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Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany

Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany The invasive mosquito species Aedes japonicus japonicus (Ae. japonicus) is widely distributed in Central Europe and is a known vector of various arboviruses in the laboratory, including flaviviruses such as Japanese Encephalitis virus or West Nile virus. However, the vector competence of Ae. japonicus for the recently emerging Zika virus (ZIKV) has not been determined. Therefore, field-caught Ae. japonicus from Germany were orally infected with ZIKV and incubated at 21, 24, or 27 °C to evaluate the vector competence under climate conditions representative of the temperate regions (21 °C) in the species’ main distribution area in Europe and of Mediterranean regions (27 °C). Aedes japonicus was susceptible to ZIKV at all temperatures, showing infection rates between 10.0% (21 °C) and 66.7% (27 °C). However, virus transmission was detected exclusively at 27 °C with a transmission rate of 14.3% and a transmission efficiency of 9.5%. Taking into account the present distribution of Ae. japonicus in the temperate regions of Central Europe, the risk of ZIKV transmission by the studied Ae. japonicus population in Central Europe has to be considered as low. Nevertheless, due to the species’ vector competence for ZIKV and other mosquito-borne viruses, in combination with the possibility of further spread to Mediterranean regions, Ae. japonicus must be kept in mind as a potential vector of pathogens inside and outside of Europe. Introduction clinical symptoms to severe diseases, including neonatal Zika virus (ZIKV) is an emerging mosquito-borne virus microcephaly and neurological disorders such as Guillain- within the family Flaviviridae that was first isolated from Barré syndrome . The mosquito species Aedes aegypti and sentinel rhesus macaques in Uganda in 1947 . After Aedes albopictus are considered the primary and sec- decades of silent circulation, unprecedented ZIKV epi- ondary vectors of ZIKV; however, a wide variety of other demics occurred in Micronesia, Polynesia, and, finally, in Aedes species have been identified as potentially suscep- the Americas in 2015; the hundreds of thousands of tible to ZIKV infection . Recent experimental studies human cases finally resulted in the announcement of a suggested that only Ae. albopictus might play a role in Public Health Emergency of International Concern ZIKV transmission in Central Europe, while common through the World Health Organization . Clinical courses members of the genus Culex are probably not impor- 5,6 associated with ZIKV infections can range from mild tant . However, north of the Alps, the Asian tiger mos- quito is currently established at only a few sites, with 7,8 relatively low abundance . By contrast, the invasive Asian bush mosquito Aedes japonicus japonicus (Ae. Correspondence: Anna Heitmann (heitmann@bnitm.de) Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany japonicus) is widely distributed in Central Europe and is German Centre for Infection Research (DZIF), Partner site Hamburg-Luebeck- currently established in at least 10 countries, including Borstel-Riems, 20359 Hamburg, Germany large parts of Germany . In 2008, the first invasive Full list of author information is available at the end of the article. These authors contributed equally: Stephanie Jansen, Anna Heitmann © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 2 of 6 spreading of Ae. japonicus in Europe was reported in Switzerland , and Ae. japonicus is now listed as one of the nine most dominant mosquito species in Switzerland. Shortly after its introduction in Switzerland, Ae. japonicus was first reported in Germany, followed by the estab- lishment of populations at several sites. In the Nether- lands and Belgium, mosquito control programs have been 9–12 initiated due to the massive Ae. japonicus populations . Ae. japonicus is a container-dwelling species, colonizing both natural (e.g., bamboo stubs and tree holes) and man- made (e.g., tires and barrels) breeding sites . Due to its tolerance of rather low temperatures, Ae. japonicus has a relatively long seasonal activity compared to other container-breeders . Ae. japonicus has an opportunistic feeding pattern with a preference for mammals, including humans, although avian host species have also been 15,16 reported . Thus, Ae. japonicus could potentially serve as a bridge vector for zoonotic arboviruses. The species is an experimentally proven vector of several flaviviruses, including Japanese Encephalitis virus (JEV), West Nile Fig. 1 Means and 95% confidence intervals for the ZIKV infection, 17–19 virus (WNV), and Saint Louis encephalitis virus ,as dissemination, and transmission rates as well as the transmission well as arboviruses of other families, such as La Crosse efficiency of Ae. japonicus from southwestern Germany as revealed by analyses of bodies (IR), legs (DR), and saliva (TR and TE) following virus (LACV, Peribunyaviridae) and Chikungunya virus 20,21 challenge of mosquitoes with infectious blood meals and incubation (CHIKV, Togaviridae) . Previous