INTRODUCTION
Recurrent outbreaks of human pathogenic viruses resulting in epidemics or pandemics are due to their ability to rapidly evolve and adapt as compared to the other microbial pathogens[1]. Generally, novel viruses or their new strains emerge when humans are exposed for the first time, to an evolved virus of zoonotic origin. Compared to DNA viruses, RNA viruses have a much more recent history of ‘genetic-evolution’ due to very high replication-fidelity rate (approximately 10-4 error/site/cycle) of their RNA-dependent RNA polymerases (RdRp) and therefore, ‘human-adaptation[1,2]. In the evolutionary and adaptive process of RNA viruses, genetic mutations, re-arrangement or assortment, and virus-host genetic recombination are the major events towards establishing new and stable strains. Therefore, it is very much expected that such newly human-adapted strains would persist in specific populations (endemics) or spill across populations (epidemics) or eventually spread globally (pandemics[2]).
Rotaviruses are segmented, double-stranded RNA (dsRNA) viruses that mainly cause acute gastroenteritis or diarrhoeal disease in pediatric population (< 5 years). Of the nine species of rotavirus (group A, B, C, D, F, G, H, I and J), rotavirus A (RVA) primarily infects humans[3]. Six strains of RVA, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] have been shown to dominate about 90% of global rotavirus transmission in humans[4,5]. In 2019, there were 9.1% of under-five global mortality due to diarrhea as clinical presentation of rotavirus infection[6]. The mortality caused by rotavirus infection is predominantly occurred in the developing countries with middle to low income[7].
Rotavirus evolution, especially a dynamic replacement of circulating RVA from one strain into another, has been observed in Asia, Africa, Australia, America, and Europe[8,9]. In addition, the emergence of the unprecedented strains as the results of intra- or intergenogroup combinations which subsequently become the predominant strains, were also observed[10]. This genotype alteration was created by the mechanism of reassortment between strains with a similar genotype constellation[11]. However, many novel rotavirus strains have emerged by the mechanism of inter-genogroup multiple reassortment[8,12].
In addition to achieving the lower incidences of rotavirus associated-acute gastroenteritis, vaccination is also an important driving factor for the dynamic evolution of rotaviruses. The vaccine will exert selection pressure on the rotavirus genotypes and consequently, trigger the emergence of novel strains through reassortment (genetic shift)[13]. The accumulation of point mutations (genetic drift) may also reduce the effectiveness of rotavirus vaccines[14]. Furthermore, the RVA P[II], which has a wider host range due to glycan specificity, is known to have evolved from RVA P[I][15]. This event also shows the zoonotic mechanism of RVA[16].
Other factors that also influence the evolution of rotaviruses are the host immune responses[17]. RVA has a specific strategy to evade the innate and adaptive immune responses[18,19]. This strategy is essentially required by rotavirus to continue to survive and is the key to its continuous evolution[18]. Although no anti-rotavirus drug has yet been approved by the World Health Organization (WHO), this antiviral drug could also be a driving factor in the evolution of rotaviruses. The pressure exerted by antiviral drugs may trigger accumulation of mutations or reassortment of circulating rotavirus strains[20-23]. In this review, we discuss the driving factors of rotavirus evolution, including vaccines and host-immune responses, towards improving our understanding of the evolutionary dynamics of its emerging strains as a foundation for developing effective preventive and therapeutic measures.
GENOMIC STRUCTURE OF ROTAVIRUSES
Rotaviruses are a non-enveloped RNA virus that has 11 segments of dsRNA encapsulated by three-layered capsid proteins (Figure 1). Rotaviruses belong to the Reoviridae family. The segmented dsRNA genome of rotaviruses encodes six structural viral proteins (VP, VP1-VP6) and six non-structural proteins (NSP, NSP1-NSP6). The VPs are present in the mature viral particle and determine the specificity of rotaviruses, along with their capacity to induce the host immune responses[4]. The NSPs regulate the specific functions for genomic replication as well as antagonistic functions toward the host innate and adaptive immune responses[24].
Figure 1 Schematic structure of rotaviruses.
Rotaviruses are a non-enveloped RNA virus that has 11 segments of double-stranded RNA (dsRNA) covered by three layers of capsid proteins. The inner layer consists of VP1 and VP2 proteins, the middle layer consists of VP6 protein, while the outer layer consists of VP4 and VP7 proteins.
