North American Culex pipiens and Culex quinquefasciatus are competent vectors for Usutu virus

Background Usutu virus (USUV) is a member of the Japanese encephalitis virus (JEV) serocomplex in the Flaviviridae family. Emergence of USUV in Europe has led to disease burdens in birds and created increasing concern for the potential zoonotic transmission to humans. Whilst USUV has not been detected in the New World, the identification of competent vector species in North America is critical in the assessment of the likelihood of its dispersal and establishment of enzootic transmission cycles. The objective of this study was to determine vector competence of potential mosquito vectors in North America for USUV. Three medically important mosquito species were selected for testing because of their involvement in the transmission of West Nile virus and St. Louis encephalitis virus, two related JEV serocomplex flaviviruses in the New World. Methodology/Principal findings Oral challenge of Culex pipiens, Culex quinquefasciatus, and Aedes albopictus was performed to determine the susceptibility and vector competence of North American mosquitoes for USUV. Infection status was monitored by the isolation of virus from homogenized mosquito tissues. The disseminated form of infection was demonstrated by the detection of infectious virus in the head, wings, and legs of infected mosquitoes. The presence of viral RNA in saliva of infected Cx. pipiens and Cx. quinquefasciatus indicated that both species are competent for transmission of USUV. Conclusions/Significance Results indicate that members of the Cx. pipiens complex are susceptible to USUV and competent for its transmission potential in North America in the event of its introduction. In contrast, Ae. albopictus were highly refractory to USUV infection, suggesting that this species is unlikely to contribute to USUV transmission in North America.


Introduction
Usutu virus (USUV) is a mosquito-borne flavivirus classified under the Japanese encephalitis virus (JEV) serocomplex in the Flaviviridae family. Usutu virus was originally isolated in 1959 in South Africa [1]. The earliest dispersal of USUV from the African continent was through the detection of viral RNA in tissues of the common European blackbird, Turdus merula, in Italy in 1996 [2]. Since 2001, the emergence of USUV has been continuously reported throughout Europe. It has been reported in Belgium, Czech Republic, England, Hungary, Italy, Germany, Poland, Spain, and Switzerland; and has been responsible for the deaths of thousands of birds with an estimated 15.7% blackbird population decline attributed to the virus [3,4]. It has also become a potential public health threat after reports of neurotropic diseases in humans.
Exposures to USUV typically lead to mild diseases and seroconversion in incidental hosts as observed in humans and horses [5]. Historically, human USUV infections have been primarily associated with self-limiting febrile illness and rash followed by the development of neutralizing antibodies. USUV infection leading to neurotropic disease was only documented in immunocompromised individuals [6,7]. However, more recent evidence indicates that severe disease could also take place in healthy adults which has considerably increased the public health significance of USUV [8,9].
As historically observed with the dispersal of multiple flaviviruses in the JEV serocomplex, enzootic transmission can be quickly established in the presence of competent vectors and amplification hosts. The emergence of USUV in Europe during the last two decades has significantly increased the likelihood of its further dispersal into other geographic regions including the United States. In this study, oral challenge with USUV was performed in North American Cx. pipiens, Cx. quinquefasciatus, and Ae. albopictus to determine their vector competence for USUV. The three species were selected based on their known involvement in the transmission of West Nile virus, a flavivirus within the JEV serocomplex, in North America.

Cells and virus
African green monkey kidney epithelial Vero76 cells maintained in L-15 medium supplemented with 10% fetal bovine serum, 10% tryptose phosphate, penicillin, streptomycin, and Lglutamine were used in this study for propagation of virus stocks and titration of all infectious materials as previously described [19]. The prototype SAAr1776 strain was used for all per os infection of mosquitoes. The strain was originally isolated from a pool of Cx. neavei in South Africa in 1959. Prior to the experiment, the strain obtained from the Division of Vector-Borne Diseases, Centers for Disease Control was passaged seven times in suckling mouse brain and twice in Vero cells. Two additional passages in Vero76 cells were performed in our laboratory to generate stocks for per os challenge. Phylogenetically, the SAAr1776 strain forms a monophyletic cluster with recent isolates of USUV in Europe and has the closest relatedness to the sequence of USUV MB119/06 isolated in Spain in 2006 [20].

