An overview of Zika virus genotypes and their infectivity

ABSTRACT Zika virus (ZIKV) is an enveloped, single-stranded RNA arbovirus belonging to the genus Flavivirus. It was first isolated from a sentinel monkey in Uganda in 1947. More recently, ZIKV has undergone rapid geographic expansion and has been responsible for outbreaks in Southeast Asia, the Pacific Islands, and America. In this review, we have highlighted the influence of viral genetic variants on ZIKV pathogenesis. Two major ZIKV genotypes (African and Asian) have been identified. The Asian genotype is subdivided into Southwest Asia, Pacific Island, and American strains, and is responsible for most outbreaks. Non-synonymous mutations in ZIKV proteins C, prM, E, NS1, NS2A, NS2B, NS3, and NS4B were found to have a higher prevalence and association with virulent strains of the Asian genotype. Consequently, the Asian genotype appears to have acquired higher cellular permissiveness, tissue persistence, and viral tropism in human neural cells. Therefore, mutations in specific coding regions of the Asian genotype may enhance ZIKV infectivity. Considering that mutations in the genomes of emerging viruses may lead to new virulent variants in humans, there is a potential for the re-emergence of new ZIKV cases in the future.


INTRODUCTION
Zika virus (ZIKV) is a Flavivirus transmitted through the bite of female mosquitoes of the Aedes, Culex, and Anopheles genera 1 . Zika was first isolated from a Rhesus monkey in 1947 in Zika Forest, Uganda 2 . In 1954, the first case reported in humans was described on the African continent 3 . ZIKV was also detected in Asia in 1966 and has remained restricted to this region for almost five decades 4

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In the early 2000s, ZIKV outbreaks were reported in regions of Southeast Asia, the Pacific Islands, and the Americas, with a proportional increase in infection rates. Outbreaks from Pacific Island and the Americas present higher numbers of cases 5 . In general, ZIKV had a higher epidemiological impact in tropical and subtropical countries once the mosquito Aedes spp. became a "cosmopolitan" vector, being widely distributed in tropical areas 1 . The first reported ZIKV outbreak occurred on Yap Island, Federated States of Micronesia, in 2007 6 . In 2013, ZIKV was associated with the development of Guillain-Barre syndrome (GBS) in the Pacific Islands of French Polynesia 7 . In 2016, Brazil recorded 440,000-1,300,000 suspected cases and 2,975 cases of ZIKVassociated microcephaly 8 , which led the World Health Organization to declare a worldwide state of public health emergency 9 .
Acute ZIKV infections, known as Zika fever, generally result in mild illness in adults. The viral incubation period varies from 3 to 10 days, and most patients do not require hospitalization 5 . Zika fever is clinically characterized by fever, rash, fatigue, conjunctivitis, arthralgia, headache, myalgia, and retroorbital pain. These symptoms manifest in about 20-25% of symptomatic individuals. However, a small percentage of cases have been associated with neurological disorders in neonates (mainly microcephaly), a condition later named congenital Zika syndrome (CZS) 9 .
Decades later, efforts of the scientific community to identify a vector control method, as well as vaccines and treatments to combat ZIKV infection, continue. Similarly, elucidating the pathophysiological mechanisms underlying this infection remain a challenge. During infection, host cells demonstrate morphological and molecular alterations 10,11 that eventually culminate in mitotic abnormalities and cell death 12 , leading to tissue loss and neurological injury 13 .
Many studies have shown that structural and nonstructural proteins are crucial components of viral pathogenesis 10,11 . However, it remains unclear which genetic factors of ZIKV may increase infection rate and virulence in humans. Here, we discuss the latest findings related to ZIKV genetic variants in terms of the infection process, cellular permissiveness, and tissue persistence.

