|Year : 2018 | Volume
| Issue : 4 | Page : 305-309
First field evidence infection of Culex perexiguus by West Nile virus in Sahara Oasis of Algeria
S Benbetka1, A Hachid2, KE Benallal3, C Benbetka4, A Khaldi4, I Bitam5, Z Harrat3
1 Laboratoire d'Eco-épidémiologie Parasitaire et Génétique des Populations; Université Mohamed Bouguerra, Boumerdesse, Algeria
2 Laboratoire des arbovirus et des virus émergents, Institut Pasteur d'Algérie; Faculté de Médecine, Université d'Alger, Algeria
3 Laboratoire d'Eco-épidémiologie Parasitaire et Génétique des Populations, Boumerdesse, Algeria
4 Laboratoire des arbovirus et des virus émergents, Institut Pasteur d'Algérie, Algeria
5 Ecole Supérieure en Science de l'Aliment et des Industries Agroalimentaires d'Alger, Algeria
|Date of Submission||07-Nov-2017|
|Date of Acceptance||18-May-2018|
|Date of Web Publication||18-Apr-2019|
2 Route Petit Staouéli, Dely Ibrahim, Algiers
Source of Support: None, Conflict of Interest: None
Background & objectives: West Nile virus (WNV) is considered one of the most widely distributed arboviruses in the world which is transmitted by several mosquito species including the Culex genus. Culex pipiens is the major vector of this virus in Europe and USA whereas in African countries, other species such as Cx. perexiguus is considered as an important vector. This paper aimed to study the mosquito species involved in WNV transmission in Aougrout, one of the highly populated Oasis of Timimoun Province in Algeria where human WNV neuroinvasive diseases are prevalent.
Methods: CDC light-traps were installed in animal and human shelters for three nights. Collected mosquitoes were pooled and real-time PCR was performed to detect and identify WNV lineages 1 and 2 in the samples. Results: CDC light-traps collected 270 mosquitoes belonging to three genera. Culex genus was predominant with Cx. perexiguus as main species followed by Aedes and Anopheles genus. A total of 33 pools were tested; one pool containing Cx. perexiguus was found positive for WNV lineage 1.
Interpretation & conclusion: This study reports for the first time a WNV natural infection of Culex perexiguus in the study region indicating that species other than Cx. pipiens should be taken into consideration in WNV surveillance, especially in specific environments like Saharan Oasis ecosystem.
Keywords: Algeria; Culex perexiguus; Cx. pipiens; real-time RT-PCR; West Nile virus
|How to cite this article:|
Benbetka S, Hachid A, Benallal K E, Benbetka C, Khaldi A, Bitam I, Harrat Z. First field evidence infection of Culex perexiguus by West Nile virus in Sahara Oasis of Algeria. J Vector Borne Dis 2018;55:305-9
|How to cite this URL:|
Benbetka S, Hachid A, Benallal K E, Benbetka C, Khaldi A, Bitam I, Harrat Z. First field evidence infection of Culex perexiguus by West Nile virus in Sahara Oasis of Algeria. J Vector Borne Dis [serial online] 2018 [cited 2022 May 19];55:305-9. Available from: https://www.jvbd.org/text.asp?2018/55/4/305/256566
| Introduction|| |
West Nile virus (WNV) was first isolated from humans in the West Nile district of Uganda in 1937 and later from many other vertebrate hosts including horses, dogs, rodents and bats. Since then, its distribution has extended to all continents except the arctic regions,. This arbovirus belongs to Flavivirus genus within Flaviviridae family. It is an enveloped virus with single stranded positive sense 11 kb RNA genome coding for seven non-structural and three structural proteins. Molecular studies have allowed the classification of WNV into at least seven putative genetic lineages, although among them only lineages 1 and 2 are associated with human and animal disease. Furthermore, WNV has the potential to cause severe illness characterized by neurological disorders in some mammals such as horses and humans,.
The transmission cycle of WNV involves several bird species as primary hosts and mosquitoes as vectors. About 70 mosquito species have been identified as competent for virus transmission mainly among Culex, Aedes, Ochlerotatus and Anopheles genus,,. Human, horses and other mammals are considered as accidental dead-end hosts.
The principal vector of WNV in Europe and USA is Culex pipiens,, whereas in Africa and Middle East countries the most important vector is Culex perexiguus,,,,, In Algeria several studies and serological surveys between 1973 and 1976 have documented WNV circulation in human populations, predominantly in Sahara Oasis and steppe areas; however, data availability about the virus vector is limited. Indeed, WNV was first isolated in Algeria in 1968 from a pool of Culex mosquitoes in the District of Djanet, a Saharan Oasis in the southeastern part of the country. In 1994, >50 human cases and eight fatalities were documented during an outbreak of WNV encephalitis in Tinerkouk Oasis in the Province of Timimoun, in southwest of Algeria. Recently in the same province, a WNV meningo-encephalitis case was reported in Aougrout Oasis.
