Diversity and molecular characterization of insect-specific flaviviruses in mosquitoes (Diptera: Culicidae) collected in central and northern Argentina

Abstract

The genus Flavivirus comprises approximately 80 different viruses. Phylogenetic relationships among its members indicate a clear ecological separation between those viruses transmitted by mosquitoes, ticks, with no known vector, and insect-specific Flaviviruses. The diversity and phylogenetic relationships among insect-specific flaviviruses circulating in the central and northern regions of Argentina were studied by performing molecular detection and characterization of the NS5 protein gene in mosquitoes collected in Córdoba, Chaco and Tucumán provinces. Overall, 68 out of 1776 pools were positive. CxFV, KRV and CFAV circulate in the 3 studied provinces. Several mosquito species (Aedes aegypti, Culex bidens, Cx. dolosus, Cx. interfor, Cx. quinquefasciatus, Cx. saltanensis, Haemagogus spegazzini) were found infected. A wide circulation of CxFV was observed in the central-northern region of Argentina. CxFV strains detected in our study clustered with strains circulating in Santa Fe and Buenos Aires provinces (Argentina), and other countries such as Indonesia, Mexico, Uganda and Taiwan. The presence of these viruses in mosquitoes could play an important role from the public health perspective, because it has been shown that previous CxFV infection can increase or block the infection of the mosquito by other pathogenic flaviviruses.

Key words
ISF; Flavivirus; Culicidae; PCR; molecular characterization

INTRODUCTION

Members of the genus Flavivirus, family Flaviviridae are enveloped viruses that have a positive-sense single-stranded RNA genome. During recent years cell culture virus isolation and molecular screening of mosquitoes for flaviviral pathogens has resulted in the isolation and detection of numerous novel flaviviruses from various locations that seem to be specific for mosquito hosts. These viruses were previously defined as mosquito-only flaviviruses or insect-specific flaviviruses (ISFs), and have been widely detected in mosquitoes in different countries in the last decades (Blitvich et al. 2015, Calisher & Higgs 2018, Sang et al. 2003, Crabtree et al. 2003). The first virus characterized within the ISF group was cell fusing agent virus (CFAV), which was isolated from an Aedes aegypti cell line in 1974 (Stollar et al. 1975, Crabtree et al. 2003). Besides CFAV, many other ISFs, including Culex flavivirus (CxFV), Kamiti River virus (KRV) and Aedes flavivirus (AeFV), have been isolated and characterized (Blitvich et al. 2015, Calisher & Higgs 2018). CxFV is the most frequently reported ISF to date (Fernandes et al. 2018). Based on their phylogenetic and antigenic relationships, ISFs can be separated into two distinct groups. The largest group consists of the classical insect-specific flaviviruses (cISFs), such as CFAV, CxFV, and KRV. cISFs form a separate clade from the vertebrate pathogenic flaviviruses. The other ISFV group consists of the arbovirus-related or dual host affiliated insect-specific flaviviruses (dISFs) (Blitvich et al. 2015). dISFs are phylogenetically more similar to flavivirus vertebrate pathogens than to cISFs. These similarities raise the possibility that some of the dISFs might modulate arbovirus infection and transmission in a dually infected mosquito host; in addition, they could be useful in the development of potential flavivirus vaccines or reagents (Blitvich et al. 2015, Guzman et al. 2018). However, evidence is scarce, unclear, and in some cases contradictory (Bolling et al. 2011, Huanyu et al. 2012). Despite the search and detection of ISFs in different regions of the world, knowledge for South American countries is scarce. Like studies carried out in Brazil, in which CxFV was isolated from Culex sp. (Machado et al. 2012), CFAV in mosquitoes from the Amazon Region (Fernandes et al. 2018), newly Alfavirus (Tscha et al. 2021) and DNA similar to flavivirus from Ae. in Argentina (Bonica et al. 2021). The aim of the present study was to analyze the natural diversity and phylogenetic relationships among ISFs circulating in mosquitoes collected in the central and northern region of Argentina.