ZIKV genome and life cycle
The ZIKV genomic organization is similar among members of the Flavivirus genus (Flaviviridae family) such as dengue virus (DENV), yellow fever virus (YFV), and West Nile virus (WNV) 14 . The ZIKV genome consists of 10,794 nucleotides in a single-stranded positive-sense RNA that encodes a polyprotein of 3,424 amino acids and 10 proteins crucial for the viral life cycle 10 . ZIKV RNA has two untranslated regions (UTRs) and a single open reading frame (ORF).
The 5′ and 3′ UTRs exhibit methylated nucleotides and nonpolyadenylated forms, respectively, forming a loop structure. Moreover, the 5′ and 3′ UTRs have an essential function in virus replication. The 5′ UTR mediates the "start" signal for reading through the CAP AUG type 1 structure. Meanwhile, the 3′ UTR has a poly(A) tail that functions as a "stop" signal for the final step in polyprotein processing 15,16 . The ORF encodes three structural proteins (E, prM, and C) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) 10 .
ZIKV must undergo attachment, entry, replication, and exocytosis to successfully infect human cells. ZIKV cell attachment is mediated by attachment factors such as negatively charged glycosaminoglycans 17 . These molecules retain viral particles on the cell surface, providing conditions for membrane fusion. The entry process occurs via ZIKV envelope protein E 18 , which interacts with entry receptors in the host cell, such as C-type lectin 19 and phosphatidylserine (PS) receptors 20 . These interactions cause conformational changes in the cell membrane and induce clathrin-mediated endocytosis, allowing the release of the viral genome into the cytoplasm 21 .
ZIKV protein E is the largest antigenic glycoprotein in flaviviruses and plays a role in adhesion, recognition, and fusion to the host cell. The dimeric structure of protein E contains an ectodomain with three domains: DI, DII, and DIII 18,19,22 . DI has a structural function in that it acts as a binder and chemical support for other domains. DII interacts and promotes fusion on the cell membrane through a loop-shaped structure located on the support loop with DI 18 . DIII is an immunoglobulin-like domain with the capacity to bind extracellular receptors 23,24 . Protein E contains a glycosylation site in an asparagine residue (Asn154), which may be associated with ZIKV virulence.
This pattern of N-glycosylation is conserved among DENV, YFV, and WNV. In DENV, glycosylation follows the Asn154 and Asn67 residues 19 . According to Wen 18 , N-glycosylated residues on protein E may enhance ZIKV infectivity by increasing the affinity of protein E to the entry receptors.
Once inside the cell, the low pH within the endosome enables the native state of protein E, which subsequently fuses to the endosome membrane and releases the viral RNA into the cytoplasm. Once in the cytoplasm, ZIKV undergoes particle assembly, followed by RNA replication and translation into viral proteins 25 . During maturation, newly assembled viral particles enter the endoplasmic reticulum (ER) and acquire PS. Viral particles then migrate from the ER to the Golgi complex where viral maturation occurs 26 . This process is mediated by the protein furin in the host, which cleaves the prM protein into the "pr" and "M" portions 22,25 . Finally, new mature ZIKV viral particles are released into the extracellular environment 22 .