This paper aimed to study the mosquito species involved in WNV transmission in Aougrout, one of the highly populated Oasis of Timimoun where human WNV neuroinvasive diseases are prevalent.
| Material & Methods|| |
Study area and mosquito collections
Aougrout is a village of Timimoun Province, Department of Adrar located in the southwest region [Figure 1], about 1400 km from the capital city of Algeria (28° 42.789′N, 0° 19.881′E). This region is characterized by a Saharan climate; 14–15 mm rainfall throughout the year with a mean temperature of 26–27 °C and 14% of relative humidity. This area is known to harbour many wetlands and a great number of migratory birds.
The mosquitoes were captured using CDC light-traps near human habitations and animal shelters for three consecutive nights in September 2015. Live mosquitoes were morphologically identified using identification keys. They were pooled, with a maximum of 15 specimens per pool, by sex, species, collection sites and date. Mosquito pools were directly stored at –80 °C in the laboratory facilities until they were processed by real-time RT-PCR assay.
West Nile virus detection and differentiation
Each pool was crushed in 1 ml of Leibovitz's L15 medium (supplemented with 20% of FBS, 10% of tryptose phosphate, 1% of penicillin/streptomycin and 0.005% of fungizone) using pellet pestle motor KONTES (Sigma-Aldrich, USA). The homogenates were centrifuged at 10,000 rpm for 20 min at 4 °C. The supernatants were recovered and divided into three aliquots. Viral RNA was extracted from 200 μl mosquito homogenates using PureLink Viral RNA/DNA kit (Invitrogen, Thermo-Fisher Scientific, USA) according to the manufacturer's instructions. A rapid multiplex real-time RT-PCR was carried out as described earlier for simultaneous detection and differentiation of WNV lineages 1 and 2, with some modifications. Real-time RT-PCR was performed using SuperScript™ III Platinum® One-Step qRT-PCR kit (Invitrogen, ThermoFisher Scientific, USA). Briefly, reaction mix contained (per vial) 5 μl of sample RNA, 0.8 μM of each primer (F1: 5′-GTCTGACATTGGGTTTGAAGTTA-3′; R1: 5′-GTGATCCATGTAAGCCCTCAGAA-3′) and 0.4 μM of each probe (L1: FAM-AGGACCCCACATGTT-MGB; L2: VIC-AGGACCCCACGTGCTMGB), 2.5 μM ROX, 12.5 μl 2x reaction mix, 0.5 μl of SuperScript® III/Platinum® Taq mix and nuclease free water (final volume 25 μl). Amplification included a first reverse transcription step at 50 °C for 30 min, followed by 2 min at 95 °C for retro-translation (RT) inactivation and 40 cycles of 15 sec at 95 °C and 30 sec at 60 °C. PCR amplification was carried out on ABI 7500 real-time PCR system (Applied Biosystems ABI 7500, Foster City, USA). WNV lineages 1 and 2, RNA were used as positive controls. Additionally, each positive pool was tested again by another real-time RT-PCR assay targeting a conserved region of WNV lineages 1 and 2 as described earlier.
Isolation of live virus in cell culture
Culture media was decanted and 200 μl of mosquito positive pool homogenates were inoculated onto Vero cells. Cultures were incubated for 2 h at 37 °C and shacked gently every 15 min to allow virus adsorption. About 5 ml of maintenance medium (DMEM medium with 2% fetal bovine sera) was added to T-25 flask and cultures were incubated at the same temperature. Flasks were examined daily for the cytopathic effect (CPE). At Day 7 post-infection, supernatant was harvested and clarified by centrifugation at 2800 rpm for 20 min at 4 °C. A blind passage was done systematically. An aliquot of each passage was tested by real-time RT-PCR to confirm the presence or absence of WNV.
| Results|| |
A total of 270 mosquito specimens were collected, belonging to three genera; Culex, Aedes and Anopheles. Culex perexiguus was the dominant species (66.30%) followed by Aedes caspius (27.03%), Cx. pipiens (4.45%), Anopheles d'thali (1.85%) and An. rhodesiensis rupicolus (0.37%), as shown in [Table 1].
