Genomic studies of envelope gene sequences from mosquito and human samples from Bangkok, Thailand

Dengue virus (DENV) is a single-stranded positive-sense RNA virus belonging to genus Flavivirus of family Flaviviridae (De la Guardia and Lleonart 2014). This virus is the causative agent of dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), which is transmitted by infected female Aedes aegypti mosquitoes and seasonally epidemic in Thailand. In 2012, for example, total numbers of DF, DHF, and DSS cases were 39,392 (61.5 cases per 100,000 people), 37,798 (59/100,000), and 1321 (2.1/100,000), respectively (Corbel et al. 2013). DENV comprises a set of four closely related but genetically distinct serotypes (DENV1–4). These four serotypes show 25–40% variation based on amino acid sequences (Thai et al. 2012). Additional variation is present within each serotype, namely, 6–8% at nucleotide and 3% at amino acid levels, thereby giving rise to a diverse set of genotype lineages (Thai et al. 2012). Because of the nature of RNA viruses, DENV is composed of populations of closely related sequences, known as quasispecies, that display genetic variation relative to their master sequences (Kurosu et al. 2014).

The DENV genome is a single 11-kb RNA strand coding for capsid, membrane, and envelope (E) proteins and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Qi et al. 2008). Of these 10 proteins, the E protein with its higher sequence heterogeneity is most frequently used to study DENV variation. The E protein consists of three functionally different structural domains (EDI–III). Because it is mainly involved in cell receptor binding, the EDIII domain is the focus of the present study. As the primary target under immune selection pressure, EDIII is the most heterogeneous region and is influenced by positive selection (Chao et al. 2005). Consequently, the variation in the EDIII region has been exploited for the characterization of dengue virus evolution in both human hosts and mosquito vectors (Thai et al. 2012; Kurosu et al. 2014; Lin et al. 2004; Wang et al. 2002).

Several previous studies have revealed that DENV isolated from different infection phases, patients, or hosts shows different levels of variation. For example, the mean diversity of DENV3 existing in human hosts and mosquitoes was found to be 0.38% (ranging from 0.15 to 0.59%) and 0.21%, respectively (Lin et al. 2004). In a study of DENV1-infected patient plasmas, however, intra-host variation as low as 0.0072 was observed (Thai et al. 2012). In 2014, DENV2 isolated from samples collected from acute patients from several provinces of Thailand showed some variations, with an average diversity of 0.145 in primary infections and 0.020 in secondary infections (Kurosu et al. 2014). Compared with the amount of information collected from human hosts, especially those isolates circulating in Thai DENV patients, knowledge of genetic variation in DENV2 obtained from A. aegypti mosquitoes, the primary transmission vector, is still limited.

In this study, we used clonal sequencing to identify the sequence variation of DENV2 isolated from Aedes mosquitoes comparing to that found in DENV2 isolated in 2010 from six dengue patients at the Hospital for Tropical Diseases, Bangkok, Thailand. The results of this comparative study of genetic variation between humans and mosquitoes may have implications for DENV evolution, overall fitness during viral transmission, and pathogenesis.

Flaviviruses, including DENV, are RNA viruses known to exist within hosts as a mixture of genetically related variants. When undergoing replication, these viruses may generate new progeny having nucleotide and amino acid mutations relative to the master sequence. A correlation is hypothesized to exist between these variations and viral pathogenesis as well as viral evolution (fitness) within and between hosts (Lin et al. 2004; Kurosu 2011; Jerzak et al. 2005). In this study, we therefore compared both inter- and intra-host genetic diversity of the EDII–III region of DENV2 from mosquito and human samples.

Six human plasma samples and two (mos56 and mos275) of four collected mosquito pools displayed 346-bp PCR products specific to the DENV2 E gene target region. The sequence populations represented in these human and mosquito samples were then identified by clonal sequencing. In contrast to a previous study of human plasma samples (Wang et al. 2002), we found a higher number of silent than non-silent mutations in the 83 sequences analyzed. None of the clones had a stop codon in this region.

Comparison of DENV2 sequences obtained from humans and mosquitoes revealed a slightly lower diversity (inferred from the overall mean diversity) in the mosquito samples (Table 1), a finding similar to observations of previous studies (Chao et al. 2005; Lin et al. 2004). From the spectrum of viral sequence variation found in mosquitoes, this genetic diversity was apparent even during a short incubation period of 8–12 days (Lin et al. 2004). In the future, determination of genetic variation within individual mosquitoes might be required, but the occurrence of genetic mutations during viral amplification of single mosquitoes during cell culture experiments will need to be considered (Craig et al., 2003).

