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      A rhesus macaque model of Asian-lineage Zika virus infection

      Nature Communications
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          Zika Virus Infection and Stillbirths: A Case of Hydrops Fetalis, Hydranencephaly and Fetal Demise

          Background The rapid spread of Zika virus in the Americas and current outbreak of microcephaly in Brazil has raised attention to the possible deleterious effects that the virus may have on fetuses. Methodology/Principal Findings We report a case of a 20-year-old pregnant woman who was referred to our service after a large Zika virus outbreak in the city of Salvador, Brazil with an ultrasound examination that showed intrauterine growth retardation of the fetus at the 18th gestational week. Ultrasound examinations in the 2nd and 3rd trimesters demonstrated severe microcephaly, hydranencephaly, intracranial calcifications and destructive lesions of posterior fossa, in addition to hydrothorax, ascites and subcutaneous edema. An induced labor was performed at the 32nd gestational week due to fetal demise and delivered a female fetus. ZIKV-specific real-time polymerase chain reaction amplification products were obtained from extracts of cerebral cortex, medulla oblongata and cerebrospinal and amniotic fluid, while extracts of heart, lung, liver, vitreous body of the eye and placenta did not yield detectable products. Conclusions/Significance This case report provides evidence that in addition to microcephaly, there may be a link between Zika virus infection and hydrops fetalis and fetal demise. Given the recent spread of the virus, systematic investigation of spontaneous abortions and stillbirths may be warranted to evaluate the risk that ZIKV infection imparts on these outcomes.
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            Mosquitoes Inoculate High Doses of West Nile Virus as They Probe and Feed on Live Hosts

            Introduction West Nile virus (WNV) has become the most prevalent arbovirus in the United States, causing more than 24,000 reported human cases and 960 deaths since it was first detected in New York in 1999 [1]. The virus is maintained in an enzootic cycle involving birds and mosquitoes (primarily Culex species) [2]. Most humans become infected with WNV through the bite of an infected mosquito. After locating a suitable host, a mosquito probes throughout the dermis with her mouthparts and imbibes blood once a blood vessel is pierced. Saliva (and virus, if a mosquito is infectious) is deposited into the host throughout the feeding process. A fundamental component of the mosquito transmission process, namely how much virus mosquitoes inoculate into a host while feeding, is not known. Previous studies have used in vitro methods to estimate the WNV dose inoculated by mosquitoes. Depending on mosquito species, mean WNV titers ranged from 101.2 to 104.3 plaque forming units (PFU) [3–6]. Although in vitro methods are convenient and relatively easy to perform, they do not allow mosquitoes to probe or feed naturally. Most of the saliva deposited by mosquitoes while blood feeding is re-ingested [7,8]. Therefore, mosquitoes inoculate most of the saliva, and thus virus, during the probing phase. Because in vitro techniques do not allow mosquitoes to probe naturally, these techniques are likely to underestimate the dose of virus inoculated. We developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a live host. We used this assay to determine the dose of WNV inoculated by two important enzootic vectors, Cx. tarsalis and Cx. pipiens, and two potential bridge vectors, Ae. japonicus and Ae. triseriatus, as they probed and fed on a mouse tail, mouse ear, or chick toe. In addition, we examined the movement of virus from the probing/feeding site, determined whether the amount of virus inoculated was related to mosquito probing time, compared in vitro and in vivo estimates of the dose of WNV inoculated by mosquitoes, and examined clearance of the virus from the blood of mice following intravenous inoculation. We found that mosquitoes inoculate high doses of WNV into hosts while probing and feeding, doses that are 10- to 1,000-fold higher than previous estimates. Additionally, we found that mosquitoes inoculate low amounts of WNV directly into the blood while feeding. Results Efficiency of Virus Recovery from Mouse Tissues We determined our ability to recover a known amount of WNV (∼105 PFU) inoculated subcutaneously into the tail and