MATERIALS AND METHODS

Mosquitoes were sampled using CDC light traps baited with CO₂. Sampling was performed in the provinces of Chaco (Resistencia, Pampa del Indio and Monte Alto), Córdoba (Córdoba, Mar Chiquita) and Tucumán (San Miguel de Tucumán) from 2001 to 2012 (Table I). Between December 8, 2009 and March 18, 2010, 177 ovitraps were placed in different neighborhoods of Córdoba city. Collected adult mosquitoes and larvae were transported under refrigerated conditions to the Laboratory of Arbovirus (Institute of Virology “Dr. JM Vanella”, Faculty of Medical Sciences, National University of Córdoba), where they were taxonomically identified on top of a cold plate and stored at -20 °C (Darsie et al. 1985). Mosquitoes were sorted by species, sex, collection site, and date, and gathered in pools of a maximum of 50 individuals each. Each pool was homogenized with MEM in sterile mortars and pestles, centrifuged and stored at -80 °C until processing for virological studies.

Table I
Mosquito collection carried out in central and northern area of Argentina during 2001 and 2012.

Province	City of collection	Time period/Year	Pools Collected (n)	Total (n)
Córdoba	Córdoba	2001-2004	331	1259
		2006	36
		2008-2013	696
	Altos de Chipión	2008-2009	112
		2004-2006	84
Chaco	Resistencia	2001-2003	188	388
	Monte Alto	2001-2003	129
	Pampa del Indio	2009	71
Tucumán	San Miguel de Tucumán	2005	129	129

Molecular screening was performed using a generic flavivirus RT-Nested PCR that amplifies a 143-base pair (bp) fragment of the flavivirus NS5 region (Sánchez-Seco et al. 2005). To test whether the positive pools were the results of genomic RNA amplification or DNA forms, nucleic acid extracts were treated with RNAsa (Promega) before amplification. Then, positive pools were analyzed using a generic RT-Nested PCR technique for the amplification of an 860-bp fragment of the NS5 region of the Flavivirus genome for phylogenetic analysis (Vázquez et al. 2012). This region of the genome was chosen because it provides useful information for phylogenetic studies, allowing a clear molecular differentiation of the members of the genus Flavivirus; it is also one of the most representative regions in GenBank (Cook et al. 2012). All the amplified and purified fragments were sequenced in both directions through automatic sequencers (MACROGEN, Korea).

The genomic sequences obtained were initially identified by comparing them with all the sequences available in GenBank using BLASTn software (http://www.ncbi.nlm.nih.gov/BLAST/). The blast coverage percentages are greater than 98%. Multiple sequence alignments were generated with the relevant ISFV genomes available in GenBank. The final dataset contained 824 positions. Consequently, the construction of the maximum likelihood (ML) was completed under the GTR + I + G model for the NS5 gene. The ML tree was performed using MEGA XI software, with 1000 bootstrap replications (Kumar et al. 2018).

RESULTS

A total of 1776 mosquito and larva pools were analyzed, which were collected in the city of Córdoba (center of the province) between 2001 and 2012 (except for 2005) (n = 800); in the town of La Para (northeast of Córdoba province) in 2008, 2009 and 2010 (n = 129), in Resistencia city (Chaco province) in 2003, 2004 and 2009 (n = 143 ), in the town of Pampa del Indio (Chaco province) in 2009, 2010 and 2011 (n = 71); and in San Miguel de Tucumán city (Tucumán province) in 2005 and 2006 (n = 129). CxFV was detected in 2 of the 3 provinces. In Córdoba city, the 3 ISF species were detected: CxFV, CFAV and KRV, with CFAV being the predominant one, whereas in Chaco CFAV and CxFV were detected (Figure 1). Only one virus was detected in La Para (CxFV) as well as in Tucumán (KRV). The presence of CxFV, KRV and CFAV was not constant during the collection period (2001 - 2012) in the three provinces (Figure 1).

Figure 1
Geographical distribution and proportion of insect-specific flavivirus positive pools detected in 3 provinces of Argentina. a- Tucumán, b- Chaco, c- Córdoba.

We found that 68 of the 1776 pools were positive by generic RT Nested PCR using the short fragment (Sánchez Seco PCR). According to the BLASTn analysis, 14 pools were classified as CFAV, 4 pools as KRV and 19 pools as CxFV. Only 27% of the pools (10/37) were amplified by PCR using the long fragment of NS5 (Vázquez PCR) and then sequenced. The identity of all (100%) of these sequences of the viral species agreed between both fragments. CFAV was found mainly (85.71%) in Ae. aegypti (12/14); it was also found in a pool of Ochlerotatus scapularis and in another pool of Mansonia titillans, whereas KRV was found in a 3:1 ratio in Ae. aegypti and Culex interfor. On the other hand, 94.73% (18/19) of CxFV was found in mosquitoes of the genus Culex, except for one positive pool of Haemagogus spegazzini collected in Chaco province. It should be noted that ISFs were found in both adult male and female mosquitoes as well as in larvae. Detailed information on species distribution, geographic origin, collection year, sex and stage of development of positive pools are shown in Table II.