ZIKV genotypes
To date, two major ZIKV genotypes have been identified: African and Asian. The African-ZIKV genotype has caused sporadic or recurrent infections in West African countries, with clinical manifestations of fever, conjunctivitis, and myalgia 3,27 . Nevertheless, the Asian-ZIKV genotype has circulated in Southeast Asia, the Pacific Islands, and the Americas, causing major outbreaks characterized by fever, arthralgia, conjunctivitis, CSZ, GBS, and ophthalmological anomalies 6,7,9,28. Through the timespan of these major outbreaks, it has been reported that the number of people with severe symptoms has increased as the Asian-ZIKV epidemic has disseminated among continents 29,30 .
The African-ZIKV genotype is subdivided into East African and West African strains. The Asian-ZIKV genotype is subdivided into Southwest Asia, Pacific Island, and American strains 31 . The African and Asian genotypes exhibit few different amino acid sequences 14 . Nevertheless, they share subcellular locations in host cells and protein function. ZIKV polyprotein from African and Asian genotypes are schematized in Figure 1. than the Asian genotype, with 18 nonsynonymous mutations and only one synonymous mutation. This may explain why both African strains remained restricted to the African continent 32 .
Taking into consideration the coding region sequences in the Asian genotype, Faria 35 and Ye 14 found great genetic similarities between ZIKV strains from the Pacific Islands and the Americas. However, these ZIKV strains exhibited a phylogenetic distance of decades compared to strains from Malaysia, which were later identified as a Southeast Asian strain 35 . In addition to phylogenetic differences, dissimilar nucleotides were also found between strains from the Pacific Islands and Malaysia 14,35 , indicating that these Asian strains do not share the same lineage. ZIKV strains from the Pacific Islands and Americas constitute only one lineage within the Asian genotype 14,35 . Among the lineages of the Asian genotype, Malaysian strains sampled in 1966 were the oldest 35 .
Ye 14 suggested that the American strain constitutes a new clade within the Asian-ZIKV genotype. Reports also indicated a common origin among ZIKV strains from Micronesia, French Polynesia, and Brazil during outbreaks in 2007, 2013, and 2016, respectively 14,31,36 . However, many reports indicate that there are variations among amino acids throughout the Asian-ZIKV genome, which can lead to viral adaptations ( Table 1). In this context, a study conducted by Kawai 31 evaluated the pathogenicity of Southern Asian, Pacific Island, and American strains in vitro and in vivo. It has been shown that the American strain induces strong pathogenicity 31 .
Other in vitro and in vivo studies have been conducted to elucidate the impact of nonsynonymous mutations on the Asian-ZIKV genome. Yan 40 demonstrated that the mutation S139N in prM of the Asian genotype may contribute to the development of CZS. This mutation in the prM protein was detected before the outbreak in French Polynesia, and it remained stable during ZIKV spread until the outbreak in the Americas in 2015 40 . In the viral protein NS4B, the substitution E2587D was observed in an Asian strain from China, in 2016 41 . Moreover, two substitutions in protein E (D67N and V473M) may have increased ZIKV replication and neurovirulence as well as its transmission during pregnancy and viremia after the American epidemic 42,43 . In an Asian isolate from a Thai patient in 2021, unique nonsynonymous mutations were detected in proteins E (A310E and E393K) and NS3 (H355Y) 24 . These findings suggest that after the outbreak in French Polynesia and before the outbreak in the Americas, ZIKV strains might have mutated and acquired higher infectivity.
Moreover, Li 44 proposed that proteins E, C, and prM contribute to Asian-ZIKV attachment, permissiveness, and cytopathic effects in human glial cells. In addition, NS2A recruits unprocessed proteins to be cleaved by NS2B/NS3 serine-protease at the E-prM-C site 45 . NS2A and NS4B also play a role in the assembly of new particles 11 . Haddow 36 demonstrated that ZIKV genotypes can exhibit different N-glycosylation sites, whereas Bos 46 found new glycosylated residues in protein E (I152, T156, and H158) in Brazilian ZIKV strains. Highly glycosylated residues may influence ZIKV attachment, entry, and fusion with host cells 46 .

Cellular permissiveness of ZIKV
ZIKV is known to infect different hosts, ranging from mosquitoes to mammals, as well as many cell types and tissues (Figure 2). Rat mesenchymal stem cells, mouse embryonic fibroblasts, murine macrophages, monkey kidneys, and mosquito larvae cells are some non-human cellular models that have been described as susceptible to ZIKV entry, replication, and release 47 .
The entry processes of African and Asian genotypes in humans share a highly conserved mechanism that requires clathrin-mediated endocytosis 21 . Among human cells, ZIKV is known to infect dermal fibroblasts 48 , fetal neurons 49 , primary Hofbauer 50 and mesenchymal stem cells 47 , epidermal keratinocytes 48 , fetal cortical astrocytes 49 , primary trophoblasts 50 , embryonic kidney cells 47 , and sperm cells 51 . Furthermore, some types of innate immune cells (such as primary monocytes and plasmacytoid dendritic cells) have been identified as permissive to viral infectivity 30 .   During ZIKV infection, the skin cells mediate an early innate immune response 48 . In vitro studies have evaluated the persistence of ZIKV infection in human skin cells in an attempt to understand the infection route following mosquito bites in mammalian hosts. Hamel 52 observed that human epidermal keratinocytes, dermal fibroblasts, and immature dendritic cells were fully permissive to French Polynesia isolates. However, Hou 26 showed that fibroblasts and epidermal human lineages did not display any differences in permissiveness, infection rate, and replication modes between isolates from Uganda and Puerto Rico.
According to Hou 26 , immunological cells did not demonstrate a difference in permissiveness between African-and Asian-ZIKV genotypes. However, Osterlund 53 observed differences in replication rates among these genotypes, although both showed great replication in human dendritic cells. Unlike the African genotype, viral replication in the Asian genotype is attenuated in human macrophages 53 . These findings suggest that the Asian-ZIKV genotype may use immunological cells as a viral reservoir.