studies with an Ae. at 21 °C (n = 30), 24 °C (n = 29), or 27 °C (n = 21) for 2 weeks. Three japonicus population from southwestern Germany also independent trials were performed for each temperature. *Infection revealed a vector competence for JEV under laboratory rate (IR): number of ZIKV-positive mosquito bodies per number of fed conditions . females. **Dissemination rate (DR): number of mosquitoes with ZIKV- positive legs per number of ZIKV-positive mosquito bodies. In light of the continuing spread of Ae. japonicus in ***Transmission rate (TR): number of mosquitoes with ZIKV-positive Europe and the ongoing circulation of ZIKV in America, saliva per number of ZIKV-positive mosquito bodies. ****Transmission the aim of this study was to evaluate whether Ae. japo- efficiency (TE): number of mosquitoes with ZIKV-positive saliva per nicus has vector competence for ZIKV under climate total number of fed females conditions representative of tropical and temperate regions. Table 1 Calculation of the virus titers from bodies or legs Results for Ae. japonicus specimens from southwestern Germany To assess the suitability of the collected Ae. japonicus following challenge of mosquitoes with infectious blood for vector competence studies, a small number of speci- meals and incubation at 21 , 24, or 27 °C for 2 weeks mens were challenged with JEV in a preliminary study. In agreement with previous findings , the Ae. japonicus Temperature in °C Body titer, mean (SD) Leg titer, mean (SD) specimens from southwestern Germany were susceptible log10 RNA copies/ log10 RNA copies/ to JEV. The infection rate (IR) was 51.9%, with an average specimen specimen amount of viral RNA of 5.6 × 10 copies/specimen (n = 21 4.6 (1.0) 0.0 (0.0) 27). In addition to the previous experiment, we also 24 4.9 (1.5) 2.9 (2.9) investigated the transmission of infectious virus particles by analyzing mosquito saliva following incubation of 27 5.9 (1.8) 4.2 (4.2) infected mosquitoes at 27 °C for 14 days. The results indicated a transmission rate (TR) of 78.6%. Subsequently, mosquitoes were analyzed for ZIKV to 24.1% (7/29) at 24 °C and to 66.7% (14/21) at 27 °C. infection. Fourteen days post infection, ZIKV RNA was This pattern is also reflected in the amount of virus RNA present in the bodies of challenged Ae. japonicus at all of within the mosquito bodies, which increased from 1.2 × 4 6 the tested temperatures (Fig. 1). The relative numbers of 10 RNA copies/specimen at 21 °C to 2.6 × 10 RNA ZIKV-positive mosquitoes (Fig. 1) and the amount of viral copies/specimen at 24 °C to 6.4 × 10 RNA copies/speci- RNA (Table 1) increased with increasing incubation men at 27 °C (Table 1). Dissemination of the virus was temperatures. The IR increased from 10% (3/30) at 21 °C found in mosquitoes kept at 24 and 27 °C but not in Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 3 of 6 6,24–28 mosquitoes incubated at 21 °C. However, the averaged leg albopictus (18–77%) . Nevertheless, both species titers were substantially higher at 27 °C (6.4 × 10 RNA showed considerably higher TRs than Ae. japonicus; copies/specimen) than at 24 °C (8.4 × 10 RNA copies/ whereas Ae. vexans also has a low vector competence for specimen) (Table 1). This is also reflected by the detection ZIKV, with a TR between 2 and 7% . Likewise, the of infectious virus particles in the saliva of two mosquitoes transmission efficiency of 9.5% for Ae. japonicus at the kept at 27 °C, resulting in a TR of 14.3% (2/14) and a tropical temperature is lower than the known transmis- transmission efficiency of 9.5% (2/21). sion efficiency for Ae. aegypti (26%) under similar con- ditions . Previous vector competence studies with Ae. Discussion japonicus and various arboviruses were only performed 17–21 To the best of our knowledge, this is the first study on under tropical temperature conditions (i.e., 25–28 °C) . the vector competence of Ae. japonicus for ZIKV (see Therefore, it is unknown if the lack of virus transmission review by Epelboin et al. ), and our results show a low at lower temperatures is a general feature for viral transmission efficiency at high temperatures. The species transmission by Ae. japonicus or if this observation is is a known competent vector of a variety of arboviruses, specific for ZIKV. By contrast, the primary and secondary including flaviviruses (e.g., WNV, JEV, or Dengue virus), as vectors Ae. aegypti and Ae. albopictus can transmit ZIKV well as members of other virus families, including Peri- below 27 °C. Viable ZIKV virus particles were detected in 17,19–21 30 bunyaviridae (LACV) and Togaviridae (CHIKV) . the saliva of Ae. aegypti even at 20 °C . Therefore, the Over the last two decades, Ae. japonicus has successfully lack of transmission by Ae. japonicus is a species-specific invaded Central European countries as well as large parts observation and not a general pattern for ZIKV. of North America, and it is found primarily in areas with One explanation for the lack of ZIKV transmission by 8,13,14,22,23 predominantly temperate climate conditions . Ae. japonicus at temperatures below 27 °C might be a Ae. japonicus eggs are resistant to frost and desiccation. combination