Despite the nationwide serological evidence on the prevalence of WNV since 1976  and the establishment of Arbovirus monitoring system since 2000, there is a paucity of information about WNV vectors in Iran . Also, the distribution map of the globally spreading Lineage 1a of West Nile Virus as a serious public health problem still not clear at national level . The present study aimed to contribute towards WNV vectors identification and surveillance. This information can help to anticipate new endemic foci, monitor transmission dynamics and identify different mosquito vectors that could transmit the virus to animal hosts, reservoirs and people.
Our results confirmed the vast geographical distribution of Lineage 1a of WNV in mosquito vectors. For the first time, this study reported the infection of four Culicidae species namely Cx. pipiens, Cx. sitiens, An. stephensi and Cs. longiareolata with Lineage 1a WNV strains in Bandar Abbas County including the provincial capital in winter and spring seasons. Hormozgan Province had been previously designated to accommodate WNV seropositive horses . Ziyaeyan et al reported the circulation of WNV in people and mosquitoes in Hormozgan Province .
In agreement with reports on mosquito species which were found infected with WNV, we detected the virus in three mosquito genera namely Culex, Culiseta and Anopheles [35, 36]. It is stated that the main transmission cycle of WNV in nature involves Culex spp mosquitoes and birds. The species Cx. pipiens, is also known to be the main vector of the virus and responsible for its both enzootic and epizootic transmission cycles in urban areas of the United States , Europe and Russia [37, 38]. But the species Cx. sitiens can play a regional and complementary role in the transmission . The species Cx. sitiens was introduced as the new WNV vector in India . Although the species has shown relatively weak vectorial capacity for transmitting WNV NY-99 in laboratory, it is still important for being ornithophagic and prevalent in coastal areas. It can also build up its population very fast under favorable conditions . The species Cs. longiareolata is also known to transmit WNV and feed predominantly on birds and rarely on humans . However, so far there has been no report on An. stephensi involvement in WNV transmission to humans. Nonetheless, as an anthropophilic mosquito, the anopheline may contribute to the disease outbreak in seasons coinciding with its high population and peak activity when infected birds become its immediate feeding sources.
This study demonstrated the presence of WNV in Cx. pipiens in Bandar Abbas City and the neighboring Sagh Village as a primary health concern, In addition the viral infection of Cx. sitiens and Cs. longiareolata in Siahoo village and the surrounding plain areas was shown as a secondary hazard. More importantly, the present study reports for the first time the vertical transmission of WNV Lineage 1a in Cx. sitiens, An. stephensi and Cs. longiareolata, though the vertical transmission of the virus has been already shown in 14 mosquito species, including Cx. pipiens [41–50]. The vertical transmission of WNV through ornithophagic mosquitoes may not only stabilize the endemicity of the disease in Hormozgan Province, but also complicate its epidemiology and control measures.
The minimum filial infection rate (MFIR) obtained in the current study for Cx. pipiens s.l. is 2.8/1000, exactly similar to that reported by Anderson et al. . Other researchers have obtained MFIR values for Cx. pipiens s.l., which were between 0.52/1,000 and 4/1,000 [44, 47, 51]. The filial infection rate of 4.3/1000 in An. stephensi and 4.6/1000 in Cs. Longiareolata. are comparable to those stated by Baqar et al. . In our study, Cx. sitiens from Siahoo village has shown the highest MFIR equal to ≈ 45/1,000, since 1 of 3 pools was found infected with detectable virus. The discrepancy in vertical transmission rates between Cx. sitiens and other species may be attributed to its higher susceptibility to WNV infection as well as the small sample size collected and tested compared to other species. The overall MFIR value of WNV infected mosquito species in Hormozgan Province was equal to 3.1 infected progeny per 1000 individuals. This value is similar to both field and laboratory results reported by other researchers for Cx. pipiens, Cx. quinquefasciatus, and Cx. tarsalis [44, 47, 52]. Our results emphasize the findings that vertical transmission rates depend on various combinations of vector mosquito species, viral strains, and environmental conditions . Over the study area, which included different biotopes, the infected mosquitoes were detected only in autumn. On the other hand, except for Cx. sitiens, the vertical infection rates of field collected mosquitoes were less than 20–30 % which hardly satisfy the least required rates for viral persistence in nature . These evidences may indicate the importance of re-introduction of viral load by migratory birds in addition to vertical transmission of WNV to maintain the virus in Hormozgan Province. Both vertical transmission and re-introduction of WNV in the region not only support the detectable virus in autumn but also the virus persistence in overwintering female mosquitoes. The effective vertical transmission of WNV by ornithophagic mosquitoes corroborates the endemicity of WNV and complicates intervention measures in Hormozgan Province.
In plain sites of Siahoo Village, three mosquito species were found infected with the virus, whereas no infection was detected in mosquitoes trapped in slopes and mountainous areas. This can be attributed to more favorable climate conditions, and richer wildlife and vegetation diversity in Siahoo plain compared to elevations. In addition, WNV was isolated from mosquitoes of Bandar Abbas City in three consecutive seasons of autumn, winter and spring during 2015–2016 but not in summer. This may be due to the fact that every year from autumn to spring, Hormozgan Province provides wetland and coastal refuges for migratory bird flocks arriving from cold regions of Siberia and Europe, a number of which harbor and transmit WNV to local ornithophagic mosquitoes .
Based on phylogenetic analysis, only West Nile virus Lineage 1a was detected in infected mosquitoes of Bandar Abbas County in Hormozgan Province, while Lineage 1b strains were reported from Aedes caspius mosquitoes and from a patient with encephalitis in northwestern and central Iran respectively. [29, 56] However, the WNV isolated from Cx. pipiens in northern Iran was shown to be a Lineage 2 isolate. So far, there is no comprehensive map indicative of geographical distribution and epidemiological patterns of dominance for various lineages of West Nile virus in Iran. In fact, a nationwide surveillance is still lacking to determine the phylogenetic status of native and exotic WNV strains in the country. However, the involvement of birds especially the migratory ones as amplifying hosts and various mosquitoes as vectors in the natural cycling of WNV add more complications to distribution and transmission dynamics of the viral strains. This is particularly true given the locomotion of the components of epidemiologic triangle of WNV under rapidly changing global environment [57, 58]. Therefore, the phylogenetic variation of WNV strains at national level appears to be plausible. The presence of migratory birds harboring WNV from their origin in Europe and Russia in Hormozgan wetlands and coastal areas over 3 seasons of the year may stand behind the similarity of the strain isolated in this study with those reported from Russia, Italy and Spain. By the same token, the same argument can be put forward for the presence of WNV Lineage 1a in Dubai, United Arab Emirates , for their neighborhood to Hormozgan Province and shared commonality. (Fig. 2)
West Nile Lineage 1a is the most serious strain for its association with encephalitis epidemics as well as epizootics of high mortality to birds and horses in many parts of the worlds.  Although, no WNV outbreak has been reported in Iran, the serological studies have already confirmed its presence in humans, equines and birds in 26 provinces of Iran. . Therefore, public health officials in Iran should pay more attention to vector control measures to reduce the risk of WNV epidemics in southern region of Iran.