Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia

Dengue virus infection has been an important impact on humans over the last several years, with an estimated 50 million dengue infections and an average of 1 million cases reported annually in more than 100 countries in tropical and subtropical regions [1‐5]. This mosquito-borne flavivirus causes a wide spectrum of clinical manifestations in humans, which include an acute self-limited flu-like illness known as dengue fever (DF). DF is characterized by headache, myalgia, arthralgia, retro-orbital pain and sometimes maculopapular rash. Dengue haemorrhagic fever (DHF) is a severe illness documented by haemoconcentration (haematocrit increase by 20%) and evidence of plasma leakage such as pleural effusion and ascites as the major pathophysiological features. In some patients, DHF may progress to hypovolemic shock (Dengue Shock Syndrome, DSS) with circulatory failure [2, 6‐8].

Dengue virus (DENV) is an enveloped virus with a positive sense ssRNA of about 11 kb coding a single open reading frame for three structural proteins, core (C), pre-membrane/membrane (prM/M) and envelope (E), and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Based on serological analysis, DENV can be differentiated as four distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4), each one with the capacity to infect and cause even the more severe manifestation, although some serotypes have been isolated more frequently in DHF epidemics. On the other hand, evolution studies and molecular epidemiology using nucleotide sequences from the DENV genome have demonstrated the occurrence of genotype clades within each serotype [9‐28]. For this reason, genetic characterization of DENV has become a critical issue for understanding epidemic patterns of viral spread. At the same time, the important role of DENV itself in disease severity has also been proposed rather than the immune enhancement developed after subsequent infection with heterologous serotypes [1, 7, 29]. The increase in virus transmission over the last 50 years has possibly increased its adaptive potential. In addition, host factors such as the age, race, presence of non-neutralazing cross-reactive antibodies and possibly chronic diseases could act as selective pressures, resulting in more virulent genotypes that may be associated with DHF/DSS [9, 17, 29‐34].

Four DENV serotypes have been involved in Colombian epidemics, although DENV-1 and DENV-2 have the higher circulation rate since 1971[5, 6, 21]. Moreover, since the first case of DHF in Colombia at the end of 1989, these two serotypes have been associated with severe disease. To date, DENV-1 falls into five clades designated as genotype I (Southeast Asia, China and East Africa), genotype II (Thailand), genotype III (Malaysia), genotype IV (South Pacific) and genotype V(America, Africa). Additionally, the existence of lineages with distinctive geographical and temporal relationships had been suggested [12, 20, 26, 28, 35‐38]. Due to the importance of DENV in public health, the particular goals of this research were to reconstruct the phylogenetic history of DENV-1 and to date the phylogenetic tree using isolation time as calibration points to establish date of introduction of virus and rate evolution patterns of virus in Colombia.

Emerging and re-emerging diseases have become a public health major concern in developing countries, where dengue is perhaps the most important vector-borne viral disease in terms of morbidity. In Colombia, DF and DHF had been associated to the four DENV serotypes with DENV-2 and DENV-1 predominating since 1971 after the re-appearance and spread of Aedes (Stegomyia)aegipty[6]. DENV-3 circulated for a short time in 1975 and then it was not detected until 2002 when re-introduction occurred probably from Venezuela [27]. DENV-4 has been detected sporadically every year since 1984, when it was involved in several DF cases.

The huge genetic diversity of DENV has been vastly documented, starting perhaps with the Rico-Hesse proposal of different "genotypes" comprising serotypes 1 and 2 [10], following by several studies and genotype definition of DENV-3 and DENV-4. In this way, five different genotypes has been previously defined for DENV-1 (genotypes I to V) suggesting a significant genetic variation. In fact, various lineages had been proposed based on time-spatial clustering and clade distribution [26, 28, 35‐38]. In the present study, 74 Colombian DENV-1 sequences were analyzed to try to reconstruct the phylogenetic history of the virus in this country. Different genome regions have been used to infer DENV phylogeny including those with short fragments [10, 27, 28]. Here we employed a sequence from the carboxi terminal of the envelope (E) protein which has demonstrated to provide a useful phylogenetic signal to define genotype clustering [26]. It is important to note that the better resolution of evolutionary patterns should be obtained from complete genomes. However, it was not possible to obtain largest fragments from the oldest isolates, probably because of RNA degradation across the time. As expected, all strains were clustered with those from Brazil, Paraguay, Argentina, and different Caribbean Islands, corresponding to the formerly named genotype V (America/Africa), showing a well supported clade clearly separated from the others genotypes. Colombian strains DENV-1/CO/261_Atlantico/1978, DENV-1/CO/263_Choco/1979 and DENV-1/CO/150_Choco/1979, were separated from the remaining isolates and appeared closer to those from the Caribbean islands, which represent the entrance of serotype 1 into the Americas. It was reported for the first time in 1977 in Jamaica and rapidly spreading to the Antilles including Cuba, Antigua & Barbuda, Aruba, Bahamas, Barbados, Curaçao, Dominica, Grenada, Guadaloupe, Guyana, Haiti, Martinique, Montserrat, Puerto Rico, St. Kitts, St. Martin, St. Vincent and the Grenadines, Trinidad, Turks and Caicos, and the Virgin Islands [5]. In 1978, DENV-1 was implicated in large mainland outbreaks perhaps occurring at the same time in Colombia, Venezuela, Surinam, French Guyana, and eventually Centro America and Mexico. In Colombia, DENV-1 was isolated between 1977 and 1978, so the strain DENV-1/CO/261_Atlantico/1978 represents perhaps the first virus entrance to the country. It rapidly spread until the next isolation in Choco (DENV-1/CO/263_Choco/1979 and DENV-1/CO/150_Choco/1979) and then it fades away (or at less it was not reported) probably displaced by DENV-2 (maintaining DENV-1 in a silent low circulation) until 1985 when it established in different localities. It is important to note that even with the mobility between countries and increasing opportunity of viral introduction, only one DENV-1 genotype is circulating in America, different to DENV-2 and DENV-3 of which at least 2 genotypes has been detected (America/Asia genotypes and I/III genotypes respectively) suggesting perhaps, dissimilar patterns of viral spread and transmission between DENV genotypes and even different adaptation capacity.