Rotaviruses are classified based on the nucleotide sequence as well as antigenic epitopes in the VP6 protein. To date, there are 10 species of rotaviruses, namely species A-J. The most common species causing infection in children is RVA. RVA can be further classified according to the RNA sequences in the RNA segments 7 and 4, which are responsible for encoding the VP7 and VP4 proteins, respectively. VP7 is a glycoprotein and is employed to classify the G-type of rotavirus, while VP4 is a protein cleaved by a protease (protease-cleaved protein) and is employed to classify the P-type of rotavirus. To date, 41 G genotypes and 57 P genotypes of rotaviruses have been successfully identified[4,25].
However, there are about six G-types and three P-types of RVA which are predominantly found circulating worldwide, namely G1, G2, G3, G4, G9, and G12, as well as P[4], P[6], and P[8]. Of the many combinations between different G and P genotypes that have been identified, there are 6 strains of RVA that play a dominant role in more than 90% of the global rotavirus spread, namely G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8][4].
Classical binomial genotyping of rotaviruses into G (VP7) and P (G4) genotypes which is commonly used for rotavirus surveillance limits our knowledge on the rotavirus genome characteristics. Thus, a whole-genome classification system based on their 11 RNA segments has been introduced[26]. The segment of the RVA genome that encodes VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/NSP6 will be expressed using the abbreviations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x is the genotype number, an Arabic number starting from 1. The RVA grouping based on the 11 genome segments therefore, forms a constellation of viral genotypes that distinguishes between its various strains[9].
EPIDEMIOLOGY OF ROTAVIRUSES
In general, rotaviruses infect children under five years old and are transmitted by the fecal-oral route[4]. However, rotavirus infection predominantly occurs in children under two years of age, with the highest incidence in infants aged 7-12 months. Maternal antibodies obtained from the mother can only protect the baby from viral infection for up to 6 months and will decrease significantly thereafter. This is the main reason why the incidence of rotavirus infection often occurs in children older than six months old. The incidence of rotavirus infection decreases in children of over two years age because of increased exposure to infections and stronger immunity[27].
A total of 115 million cases of rotavirus infection in children aged less than five years were reported in 2003, with 2.3 million of whom required hospitalization. In 2013, the global mortality of children under-five years of age due to rotavirus infection reached 215000 deaths. Of this large mortality rate, more than 95% occurred in developing countries, such as countries located on the African and Asian continents[27]. During the period of 2019, 9.1% of under-five mortality in the world was caused by diarrhea as manifestation of rotavirus infection[6]. In one of Asia countries, such as Indonesia, mortality case due to diarrhea in 2020 among children aged 29 days until 11 months and 1 to 4 years old were 9.8% and 4.5%, respectively[28]. The Global Rotavirus Surveillance Network under the coordination of the WHO reported that before the introduction of the rotavirus vaccine, nearly one-third of hospitalized diarrheal patients were caused by rotavirus infection[7]. After implementation of rotavirus vaccine into the national immunization program, the prevalence of rotavirus infection among children under-five who were hospitalized or admitted in emergency department declined by approximately 40%[29].
Rotavirus infection in children (< 5 years) causes acute diarrhea, dehydration, electrolyte imbalance, and metabolic acidosis. In addition, rotavirus infection also causes vomiting and fever, and can lead to death if not appropriately treated[27]. Children (< 5 years) in low-income countries have a higher percentage of deaths from rotavirus infection than children in high-income countries. This can be caused by several factors, such as limited coverage of health care centers, lack of hygiene and sanitation, the absence of rehydration therapy, as well as the influence of other diseases, such as malnutrition[4].
Rotavirus infection is generally non-seasonal, but the incidence rate can increase in certain seasons. In subtropical countries, for instance countries in Asia and Africa, rotavirus infection is erratic and varies throughout the year. However, the incidence rate of rotavirus infection increased significantly during the wet and dry seasons in some subtropical countries, e.g., Benin, West Africa. Meanwhile, rotavirus infection in countries on the European continent tends to depend on the season where the incidence rate of rotavirus infection will increase in the winter[27].
Diarrheal disease due to rotavirus infection in a certain area is often caused by infection with rotavirus strains that are rarely found, for example the G9P[6] strain which prevalence is only 9.5% in India[4]. The selection pressure on the spread of natural rotavirus strains by the introduction of the vaccine will cause genotype shifting through the reassortment process. However, changes in the rotavirus genotype are not always associated with the introduction of the vaccine. For example, there were genotype changes that occurred in Indonesia which had not implemented the national (universal) rotavirus immunization program. There was a change in the genotype of the horse (equine-like strain) G3P[8]/[6] into typical human rotavirus strains G1P[8]/[6] and G2P[8]/[6][30].