Tissue persistence and viral tropism
During ZIKV infection, some cells and tissues may become viral reservoirs, contributing to the dissemination of Asian-ZIKV to nearby tissues. It was observed in vitro that both ZIKV genotypes have the capacity to infect human peripheral blood mononuclear cells 26 , indicating that these cells may act as an "entry door" for ZIKV spread.
Moreover, ZIKV-infected monocytes exhibited a quicker transmigration process than cell-free viruses on endothelial barriers in studies using in vitro, in vivo, and ex vivo models 30 . ZIKV-infected mast cells were also detected in situ in the placental tissue of pregnant Brazilian women 54 . These reports indicate that ZIKV-infected immunological cells might circulate throughout the host's blood tissue, promoting Asian-ZIKV spread and contributing to vertical transmission.
Asian-ZIKV has also been found to be transmitted by the sexual route. For instance, Rashid 55 observed the infection and replication of ZIKV (isolates from Puerto Rico) in primary human Sertoli cells in vitro, confirming ZIKV persistence in the reproductive tract and high cellular permissiveness. In addition, Matulasi 51 demonstrated that ZIKV isolates from French Polynesia infect reproductive and somatic testicular cells in vitro, as well as, replicates in human testes ex vivo. These studies suggest that American ZIKV strains can replicate in the male reproductive system.
In this context, ZIKV-infected sperm cells can also infect tissues of the female reproductive system during sexual encounters. Using an in vitro approach, studies have demonstrated that human primary endometrial 56 , Hofbauer, and trophoblast cells 50 are vulnerable target cells of American ZIKV strains. Thus, once ZIKV infects and replicates in reproductive tissues, it poses a risk at different stages of pregnancy.
Considering that neuronal progenitor cells and glial cells, which are crucial for neurogenesis, can also be targeted by ZIKV, the central nervous system (CNS) inflammatory process during gestation can significantly impact brain development. Hence, diverse studies have shown positive tropism between ZIKV genotypes and cells in the CNS. Li 57 demonstrated that both African and Asian genotypes can infect and replicate in neurons and glial cells in vitro. In parallel, in vitro astrocytes have a good tolerance for high viral load rates for both viral genotypes 49 .
However, according to Goodfellow 58 and Aguiar 59 , loss of cellular proliferation, neuronal migration, and abnormal extracellular matrix have been observed only in infections caused by the Asian genotype. In addition, Cugola 60 proposed that ZIKV strains that circulate in Brazil can trigger autophagy and apoptotic pathways, leading to cell death in cortical progenitor cells.
Thus, compared to African isolates, Brazilian ZIKV isolates exhibited higher neurotropism for neural cell lineages. These data led us to believe that the Asian genotype has greater virulence because its strains have accumulated large nonsynonymous mutations over the time of dissemination.

CONCLUSIONS
We gathered information on the genetic variants of ZIKV and their influence on the viral life cycle, cellular permissiveness, and tissue persistence. Based on the reviewed papers, we found that nonsynonymous mutations in the ZIKV genome may increase viral entry, RNA replication, particle assembly, and viral load. Considering that mutations in the genomes of emerging viruses may lead to new virulent variants in humans, this might be a possibility for the future re-emergence of new cases. Further in vitro and in vivo experiments are required to better evaluate these mutations.