Table II
Summary of the insect-specific flaviviruses detected in different mosquito pools captured in Central and Northern Region of Argentina, during 2001 to 2012.

Pool	Host	Collection date	Stage	Province	City of collection	Virus	GenBank ID	Sex
LP2_045	Culex saltanensis	04/2009	Adult	Córdoba	La Para	CxFV2	KF316946	Female
LP2_047	Culex dolosus	04/2009	Adult	Córdoba	La Para	CxFV2	KF316947	Male
CHPI 79	Haemagogus spegazzini	12/2009	Adult	Chaco	Pampa del Indio	CxFV2	KF316948	Female
819	Culex interfor	12/2009	Adult	Córdoba	Córdoba	CxFV2	KF316939	Female
CA13	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV2	KF316941	Female
CA18	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV2	KF316942	Female
CA19	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV2	KF316943	Female
CA26	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV2	KF316944	Female
CA28	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV2	KF316945	Female
925	Culex interfor	01/2010	Adult	Córdoba	Córdoba	CxFV2	KF316940	Female
3_95	Aedes aegypti	02/2004	Adult	Córdoba	Córdoba	CFA1		Female
3_86	Culex interfor	02/2004	Adult	Córdoba	Córdoba	KRV1		Female
3_76	Culex quinquefasciatus	03/2004	Adult	Córdoba	Córdoba	CxFV1		Female
3_75	Ochlerotatus scapularis	03/2004	Adult	Córdoba	Córdoba	CFA1		Female
AR374	Aedes aegypti	03/2004	Adult	Córdoba	Córdoba	CFA1	DQ335466	Male
AR336	Aedes aegypti	03/2004	Adult	Córdoba	Córdoba	KRV1	DQ335465	Female
AR349	Mansonia titillans	04/2004	Adult	Córdoba	Córdoba	CFA1	DQ335467	Female
AR310	Aedes aegypti	04/2004	Adult	Córdoba	Córdoba	CFA1	DQ431718	Female
ChRS7	Culex quinquefasciatus	03/2009	Adult	Chaco	Resistencia	CxFV1		Female
ChRS20	Aedes aegypti	03/2009	Adult	Chaco	Resistencia	CFA1		Female
LP2_046	Culex bidens	04/2009	Adult	Córdoba	La Para	CxFV1		Female
CA21	Culex spp.	12/2009	Adult	Córdoba	La Para	CxFV1		Female
CA23	Culex sp.	12/2009	Adult	Córdoba	La Para	CxFV		Female
CHPI 158	Culex sp.	05/2011	Adult	Chaco	Pampa del Indio	CxFV1		Female
SMT105	Aedes aegypti	12/2005	Adult	Tucumán	San Miguel de Tucumán	KRV1		Female
SMT114	Aedes aegypti	11/2005	Adult	Tucumán	San Miguel de Tucumán	KRV1		Female
933	Culex interfor	01/2010	Adult	Córdoba	Córdoba	CxFV1		Female
942	Culex interfor	01/2010	Adult	Córdoba	Córdoba	CxFV1		Female
cba_52	Aedes aegypti	01/2011	Adult	Córdoba	Córdoba	CFA1		Female
cba_43	Aedes aegypti	01/2011	Adult	Córdoba	Córdoba	CFA1		Female
cba_103	Aedes aegypti	02/2011	Adult	Córdoba	Córdoba	CFA1		Female
cba_104	Aedes aegypti	02/2011	Adult	Córdoba	Córdoba	CFA1		Male
cba_86	Aedes aegypti	02/2011	Adult	Córdoba	Córdoba	CFA1		Female
cba_87	Aedes aegypti	02/2011	Adult	Córdoba	Córdoba	CFA1		Male
cba_132	Culex saltanensis	03/2011	Adult	Córdoba	Córdoba	CxFV1		Female
L897	Aedes aegypti	01/2010	Larvae	Córdoba	Córdoba	CFA1		Female
L497	Aedes aegypti	01/2010	Larvae	Córdoba	Córdoba	CFA1		Female

• ¹Small segment sequenced is showed in Supplementary Material – Appendix 1, ²Large Fragment sequenced, CFAV = Cell Fusing Agent virus, CxFV = Culex Flavivirus virus, KRV = Kamiti River virus.