During the coronavirus disease 2019 (COVID-19) pandemic, there were reports on decreasing vaccination coverage against rotavirus[31]. This trend was due to disruption of routine childhood immunisation during the pandemic[32]. However, nonpharmaceutical interventions implemented during the pandemic have significantly impacted on decreasing the prevalence of rotavirus infection in children[33,34]. Interestingly, water-based surveillance in Japan of enteric viruses during the COVID-19 pandemic, identified rotavirus as the most frequently detected viral pathogen, indicating its continuous transmission in the community[35]. A modeling study further predicted re-surgence of rotavirus in post-pandemic period; however therein, a thorough epidemiological study is needed to confirm the findings[36].
GENETIC REASSORTMENT, MUTATION RATE, AND GLYCAN SPECIFICITY
Genetic reassortment (genetic shift)
The segmented genomes of rotaviruses can derive from two different mechanisms, either through the process of accidentally exchanging two genomes from two different strains (genetic reassortment), or through the process of diploidy or polyploidy where more than one genome have been randomly packed into the viral progeny. Rotaviruses have a unique characteristic, which is their ability to sort and exchange its genetic material during the co-infection process of two different strains, known as the reassortment. These viruses are able to coinfect a single host cell, then exchange their segmented RNA genetic materials, resulting in a single “hybrid” virion. The reassortment process in RVA is the main mechanism for the virus to evolve and increase its diversity[11]. Reassortment is best-described in influenza virus as a primary mechanism for interspecies (animal-to-human) transmission and the emergence of novel pandemic strains[37].
The reassortment mechanism of RVA has not yet to be comprehensively elucidated due to the limitations of in vitro experimental systems. However, available data indicate that there is a similar process between the reassortment mechanism of RVA with ϕ6 (Pseudomonas phage phi6) and influenza A viruses. Reassortment in RVA begins with the transcription of double-stranded RNA (dsRNA) by viral polymerases which produces positive-sense RNA. A total of 11 positive segments of RNA that have been transcribed will form a complex of supramolecular structures and be immediately assembled by virion particles that grow through the encapsidation process. The polymerase enzyme then converts positive RNA into dsRNA shortly after the encapsidation process takes place (Figure 2)[11].
Figure 2 Reassortment mechanism of rotaviruses group A (RVA).
A: Diagrammatic representation of the emergence of a novel reassortant strain with genes derived from two parental strains; B: Reassortment in RVA begins with the synthesis of positive-sense RNA. A total of newly-synthesized 11 positive segments of RNA will form a complex of supramolecular structures and be immediately assembled by virion particles that grow through the encapsidation process. The polymerase enzyme then converts positive RNA into dsRNA shortly after the encapsidation process.
The reassortment pattern of RVA is strongly influenced by the constellation of the genome[38]. In general, the RVA genome is divided into three constellations of genotypes: Genogroups 1, 2, and 3. Genogroup 1 or Wa-like has a genotype constellation G1/3/4/9/12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1; genogroup 2 or DS-1-like has the genotype constellation G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2; and genogroup 3 or AU-1-like has a genotype constellation G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3[12]. Reassortment that occurs in intra-genogroups will produce reassortants that have variations at the subgenotype level[39,40]. Rotavirus reassortment rarely occurs across genogroups because the viral progeny tends to have low survival. This can be influenced by the possible incompatibility of RNA or protein encoded by RNA segments in viruses that undergo reassortment across genogroups. Consequently, it will lead to the constraints in viral replication[11]. Interestingly, full-genome characterization of rotaviruses from 2022-2017 in Vellore, India showed that most strains had stable classical genotype constellation of Wa-like and DS-1-like and only a small number had reassortant constellations. A similar finding was reported from 6-year surveillance (2010-2016) of rotaviruses in northern Brazil[41].
Although the evolution of rotavirus is highly dependent on reassortment associated with the constellation of the genome, several studies have reported that there are several types of RVA identified to have evolved through intergenogroup reassortments. RVA strain DS-1-like G1P[8] which infected 14% of RVA patients in Vietnam was reported to be rotavirus strains that evolved through an intergenogroup reassortment mechanism. G2P[4], a local RVA strain in Vietnam, obtained the VP7 gene from a strain similar to G1P[8] and the VP4 gene of a strain similar to G3P[8] circulating in China. However, this double-gene reassortment of the RVA G1P[8] did not cause more severe diseases than the original G1P[8] strain[42].