ear of three mice using our tissue grinding protocol (see Materials and Methods). As a control, the same volume of virus was inoculated directly into each of five microcentrifuge tubes. Control samples contained an average of 106,000 PFU, whereas mouse tails contained 30,900 PFU, and mouse ears contained 34,500 PFU. Assuming the same dose was inoculated into the ear and tail as was inoculated into the control samples, we recovered 29.2% of the inoculated virus from the mouse tail and 32.5% from the mouse ear. These results indicate that subsequent amounts of virus recovered from mouse tissues need to be multiplied by ∼3 to provide the actual amount of virus inoculated into these tissues by mosquitoes. Unless otherwise indicated, titers mentioned in the text or shown in figures are not adjusted for “unrecovered” virus. Amount of Virus Inoculated into Host Tissues by Mosquitoes We determined the amount of virus inoculated by WNV-infected mosquitoes while probing and feeding on a mouse tail, mouse ear, or chick toe. Eight independent trials of this experiment were conducted, with orally infected or intrathoracically inoculated mosquitoes of four species (Cx. tarsalis, Cx. pipiens, Ae. japonicus, and Ae. triseriatus) (Table 1). Cx. tarsalis and Cx. pipiens are important enzootic vectors of WNV in the United States, and Ae. japonicus and Ae. triseriatus have been implicated as bridge vectors for WNV [9]. There was variation in the age of the hosts used in these studies and mosquito extrinsic incubation period following intrathoracic inoculation (Table 1); however, these variables did not have a significant effect on the amount of virus inoculated by mosquitoes (Spearman's rank correlation coefficient p > 0.05). Table 1 Details of Experiments to Determine the Dose of Virus Inoculated by Mosquitoes while Probing and Feeding on Live Hosts Mosquitoes inoculated high doses of WNV into host tissues while probing and feeding (Figure 1A). The amount of virus recovered from tissues ranged from below the limit of detection (5 PFU) to 106.6 PFU. Mean and median values of the groups ranged from 102.9 PFU to 105.5 PFU. Although there was variation in mean and median inoculated doses among the groups, these differences were not statistically significant (Kruskal-Wallis test, p > 0.05) (Figure 1A). Mosquito infection method (orally infected or intrathoracically inoculated) also had no effect on the amount of virus recovered from tissues when the analysis was restricted to mouse tissues fed upon by Cx. tarsalis (Wilcoxon test, p > 0.5) or when the data were pooled (Figure 1B). Similarly, the type of host tissue fed upon by Cx. tarsalis (mouse tail, mouse ear, or chick toe) had no effect on inoculation dose (Kruskal-Wallis test, p = 0.46). However, when the data were pooled, more WNV was recovered from the mouse ear and chick toe than from the mouse tail (Kruskal-Wallis test, p = 0.03) (Figure 1B). The amount of virus recovered also varied by mosquito species; Cx. pipiens feeding on chick toes inoculated higher amounts of virus than did Cx. tarsalis feeding on chick toes (ANOVA of ranked data, p = 0.04). When the data were pooled, Cx. pipiens inoculated higher doses than did Cx. tarsalis or Ae. triseriatus (Kruskal-Wallis test, p = 0.01). Mean and median doses inoculated by Cx. pipiens were 105.4 and 105.6 PFU, Cx. tarsalis inoculated mean and median doses of 103.8 and 104.5 PFU, Ae. japonicus inoculated mean and median doses of 104.2 and 104.2 PFU, and Ae. triseriatus inoculated mean and median doses of 103.1 and 102.9 PFU. Considering that we recovered approximately one-third of the virus from mouse tissues, and assuming that we would have recovered a similar proportion from chick tissues, these mean and median values should represent the minimum average values inoculated by mosquitoes. Adjustment of these values to account for unrecovered virus suggests that Cx. pipiens inoculated mean and median doses of 105.9 and 106.1 PFU, Cx. tarsalis inoculated mean and median doses of 104.3 and 105.0 PFU, Ae. japonicus inoculated mean and median doses of 104.7 and 104.7 PFU, and Ae. triseriatus inoculated mean and median doses of 103.6 and 103.4 PFU. Figure 1 Mosquitoes Inoculate High Doses of WNV In Vivo under Various Experimental Conditions (A) WNV doses inoculated extravascularly by mosquitoes. x-Axis labels indicate mosquito infection method (Inoc = intrathoracic inoculation, Oral = orally infected), mosquito species (Tar = Cx. tarsalis, Pip = Cx. pipiens, Jap = Ae. japonicus, Tri = Ae. triseriatus), and tissue in which the mosquito probed or fed in (Tail = mouse tail, Ear = mouse ear, Toe = chick toe). Limit of detection (LOD) of plaque assay is shown. (B) Same data as in (A) but pooled by tissue type, mosquito species, and infection method. Within each larger grouping (tissue type, mosquito species, or infection method), groups designated with different lower case letters (above graph) are significantly different from one another (p 7 min on the shaved back of a mouse [14]. Studies with Ae. aegypti and Anopheles stephensi reported average probing times of 99%) recovered from the mouse tail was recovered from the 1-cm section that the mosquito had probed or fed in. This result suggests that most virus is inoculated extravascularly while the mosquito is probing, and it further suggests that virus does not spread very quickly within the tissues. Extravascular inoculation of virus by mosquitoes has been demonstrated previously for Rift Valley fever virus, Saint Louis encephalitis virus, and VEEV [12,20,21]. Although mosquito inoculation of WNV is primarily extravascular, our results also indicate that some mosquitoes inoculate a small amount of virus directly into the blood while blood feeding. Virus (average titer = 102.0 PFU/ml) was detected in the sera of 22 out of 29 animals when mosquitoes imbibed blood. However, when mosquitoes only probed and did not blood feed, virus was detected in the serum of only two of 20 animals. Direct inoculation of virus into the blood by mosquitoes could alter viral tropism and kinetics, and may explain the earlier development of viremia in hosts infected with WNV by mosquito bite compared to infection by needle inoculation [3]. In addition, our results suggest that the recent finding of non-viremic transmission of WNV by Cx. pipiens quinquefasciatus could have been due to infected mosquitoes inoculating a small amount of virus directly into the blood, which is imbibed by recipient mosquitoes, resulting in a low infection rate [22,23]. A recent publication supports this hypothesis. Low viremia levels (102.9–104.2 PFU/ml) were detected in house finches 30 to 45 min after infected mosquitoes fed, resulting in low infection rates in recipient mosquitoes [24]. At low viremia levels ( five virions, if virus is distributed at random in the blood [25]. Therefore, mosquito infection rates would be expected to be low (not zero), as long as some proportion of the mosquito population was able to become infected after ingesting low numbers of virions (i.e., a highly competent population). Additionally, our intravenous clearance study indicates that virus is cleared from the blood of mice at a rate of 0.7 log10 PFU/ml per hour. Therefore, 100 PFU of virus inoculated directly into the blood by a donor mosquito could theoretically circulate in the blood for 1–2 h and could infect recipient mosquitoes. Most of the virus (50%–75%) in a mosquito was recovered from the thorax; amounts ranged from 103.7 to 107.7 PFU. The thorax contains not only musculature for locomotion, but also the salivary glands. Large aggregations of WNV virions were observed in salivary glands of orally infected mosquitoes at 14 d PI by electron microscopy [18,23]. Salivary glands of intrathoracically infected Cx. pipiens quinquefasciatus contained high titers of WNV (up to 107 PFU equivalents) [23]. Assuming that much of the virus we detected in the thorax is contained within the salivary glands, thoracic viral load correlates well with the high doses inoculated into hosts by mosquitoes. In conclusion, we found that mosquitoes inoculate high doses (104–106 PFU) of WNV extravascularly and low amounts (∼102 PFU) intravascularly while probing and feeding on a live host. Direct inoculation of WNV into the host's blood during feeding may alter viral tropism, lead to earlier development of viremia, and result in infection of co-feeding mosquitoes. In a direct comparison, the amount of virus inoculated by a mosquito while feeding on a live host was ∼600-fold higher than that recovered during an in vitro capillary tube assay. These results suggest that the use of an in vitro capillary tube assay will result in lower estimates of the dose inoculated by mosquitoes and may also underestimate transmission rates (the proportion of mosquitoes that are capable of transmitting a pathogen once infected). Use of an accurate dose to infect animals is important in vaccine, host competence, and pathogenesis studies, especially because viral dose has been shown to affect WNV viremia