of higher virus replication rates and species- Furthermore, the seasonal activity of the species is longer specific, temperature-dependent effects on the mosquito than that of other container-breeding species. Due to microbiome or immune regulatory pathways . However, these attributes, Ae. japonicus has some developmental as shown before, vector competence is influenced by a advantages over native species that could affect mosquito three-way interaction between the vector population, the 32,33 population patterns as well as pathogen transmission in virus strain, and temperature . Only a suitable com- newly colonized regions. Likewise, it is of considerable bination of these factors allows the virus to replicate and interest to collect information on the vector competence disseminate to the salivary glands to enable transmission of Ae. japonicus for newly emerging viruses such as ZIKV, through the next bite. However, studies with a combina- with a special emphasis on the temperate climate condi- tion of one specific mosquito population with one specific tions of the species’ current distribution range. virus strain must be interpreted with caution. Even studies Previous studies described relatively low feeding rates of with the same combination of vector species and virus can field-caught Ae. japonicus using saturated cotton sticks or come to varying TRs or even to inconsistent results 19,21 feeding systems with chicken skin . The experiments regarding the species’ susceptibility to a virus. Studies presented here demonstrate that artificial feeding via performed with either field-caught Ae. aegypti or Ae. blood drops seems to be an efficient alternative for field- albopictus populations from different sites within one caught Ae. japonicus mosquitoes, resulting in a feeding country revealed clearly varying TRs for the same ZIKV 34,35 rate of 75%. The experimental results clearly indicate strains . In addition, the vector competence of the temperature-dependent variations in the susceptibility of same mosquito population can be highly strain-specific. Ae. japonicus to ZIKV. Following the experimental chal- Ae. aegypti from Mexico showed an increased vector lenge with ZIKV-containing blood meals, the number of competence for African strains of ZIKV compared with 36,37 infected specimens as well as the amount of ZIKV RNA an American ZIKV strain . Studies with the WNV copies per mosquito increased with increasing incubation strain NY99 and Ae. japonicus mosquitoes from northern temperatures (21 °C < 24 °C < 27 °C). This result is con- Switzerland revealed transmission of WNV . By contrast, sistent with our previous studies performed with ZIKV, Ae. japonicus mosquitoes from southwestern Germany where the IRs of various mosquito species from Central were shown to be refractory to the same WNV strain . Europe were also temperature-dependent . Dissemination Similar contradictions have been discussed regarding the was only observed at 24 and 27 °C. Infectious virus par- vector competence of Culex quinquefasciatus for ZIKV, ticles were exclusively detected in two mosquito speci- where some studies detected transmission while others 39–41 mens incubated at an elevated temperature of 27 °C, did not . These differences might be explained by resulting in a TR of 14.3%. There is a high variability in variations in the experimental setup, e.g., the origin of the the range of TRs at tropical incubation temperatures mosquito population, the virus strain, or vector main- (26–28 °C) for both Ae. aegypti (21–87%) and Ae. tenance protocols in the laboratory . Thus, standardized Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 4 of 6 studies to investigate the vector competence for different is known to positively correlate with increasing tem- local mosquito population/virus strain combinations must peratures up to 30 °C. As the pupation limit is reached at be considered to allow a thorough risk assessment. In 28 °C, we chose 26 °C as the incubation temperature for 14,50 particular, further analysis of the Ae. japonicus popula- successful and rapid development of the mosquitoes . tions from northern America/Asia should be performed Species identification was performed using the morpho- to assess the risk of ZIKV transmission in these regions. logical key in the “Guidelines for the surveillance of Currently, the distribution of Ae. japonicus in Europe is invasive mosquitoes in Europe” . To exclude natural primarily restricted to regions with temperate climates . flavivirus infections that could potentially interfere with The lack of vector competence at temperatures below the experimental outcome, 10 randomly selected adult 27 °C suggests a limited risk for ZIKV transmission by Ae. specimens were tested with pan-Flavi-, pan-Bunya-, and 52–54 japonicus in Europe. However, the rapid spread of Ae. pan-Alphavirus PCRs, but these tests were negative . japonicus in Northeast America (as far as 30°N′ latitude in Groups of 20 females (4–14 days old) were placed in Florida) and the native distribution in Asia at the same plastic vials, starved for 24 h, and challenged with infec- latitude illustrate the risk for the species to