3.5 Amino acid comparison and homology modeling of the VP7 of G8 strains
The amino acid sequences of the VP7 antigenic site were analyzed to better understand the amino acid variation and antigenic properties among human and animal G8 strains. The atomic structure and conformation of the outer capsid VP7 trimer of RVA generate the neutralizing antigenic epitopes 7-1, which is sub-divided into 7-1a and 7-1b and 7-2, containing 29 amino acid residues. Interestingly, 14 G8 strains analyzed in this study and 13 G8 strains in the same sublineage A demonstrated conservative amino acid substitutions at A125S and N147D (Figure 3), whereas these changes were not discovered in other human and animal G8 reference strains. The homology modeling of the capsid protein VP7 confirmed that these two amino acid substitutions were present on the surface exposed area of the G8 virion.
4 DISCUSSION
Rotavirus A (RVA) is one of the leading viral pathogens of acute gastroenteritis in young children less than five years of age. Detection and genetic analysis of RVA in Chiang Rai, Thailand had been continuously performed since 2015.3,4 In the present following-up surveillance, molecular characterization of RVA in Chiang Rai was carried out by RT-PCR in children hospitalized with acute gastroenteritis during 2018-2020. In Thailand, RVA infection rates during 2000-2019 ranged from 15 to 44.5%.3,4,23-26,32-37 In this study, RVA infection rates were 11.3% in 2018-2019 and 11.9% in 2019-2020 (Table 1), which declined around 2-3 folds (23.2 to 37.8%) from the previous studies during 2015-2018.3,4 This declining trend is also observed in recent RVA surveillances in different areas of Thailand during the time overlapping with this study, 15% in 2016-201924 and 17.9% in 2018-2019.37 It has been demonstrated that the implementation of rotavirus vaccines has reduced the disease burden associated with RVA in a wide range of settings.38-41In Thailand, rotavirus vaccines have just recently been included in the national immunization program since 2020, although the vaccines were introduced in a few provinces before 2020.42 Thus, RVA identification and characterization should be monitored in the future whether the rotavirus vaccine significantly reduces diarrhea-related hospitalization and detection rate in Thailand.
RVA commonly causes acute gastroenteritis in infants and young children under the age of three years.3,4 In line with this, the current study revealed that patients under three years of age (<36 months old) were at the highest risk group for RVA infection (85.7%). Even though RVA infection or reinfection can occur at any age, protective immunity against the virus acquired from natural infection or vaccination can prevent older children from the disease resulting in a decreasing infection rate.
Distribution of RVA genotypes in Thailand between 2000 and 2019 showed that G1P[8] was the most prevalent, following by G9P[8] and G3P[8]. 3,4,23-26,32-37 In Chiang Rai, G9P[8] was predominant in 2015-2016, but the genotype shifted to the equine-like G3P[8] in the following two seasons during 2016-2018.3,4 Distribution of RVA genotypes in Thailand between 2000 and 2019 showed that G1P[8] was the most prevalent, followed by G9P[8] and G3P[8]. 3,4,21-24,32-35 In Chiang Rai, G9P[8] was predominant in 2015-2016, but the genotype shifted to be the equine-like G3P[8] in the following two seasons during 2016-2018.3,4 According to previous reports since 2010 in Thailand, G8P[8] strains have been identified with the incidence rates ranging from 3.1 to 22%.4,23-26,36,37Interestingly, G8P[8] strains emerged as the most common genotype for the first time in Chiang Rai, Thailand with high incidence rates of 68.4% in 2018-2019 and 81.2% in 2019-2020 (Table 1).
Full genome analysis of four representative G8P[8] strains in the present study revealed the DS-1-like constellation II, G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Similarly, these DS-1-like G8P[8] strains have also been identified formerly in many countries such as Argentina, Chile, China, the Czech Republic, Japan, Korea, Singapore, Thailand, and Vietnam. Only one Croatian strain RVA/Human-wt/CRO/CR2006/2006/G8P[8] presenting the Wa-like constellation I, G8-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, has been described so far, showing a close evolutionary relationship to the prototype G1P[8] human strain Wa.43 This implies that the G8P[8] strains with whole-genome sequences published globally between 2013 and 2021 process the DS-1-like constellation II.
Genetic analysis of the VP7 gene revealed that DS-1-like G8P[8] strains in this study and 13 G8P[8] strains in the sublineage A contained the conserved two amino acid substitutions on the antigenic epitopes (A125S and N147D) (Figure 2A, 3). Of the 13 G8P[8] strains in the sublineage A, some G8P[8] strains were first reported from hospitalized and outpatient adults in Bangkok, Thailand in 2017-2018.44 Next, three strains (THA/CMH-ST260-18/2018/G8P[8], THA/CMH-N065-18/2018/G8P[8], THA/CMH-N005-19/2019/G8P[8]) were detected from hospitalized children in Chiang Mai, Thailand in 2018-2019.24 And, the latest study reported two strains of DS-1-like G8P[8] strains (RVA/Human-wt/CHN/GZ-0005/2021/G8P[8], RVA/Human-wt/CHN/GZ-0013/2021/G8P[8]) from hospitalized children in 2021 in China.43 This indicates that the G8P[8] strains bearing the amino acid substitutions have circulated in Asian countries, Thailand and China, at the same time during 2017-2021 and caused acute gastroenteritis in both children and adults.
Phylogenetic trees of the VP1, VP6, NSP2, NSP3, and NSP4 genes have demonstrated that Chiang Rai DS-1-like G8P[8] strains and 51 DS-1-like G8P[8] reference strains published previously clustered in different lineages (Figure 2C-2G), suggesting that the origin of the Chiang Rai DS-1-like G8P[8] strains might not derive from one of the DS-1-like G8P[8] reference strains. Moreover, the phylogenetic analysis also confirmed that DS-1-like G8P[8] strains detected in 2013 in Thailand,25,26 formed a unique lineage of the VP6 gene, closely related to the Vietnamese G10P[14] strain. These findings indicate the genomic diversity between the DS-1-like G8P[8] strains detected in Thailand in 2013 and the Chiang Rai DS-1-like G8P[8] strains identified in this study.
There are only two Chinese DS-1-like G8P[8] strains belonging to sublineage A that had whole genome sequences available in the GenBank database15, whereas other 11 Thai G8P[8] strains had no whole genome sequences for the genetic analysis. Phylogenetic trees confirmed that these Chinese DS-1-like G8P[8] strains were not derived from the DS-1-like G8P[8] found in the present study because the NSP3 and NSP4 genes of the Chinese strains formed lineages that separated from the Chiang Rai DS-1-like G8P[8] strains (Figure 2F, 2G).
In conclusion, molecular and epidemiological identification of RVA in Chiang Rai, Thailand during 2018-2020 demonstrated that novel DS-1-like G8P[8] strains containing specific amino acid substitutions on the antigenic epitopes of the VP7 capsid protein had different VP1, VP6, NSP2, NSP3, and NSP4 genes compared with the DS-1-like G1P[8] strains previously reported globally. Taken together, genetic analysis clearly defined the Chiang Rai DS-1-like G8P[8] strains appeared to have evolved genetically through reassortment events with locally or globally circulating genotypes. Finally, the detection of the novel DS-1-like G8P[8] rotavirus reassortant strains highlights the variety of RVA strains in Northern Thailand. Thus, genetic characterization of RVA is essential for better assessing whether such unusual strains will impact the efficacies of the vaccines.