Not only in Asia, RVA strain DS-like G1P[8] double-gene reassortant is also found in various other continents, such as Europe, Africa, Australia, and America[8]. RVA strain DS-1-like G1P[8] in Brazil was reported to have a phylogenetic similarity with strain DS-1-like G1P[8] in Asia and has a distant relationship with strains found in Africa. Thus, RVA strain DS-1-like G1P[8] in Brazil may have originated in Asia. Further sequence analyses demonstrated that RVA strain DS-1-like G1P[8] in Brazil also has a close relationship with the equine-like strain DS-1-like G3P[8] which indicates that they have the same origin[12].
Another DS-1-like intergenogroup reassortant of RVA strains has also been reported, such as DS-1-like G8P[8] in Thailand which is known to evolve through multiple reassortment mechanisms. The 11 genes were derived from the rotavirus strain of DS-1-like, bovine, bovine-like human, human, and locally circulating DS-1-like G2P[4][8]. This shows that the evolution of RVA is not only able to take place through intragenogroup reassortment, but also through intergenogroup reassortment[8,12]. The emergence of RVA which evolved through the intergenogroup reassortment mechanism is capable of causing outbreaks, as occurred in Singapore in 2016 where the reassortant intergenogroup DS-1-like G8P[8] was found to be the main cause of gastroenteritis outbreaks[10].
The evolution of the RVA G12 strain was also reported. This G12 strain was initially very rare until 2008, but experienced a significant increase thereafter[9]. RVA strain G12 is the dominant strain in neonatal infection, for example in India, where the prevalence is about 24.31% of the neonates, without showing any symptoms[43]. This is similar to what reported in Spain, where the G12 strain was first detected in 2004, but went undetected again 4 years later. The epidemic of the Wa-like G12P[8] strain in Spain, particularly in the province of Gipuzkoa, occurred from early 2010 to 2018 and was able to exceed the number of cases of the G1 strains which had become the dominant strain from 1989 to 2009. These cases could be affected either by the migration of G12 strains from other countries to Spain which eventually caused epidemics, or by the local evolution of the G12 strains. This indicates the possibility of a reassortment event of G12 circulating in various countries and making contact with various other RVA strains causing the emergence of new RVA strains[9].
Point mutations (genetic drift)
In addition to the reassortment event of G12, the local evolution of this G12 strain also indicates the possible influence of other factors, such as interspecies recombination and accumulation of point mutations[44]. Generally, in RNA viruses, accumulation of point mutations is caused by their error-prone RdRp enzymatic activity, resulting in an estimated mutation rate (1.0 × 10-3 to 1.0 × 10-6 base substitution/site/year)[45]. Meanwhile, recent studies reported that intersegment recombination did not affect the long-term evolution of RVA[46]. The evolution of G12 strain was also found in Africa, where the African G12 rotaviruses which belongs to line III rotavirus has evolved and produced 2 sub-lines, namely the West African G12 rotavirus group and the South African G12 rotavirus group. This shows that the evolution of rotavirus can also be influenced by geographical location, where the topographical structure can be a barrier to the movement of human populations, thus triggering genetic diversification in rotaviruses. The high genetic diversification and evolution of rotaviruses are due to the tendency for molecular evolution to occur due to a stable mutation rate, which ranges from 1.201 × 10-3 to 2.198 × 10-3 base substitution/site/year[44].
The effect of mutations on the evolution of rotavirus A was also reported in the RVA strain G4P[8] in Italy. This random point mutation in RVA strain G4P[8] led to the evolution of the strain into 3 circulating lines simultaneously. Apart from the effect of point mutations, the 3-line evolution of the G4P[8] strain was also influenced by reassortment2, particularly in the VP4, VP6, and NSP4 genes. This shows that the evolution of rotaviruses is not only influenced by genetic shifting due to reassortment mechanisms, but is also influenced by genetic drift events due to random point mutations[47].
P[II] genogroups such as P[4], P[6], and P[8] in human rotavirus are the most dominant rotavirus genotypes infecting humans. This dominance is influenced by the infectious ability of this genogroup which has higher specificity to the host as compared to other rotavirus genogroups (P[I,III-V])[15]. The human rotavirus genogroup P[II] can infect host cells by binding to two types of glycans, mucin core and type 1 histo-blood group antigen (HBGA), in addition to binding to GlcNac as a ligand binding center. The interaction with these two types of glycans is not shared by other genogroups. P[I] genogroup that infects animals only has a binding site with the GlcNac ligand. This indicates that the rotavirus genogroup P[I] is the ancestor of the rotavirus genogroup P[II]. It is characterized by the P[II] that retains its binding site with the GlcNac ligand but has two additional glycan ligands which enable the infection to spread to both animals and humans. This also explains the existence of zoonotic transmission events in rotaviruses. Changes in the binding site with the glycan residue are able to trigger the evolution of the rotavirus genogroup P[II] so that it has a dominant ability to infect various types of animal and human hosts[16].