Of the 33 pools tested, pool number 15 containing Cx. perexiguus species was positive in both the real-time PCR assays, revealing WNV lineage-1 presence, with a delayed cycle threshold (Ct = 35, and Ct = 34, respectively). In order to discard a fluorescence artefact at the end of the real-time PCR, an agarose gel electrophoresis was run and the presence of specific-WNV band was checked (data not shown). The vector infection rate is estimated to 0.56% (number of positive pools/number of the females tested). Unfortunately, attempt to isolate virus from positive pool was inconclusive after two serial passages of one-week incubation.
| Discussion|| |
Mosquitoes are responsible for transmission of several pathogens, including parasites that cause malaria; viruses that trigger dengue, chikungunya, West Nile encephalitis; as well as worms that cause lymphatic filariasis; that have high morbidity and/or mortality. Among them, genus Culex comprising about 768 taxa, includes some of the most ubiquitous species as well as important vectors of human pathogens. In Algeria, six mosquito species are reported to be involved in the transmission of arboviruses including Cx. pipiens, Cx. modestus and Cx. perexiguus. A recent study has shown that Cx. pipiens circulating in Algeria with its three forms Cx. pipiens pipiens, Cx. pipiens molestus and the hybrid form Cx. pipiens/molestus could play a major role in WNV transmission. Additionally, the risk of autochthonous cases of other arboviruses such as dengue, chikungunya and zika virus remain significant, since their vector Ae. albopictus has been recently established in two high densely populated cities of Algeria, Oran and Algiers (unpublished data).
In this study, predominance of Cx. perexiguus in Aougrout is not a surprise since it is in accordance with the geology of this region as the main breeding sites in this region are formed by salty waters which take source from the groundwater. This species is reported in Spain and also from Morocco to India, and are mainly active in summer and autumn. In Middle East, this species was confused for longtime with Cx. univittatus, until 1999 when Harbach resolved this confusion and showed that Cx. univittatus is restricted to the temperate highlands of East and South Africa and Yemen. The detection of a positive pool for WNV lineage 1 by real-time RT-PCR highlights that Cx. perexiguus might have an important role in WNV transmission in the affected region where density of Cx. pipiens is low. Interestingly, the positive result was confirmed by two separate real-time RT-PCRs. Role of Cx. perexiguus as vector for WNV has been reported in Portugal, Italy and Spain even at low abundance in some cases,, and also in Egypt, Senegal and Israel. For instance, Cx. perexiguus is considered as the main vector of WNV in Egypt and Israel. Identification of WNV lineage 1 in Aougrout confirms past findings of lineage 1 circulation in Sahara Oasis. This well-known virulent lineage could have been the origin of the fatal outbreak of WNV that occurred in Tinerkouk, Department of Adrar in 1994. Moreover, the WNV lineage 1 has been reported in neighbouring countries such as Morocco and Spain, located on the same fly way of migratory birds crossing these regions between Africa and Western Europe. Unfortunately, virus isolation and attempts of sequencing the virus from this pool were not successful, probably due to the low viral load in mosquito samples as shown by delayed Ct in real-time PCRs.
In a recent study, experimental infection of Cx. pipiens collected from Timimoun Province showed that this species could also be an efficient vector of WNV and Rift Valley fever virus. To the best of our knowledge, this is the first evidence of Cx. perexiguus natural infection by WNV in Algeria and in Maghreb regions (Morocco, Algeria and Tunisia), as till date WNV has been only isolated from Cx. pipiens in Tunisia. However, in the Mediterranean context the presence of WNV in Cx. perexiguus is not uncommon. Culex perexiguus could have an important role in the transmission of WNV in these countries, especially in Saharan villages where predominant wetlands include saltwater marshes which favour its breeding. Furthermore, Cx. perexiguus which is mainly ornithophilic could sometimes feed on humans; thus this bridge behavior could foster spillover of WNV to humans in areas where migratory birds and humans share the same environment. The detection of this virus in September coincided with the high activity of this virus from August to October as reported in several studies.
The infection rate (IR) in the present study was very low compared to IR reported from Israel and Egypt, which is probably related to the low number of Cx. perexiguus collected in the traps during the study period. Attention should also be paid to Ae. caspius which is the second most prevalent species in the study since it has been reported to transmit WNV in Israel.
| Conclusion|| |
This study reports for the first time a WNV natural infection of Cx. perexiguus in Algeria and in Maghreb regions indicating that species other than Cx. pipiens should be also taken into consideration in WNV surveillance, especially in specific environment like Saharan Oasis ecosystem. Further studies are required to confirm the results by collecting and screening more specimens in different places and at different times in Saharan villages in order to design control strategies tailored to this specific ecosystem. Isolation of the virus is also necessary so as to better understand the phylogenetic relationship between WNV strains circulating in the region and patterns of their diffusion.
Conflict of interest
The authors declare that they have no any conflict of interest.
| Acknowledgements|| |
The authors thank the local Public Health Authorities, the Communicable Diseases Service at Health Ministry and the inhabitants of Aougrout for their collaboration and excellent support. The authors are also grateful to Cellule d'Intervention Biologique d'Urgence (CIBU) at Pasteur Institute of Paris, and Laboratory of Arbovirus and Imported Viral Diseases at Instituto de Salud Carlos III, Spain for kindly providing Vero cells and WNV positive controls, respectively. Authors thank Dr Jovita Fernández-Pinero and Dr Miguel Ángel Jiménez-Clavero from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain, for their useful comments. This study was financially supported by the Internal Research Program of Pasteur Institute of Algeria, Algeria.
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