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.