DRIVING FORCES OF ROTAVIRUS EVOLUTION
The key driving force behind the evolution and emergence or re-emergence of pathogenic viruses is the intricate ‘host-pathogen-environment’ relationship[2]. Therein, the relative importance of zoonosis is a function of the prevalence of reservoir animal species and the probability of close contact (direct or indirect) with the susceptible hosts. In addition, because RNA viruses are known to incorporate drastic mutations in their genomes, their new strains or genotypes can spread to different geographical regions with immunologically naive populations. It is also quite possible that such newly human-adapted viral strains could circulate asymptomatically and remain undetected until they manifest clinically. Nonetheless, to understand evolutionary dynamics as well as to accurately predict such epidemics or pandemics, few well-known computational and mathematical models have been developed[2].
Similar to other pathogenic RNA viruses, the incidence of rotaviruses in various countries which shows genotypic differences fluctuating over time has also shown its dynamic evolution. The evolution of rotavirus is influenced by a number of driving factors, such as vaccine introduction, the host immune responses, and antiviral drugs[17,21,39,40,42,43].
Vaccine introduction
The introduction of rotavirus vaccine has an important role in reducing the incidence of pediatric gastroenetritis. The national immunization program for rotavirus vaccine has been implemented in 123 countries as of January 2023. In addition, twelve countries have planned to introduce rotavirus vaccine for the national immunization program[48]. The vaccines can significantly reduce the morbidity and mortality of children under-five due to acute diarrhea[49]. There are two types of live attenuated, oral rotavirus vaccines that have received WHO approval and have been used in more than 100 countries: Rotarix (GSK Biologics) and RotaTeq (Merck and Co.). Rotarix is a monovalent rotavirus vaccine containing human rotavirus strain G1P[8], while RotaTeq is a pentavalent rotavirus vaccine containing human rotavirus reassortant strains G1, G2, G3, G4, and P[8][4]. In high-mortality countries (i.e., Africa and Asia), Rotarix and RotaTeq vaccines have equal vaccine efficacy against severe rotavirus gastroenteritis with 57% during one-year after vaccination. Meanwhile, after two-years follow-up, the efficacy of Rotarix and RotaTeq vaccines are 29% and 44%, respectively[50]. Safety data obtained from the clinical trials showed that rotavirus vaccines are well tolerated. A Cochrane systematic review of available evidence indicated no increase in serious adverse events associated with Rotarix and RotaTeq vaccines[51]. There is no increased risk of intussusception among vaccinated children, although safety surveillance should be continuously conducted[51]. In addition, epidemiological and immunological studies show a possible association between rotavirus infection and autoimmune diseases, most commonly celiac disease. Thus, the rotavirus vaccines should be able to decrease this autoimmune disease-associated rotavirus infection, although it is still controversial[52,53].
In addition to these two vaccine being used globally, there are several rotavirus vaccines that have been approved in specific countries, such as Rotasiil and Rotavac (India), Lanzhou Lamb Rotavirus (China), and Rotavin-M1 (Vietnam)[43,54-56]. Rotasiil contains human reassortant rotavirus strains G1, G2, G3, G4, and G9, while Rotavac contains G9P[11] strains[54,56]. LLR and Rotavin-M1 are monovalent rotavirus vaccines for the animal rotavirus strain G10P[12] and the human rotavirus strain G1P[8], respectively[43,55]. Within one-year follow-up, the efficacy of Rotavac and Rotasiil vaccines against severe rotavirus gastroenteritis are 57% and 48% respectively. While, two years after vaccination, the efficacy of Rotavac and Rotasiil vaccines decline to 54% and 44%, respectively[50]. Although it has been implemented by several countries, these four types of vaccines are still in the WHO pre-qualification stage[4].