As reported previously, DENV populations during secondary infection are more homogeneous (0.010–0.037% variation) than viruses isolated from primary infections (0.067–0.222%) (Kurosu et al., 2014). In the present study, human samples were all derived from secondary DENV infections (Setthapramote et al. 2012). Nevertheless, the mean diversity of DENV2 nucleotide sequences isolated from our human samples ranged from 0.058 to 3.47%, indicating higher variation between different samples than that previously reported (Kurosu et al. 2014; Lin et al. 2004; Wang et al. 2002). This higher variation, however, was inferred from mean diversity compared with master sequences. When individual human samples were considered, we found that the different human samples were infected with DENV2 strains belonging to different clades. Within-group mean distance was then calculated. More homogeneous populations were subsequently identified within each individual human sample (0.00–0.19) (Table 1). In regard to mosquito-derived samples, heterogeneous populations were identified (mean distance 0.89 for Mos275 and 1.69 for Mos56) from the individual samples resulting from a collection of mosquitoes in each pooled sample.

As most uncovered substitutions were silent, the variation in amino acid sequences was less heterogeneous. In particular, a mean diversity of 0.00–1.83 and 0.31–0.75 were calculated for human- and mosquito-derived samples, respectively.

Notably, amino acid substitutions observed in DENV from mosquito samples were different from those obtained from human samples (Table 3). This difference suggests that the amino acid substitutions occurred at random positions in both host types. Interestingly, substitution at H346Y, previously found in DENV2 from human plasma samples (Puiprom et al. 2010), was also observed in our study in both human- and mosquito-derived sequences. This occurrence suggests that the variation found at this position is not due to the natural escape of DENV2 from immune selection pressures occurring in the hosts; instead, it is probably derived from variation in DENV2 itself. This H/Y variant may thus have been transmitted to human hosts by infected mosquitoes.

Regarding the 13 and 10 nucleotide variants respectively cloned from human- and mosquito-obtained samples, the majority of sequences were identical to the consensus sequence. Hu2 variant which constituted the largest population and was mostly derived from two dengue patients (ID36 and ID47). Regarding mosquito-derived sequences, variant m1 was the master sequence among two mosquito samples (11 of 28: 8 from mos275 and 3 from mos56). Both of the largest populations, hu2 and m1, were classified in the same clade (C1) (Fig. 3). Several other minor variants were identified within this clade: hu1, hu3, hu4, hu5, m2, m3, m4, m6, and m7. Variants hu6 and hu7, derived from dengue patients ID37 and ID41, were the second and the third most abundant variants, respectively, while m8 was the second most abundant variant (8/28) isolated from mosquito samples. These variants from humans (hu6 and hu7) and mosquitoes (m8) were grouped in clade C2 together with minor variants m9 and m10 (Fig. 3). Among 14 sequences obtained from mos56, two major lineages, classified into clades C1 and C2, were responsible for the greater variation and higher mean diversity of mos56 (Tables 1, 2). The third clade (C3) contained only one variant from mosquitoes (m5) along with six human variants (hu8, hu9, hu10, hu11, hu12, and hu13) derived from the other two dengue patients (ID45 and ID50). Among six dengue patients, three major clusters (C1, C2, and C3), each representing samples from two dengue patients, were clearly identified. Of the six individual dengue patient samples, four (ID36, 45, 47, and 50) contained three variants (Table 2) and two (ID37 and ID41) were represented by identical sequence among all clones analyzed. Consequently, dengue virus quasispecies were present in all individual human samples except for ID37 and ID41. To further confirm the existence of dengue quasispecies in human samples, however, a higher number of clonal sequences need to be isolated from each sample.

To test for genetic fitness on the basis of positive selection, we used MEGA to determined dN/dS of each human and mosquito variant. Selection for variants m1 and hu2 was shown to be neutral (dN/dS = 1.00), suggesting that these two variants are the optimal DENV2 sequences circulating among the two hosts. Some other variants (hu1, hu3, hu4, hu5, m1, m2, m3, m4, m6, and m7) derived from these two variants were found to have minimal fitness, a conclusion inferred partly from the deleterious effect implied by a dN/dS lower than 1 (Table 2). We found that all variants in the C2 cluster were experiencing neutral selection (dN/dS = 1) (Table 2; Fig. 3). Similarly, members of the C3 cluster, namely, hu8, hu9, hu10, hu11, hu12, and hu13, also showed evidence of neutral selection (Table 2).

Even though the E-gene region is frequently used to study quasispecies, other structural and nonstructural genes, such as those encoding NS1, NS3, NS5 or the capsid, should not be excluded from consideration in future studies (Chao et al. 2005; Lin et al. 2004). To our knowledge, however, this is the first reported comparison of the genetic variation of DENV2 quasispecies circulating in humans and mosquitoes in Bangkok, Thailand. To verify the presence of quasispecies within these two hosts, a larger sampling of both humans and mosquitoes will be required in future studies.