To analyze the phylogenetic relationships between CxFV and other ISFs, we used the long NS5 Flavivirus fragment. The analysis revealed two well-differentiated groups, one including viruses associated with Aedes spp. and Ochlerotatus spp., and the other including viruses associated with Culex species. The latter group includes Quang binh (QBV), Calbertado (CLBV), Spanish Culex flavivirus (SCxFV), Nakiwogo (NKV) and CxFV. The group associated with CxFV is the most heterogeneous and diverse one, and shows a clear separation into two subgroups at the host species level: one subgroup that is not clearly defined due to the scarcity of samples and the lack of details of the identified species (subgroup 1) and the other composed of Culex pipiens, Culex quinquefasciatus and Culex tritaeniorhynchus (subgroup 2) (Figure 2). CxFV strains detected in our study clustered with strains circulating in Santa Fe and Buenos Aires provinces (Argentina), and other countries such as Indonesia, Mexico, Uganda and Taiwan.

Figure 2
Phylogenetic tree estimated using the Maximum Likelihood method and GTR +I+G model. This analysis involved 68 nucleotide sequences. The final dataset contained 824 positions. Evolutionary analyses were conducted in MEGA XI. The tree was constructed based on the NS5 target gene of mosquito samples positive for Culex flavivirus and ISF sequences from GenBank. This figure represents the Maximum Likelihood cladogram; bootstrap values are superimposed to indicate nodal support

DISCUSSION

Recently, several ISFs have been sequenced, characterized, and identified in arthropods (Bonica et al. 2021, Guarido et al. 2021, Tscha et al. 2021). In this study, we aimed to investigate the presence of ISFs in mosquitoes and to analyze the natural diversity and phylogenetic relationships among them in the central-northern region of Argentina.

The temporal analysis of CxFV, KRV and CFA in Córdoba city provides evidence of an erratic maintenance, with the occurrence of presence-absence events and with a permanence of the genetic identity of the circulating CxFV over time. This is consistent with the seasonal fluctuations detected in the Culex mosquito populations in the temperate regions of Argentina. The absence of this virus during certain periods could be explained by the low number of mosquitoes collected and analyzed.

The screening performed using Sánchez Seco PCR for Flaviviruses (Sánchez-Seco et al. 2005) allowed us to identify the activity of three ISF species, CFAV, CxFV and KRV, in central-northern Argentina. Although only 10 pools belonging to CxFV were amplified using Vázquez PCR technique (Vázquez et al. 2012), our results indicate 100% of coincidence between the short and long fragments of the NS5 gene. This result shows that this PCR screening technique is an appropriate tool for surveillance and identification of circulating flaviviruses. The NS5 gene is widely used as target for molecular characterization and identification of flaviviruses, providing useful information about phylogenetic relationships (Sánchez-Seco et al. 2005, Vázquez et al. 2012, Fang et al. 2018, Talavera et al. 2018). The obtained sequences strongly suggest the presence of other still not isolated putative viruses; this was the case of OcFV, which was isolated after the detection of field sequences (Calzolari et al. 2012, 2016).

The group of ISFs is highly divergent within the genus Flavivirus and shares a common ancestor with all the other members, including those capable of infecting mammalian hosts. A possible explanation for the origin of ISFs is the existence of a co-divergence between vectors and viruses; this hypothesis is supported by the virus-vector association pattern observed in this work (Figure 2). In agreement with other studies (Huanyu et al. 2012, Bittar et al. 2016), there is a clear divergence between Clade 1: ISFs associated with mosquitoes of the genus Aedes / Stegomyia (CFAV, KRV, AeFV and Spanish Ochlerotatus flavivirus (SOcFV)) and Clade 2: ISFs associated with Culex (CLBV, QBV, CxFV, SCxFV). ISFs would have undergone multiple introductions with frequent vector changes and occasional genetic reassortments, particularly in CFAV. However, there are no studies to support that CxFV was generated by codivergence between these viruses and their vectors (Cook et al. 2009, Bittar et al. 2016, Chatterjee et al. 2021). As expected, based on previous evidence, CFAV (Clade 1) was found in mosquitoes of the genera Mansonia and Ochlerotatus which, according to the phylogeny of mosquitoes, are more related to the genus Aedes / Stegomyia than to Culex (Cook et al. 2009).