Notably, while vaccines have an important role in preventing rotavirus infection, they are also an important driver of rotavirus evolution. The evolution of rotavirus can be characterized by the emergence of an unusual strain of RVA which is characterized by the ineffectiveness of the introduced vaccine in preventing infection[13]. The ineffectiveness of this vaccine causes children who have been fully or partially vaccinated to remain susceptible to the symptomatic rotavirus infection[57]. The introduction of RotaTeq and Rotarix in Korea in 2007 and 2008, respectively, was associated with an alteration of dominant genotype constellation of genogroup 1 to 2. Prior to the introduction of the vaccines, RVA strain G1P[8] was the dominant RVA genotype circulating in Korea. However, after the introduction of the vaccine, G2P[4] which was very rarely detected before the introduction of the vaccine became the dominantly circulating RVA strain. Subsequent genetic analysis showed that the G2P[4] genotype which emerged after the introduction of the vaccine in Korea in 2007, underwent multiple interspecies reassortment. This G2P[4] reassortant strain has a constellation of DS-1-like genotypes and acquired NSP4 genes from cattle and buffalo, and VP1 and VP3 genes from goats. In addition to being a reassortment, a mutation in the VP7 gene also potentially causes the RotaTeq or Rotarix vaccines to be less effective[58].
Interspecies reassortment following the introduction of the vaccines was also reported in Belgium, where RVA strain DS-1-like G2P[4] acquired a bovine-like NSP4 gene segment of animal origin. In addition, there are six other gene segments that are also involved in interspecies reassortment of this G2P[4] strain, i.e. VP6, VP1-3, NSP2, NSP4 and NSP5 genes, where two NSP4 clusters and one VP3 cluster tend to be preserved in human-to-human viral transmission. Interspecies reassortment causes dead-end infection since the majority of animal-human interspecies reassortants rarely show human-to-human transmission. Although the main factor in the emergence of RVA G2P[4] strain in Belgium was the selection pressure from the vaccine introduction, another influencing factor was the migration of G2P[4] from other regions, which was supported by the presence of a bovine-like NSP4 gene segment that tends to be retained in human-to-human transmission[59].
In addition to interspecies reassortment, intragenogroup reassortment of RVA strain Wa-like G1P[8] was also reported in Belgium after the introduction of the Rotarix vaccine. Reassortment can also occur between circulating rotavirus strains and the introduced vaccine strains. The evolution of G1P[8] is also observed from the notable difference between the circulating G1P[8] gene segment after vaccine introduction and the Rotarix vaccine strains, such as VP6, VP2, and NSP2 genes. This difference may be due to the accumulation of point mutations that make Rotarix vaccine less effective in preventing the infection due to circulating rotavirus strain G1P[8] [60].
Similar phenomena were observed following rotavirus vaccine introduction in Venezuela, where there was a change in dominance from G1P[8] to G2P[4], followed by the emergence of various unusual genotype combinations, such as G8P[14], G1P[4], G4P[4], and G8P[4]. Mutations in VP7 and reassortment are also known to be essential factors driving the evolution of the rotaviruses. A mutation in the VP7 neutralization domain in the form of a D96N substitution is known to have an important role in the emergence and dominance of the G2 rotavirus strain. However, the dominance of G2P[4] in Venezuela only lasted for one year, with the emergence and re-dominance of the G1P[8]. This could be due to differences in the viral fitness between susceptible and resistant hosts, thus giving rise to dynamics between rotavirus strains. The re-emerging G1P[8] is known to have mutations in VP7, resulting in escape of the antibodies induced by the rotavirus vaccine[14]. The predominance of G2P[4] strain after vaccine introduction was also found in Botswana[61].
Generally, the introduction of vaccines does not necessarily affect the changes in the dominance of circulating strains in a country. The introduction of the vaccine in east and south African countries did not result in any changes in the circulating strains. RVA strain G1P[8] was the dominant circulating strain, either before or after vaccination. However, some unusual combinations of RVA genotypes were found in low frequency, such as G1P[4], G2P[8], G9P[4], and G12P[4]. These strains arise due to intragenogroup or intergenogroup reassortment mechanisms[62]. The introduction of other rotavirus vaccines, such as Rotavac in India, has also led to the emergence of unusual combinations of RVA genotypes, such as G9P[4], G2P[6], G2P[8], G12P[4], and G1P[11][63]. Furthermore, the introduction of the rotavirus vaccine was able to significantly reduce the incidence of acute gastroenteritis due to rotavirus infection in pediatric population (< 5 years). However, other viral infections such as norovirus pose new problems because they are the main disease agent of acute gastroenteritis with higher severity after rotavirus vaccine introduction[64,65].
