Experimental transmission of Zika virus by mosquitoes from central Europe

A Heitmann 1 2 , S Jansen 1 2 3 , R Lühken 1 , M Leggewie 1 3 , M Badusche 1 , B Pluskota 4 , N Becker 4 5 , O Vapalahti 6 , J SchmidtChanasit 1 3 , E Tannich 1 3 1. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany 2. These authors contributed equally to this work 3. German Centre for Infection Research (DZIF), partner site Hamburg-Luebeck-Borstel, Hamburg, Germany 4. Institute for Dipterology (IfD), Speyer, Germany 5. University of Heidelberg, Heidelberg, Germany 6. University of Helsinki and Helsinki University Hospital, Helsinki, Finland


Mosquitoes collected in Germany in 2016, including
Culex pipiens pipiens biotype pipiens, Culex torrentium and Aedes albopictus, as well as Culex pipiens pipiens biotype molestus (in colony since 2011) were experimentally infected with Zika virus (ZIKV) at 18 °C or 27 °C.None of the Culex taxa showed vector competence for ZIKV.In contrast, Aedes albopictus were susceptible for ZIKV but only at 27 °C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.
In 2015, Zika virus (ZIKV) emerged in Columbia and Brazil and spread rapidly across the American continent and the Caribbean, causing an epidemic with notable numbers of associated clinical cases of microcephaly and Guillain-Barré syndrome [1].Mosquitoes of the species Aedes aegypti and Ae.albopictus are considered the primary and secondary vectors of ZIKV [2].However, with transmission rates below 50%, their vector competence for ZIKV in the laboratory is low [3].The question therefore remains whether other common mosquito species such as Culex spp.play a role in the transmission cycle of ZIKV.The few studies performed so far have provided inconclusive results and suggested that at least Culex quinquefasciatus might be able to transmit ZIKV [4][5][6][7][8][9].In addition, for an assessment of the risk of possible spread to regions with temperate climate such as central Europe, information is lacking on ZIKV vector competence of mosquitoes under reduced temperature conditions (< 20°C).
Between 150 and 200 female mosquitoes 4-14 days-old were starved for 24 h before application of infectious blood meals containing ZIKV (strain ZIKV_ FB-GWUH-2016, GenBank KU870645, fifth passage) [13] at a final concentration of 107 plaque-forming units (PFU)/mL.Artificial feeding was performed using a Hemotek Feeder (Aedes spp.) or by cotton sticks (Culex spp.).Engorged females were incubated at 80% humidity at either 18 °C or 27 °C.Analyses for ZIKV were done 14 and 21 days post infection (dpi) for approximately 35 randomly selected females and twice the number for Ae.aegypti at 27°C.For salivation, mosquitoes were anaesthetised and the proboscises were inserted into cropped 10 µL filter tips containing 10 µL phosphatebuffered saline (PBS).After 30 min, tips were removed and saliva-containing PBS was analysed for the presence of infectious virus particles by measuring its cytopathic effect (CPE) on Vero cells within the following 8 days.ZIKV in the supernatant of cytopathic cells was confirmed by qRT-PCR using Real Star Zika Virus RT-PCR Kit (Altona diagnostics, Hamburg, Germany).In addition, bodies of all challenged mosquitoes, www.eurosurveillance.orgexcluding legs and wings, were analysed for ZIKV RNA by qRT-PCR.

Results
At 14 or 21 dpi, ZIKV RNA was detected in the bodies of all challenged mosquito taxa, with infection rates ranging between 3 and 72% in the species-temperature combinations with ZIKV-positive bodies.Infection rates and virus titres were substantially higher in Aedes species, with viral RNA copies ranging from 10 2 to 10 4 in Culex spp.and from 10 4 to 10 9 in Aedes spp.(Table ).
Virus load was generally higher at elevated incubation temperature (27 °C vs 18 °C).However, transmission of infectious virus particles as measured by CPE of Vero cells incubated with mosquito saliva was not detected in any of the Culex taxa.In contrast, saliva was positive for infectious virus particles in all Aedes species, but only at 27 °C incubation temperature.Interestingly, transmission rates at 21 dpi were similar in Ae. aegypti and Ae.albopictus from Germany but were substantially lower in Ae. albopictus from southern Italy (30% vs 13%).

Discussion
Culex species from central Europe are known as established vectors, able to transmit numerous viruses including West Nile, Sindbis and Usutu virus [14,15].The results presented here indicate that the three most common Culex taxa in central Europe (Cx.p. pipiens, Cx. p. molestus and Cx.torrentium) do not have vector competence for ZIKV.This is in agreement with results from other parts of the world including Italy [4][5][6][7]9], which all showed a low degree of compentence of the Cx.pipiens complex for ZIKV transmission.
The invasive mosquito Ae. albopictus is established in large parts around the Mediterranean Sea and is considered to be the main vector in Europe for autochthonous human infections with chikungunya and dengue virus [16].Aedes albopictus are regularly introduced into Germany as accidental cargo via road traffic from southern Europe [17].In the winter 2015/16, successful overwintering of the species was observed for the first time in southern Germany [18].The results presented here indicate that specimens of this overwintering population have considerable susceptibility to ZIKV, although only at elevated temperature of 27 °C.

Table
[19]eptibility and transmission rates of mosquitoes experimentally infected with Zika virus (n = 856) Moreover, the transmission rate in this overwintering population was substantially higher than in Ae. albopictus from the Calabrian region in southern Italy.Whether the difference in virus susceptibility between German and Italian Ae. albopictus populations is due to an ongoing process of adaptation to a new environment or to experimental conditions remains to be determined.Nevertheless, the susceptibility of European Ae. albopictus to ZIKV demonstrates the risk of arbovirus transmission associated with the establishment and ongoing spread of this invasive mosquito species in Europe.Of note, none of the tested Aedes populations were susceptible to ZIKV at 18 °C, which may limit the spread of ZIKV in central Europe to short summer periods with high temperatures.However, for a comprehensive risk assessment of ZIKV transmission in central Europe, further infection studies are needed at intermediate temperatures (e.g.21 °C and 24 °C) as well as with other common Aedes species such as Ae.vexans or the newly established Ae. japonicus[19].
GER: from Germany; IR: infection rate; ITA: from Italy; NA: not available; SD: standard deviation; T: temperature; TR: transmission rate.a Infection rate: number of ZIKV-positive mosquito bodies per number of fed females.b RNA copies were averaged over all ZIKV-positive mosquito bodies excluding the zeros of ZIKV-negative mosquito bodies.c Transmission rate: number of mosquitoes with ZIKV-positive saliva per number of ZIKV-positive mosquito bodies.d Not available: Mean viral RNA copies and transmission rate could not be calculated for the species-temperature combinations with no ZIKVpositive bodies.