spread to the tious blood meals. The feeding, incubation, and analysis of 14,43 Mediterranean region . Ae. japonicus may adapt to the mosquitoes were performed in the BSL-3 insectary in new environmental conditions and might also have the the Bernhard Nocht Institute for Tropical Medicine, potential to invade areas of higher temperatures in the Hamburg, Germany. To support a high feeding rate, we Mediterranean region, as has already happened in North provided infectious blood in 50 µl drops at the bottoms of America . Nevertheless, for a comprehensive risk the vials (two drops per vial). Thus, a feeding rate of 75% assessment of ZIKV transmission in Central Europe, (i.e., the percentage of engorged females to total females) Aedes species such as the native mosquito species Aedes was reached. vexans and the invasive species Aedes koreicus should also For validation of the salivation assay for Ae. japonicus, be considered and investigated as potential ZIKV vectors an infection experiment with JEV was first performed. under temperate climate conditions. Ae. vexans from Female mosquitoes were infected via an infectious blood North America was proven to have a transmission meal using the SA-14 strain of JEV (GenBank accession 55 7 potential for ZIKV of approximately 1–5% at incubation number EU073992) at a final concentration of 10 29,44 temperatures of 28 or 27 °C . These low TRs must be plaque-forming units/milliliter (PFU/ml) and were kept at considered in light of the locally very high mosquito 27 °C for 14 days. abundance and aggressive human-biting behavior along Subsequently, a total of 381 female mosquitoes were rivers in Central Europe . Therefore, further studies challenged with blood meals containing ZIKV, strain should investigate whether this species can transmit ZIKV ZIKV_FB-GWUH-2016 (GenBank accession number 56 7 at lower temperatures. Another candidate of interest is KU870645, fifth passage) at a final concentration of 10 the invasive mosquito species Ae. koreicus, which is clo- PFU/ml. Two hundred forty-three engorged females were sely related to Ae. japonicus. Ae. koreicus is also a vector incubated at 80% humidity and temperatures of 21, 24, or 46 23 for arboviruses such as JEV or CHIKV and was quite 27 °C. recently introduced into Central Europe, including in 47–49 Germany . Assessment of ZIKV infection, dissemination, and In conclusion, transmission of ZIKV by Ae. japonicus transmission appears to be limited to elevated temperatures. Never- Fourteen days post infection, mosquitoes were analyzed theless, due to the demonstrated species’ vector compe- for JEV (n = 27) or ZIKV (n = 79) infection, dissemina- tence for ZIKV and for other mosquito-borne viruses, in tion, and transmission. Infection, dissemination, and virus combination with a possible further spread to southern titers were determined by separate analyses of mosquito Europe, Ae. japonicus must be considered a potential bodies and heads without legs and wings (infection and vector of pathogens, including ZIKV. body titer) and of legs (dissemination and leg titer) for the presence of viral ZIKV RNA using a quantitative real-time Materials and methods PCR assay (qRT-PCR; Real Star Zika Virus RT-PCR Kit, Source, rearing, and experimental infection of mosquitoes Altona Diagnostics, Hamburg, Germany). ZIKV trans- Ae. japonicus eggs were collected with ovitraps in mission was assessed by testing mosquito saliva for the southwestern Germany (49°31′26.26″N, 8°40′16.88″E) in presence of infectious virus particles using the salivation summer 2017. Approximately 1200 eggs were flooded in assay as previously described . In short, mosquitoes were the laboratory, and the larvae and adults were maintained immobilized and the probosces were placed into a filter at 26 °C, with a relative humidity of 80% and a 12:12 light: tip containing 10 µl of phosphate-buffered saline (PBS). dark photoperiod. These temperature conditions were After 30 min, saliva-containing PBS was pipetted into the selected because the larval development of Ae. japonicus media of Vero cells seeded in a 96-well plate to measure Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 5 of 6 the cytopathic effect, i.e., the presence of infectious virus At <http://www.who.int/en/news-room/detail/01-02-2016-who-director- general-summarizes-the-outcome-of-the-emergency-committee-regarding- particles, after 7 days. The presence of ZIKV in the clusters-of-microcephaly-and-guillain-barr%c3%a9-syndrome> (2016). supernatant of cytopathic cells was subsequently tested by 3. Musso, D. & Gubler, D. J. Zika virus. Clin. Microbiol. 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Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany

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Biomedicine; Biomedicine, general; Immunology; Medical Microbiology; Microbiology; Antibodies; Vaccine
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Abstract