Host immune responses in rotavirus infection
Humans have innate and adaptive immune defense systems to fight microbial infections, inc2luding viruses. The host cells have a number of membrane-bound and cytoplasmic receptors that function to recognize viral-derived nucleic acids, including toll-like receptors and RIG-I-like receptors. The binding of the ligand-receptor will trigger the activation of a series of downstream signaling to increase the expression of pro-inflammatory cytokines and chemokinesto activate the antiviral response and provide danger signals to neighboring cells, generating antiviral states[66]. Interferons (IFNs) that play a role in the antiviral responses in the infected cells are type I and III IFNs. Type I IFN was found in high concentrations in the serum of rotavirus-infected hosts, while type III IFN had a more specific role in the antiviral response in the epithelial cells, including in the intestines, lungs, and skin. IFN signaling induces the expression of interferon-stimulated genes (ISGs) as an antiviral effector protein which functions to limit viral replication[67,68].
In addition to IFN-λ which belongs to type III IFN, interleukin 22 (IL-22) is also known to have an essential function in protecting the surface barrier of intestinal epithelial cells from rotavirus infection. IL-22 produced by innate lymphoid cell group 3 (ILC3) functions as an amplifier of IFN-λ. IL-22 and IFN-λ work in synergistic manner to prevent infection and viral replication. The presence of these two molecules is required for optimum activation of the STAT2 transcription factor[69]. Besides ILC3, macrophages are also known to play a role in producing cytokines after rotavirus infection, particularly IFN I and other antiviral cytokines[70]. Furthermore, microbiota that have immunomodulating and antiviral abilities such as Bifidobacteria are known to enhance the innate immune response to fight infection and rotavirus replication[71].
In addition to innate immunity, the adaptive immune response obtained following vaccination or natural infection also plays an important role in eliminating rotavirus infection[72]. The immunological defense mechanism against rotavirus has yet to be comprehensively elucidated up to this moment. However, it is known that IgA, neutralizing antibodies, and T cells have an important role in fighting rotavirus infection[73]. The essential role of IgA in defending rotavirus infection was shown in mice models deficient of the IgA gene. When compared to the wild-type mice, IgA-/- mice had a considerable and significant delay in clearing rotavirus infection[74].
The B and T cells have important roles in preventing rotavirus infection and controlling rotavirus replication, respectively[17]. In addition to an increase in IgA, rotavirus infection or the introduction of vaccines can induce an increase in antibody-secreting B cells that reside in the intestines[75]. Rotavirus infection is able to induce a T-cell immune response in the serum of children under 5 years of age[73]. However, rotavirus has the ability to escape from the T cells through inhibition of the expression of MHC class I which is required by T cells to kill virus-infected cells[19].
Although the host has mechanisms to limit and prevent infection, rotaviruses have a number of strategies to evade the host's innate immune system in order to survive and reproduce. The existence of this immune system evasion strategy is the main key for rotavirus to be able to evolve and have high genetic diversity. There are three rotavirus proteins involved in the evasion mechanisms of the host innate immune system, i.e. NSP1, VP3, and NSP3 (Figure 3)[18].
Figure 3 Cartoon representation of Rotavirus strategy to evade the host innate immune system.
OAS: Oligoadenylate synthase; β-TrCP: Beta-transducin repeats-containing proteins; IRF: Interferon regulatory factor; STAT: Signal transducer and activator of transcription; PABP: Poly(A) binding protein; ISRE: Interferon-stimulated response element; eIF4G: Eukaryotic translation initiation factor 4G.
NSP1 can trigger rotavirus evasion of the host innate immune response in two ways: proteasome-dependent or proteasome-independent. The proteasome-dependent innate immune response evasion strategy in animal rotavirus strains occurs via degradation of IRF by the NSP1-induced proteasome. In human and porcine rotavirus strains, it occurs via degradation of β-TrCP by the NSP1-induced proteasome. In addition, NSP1 is also involved in the proteasome-mediated degradation of a number of other proteins involved in the antiviral immune response, for example the TRAF protein family involved in the NFκB pathway. Proteasome-independent mechanisms of NSP1 induces the blocking of IRF3 transcriptional activity or preventing RIG1 and MAVS signaling which leads to prevention of IFN and ISG activation in the host cells. Prevention of ISG transcription in the host cells by rotavirus also occurs through the blocking mechanism of STAT1 phosphorylation and inhibition of the translocation of STAT1 and STAT2 to the nucleus[76].
