To determine the predominance of the unusual G9P[4]
circulating in Indonesia, we performed aa analyses of RVA interaction
sites on host cells, focusing on the outer and inner capsid proteins
G9-VP7 and I2-VP6. Aa substitutions were deduced from 9 VP7-G9 and
VP6-I2 reference strains from the GenBank database (2009-2015). The aa
substitutions of T78I and T108I were determined in the VP7 gene (Table
3). Additionally, a single aa substitution of L291S was detected in the
VP6 gene (Table 4).
DISCUSSION
Previously, we reported dynamic changes in RVA genotypes from
equine-like G3 to human G1/G312,25. Complex RVA
genotype diversity is more common in developing countries than in
developed countries21,26,27. Inter-genogroup
reassortant strains highlight the ongoing spread of the unusual RVA
strains throughout Asia and other countries28. It has
been suggested that evolution occurs primarily through the selection of
point mutants in the antigenic site or by the reassortant of each
segment of the RVA genome3. The mutation rate of RVA
was estimated to be around the value of 5×10-5 per
nucleotide during genome synthesis, suggesting that approximately one
mutation emerges in each new copy10.
In this study, we examined the whole genome sequences of the unusual
G9P[4] RVA detected in 2021 in Indonesia. This is the first report
of the whole genome sequence of the unusual G9P[4] RVA in Indonesia.
The newly identified unusual Indonesian G9P[4] strains exhibit
genotypes within the G9P[4] constellation distinct from the sporadic
G9P[4] strains detected in India, Bangladesh, Japan, and South Korea
from 2007 to 2012. The more prevalent G9 genotype in the Americas (18%)
and Europe (13%) underwent recombination with the P[8] genotype in
2003 –200410. Human RVA G9P[4] was first
identified in Brazil in 1999 as an uncommon
genotype14. Reassortant in the G9P[4] genotype
variation was found in Paraguay with mixed infection of G2G9P[4]
from Bangladesh in 2005 and from India in 20081,18.
The unusual G9P[4] with a genomic constellation backbone of
DS-1-like was reported in Indonesia and was detected in several
countries such as the USA, Japan, India, Italy, Mexico, and
Bangladesh21,27-31. Human RVA reassortant with genomic
constellation backbone of Wa-like or DS-1-like were found more
frequently16. The double-reassortant strain
(G9-P[4]-I2-R2-C2-M2-A2-N2/N1-T2-E6-H2) detected in India and in the
Czech Republic have the same genotypic constellation as the Indonesian
strain15,32. In 2013, triple-reassortant strains
(G9-P[4]-I2-R2-C2-M2-A2-N1-T2-E6-H2) were found, following the
detection of unusual G9P[4] strains in India during
2011-201315.
A time-scaled Bayesian phylogenetic tree was constructed to examine the
evolution of the RVA genotype14,32. The evolutionary
rate of VP7-G9 in this study was estimated to be
6.43×10-3 nucleotide substitutions/site/year, which
was similar to those of the VP7-G9 genotype with
1.38×10−3, 1.87×10−3, and
1.609×10−3 nucleotide substitutions/site/year in China
and Ghana3,16,33. The evolutionary rate of the
VP4-P[4] in this study was 2.53×10-3 nucleotide
substitutions/site/year, comparable to the Chinese strain’s evolutionary
rate of 1.172×10-3 nucleotide
substitutions/site/year33. Conversely, the VP4 gene in
Ghana showed a higher evolutionary rate at 8 ×10-4nucleotide substitutions/sites/year21. These results
align with the evolutionary rates typical of many RNA viruses, which
evolve at a rate of approximately 1×10-3 nucleotide
substitutions/site/year4. The parent viruses spread
regionally within 1-3 years, whereas the ancestral virus takes 2-6 years
to propagate13. In the present study, the evolutionary
rates of both the VP7/VP4 G9P[4] genes of RVA show similar
evolutionary speed to those of G9 from China and Ghana. However, the
evolution of the P[4] genotype happened more rapidly than previously
observed in Ghana. It is essential to monitor the evolutionary rates of
RVA genotypes to identify trends in RVA genotype evolution.