The invasive mosquito species Aedes japonicus japonicus (Ae. japonicus) is widely distributed in Central Europe and is a known vector of various arboviruses in the laboratory, including flaviviruses such as Japanese Encephalitis virus or West Nile virus. However, the vector competence of Ae. japonicus for the recently emerging Zika virus (ZIKV) has not been determined. Therefore, field-caught Ae. japonicus from Germany were orally infected with ZIKV and incubated at 21, 24, or 27 °C to evaluate the vector competence under climate conditions representative of the temperate regions (21 °C) in the species’ main distribution area in Europe and of Mediterranean regions (27 °C). Aedes japonicus was susceptible to ZIKV at all temperatures, showing infection rates between 10.0% (21 °C) and 66.7% (27 °C). However, virus transmission was detected exclusively at 27 °C with a transmission rate of 14.3% and a transmission efficiency of 9.5%. Taking into account the present distribution of Ae. japonicus in the temperate regions of Central Europe, the risk of ZIKV transmission by the studied Ae. japonicus population in Central Europe has to be considered as low. Nevertheless, due to the species’ vector competence for ZIKV and other mosquito-borne viruses, in combination with the possibility of further spread to Mediterranean regions, Ae. japonicus must be kept in mind as a potential vector of pathogens inside and outside of Europe. Introduction clinical symptoms to severe diseases, including neonatal Zika virus (ZIKV) is an emerging mosquito-borne virus microcephaly and neurological disorders such as Guillain- within the family Flaviviridae that was first isolated from Barré syndrome . The mosquito species Aedes aegypti and sentinel rhesus macaques in Uganda in 1947 . After Aedes albopictus are considered the primary and sec- decades of silent circulation, unprecedented ZIKV epi- ondary vectors of ZIKV; however, a wide variety of other demics occurred in Micronesia, Polynesia, and, finally, in Aedes species have been identified as potentially suscep- the Americas in 2015; the hundreds of thousands of tible to ZIKV infection . Recent experimental studies human cases finally resulted in the announcement of a suggested that only Ae. albopictus might play a role in Public Health Emergency of International Concern ZIKV transmission in Central Europe, while common through the World Health Organization . Clinical courses members of the genus Culex are probably not impor- 5,6 associated with ZIKV infections can range from mild tant . However, north of the Alps, the Asian tiger mos- quito is currently established at only a few sites, with 7,8 relatively low abundance . By contrast, the invasive Asian bush mosquito Aedes japonicus japonicus (Ae. Correspondence: Anna Heitmann (heitmann@bnitm.de) Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany japonicus) is widely distributed in Central Europe and is German Centre for Infection Research (DZIF), Partner site Hamburg-Luebeck- currently established in at least 10 countries, including Borstel-Riems, 20359 Hamburg, Germany large parts of Germany . In 2008, the first invasive Full list of author information is available at the end of the article. These authors contributed equally: Stephanie Jansen, Anna Heitmann © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 2 of 6 spreading of Ae. japonicus in Europe was reported in Switzerland , and Ae. japonicus is now listed as one of the nine most dominant mosquito species in Switzerland. Shortly after its introduction in Switzerland, Ae. japonicus was first reported in Germany, followed by the estab- lishment of populations at several sites. In the Nether- lands and Belgium, mosquito control programs have been 9–12 initiated due to the massive Ae. japonicus populations . Ae. japonicus is a container-dwelling species, colonizing both natural (e.g., bamboo stubs and tree holes) and man- made (e.g., tires and barrels) breeding sites . Due to its tolerance of rather low temperatures, Ae. japonicus has a relatively long seasonal activity compared to other container-breeders . Ae. japonicus has an opportunistic feeding pattern with a preference for mammals, including humans, although avian host species have also been 15,16 reported . Thus, Ae. japonicus could potentially serve as a bridge vector for zoonotic arboviruses. The species is an experimentally proven vector of several flaviviruses, including Japanese Encephalitis virus (JEV), West Nile Fig. 1 Means and 95% confidence intervals for the ZIKV infection, 17–19 virus (WNV), and Saint Louis encephalitis virus ,as dissemination, and transmission rates as well as the transmission well as arboviruses of other families, such as La Crosse efficiency of Ae. japonicus from southwestern Germany as revealed by analyses of bodies (IR), legs (DR), and saliva (TR and TE) following virus (LACV, Peribunyaviridae) and Chikungunya virus 20,21 challenge of mosquitoes with infectious blood meals and incubation (CHIKV, Togaviridae) . Previous studies with an Ae. at 21 °C (n = 30), 24 °C (n = 29), or 27 °C (n = 21) for 2 weeks. Three japonicus population from southwestern Germany also independent trials were performed for each temperature. *Infection revealed a vector competence for JEV under laboratory rate (IR): number of ZIKV-positive mosquito bodies per number of fed conditions . females. **Dissemination rate (DR): number of mosquitoes with