and viral shedding [3]. Materials and Methods Virus. All experiments were conducted with WNV strain 3356 isolated in 2000 from the brain of a crow collected in Staten Island, New York [26]. This isolate was passed twice in Vero cells and had a titer of 109.5 PFU/ml, as determined by plaque assay on Vero cells. Animals. We used a Cx. pipiens colony established in 2004 from mosquitoes collected in Pennsylvania. We used the HVP Cx. tarsalis colony, which was derived from the WS colony, a colony that consisted of a mixture of field populations from California selected for high susceptibility to Western equine encephalitis (kindly provided by William Reisen, University of California, Davis). Ae. japonicus and Ae. triseriatus eggs were collected on expanded polystyrene floats [27] in Albany, New York, and reared in the laboratory at 22 °C. Emerged females were identified to species and used in our studies. Specific-pathogen-free Gallus gallus chicks (1–2 d old) were obtained from Charles River SPAFAS (http://www.criver.com/). Mouse strains C3H/HeN and C57/BL6 were obtained from Taconic Laboratories (http://www.taconic.com/) and strain FVB was obtained from Wadsworth Center, New York State Department of Health. All animals were housed in a BSL-3 animal facility. The use of chicks and mice in this experiment was approved and conducted in accordance with the Wadsworth Center Institutional Animal Care and Use Committee. Infection of mosquitoes with WNV. Mosquitoes were infected with WNV by intrathoracic inoculation of ∼30 PFU WNV or by allowing mosquitoes to feed on an infectious blood meal. Infectious blood meals were obtained from an infected 5-d-old chick inoculated subcutaneously 3 d earlier with 103 PFU/0.1 ml WNV or a Hemotek membrane feeder (Discovery Workshops, Accrington, UK, hemotek@discoveryworkshops.co.uk) that contained an infected blood meal consisting of one part virus, one part 50% sucrose, and 19 parts defibrinated goose blood (Hema Resource and Supply, http://www.hemaresource.com/). The titer of WNV in the chick was 106.1, and the titer in the feeder blood meals was 107.6 PFU/ml. Mosquitoes, starved for 24–48 h, were exposed to a lightly restrained chick or membrane feeder for ∼1 h. Fully engorged mosquitoes were removed and maintained at 27 °C, high humidity, and with a photoperiod of 16:8 (L:D), until used in experiments. Because only ∼50% of Cx. tarsalis females orally exposed to WNV become infected, we screened mosquitoes for the presence of a disseminated infection prior to use in experiments. On days 13–14 post-feeding, mosquitoes were anesthetized with CO2 and wet ice; one metathoracic leg was removed and placed into a microcentrifuge tube with a BB (Daisy Zinc Plated BB, Rogers, Arkansas, United States) and 1 ml of mosquito diluent (20% heat-inactivated fetal bovine serum in Dulbecco's prosphate-buffered saliva plus 50 ug/ml penicillin/streptomycin, 50 ug/ml gentamicin, and 2.5 μl/ml fungizone). Legs were homogenized in a mixer mill (QIAGEN, http://www.qiagen.com/) at 24 cycles/s for 30 s and then clarified by centrifugation. Virus was detected in clarified homogenate by plaque assay on Vero cells. Presence of virus in the leg indicated a disseminated infection. Efficiency of virus recovery from mouse tissues. To determine the efficiency of virus recovery from mouse tissues, we inoculated a known amount of virus (105 PFU in 1 μl) subcutaneously into the tail (∼2 cm from tip) and ear (∼1 cm from base) for each of three deeply anesthetized C3H mice, using a 30G needle and 100-μl glass syringe (Hamilton, http://www.hamiltoncompany.com/). Immediately following inoculation, the tail and ear were cut off at the base, and the mouse was euthanized. The tail was further divided into 1-cm sections, starting at the tip. Each tissue (or tissue section) was placed into an individual microcentrifuge tube containing 500 μl of BA-1 diluent (M199H, 1% bovine serum albumin, 0.05 M Tris [pH 7.6], 0.35 g/l sodium bicarbonate, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml fungizone). As a control, virus (105 PFU in 1 μl) was inoculated directly into each of five microcentrifuge tubes containing 500 μl of BA-1 diluent, using the same needle and syringe. All samples (tissues and controls) were then processed as described below. Amount of virus inoculated into host tissue by mosquitoes. We determined the amount of WNV inoculated by infected female mosquitoes while probing and feeding on a chick toe, mouse tail, or mouse ear. Individual mosquitoes, infected with