviruses in Korean calves. Between 2014 and 2018, 35 out of 138 samples of calf diarrhea feces collected nationwide were positive for BoRVA. Further genetic characterization of the VP7 and VP4 genes of 35 BoRVA isolates identified three different G-genotypes (G6, G8, and G10) and two different P genotypes (P[5] and P[11]). The G6 genotype was most common (94.3%) in BoRVA-positive calves, followed by the P[5] genotype (82.9%). Four genotypes comprised combinations of VP4 and VP7: 80% were G6P[5], 14.2% were G6P[11], 2.9% were G8P[5], and 2.9% were G10P[11]. Susceptibility to infection was highest in calves aged < 10 days (35%) and lowest in calves aged 30–50 days (15.4%). The data presented herein suggest that the G6P[5] genotype is the main causative agent of diarrhea in Korean calves. In addition, it is predicted that G6P[5] will continue to act as a major cause of diarrhea in Korean calves.


Introduction
Rotavirus A (RVA) is the leading cause of acute gastroenteritis in children and young animals worldwide. Bovine group A rotavirus (BoRVA) is one of the main global etiological agents of neonatal diarrhea in calves. Morbidity and mortality rates are high, resulting in direct and indirect economic losses to the beef and dairy industries [1].
Rotavirus (RV), which is a member of the family Reoviridae, comprises a dsRNA genome surrounded by a triple-layered protein capsid. The segmented dsRNA genome encodes six structural proteins (VP1-VP4, VP6, and VP7) and five/six nonstructural proteins (NSP1-NSP5/6) [2,3]. RVs are classified into 10 distinct groups/species (A-J) [3,4]. All have two outer capsid proteins, VP7 and VP4, which are antigenic and induce neutralizing antibodies upon infection; these proteins define the G and P genotypes, respectively [2]. VP4, which is located on the surface of mature virus particles, independently elicits neutralizing antibodies and induces protective immunity [2]. VP7 is the outer capsid protein Animals 2023, 12, 3555 2 of 10 and participates in both a membrane-displacing assembly step and a membrane-disrupting entry step [2].
Here, we investigated the appearance time and diversity of the G6P [5] genotype in calf diarrhea fecal samples collected nationwide between 2014 and 2018. We also investigated the age at which calves were most susceptible to infection with BoRVA. Of the 138 calves, 46 were born to cows vaccinated against BoRVA and 92 were born to non-vaccinated cows. The calf age of the collected samples was divided into four groups: 20 from calves aged 1-10 days, 83 from calves aged 11-20 days, 22 from calves aged 21-30 days, and 13 from calves aged 31-50 days. All cattle from which samples were taken showed signs of diarrhea and dehydration, along with anorexia and inactivity. Respiratory symptoms were also observed in some calves.

RNA Extraction and BoRVA Detection
Samples were diluted 1:3 with phosphate-buffered saline (PBS) and centrifuged at 3000× g for 20 min. The supernatants were filtered through a 0.22 µm-pore-syringe filter (Minisart ® syringe filter, Sartorius, Göttingen, Germany) and stored at −80 • C until further analysis. Total RNA was extracted from each supernatant using an RNeasy mini kit (Qiagen Inc., Germantown, MD, USA) and eluted with 50 µL of elution buffer. The samples were first screened for expression of the VP6 gene using real-time, one-step RT-PCR [17], and the amplification values (Ct values) were determined for each sample. Ct values ≤ 33 indicated a positive result.

Genotyping and Sequencing
Thirty-five positive RNA samples obtained from real-time RT-PCR screening were reverse-transcribed using the HelixCript TM easy cDNA synthesis kit (Nonohelix Co., Ltd., Republic of Korea). The cDNA from BoRVA-positive samples was subjected to G and P genotyping to detect a VP7 gene fragment of 887 bp and a VP4 gene fragment of 664 bp, as described previously [18,19]. PCR assays were carried out using the Ac-cuPower ® ProFi Taq PCR PreMix kit (Bioneer Inc., Daejeon, Republic of Korea), as described previously [18,19]. The amplified PCR products were extracted using the Omega Gel kit (Omega) and subcloned into the pGEM-T vector system II (Promega, Madison, WI, USA). Cloned plasmids containing the BoRVA VP7 and VP4 genes were sequenced with T7 and SP6 sequencing primers and an ABI Prism 3730xl DNA sequencer (Cosmo Genetech Co., Seoul, Republic of Korea). Next, the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 19 October 2022)) program Animals 2023, 12, 3555 3 of 10 was used to compare nucleotide sequences with reference sequences available in the Gen-Bank database.