ZIKV- positive legs per number of ZIKV-positive mosquito bodies. In light of the continuing spread of Ae. japonicus in ***Transmission rate (TR): number of mosquitoes with ZIKV-positive Europe and the ongoing circulation of ZIKV in America, saliva per number of ZIKV-positive mosquito bodies. ****Transmission the aim of this study was to evaluate whether Ae. japo- efficiency (TE): number of mosquitoes with ZIKV-positive saliva per nicus has vector competence for ZIKV under climate total number of fed females conditions representative of tropical and temperate regions. Table 1 Calculation of the virus titers from bodies or legs Results for Ae. japonicus specimens from southwestern Germany To assess the suitability of the collected Ae. japonicus following challenge of mosquitoes with infectious blood for vector competence studies, a small number of speci- meals and incubation at 21 , 24, or 27 °C for 2 weeks mens were challenged with JEV in a preliminary study. In agreement with previous findings , the Ae. japonicus Temperature in °C Body titer, mean (SD) Leg titer, mean (SD) specimens from southwestern Germany were susceptible log10 RNA copies/ log10 RNA copies/ to JEV. The infection rate (IR) was 51.9%, with an average specimen specimen amount of viral RNA of 5.6 × 10 copies/specimen (n = 21 4.6 (1.0) 0.0 (0.0) 27). In addition to the previous experiment, we also 24 4.9 (1.5) 2.9 (2.9) investigated the transmission of infectious virus particles by analyzing mosquito saliva following incubation of 27 5.9 (1.8) 4.2 (4.2) infected mosquitoes at 27 °C for 14 days. The results indicated a transmission rate (TR) of 78.6%. Subsequently, mosquitoes were analyzed for ZIKV to 24.1% (7/29) at 24 °C and to 66.7% (14/21) at 27 °C. infection. Fourteen days post infection, ZIKV RNA was This pattern is also reflected in the amount of virus RNA present in the bodies of challenged Ae. japonicus at all of within the mosquito bodies, which increased from 1.2 × 4 6 the tested temperatures (Fig. 1). The relative numbers of 10 RNA copies/specimen at 21 °C to 2.6 × 10 RNA ZIKV-positive mosquitoes (Fig. 1) and the amount of viral copies/specimen at 24 °C to 6.4 × 10 RNA copies/speci- RNA (Table 1) increased with increasing incubation men at 27 °C (Table 1). Dissemination of the virus was temperatures. The IR increased from 10% (3/30) at 21 °C found in mosquitoes kept at 24 and 27 °C but not in Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 3 of 6 6,24–28 mosquitoes incubated at 21 °C. However, the averaged leg albopictus (18–77%) . Nevertheless, both species titers were substantially higher at 27 °C (6.4 × 10 RNA showed considerably higher TRs than Ae. japonicus; copies/specimen) than at 24 °C (8.4 × 10 RNA copies/ whereas Ae. vexans also has a low vector competence for specimen) (Table 1). This is also reflected by the detection ZIKV, with a TR between 2 and 7% . Likewise, the of infectious virus particles in the saliva of two mosquitoes transmission efficiency of 9.5% for Ae. japonicus at the kept at 27 °C, resulting in a TR of 14.3% (2/14) and a tropical temperature is lower than the known transmis- transmission efficiency of 9.5% (2/21). sion efficiency for Ae. aegypti (26%) under similar con- ditions . Previous vector competence studies with Ae. Discussion japonicus and various arboviruses were only performed 17–21 To the best of our knowledge, this is the first study on under tropical temperature conditions (i.e., 25–28 °C) . the vector competence of Ae. japonicus for ZIKV (see Therefore, it is unknown if the lack of virus transmission review by Epelboin et al. ), and our results show a low at lower temperatures is a general feature for viral transmission efficiency at high temperatures. The species transmission by Ae. japonicus or if this observation is is a known competent vector of a variety of arboviruses, specific for ZIKV. By contrast, the primary and secondary including flaviviruses (e.g., WNV, JEV, or Dengue virus), as vectors Ae. aegypti and Ae. albopictus can transmit ZIKV well as members of other virus families, including Peri- below 27 °C. Viable ZIKV virus particles were detected in 17,19–21 30 bunyaviridae (LACV) and Togaviridae (CHIKV) . the saliva of Ae. aegypti even at 20 °C . Therefore, the Over the last two decades, Ae. japonicus has successfully lack of transmission by Ae. japonicus is a species-specific invaded Central European countries as well as large parts observation and not a general pattern for ZIKV. of North America, and it is found primarily in areas with One explanation for the lack of ZIKV transmission by 8,13,14,22,23 predominantly temperate climate conditions . Ae. japonicus at temperatures below 27 °C might be a Ae. japonicus eggs are resistant to frost and desiccation. combination of higher virus replication rates and species- Furthermore, the seasonal activity of the species is longer specific, temperature-dependent effects on the mosquito than that of other container-breeding species. Due to microbiome or immune regulatory pathways . However, these attributes, Ae. japonicus has some developmental as shown before, vector competence is influenced by a advantages over native species