WNV as described above, were placed into clear plastic 18.5-ml vials with mesh top and starved for ∼48 h prior to feeding. Prior to mosquito feeding, mice were lightly anesthetized with 0.5 mg/g of Avertin (2,2,2 tribromoethanol; Sigma-Aldrich, http://www.sigmaaldrich.com/), and the tail was marked every 1 cm, starting from the tip, with a laboratory marker. Chicks were restrained by hand, and the center toe of the left foot was marked every 0.5 cm starting from the tip. The mesh top of a vial containing one mosquito was placed in contact with the ventral side of the chick toe, ventral side of the mouse tail, or distal half of the mouse ear. Mosquitoes were observed throughout the experiment with a 5× handheld magnifying glass. Probing time, feeding time, blood engorgement status, and probing location (on the tail or toe) were recorded for each mosquito. Blood engorgement status was scored with a range from 1 (blood just able to be detected in abdomen) to 4 (fully engorged). Probing was defined as the period from when the stylets penetrated the skin and the labial sheath folded back, to the first appearance of blood in the abdomen. Feeding began when blood was first detected in the abdomen and ended when a mosquito withdrew her mouthparts from the host. We limited the probing period for each mosquito to a maximum of 10–11 min to minimize the entry of virus into host cells. Immediately after the cessation of feeding or the 10–11 min probing period, the host animal was deeply anesthetized, and the tissue that had been fed upon was cut off at the base and divided into sections (for the tail and toe) with a scalpel. A sample of blood was taken from the wing vein (chicks) or the heart (mice). Animals were then euthanized by cervical dislocation. Average time between cessation of mosquito probing or feeding and tissue excision was 5 min. Tissue and blood samples were held at 4 °C or on wet ice for up to 3 h and then frozen at −80 °C. Prior to freezing, blood samples were centrifuged at 8,000 rpm for 5 min and serum was collected. Tissue samples were processed as described below. Mosquitoes that had fed on or probed the host were killed by freezing, and the legs and body of each mosquito were dissected, placed into separate microcentrifuge tubes containing 1 ml mosquito diluent and a BB, and frozen at −80 °C. Body and leg samples were homogenized and clarified as described above. Virus was quantified in clarified mosquito homogenate by plaque assay on Vero cells. Individual host animals were included in the analysis only if the mosquito that had fed on the animal was subsequently confirmed as positive for viral dissemination (i.e., virus-positive legs). Effect of probing time on viral dose inoculated by mosquitoes. We sought to determine whether longer probing times led to higher inoculated doses of WNV. Cx. tarsalis females were infected with WNV by intrathoracic inoculation as described above. At 5 d PI, individual mosquitoes were placed into 18.5-ml clear plastic vials and starved of sucrose and water for 48 h. At 7 d PI, individual mosquitoes were allowed to probe on the tails of lightly anesthetized 12-wk-old C3H female mice for 30 s, 1 min, 2 min, 4 min, or maximum time (n = 5/group). Mosquitoes in the maximum time group were able to imbibe blood; mosquitoes in all other groups probed only. Mosquitoes were observed throughout the experiment with a 5× handheld magnifying glass. Feeding time, blood engorgement status, and probing location were recorded for each mosquito as described above. Host tail tissue and blood were harvested as described above. Comparison of in vivo and in vitro estimates of viral dose inoculated by mosquitoes. To compare in vivo and in vitro estimates of the amount of WNV inoculated by mosquitoes, we performed an in vitro capillary tube transmission assay on mosquitoes that had probed and fed on a mouse tail. Cx. tarsalis females were infected with WNV by intrathoracic inoculation of ∼300 PFU. At 5 d PI, individual mosquitoes were placed into 18.5-ml clear plastic vials and starved of sucrose and water for 48 h. At 7 d PI, individual mosquitoes were allowed to probe and feed on the tails of lightly anesthetized C3H female mice until they had taken a full blood meal. Mosquitoes were observed throughout the experiment with a 5× handheld magnifying glass. Feeding time, blood engorgement status, and probing location were recorded for each mosquito as described above. Host tail tissue and blood were harvested as described above. Within 2–4 h of host