CxFV primarily infects globally distributed mosquito species of the genus Culex, which are vectors for pathogenic flaviviruses like WNV, SLEV, and Japanese encephalitis virus (JEV) (Bittar et al. 2016). CxFV has a greater diversity of reservoirs, which might influence its phylogeny (Fernandes et al. 2018, Miranda et al. 2019). This is the first record of CxFV in the mosquito species Haemagogus spegazinii; the incorporation of this new record increases the diversity of reservoirs in this viral species. This CxFV strain was grouped in subgroup 1 (Fig. 2). The phylogenetic tree shows two clades: the first one is associated with Asian and USA strains, where Cx. pipiens is the main reservoir. However, other mosquito species, such as Culex tritaeniorhynchus and Anopheles sinensis (Liang et al. 2015), have been reported infected with CxFV belonging to the first clade (Asian/USA; Fig. 2). The second phylogenetic group of CxFV has been reported mostly in the Americas and Africa, with Culex quinquefasciatus being the main reservoir (Goenaga et al. 2014, Bittar et al. 2016, Liang et al. 2015). The sequences detected in this study are more phylogenetically related to this group (Africa/Caribbean/Latin America; Fig. 2) than to the first group. Our findings agree with this previously reported phylogeographic separation (Goenaga et al. 2014, Liang et al. 2015). Our sequences clustered with previous detections reported for Argentina (Buenos Aires, Córdoba, Chaco and Santa Fe provinces) (Goenaga et al. 2014). Moreover, all these Argentine sequences are close to that detected in Mexico and Africa. A wide circulation of CxFV was observed in the central-northern region of Argentina (Figure 1). CxFV seems to have been introduced several times in the New World in association with Culex species, which could explain the distribution of the viruses detected in this work (Bolling et al. 2011, Cook et al. 2012, Fernandes et al. 2018).

Although viruses of the genus Flavivirus share complex antigenic relationships, they can be divided into four phylogenetic/ecological groups. These divisions largely reflect the selective constraints imposed on the viruses by vertebrate hosts, invertebrate vectors, and the associated ecologies. Some authors described the evolution and possible origins of individual flaviviruses, showing a correlation of ecological and epidemiological characteristics with their phylogenies and geographic dispersal (Gould et al. 2003, Calzolari et al. 2016, de Oliveira Ribeiro et al. 2021). Many of the phylogenetic lineages that define viral species diverged relatively recently; therefore, the subsequent dispersal and epidemiology of these viruses have been significantly influenced by increasing human population densities and activities, such as recreation, urbanization, land reclamation, transportation, and deforestation (Guzman et al. 2018). In the region, DNA sequences similar to flaviviruses have been found, but in years different from this study (Bonica et al. 2021).

It has been suggested that, since these viruses do not replicate in vertebrate cells, the main route of transmission and maintenance would be the vertical and venereal routes (Bolling et al. 2011, Cook et al. 2006, Lutomiah et al. 2007, Bonica et al. 2021). However, in this study this virus was detected in another species (Haemagogus spegazzini) for the first time, suggesting other possible modes of transmission that have still not been discovered.

In conclusion, this work has gathered most of the existing information on insect-specific Flaviviruses circulating in central/northern Argentina, their diversity, phylogenetic relationships, and maintenance dynamics. More specifically, CxFV, KRV and CFAV circulate in several mosquito species in central Argentina. The presence of these viruses in mosquitoes could play an important role from the public health perspective, because it has been shown that previous CxFV infection can increase or block the infection of the mosquito by other pathogenic flaviviruses.

ACKNOWLEDGMENTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This study was supported by PICT 2013, PICT 2016- 3283 and PICT 2018-01172 grants from Agencia Nacional de Promoción Científica y Técnica (Ministerio de Ciencia y Tecnología de Argentina) and Secyt-CONSOLIDAR – Universidad Nacional de Córdoba. This article does not contain any studies with human participants or animals performed by any of the authors.

SUPPLEMENTARY MATERIAL

Appendix S1.

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Publication Dates

• Publication in this collection
  21 June 2024
• Date of issue
  2024

History

• Received
  18 Apr 2023
• Accepted
  28 Feb 2024