Phlyogenetic Analysis
The genotypes of the two major genome segments (VP7 and VP4) were determined using nucleotide BLAST and a web-based RotaC genotyping tool: http://viprbrc.org (accessed on 18 October 2022) [20]. Multiple sequence alignment (including reference sequences) was performed using CLUSTAL X (ver. 2.1) (http://www.clustal.org/clustal2/, accessed on 18 October 2022). Phylogenetic trees based on the partial nucleotide sequences of VP7 and VP4 were constructed using the maximum-likelihood (ML) method and Molecular Evolutionary Genetics Analysis X (MEGA X) software (https://www.megasoftware. net/, accessed on 18 October 2022) [21], with nucleotide distance (p-distance) and 1000 replications used for bootstrap analysis. Before each phylogenetic analysis, the model of nucleotide substitution that best fitted the data was identified using the "find best model" function in MEGA X. Phylogenetic trees were constructed based on the partial VP7 and VP6 gene sequences of BoRVA reference strains isolated in Asia, America, and Europe, including the Korean strains detected in 2004-2020. Partial VP7 and VP4 gene sequences of BoRVA reference strains were obtained from the National Center for Biotechnology Information (NCBI) GenBank database.

Phylogenetic Analysis of the G Genotype
Phylogenetic analysis of the partial nucleotide sequences of the VP7 gene (an 887 bp fragment) from the 35 Korean BoRVA strains, as well as selected reference strains from GenBank, was performed using the ML method in MEGA X (Figure 2). The phylogenetic tree for the VP7 genes revealed that the 35 Korean BoRVAs belonged to the G6, G8, and G10 types. In particular, 32 of 33 Korean BoRVAs belonging to the G6 type were closely related to USA strains UK, WC, and NCDV; the Chinese strain LNA5; the French strain V019; and the Brazilian strain 91976-SC.
Phylogenetic analysis of the partial nucleotide sequences of the VP7 gene (an 887 bp fragment) from the 35 Korean BoRVA strains, as well as selected reference strains from GenBank, was performed using the ML method in MEGA X (Figure 2). The phylogenetic tree for the VP7 genes revealed that the 35 Korean BoRVAs belonged to the G6, G8, and G10 types. In particular, 32 of 33 Korean BoRVAs belonging to the G6 type were closely related to USA strains UK, WC, and NCDV; the Chinese strain LNA5; the French strain V019; and the Brazilian strain 91976-SC.  However, and unusually, one strain (16CN07) was separated slightly, forming a more minor group closer to the USA strain MC27, the Japanese strain KN-4, and the Korean strain KNU-GC14, reported in 2020 [10]. One Korean BoRVA belonging to the G8 type was closely related to the Japanese strains GB20-25 and Sun9 and to the Korean strain KJ45, reported in 2006 [9]. One Korean BoRVA belonging to the G10 type was closely related to the USA strain B223, the Brazilian strain 1979-PR, and the Korean strain KNU-YJ8, reported in 2020 [10]. However, although the G5 genotype was detected in 2004,2006,2009, and 2020, it was not detected in 2014-2018 in the present study ( Figure 2). The accession numbers of the reference sequences reported worldwide, including the Korean strains, are displayed in Figures 2 and 3. type was closely related to the Japanese strains GB20-25 and Sun9 and to the Korean strain KJ45, reported in 2006 [9]. One Korean BoRVA belonging to the G10 type was closely related to the USA strain B223, the Brazilian strain 1979-PR, and the Korean strain KNU-YJ8, reported in 2020 [10]. However, although the G5 genotype was detected in 2004,2006,2009, and 2020, it was not detected in 2014-2018 in the present study ( Figure 2). The accession numbers of the reference sequences reported worldwide, including the Korean strains, are displayed in Figures 2 and 3.