Mosquitoes and per os infection
Colonies of Cx. pipiens (F 21 ) and Cx. quinquefasciatus (F 21 ) used in this study were established from larvae collected from Ewing Township, Mercer County, NJ and Vero Beach, FL in August 2015 as previously described [21]. The colony of Ae. albopictus (F 4 ) tested in this study was derived from eggs collected from the City of Trenton, Mercer County, NJ in July 2016. All mosquitoes were maintained with 20% sucrose solution under a 16h: 8h light: dark photo regimen at 28˚C. Eight to ten-day old mosquitoes were deprived of sugar and water 48 and 24 h before per os infection, respectively. USUV stock and defibrinated sheep blood (Colorado Serum Company, CO) were mixed in equal volumes to produce artificial viremic blood meals. Blood meals were administered to Culex species mosquitoes in gallon size cartons through cotton pledgets at room temperature for 1 h. Oral challenge of Ae. albopictus was performed with a Hemotek membrane feeding apparatus as previously described [22]. Control groups received defibrinated sheep blood mixed with tissue culture media. Ingestion of defibrinated sheep blood by control groups ensures that cytopathic effect in Vero76 cells used as an indicator for viral infection was caused by the infection of USUV and not by other pathogens present in mosquitoes. Titers of viremic blood meals were determined by titration of remaining blood meals aspirated from cotton pledgets and individual blood feeders after the completion of each per os infection experiment. Fully engorged mosquitoes were collected under cold anesthetization and returned to the cartons. Three engorged mosquitoes in each group were frozen immediately after receiving blood meals to confirm ingestion of USUV and to quantify the viral titer of the infected blood ingested by mosquitoes.
Mosquitoes were sampled at 7 and 14 days post-infection (d.p.i.) to characterize the infection process of USUV. To determine the incidence of disseminated infection, dissection of mosquitoes was performed to separate the abdomen from the head, wings, and legs. The abdomen contains the midgut where the infection of arboviruses is initially established. The head, wings, and legs are the secondary tissues, which are infected with viruses disseminated from the midgut. A second group of mosquitoes was collected, without dissection, to determine replication kinetics of USUV in whole mosquitoes. At 14 d.p.i., saliva of each mosquito was collected through forced salivation for 60 minutes prior to dissection, as previously described [19,23]. Titers of viremic blood meals and whole mosquitoes sampled at 7 and 14 d.p.i. are summarized in Table 1.

Detection of USUV in mosquito tissues and saliva
To quantify infectious viruses, all samples containing mosquito tissues were homogenized using a TissueLyser II apparatus (Qiagen, MD) at 26 Hz for 4 min and titrated by 50% tissue culture infectious dose (TCID 50 ) method with Vero76 cells as previously described [24]. The establishment of infection was defined by the detection of USUV in the body or secondary tissues from dissected mosquitoes or in homogenized whole mosquitoes. Infection rates were calculated based on the overall percentage of infected mosquitoes among mosquitoes tested. Viral dissemination was demonstrated by the detection of USUV in the secondary tissues of infected mosquitoes. Dissemination rates were derived from the incidence of positive detection of infectious viruses in secondary tissues among dissected mosquitoes infected with USUV.
A nested reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the presence of USUV in the saliva of infected mosquitoes. The transmission rate of USUV was determined by the incidence of positive detection of viral RNA among infected mosquitoes. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, MD). Complementary DNA was produced by reverse transcription of Superscript III reverse transcriptase (Life Technologies, CA) with the VD8 primer that is broadly reactive to the 3' untranslated region of all mosquito-borne flaviviruses [25]. The first amplicon was produced using Platinum Taq polymerase (Life Technologies, CA) using the VD8-EMF1 primer set as previously described [25]. The second amplicon was derived from the second RT-PCR reaction using two USUVspecific primers, USUV 10268-10287 (5'-GCAACGTGGGCTGAAAACAT-3') and USUV 10821-10802 (5'-AGTTCGCATCACCGTCTGTT-3'). The USUV-specific primer set was designed using the Primer-BLAST algorithm available from the website of National Center of Biotechnology Information. All amplicons were separated and visualized by electrophoresis using 1% agarose gel.