The tMRCA analysis predicts the rate of transmission of RVA
genotypes13,16. The tMRCA of the G9-VP7 of the unusual
G9P[4] strains in lineage-III was estimated to be in 2004 (95% HPD
interval 2001-2021) (Fig. 3), showing similar results to the unusual
G9P[4] RVA strain in India15. Monitoring of
genotype diversity revealed that rotavirus G9P[4]-VP7 lineage-III
was detected in surveillance networks worldwide in the beginning of the
millennium2. The tMRCA of the G9P[4]-VP4 in this
study was estimated to be in 1994 (95% HPD interval 1990-2021) (Fig.4).
The prevalent P[4]-VP4 genotype, G2P[4], emerged in 1959 (95%
HPD interval 1923-1984)16,33. This finding suggests
that the unusual G9P[4] strain began spreading with the onset of
reassortment in the VP4 gene in the 1990s. The NSP2 gene was generated
by reassortant between N1 and N2. The ancestor of NSP2-N1 emerged in
1986 (95% HPD interval 1981-2021), while NSP2-N2 appeared in 1998 (95%
HPD interval 1996-2021). NSP2-N1 appeared 10 years earlier than NSP2-N2,
indicating that the Wa-like strain appeared earlier than the DS-1-like
strain and that both strains are still circulating worldwide.
The outer membrane protein of the VP7 gene contains major antigenic
epitopes that induce specific neutralizing antibody
responses21. In the present study, the deduced aa
substitutions in the VP7 gene were T78I and T108I, conserved in
G9P[4] RVA in India15. Interestingly, the Indian
G9P[4] strain presents a mutation E154K in VP7, whereas the
Indonesian G9P[4] strain lacks this mutation. The aa sequence in the
VP7 has been deduced and reported2. One of the strains
was linked to the segment within aa 271-342 of VP6, which is necessary
for interaction with VP734. In addition, a pair of van
der Waals interactions between aa 279-281 on VP7 and aa 313 on VP6, as
well as a side chain hydrogen bond between aa 305 on VP7 and aa 310 on
VP6 were reported as interaction sites between VP7 and
VP613. In viral self-defense, the virus was capable of
mutating at their interaction sites of the outer capsid protein VP7 and
the inner capsid VP615. The aa substitution S291L on
the VP6 protein is linked to the interaction between VP7 and VP6
proteins. In this context, the altered relationship between G9 and I2,
with a substitution at the VP6 protein interaction site, may be more
sustainable than the unusual relationship without
substitutions15. In the present study, aa 291 did not
receive any substitutions as previously described. This phenomenon is
consistent with the notion that the unusual G9P[4] strains
circulating in Indonesia can rapidly change into G9P[6] strains.
CONCLUSIONS
The recent discovery of the unusual G9P[4] RVA strains emphasizes
the ongoing spread of RVA in Asia and other regions. In particular, the
G9P[4]-DS-1-like strain, which had been reported in several
countries outside Southeast Asia since 2011, was first detected in
Indonesia in 2018. By 2021, the majority of RVA-positive cases (19 out
of 21) were identified as G9P[4], with the remaining 3 as
G9P[6]. These Indonesian G9P[4]/P[6]-DS-1-like strains,
discovered between 2018 and 2022, show genetic variations from strains
reported in other countries between 2011 and 2015, particularly in the
NSP4 gene (which has E2 genotype instead of E6 genotype). These strains
share a common ancestry in VP7 and VP4 genes with previously reported
G9P[4] DS-1-like strains but exhibit slight genetic differences in
other genes. These results suggest that there were multiple intra-
reassortant events between the original G9P[4] strains and the
DS-1-like RVA strains that coexist in Indonesia and elsewhere.
Continuous monitoring of RVA genotype dynamics is crucial given that
G9P[4] strains emerged as the predominant genotype in Indonesia in
2021. This monitoring is essential to assess the prevalence and genetic
diversity of circulating RVA strains and to evaluate the efficacy of RVA
vaccines.