Phylogenetic Analysis of the P Genotype
Phylogenetic analysis of partial nucleotide sequences of the VP4 gene (a 664 bp fragment) from the 35 Korean BoRVA strains and selected reference strains from GenBank was also performed (Figure 3). The phylogenetic tree for VP4 revealed that the 35 Korean BoRVAs belonged to only two types (P [5] and P [11]) and that the P [5] genotype, which contains 30 of the Korean BoRVAs, was closely related to the USA strains Rota Teq-SC2-9 and WC, the Turkish strain P4 Calf TR 2, the French strain V026, the United Kingdom strain, the South Africa strain 1603, and the Thailand strain 61A (Figure 3). In addition, five Korean BoRVAs belonging to the P [11] genotype were closely related to the Japanese strains GB20-25 and GB14-45, the Iranian strain Khorasan, the Brazilian strain 1929-PR, the Chinese strain DQ-75, and the US strain B223. However, although the P[7] genotype was detected in 2004, 2006, and 2020, it was not detected in 2014-2018 in the present study ( Figure 3).

Discussion
More than 50% of the mortality observed in pre-weaned calves is related to diarrhea, and most cases occur in calves aged less than one month [22]. A previous study of BoRVApositive Korean native calves reported that 17.1% (28/164) of diarrhea samples were obtained in Gangwon and Gyeongbuk provinces between 2014 and 2016 from calves aged less than 7 months [5]. Another study of fecal samples collected in 2016-2017 from Korean calves aged up to 60 days showed that the BoRVA-positive rate for diarrhea feces was 15.2% (31/204), compared with 5.0% (17/340) for normal feces [6]. In addition, we found a BoRVA-positive rate of 25.4%, with the prevalence being above this average in four regions (Chungnam, Gyeongbuk, Gyeonggi, and Jeonbuk). Another study reported that diarrhea samples collected from calves in Jeonbuk and Gyeongbuk in 2019-2020 showed a BoRVA-positive rate of 42.5% [10]. In the present study, we found that 35.0% (7/20) of BoRVA-positive diarrheal feces were collected from calves aged 1-10 days, which is lower than the 47.2% positive rate reported in the previous study [10]; however, both studies suggest that BoRVA is most prevalent in calves under 10 days of age.
In China, which is geographically close to South Korea, several G genotypes (G6, G8, and G10) and P genotypes (P [1], P [5], P [7], and P [11]) have been detected in the bovine population [23][24][25]. The only relevant study previously conducted in China showed that G6 and P [5] were common G and P genotypes among dairy calves in some regions and that the dominant genotype combination was G6P [5] [23,25]. Similar to the G and P genotypes of BoRVA occurring in China, we found that genotypes G6, G8, G10, P [5], and P [5] were prevalent in Republic of Korea from 2014 to the present day and that G5 emerged in 2020 [10]. However, the P [7] genotype first appearing in China was detected in Korea in 2004-2005 but has not been detected since 2014. A recent study suggests that BoRVA circulates widely among dairy calves in China, and the dominant genotype is G6P[1] [26]; however, the most prevalent genotype in Korea is G6P [5], and the P[1] genotype has not yet emerged.
In a previous study in which the effect of vaccination status on the BoRVA-positive rate in pre-weaned Korean native calves was examined, 42.1% (48/114) of those born to vaccinated cows and 42.6% (147/345) of those born to non-vaccinated cows were positive for BoRVA, based on the results of real-time RT-PCR screening [10]. Interestingly, our findings are similar (26.1% and 25.0%, respectively) to those of that study, although the BoRVA-positive rate in the present study was lower. Currently, polyvalent BoRVA (G6 and G10 genotype) vaccines are used to vaccinate cattle in Korea. However, the BoRVA-positive rates of calves with diarrhea in BoRVA-vaccinated and non-vaccinated farms are similar. It is unclear whether this is due to problems in the effectiveness of the BoRVA vaccine currently used in Korea or to other problems on farms that require further investigation. In addition, a continuous and precise assessment of the prevalence of various VP7 and VP4 type-specificities is needed to evaluate vaccine effectiveness and understand whether it is necessary to construct polyvalent RVA vaccines for livestock animals.

Conclusions
Among BoRVAs, G6P [5] emerged in Korean calves from around 2014. The G6P [5] and G6P [11] genotypes are the main causative agent in 80% and 14.3%, respectively, of Korean calf infections. In general, BoRVAs infect calves aged less than one month, particularly calves younger than 10 days of age. In order to reduce the economic loss to farms caused by bovine rotavirus, continuous monitoring of VP7 and VP4 should be performed, and in addition, new preventive vaccines and treatments for epidemic rotavirus should be developed.