The mechanism of evading the host innate immune response by rotavirus VP3 protein occurs through the degradation process of 2',5'-oligoadenylate (2-5A) molecules which are known to form complexes with RNase L that are able to degrade rotavirus dsRNA. VP3 is able to degrade 2-5A because it has 2',5'-phosphodiesterase activity in its C-terminal domain, thus preventing the activation of RNase L. Meanwhile, NSP3 induces evasion of the host innate immune response by interacting with eIF4G and is involved in relocation of poly(A)-binding protein (PABP) that leads to inhibition of host cell mRNA translation. NSP3 has a higher affinity for eIF4G than PABP and is an RNA-binding protein specific for rotavirus mRNAs. Saturation of the translation initiation machinery with viral mRNAs possibly leads to cellular protein synthesis inhibition, including proteins involved in the host cell antiviral immune response[77].
Antiviral drugs
In contrast to the WHO approved rotavirus vaccines being used in > 100 countries, there is no specific anti-rotavirus drugs available to date. Currently however, the most effective treatment of rotaviral acute diarrhea is through palliative or supportive therapy in the form of rehydration therapy, by restoring the lost body fluids to prevent dehydration. However, several anti-rotavirus drugs have been extensively studied for their potential in inhibiting rotavirus infection[23]. Furthermore, one thing that should be considered is the possibility of the emergence of viral strain that resistant to the newly developed antiviral drugs. Anti-HIV drug resistance is one of the important examples of the emergence of viral strain that evolved after the introduction of the antiviral drugs for viral infection treatment[78]. Therefore, care should be taken starting from the development of the anti-rotavirus drugs.
One of the anti-rotavirus drugs that are still in the early research stages is ursolic acid (UA). UA is a pentacyclic triterpenoid which is known to have antiviral activity against rotavirus infection. UA is able to significantly reduce the amount of rotavirus viral protein and inhibit the growth of infective rotavirus progeny in the initial cycle of infection. The decrease in the number of viral proteins and infective progenies could be due to the decrease in the intraluminal calcium ion content of the endoplasmic reticulum. This will result in folding errors in VP7 protein and imperfect glycosylation of NSP4 and VP7 proteins which leads to failure of rotavirus maturation. However, at the end of the rotavirus cycle, UA did not show any significant effect on the growth pattern of rotavirus. This could be due to a number of rotavirus progenies that have been generated before the administration of UA[20]. In addition, pyrrole derivatives such as ‘pyrrolo [2,3-d] pyrimidine’ and ‘pyrrolo [3,2-e][1,2,4] triazolo [4,3-c] pyrimidine’ are also alternative antiviral drugs and have a high activity against rotavirus infection[23].
Not only synthetic drugs, but other approaches have also been done to prevent rotavirus infection by utilizing the microbiota. Segmented filamentous bacteria (SFB) inoculated in the intestines of mice was able to make the mice resistant to rotavirus infection. The mechanism that causes this to occur has not been able to be explained comprehensively. However, SFBs are capable of causing resistance to rotavirus infection by blocking an important component of rotavirus that is used to bind to host cells, and by inducing the production of IL-22 which influences enterocyte proliferation and translocation[21].
Interactions with other enteric viruses
The intestinal environment also harbors a eukaryotic virome of significant human viruses, such as RV, norovirus, astrovirus, and enteric adenoviruses, responsible for viral gastroenteritis. These viruses replicate within a spectrum of cell types, including enterocytes, lymphocytes and myeloid cells[79]. Rotavirus and norovirus recognize different HBGAs as initial receptors for attachments[80]. In pediatric patients, it was observed that viral shedding was observed significantly longer for norovirus than rotavirus[81]. Importantly, in nations where RV vaccination initiatives have been implemented, the incidence of rotavirus etiology has significantly declined. However, norovirus comparatively continues to emerge as the predominant pathogen of pediatric gastroenteritis[82]. In an Indian three-year hospital-based surveillance study of acute gastroenteritis, norovirus positivity in rotavirus -vaccinated and unvaccinated children were 16.3% and 12%, respectively[83].
Further, enteric adenoviruses replicate efficiently in human organoid models, particularly goblet cells[84]. In children with acute gastroenteritis, a high adenoviral load was noted during the first few days of infection, which rapidly declined[81]. In contrast to rotavirus, the prevalence of adenovirus infections was relatively low[85,86]. During the COVID-19 pandemic, the incidence of adenovirus infection was also lower as compared to pre-pandemic period[87]. In one study, it was reported that adenovirus was the most common diarrheal pathogen following rotavirus vaccine introduction in India[88]. Adenovirus infection was also higher in rotavirus-vaccinated than rotavirus-unvaccinated children in Venezuela[89]. All these findings suggest a dynamic coevolution of enteric viruses, especially after the introduction of nationwide rotavirus vaccination.