Statistical analysis
Infection, dissemination, and transmission rates of USUV in infected mosquitoes were analyzed using Fisher's exact test. All statistical analyses were performed using SigmaPlot version 12.0 (Systat Software Inc., CA).

Infectious viruses of USUV isolated from orally challenged Cx. pipiens and Cx. quinquefasciatus
As summarized in Table 1

Discussion
Our results demonstrate that North American Cx. pipiens and Cx. quinquefasciatus are susceptible to USUV and competent for its transmission. With the evidence demonstrating the vector competence of Cx. pipiens for USUV and its involvement of transmission in nature, the species is likely to be a critical player for the further dispersal of USUV in the future and a target species for vector control in the event of disease outbreak in North America. Whilst there was no significant difference in infectious titers of whole mosquitoes collected at 0, 7, and 14 d.p.i., a minor increase in the infectious titer and the significant increase in infection rates observed in Cx. pipiens indicate that the species is permissive for the replication of USUV. Similarly, Cx. quinquefasciatus tested in this study also allowed the replication of USUV as demonstrated with maintenance of infectious titers of whole mosquitoes at 7 and 14 d.p.i. The replication of USUV in both species ultimately led to its dissemination from the midgut into secondary tissues including salivary glands, as demonstrated by the detection of viral RNA in the saliva of infected mosquitoes at 14 d.p.i.
Whilst Ae. albopictus have been demonstrated to be competent for the transmission of multiple flaviviruses in the JEV serocomplex and a source of naturally occurring isolates of USUV  [26], the species has been demonstrated to be highly refractory to USUV based on our and others' findings [18,27]. As USUV is unable to establish infection in orally exposed Ae. albopictus, the species is unlikely to be involved in the transmission and maintenance of USUV. Isolation of infectious viruses or detection of viral RNA from Ae. albopictus may be a consequence of recent engorgement from viremic avian species as some Ae. albopictus populations have been demonstrated to have opportunistic feeding behaviors and utilize avian species as a source of blood meals [28]. Despite the known genetic differences between members of the Cx. pipiens complex in the Old and New World and differing methods for detection of USUV [29], Cx. pipiens and related species remain as the most probable vector for USUV in North America. Notwithstanding the variance in methods, our results are comparable with the previous publication on the vector competence of populations of Cx. pipiens in Northwestern Europe for USUV [17]. Our observations also do not exclude the possibility that other mosquito species can potentially serve as competent vectors for the transmission of USUV outside its endemic regions. As observed with JEV and WNV, dispersal of flaviviruses under the JEV serocomplex often involves transmission by multiple mosquito species in nature. This strategy may ultimately increase the likelihood for the permanent establishment of enzootic transmission cycles and challenge the efforts in the formulation of vector control strategies.
Whilst there have been at least two JEV-serocomplex members present in the New World including WNV and St. Louis encephalitis virus (SLEV), USUV remains likely to be established and co-circulate with related viruses in the potential event of its introduction. As co-circulation of USUV and WNV have been reported in Europe, it is highly unlikely that the potential introduction of USUV will lead to the displacement of WNV [26]. Whether or not co-circulation can ultimately result in the displacement of SLEV, as seen with the displacement of SLEV by WNV in California in 2003, remains unclear.