Many researchers have categorized DENV in non official taxonomic levels beneath genotype, based specially in cladal distribution or geographical clustering. Circulation of these "lineages" has been particularly defined for DENV-1 in India, where at least 4 different lineages had been proposed (India-1 close to American strains, India-2 related to Singapore 1993 isolate, India-3 in south India and India-4 from Delhi and Gwalior) [26, 28]. In our study, a remarkable cladogenesis event occurs around 1992 according to the molecular clock, generating two well supported clades corresponding to putative Colombian DENV-1 lineages. Despite the eco-epidemiology similarities between Colombia and neighbor countries were dengue is a major concern, lineages have not been previously demonstrated for DENV-1. In fact, according to ML phylogeny, most of the American strains (Argentina and Brazil) correspond to the lineage-1, leaving the lineage 2 restricted to Colombia. Although geographic distribution of these lineages is not clearly delimitated, it is evident that they are evolving independently and most likely in parallel at the same localities.

Despite the emergence and rapid diversification of DENV has been a matter of special concern, precise mechanisms of evolution remain unclear [45‐50]. It is a fact that human RNA viruses including Influenza, HIV, Coronavirus, etc., have particularly increased mutation and evolution rates mostly because of the lack of proofreading activity of RNA-dependent RNA-polymerase [51, 52]. Nevertheless, arthropod-borne viruses (Arboviruses) have demonstrated slower mutation rates comparing with those infecting directly human host, probably because of the trade-off effect occurring when the virus is obligated to adapt alternatively into the invertebrate vector and vertebrate host [51]. This resulting constrain has been experimentally assessed in vivo to Venezuelan Equine Encephalitis [52] and in vitro to DENV [51] demonstrating that fitness improves when virus specialize in a single cell line but decreases in virus undergoing alternative passages in different cells. In view of that, over all DENV mutation rates have been previously inferred, ranging from 4.55 × 10^-4 (DENV-1) to 9.01 × 10^-4 (DENV-3) [19]. In the present study, we found a mutation rate of 8.58 × 10^-4 substitutions per site, per year, suggesting faster evolution rates for Colombian strains, perhaps because of the high transmition occurrence especially in hyperendemic areas, where virus replicates in several human hosts, reducing the constraining effect occurred in the vector. However, this high mutation rate does not necessarily reflect a fitness advantage or a successful adaptation process. Actually, positive selection for DENV seems to be serotype/genotype dependent and even more, protein specific. In fact, envelope (E) protein apparently exhibits some adaptation evidence in DENV-3, DENV-4 and various DENV-2 genotypes, but not for DENV-1, strongly suggesting a purifying selection pressure, at least over this gene. Nevertheless, further studies have to be done to try to understand the adaptation process in DENV.

On the other hand, although mostly of Colombian strains belong to the genotype V, there is an isolate, DENV-1/CO/267_Valle/1983 placed into genotype I near to Asia, China and East Africa strains. The ML tree show this strain close to DENV-1/JP/Mochizuki/1943, a strain considered extinct. Since we do not have this virus as reference in our laboratory, we can discard cross contamination during the assay. Moreover, the presence of this virus could be explained based on the migration process occurred from Asia to America, officially starting to Colombia by 1929, and sustained until the mid XX century [53]. Thus, establishment of Asian colonies increased visitors and perhaps favored the entrance of viral strains. We can speculate that those viruses did not fit to the new environment and the adaptation events were constrained because of the selective pressures including different vectors and human immune response.

According to natural history of DENV, evolution events could bring new genetic variants and eventually increase the severity of disease. Although pathogenic markers remain unclear, hemorrhagic features on some Asian DENV-2 genotypes have been demonstrated and Asian derived DENV-3 genotypes associated to dengue fever and dengue hemorrhagic fever have been reported in Brazil [25]. Moreover, changes in clinical manifestation of disease (atypical dengue) such as viscerotropism or encephalitis may respond to the circulation of new DENV lineages with increased pathogenic potential. Consequently, epidemiological programs should include not just virological diagnosis but genotype surveillance too.

This study shows in a defined time-scale, not just the first entrance of DENV 1 in Colombia, but also the viral evolution process in a highly endemic area. As a major conclusion, only one genotype of DENV 1 has been circulating since the first epidemic reports in the continental area. Nevertheless, two different lineages have been evolving fast since the earliest 90's according to molecular clock. As these evolution events may derive in a marked pathogenic potential, surveillance programs should include molecular methodologies. In fact, unusual presentation of disease currently reported by local health care institutions may be correlated to this evolution process. Further analyses by using at least complete E gene should be done to corroborate our results.