that could affect mosquito three-way interaction between the vector population, the 32,33 population patterns as well as pathogen transmission in virus strain, and temperature . Only a suitable com- newly colonized regions. Likewise, it is of considerable bination of these factors allows the virus to replicate and interest to collect information on the vector competence disseminate to the salivary glands to enable transmission of Ae. japonicus for newly emerging viruses such as ZIKV, through the next bite. However, studies with a combina- with a special emphasis on the temperate climate condi- tion of one specific mosquito population with one specific tions of the species’ current distribution range. virus strain must be interpreted with caution. Even studies Previous studies described relatively low feeding rates of with the same combination of vector species and virus can field-caught Ae. japonicus using saturated cotton sticks or come to varying TRs or even to inconsistent results 19,21 feeding systems with chicken skin . The experiments regarding the species’ susceptibility to a virus. Studies presented here demonstrate that artificial feeding via performed with either field-caught Ae. aegypti or Ae. blood drops seems to be an efficient alternative for field- albopictus populations from different sites within one caught Ae. japonicus mosquitoes, resulting in a feeding country revealed clearly varying TRs for the same ZIKV 34,35 rate of 75%. The experimental results clearly indicate strains . In addition, the vector competence of the temperature-dependent variations in the susceptibility of same mosquito population can be highly strain-specific. Ae. japonicus to ZIKV. Following the experimental chal- Ae. aegypti from Mexico showed an increased vector lenge with ZIKV-containing blood meals, the number of competence for African strains of ZIKV compared with 36,37 infected specimens as well as the amount of ZIKV RNA an American ZIKV strain . Studies with the WNV copies per mosquito increased with increasing incubation strain NY99 and Ae. japonicus mosquitoes from northern temperatures (21 °C < 24 °C < 27 °C). This result is con- Switzerland revealed transmission of WNV . By contrast, sistent with our previous studies performed with ZIKV, Ae. japonicus mosquitoes from southwestern Germany where the IRs of various mosquito species from Central were shown to be refractory to the same WNV strain . Europe were also temperature-dependent . Dissemination Similar contradictions have been discussed regarding the was only observed at 24 and 27 °C. Infectious virus par- vector competence of Culex quinquefasciatus for ZIKV, ticles were exclusively detected in two mosquito speci- where some studies detected transmission while others 39–41 mens incubated at an elevated temperature of 27 °C, did not . These differences might be explained by resulting in a TR of 14.3%. There is a high variability in variations in the experimental setup, e.g., the origin of the the range of TRs at tropical incubation temperatures mosquito population, the virus strain, or vector main- (26–28 °C) for both Ae. aegypti (21–87%) and Ae. tenance protocols in the laboratory . Thus, standardized Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 4 of 6 studies to investigate the vector competence for different is known to positively correlate with increasing tem- local mosquito population/virus strain combinations must peratures up to 30 °C. As the pupation limit is reached at be considered to allow a thorough risk assessment. In 28 °C, we chose 26 °C as the incubation temperature for 14,50 particular, further analysis of the Ae. japonicus popula- successful and rapid development of the mosquitoes . tions from northern America/Asia should be performed Species identification was performed using the morpho- to assess the risk of ZIKV transmission in these regions. logical key in the “Guidelines for the surveillance of Currently, the distribution of Ae. japonicus in Europe is invasive mosquitoes in Europe” . To exclude natural primarily restricted to regions with temperate climates . flavivirus infections that could potentially interfere with The lack of vector competence at temperatures below the experimental outcome, 10 randomly selected adult 27 °C suggests a limited risk for ZIKV transmission by Ae. specimens were tested with pan-Flavi-, pan-Bunya-, and 52–54 japonicus in Europe. However, the rapid spread of Ae. pan-Alphavirus PCRs, but these tests were negative . japonicus in Northeast America (as far as 30°N′ latitude in Groups of 20 females (4–14 days old) were placed in Florida) and the native distribution in Asia at the same plastic vials, starved for 24 h, and challenged with infec- latitude illustrate the risk for the species to spread to the tious blood meals. The feeding, incubation, and analysis of 14,43 Mediterranean region . Ae. japonicus may adapt to the mosquitoes were performed in the BSL-3 insectary in new environmental conditions and might also have the the Bernhard Nocht Institute for Tropical Medicine, potential to invade areas of higher temperatures in the Hamburg, Germany. To support a high feeding rate, we Mediterranean