tail feeding, in vitro capillary tube transmission assays were performed using the mosquitoes that had fed on the tails and also those in the same cohort that had been starved, and not allowed to feed. For these assays, each mosquito was anesthetized with triethylamine (Sigma-Aldrich), its legs were removed, and its proboscis was placed into a glass capillary tube filled with a 1:1 solution of 50% sucrose and fetal bovine serum. After 30–40 min, the mosquito was removed from the capillary tube and contents of the capillary tube were expelled into 300 μl of mosquito diluent and frozen at −80 °C until assayed for WNV by plaque assay. Tissue sample processing. Tissue samples and virus controls were thawed on wet ice and poured into a plastic weighing boat. Tissues were macerated with a sterile scalpel. Macerated tissues were returned to their original vial, and 500 μl of BA-1 were added to the weighing boat so as to wash any remaining tissue back into the vial. A BB was added to the vial, and the sample was homogenized in a mixer mill at 24 cycles/s for 8 min and centrifuged at 14,000 rpm for 3 min. Virus was quantified in clarified tissue homogenate, control samples, and sera by plaque assay on Vero cells. WNV titer in mosquito body segments. The distribution of WNV in mosquito body segments was determined. Orally or parenterally infected Cx. tarsalis, and parenterally infected Cx. pipiens were killed by freezing at −80 °C at 16 or 7 d post-infection, respectively. Mosquitoes were later thawed on wet ice, their legs were removed, and the head, thorax, and abdomen were cut apart with a scalpel. Body segments were placed into separate microcentrifuge vials containing 1 ml of mosquito diluent and a BB. Tubes containing mosquito body parts were homogenized as described above. Virus was detected in clarified mosquito homogenate by plaque assay on Vero cells. Clearance of virus from blood following intravenous inoculation. Four adult C57/BL6 mice were inoculated intravenously in the lateral tail vein with 105 PFU in a volume of 0.1 ml. Blood samples were taken at 5, 15, and 45 min PI by tail bleeding. Mice were euthanized at 90 min PI, and a blood sample was taken from the heart. Blood samples were centrifuged at 8,000 rpm for 5 min, and serum was collected and frozen at −80 °C until tested for virus by plaque assay on Vero cells. Statistical analysis. Viral titers were log transformed and checked for normality using Shapiro-Wilk or Kolmogorov-Smirnov statistics. The limits of detection for plaque assays were 5 PFU for mosquito, mouse, and chick tissues, and 5 PFU/ml for serum. Nonparametric tests were used when we compared groups with small sample sizes or non-normal distributions. One-way ANOVA was used to test for differences between virus titers in mosquito body segments; the Tukey-Kramer method was used to adjust for multiple comparisons. Viral clearance data was normalized to the 5-min titer, and Graph Pad Prism software (http://www.graphpad.com/) was used to fit a linear regression model. Supporting Information Table S1 Amount of WNV Inoculated by Parenterally Infected Cx. tarsalis while Probing and Feeding on a Mouse Tail (57 KB DOC) Click here for additional data file. Table S2 Amount of WNV Inoculated by Orally Infected Cx. tarsalis while Probing and Feeding on a Mouse Tail (54 KB DOC) Click here for additional data file. Table S3 Amount of WNV Inoculated by Orally and Parenterally Infected Cx. tarsalis while Probing and Feeding on a Mouse Ear (37 KB DOC) Click here for additional data file. Table S4 Amount of WNV Inoculated by Parenterally Infected Cx. tarsalis while Probing and Feeding on a Chick Toe (41 KB DOC) Click here for additional data file. Table S5 Amount of WNV Inoculated by Parenterally Infected Cx. pipiens while Probing and Feeding on a Chick Toe (41 KB DOC) Click here for additional data file. Table S6 Amount of WNV Inoculated by Parenterally Infected Ae. japonicus while Probing and Feeding on a Mouse Tail (41 KB DOC) Click here for additional data file. Table S7 Amount of WNV Inoculated by Parenterally Infected Ae. triseriatus while Probing and Feeding on a Mouse Tail (37 KB DOC) Click here for additional data file.
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              Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice.

              Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.
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                10.1038/ncomms12204

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