region, as has already happened in North provided infectious blood in 50 µl drops at the bottoms of America . Nevertheless, for a comprehensive risk the vials (two drops per vial). Thus, a feeding rate of 75% assessment of ZIKV transmission in Central Europe, (i.e., the percentage of engorged females to total females) Aedes species such as the native mosquito species Aedes was reached. vexans and the invasive species Aedes koreicus should also For validation of the salivation assay for Ae. japonicus, be considered and investigated as potential ZIKV vectors an infection experiment with JEV was first performed. under temperate climate conditions. Ae. vexans from Female mosquitoes were infected via an infectious blood North America was proven to have a transmission meal using the SA-14 strain of JEV (GenBank accession 55 7 potential for ZIKV of approximately 1–5% at incubation number EU073992) at a final concentration of 10 29,44 temperatures of 28 or 27 °C . These low TRs must be plaque-forming units/milliliter (PFU/ml) and were kept at considered in light of the locally very high mosquito 27 °C for 14 days. abundance and aggressive human-biting behavior along Subsequently, a total of 381 female mosquitoes were rivers in Central Europe . Therefore, further studies challenged with blood meals containing ZIKV, strain should investigate whether this species can transmit ZIKV ZIKV_FB-GWUH-2016 (GenBank accession number 56 7 at lower temperatures. Another candidate of interest is KU870645, fifth passage) at a final concentration of 10 the invasive mosquito species Ae. koreicus, which is clo- PFU/ml. Two hundred forty-three engorged females were sely related to Ae. japonicus. Ae. koreicus is also a vector incubated at 80% humidity and temperatures of 21, 24, or 46 23 for arboviruses such as JEV or CHIKV and was quite 27 °C. recently introduced into Central Europe, including in 47–49 Germany . Assessment of ZIKV infection, dissemination, and In conclusion, transmission of ZIKV by Ae. japonicus transmission appears to be limited to elevated temperatures. Never- Fourteen days post infection, mosquitoes were analyzed theless, due to the demonstrated species’ vector compe- for JEV (n = 27) or ZIKV (n = 79) infection, dissemina- tence for ZIKV and for other mosquito-borne viruses, in tion, and transmission. Infection, dissemination, and virus combination with a possible further spread to southern titers were determined by separate analyses of mosquito Europe, Ae. japonicus must be considered a potential bodies and heads without legs and wings (infection and vector of pathogens, including ZIKV. body titer) and of legs (dissemination and leg titer) for the presence of viral ZIKV RNA using a quantitative real-time Materials and methods PCR assay (qRT-PCR; Real Star Zika Virus RT-PCR Kit, Source, rearing, and experimental infection of mosquitoes Altona Diagnostics, Hamburg, Germany). ZIKV trans- Ae. japonicus eggs were collected with ovitraps in mission was assessed by testing mosquito saliva for the southwestern Germany (49°31′26.26″N, 8°40′16.88″E) in presence of infectious virus particles using the salivation summer 2017. Approximately 1200 eggs were flooded in assay as previously described . In short, mosquitoes were the laboratory, and the larvae and adults were maintained immobilized and the probosces were placed into a filter at 26 °C, with a relative humidity of 80% and a 12:12 light: tip containing 10 µl of phosphate-buffered saline (PBS). dark photoperiod. These temperature conditions were After 30 min, saliva-containing PBS was pipetted into the selected because the larval development of Ae. japonicus media of Vero cells seeded in a 96-well plate to measure Jansen et al. Emerging Microbes & Infections (2018) 7:192 Page 5 of 6 the cytopathic effect, i.e., the presence of infectious virus At <http://www.who.int/en/news-room/detail/01-02-2016-who-director- general-summarizes-the-outcome-of-the-emergency-committee-regarding- particles, after 7 days. The presence of ZIKV in the clusters-of-microcephaly-and-guillain-barr%c3%a9-syndrome> (2016). supernatant of cytopathic cells was subsequently tested by 3. Musso, D. & Gubler, D. J. Zika virus. Clin. Microbiol. 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European Centre for Disease Prevention and Control (ECDC), European bodies, and the TR is defined as the number of virus- Food Safety Authority (EFSA). VectorNet: a European network for sharing positive saliva samples per number of virus-positive data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents. At <https://ecdc.europa.eu/en/about- bodies. Calculation of the transmission efficiency was us/partnerships-and-networks/disease-and-laboratory-networks/vector- conducted as described by Chouin-Carneiro et al. and is net> (accessed November 10, 2018). defined as the number of virus-positive saliva samples per 9. Schaffner,F., Kaufmann, C., Hegglin,D.&Mathis,A.The invasive mosquito Aedes japonicus in Central Europe. Med. Vet. Entomol. 23,448–451 (2009). total number of fed females. The R program was used 10. European Centre for Disease Prevention and Control. 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