The authors have declared that no competing interests exist.
Conceived and designed the experiments: NK BB RK GY. Performed the experiments: NK RK GY. Analyzed the data: BB RK NK. Contributed reagents/materials/analysis tools: NK RK GY BB. Contributed to the writing of the manuscript: RK BB NK GY.
Current address: Takeda Vaccines Inc., Madison, Wisconsin, United States of America
Serial samples from the same individuals may be required for certain virological studies, however, some small animals cannot easily be blood-sampled. Therefore, we evaluated the use of
The use of animals in laboratory studies is paramount to understanding biological, ecological, and clinical components of arboviral infection. Experimental infection studies have provided valuable information on viral factors such as virulence and pathogenesis, and on host factors such as susceptibility, reservoir competence and immune response. Integral to all of these types of studies is the ability to obtain blood samples from animals in a manner that both meets the specific aims of the research and minimizes stress to the animals.
Many vertebrate infection experiments require repeated blood sampling of the same individuals over specific time intervals. However, some animals such as mice, and small birds and reptiles, cannot easily be bled repeatedly due to their small size and limited blood volume or lack of sufficient vein presentation. Different groups of animals are often bled on alternating days due to limitations in the amount of blood that can safely be taken from an individual animal over a given time period
Yuill
All animal work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the CDC Institutional Animal Care and Use Committee, Protocol # 07–011. All efforts were made to minimize suffering.
Five to seven-day-old and two- to three week-old chicks (
Prior to blood sampling, hamsters were anesthetized with 0.025 mL/10 g body weight using a mixture of ketamine (dose 200 mg/kg) and xylazine (10 mg/kg), injected intraperitoneally (ip). Chicks and sparrows were administered 0.01 mL/10 g body weight ketamine (dose 50 mg/kg) and xylazine (10 mg/kg) mixture intramuscularly (im). Following experimentation, animals were euthanized by cervical dislocation performed under anesthesia.
Animals, viruses, and sampling days are presented in
Animal | N | HJV | WNV | Days post |
Inoculum | inoculum | inoculation blood samples were taken | ||
Chick (5–7 days old) | HJV:0; WNV:4 | 104.0 pfu+mosquito saliva | 104.0 pfu+mosquito saliva | HJV: Days 1 and 2; WNV: Days 2 and 3 |
Chick (2–3 weeks old) | HJV:10; WNV:10 | 104.3 pfu+mosquito saliva | 104.0 pfu+mosquito saliva | HJV: Days 1 and 2; WNV: Days 2 and 3 |
Hamster | HJV:10; WNV:10 | 104.0 pfu+mosquito saliva | 104.0 pfu+mosquito saliva | HJV: Days 1–3; WNV: Days 3 and 4 |
House sparrow | HJV:24; WNV:36 | 104.6 pfu | 102.6 pfu | HJV: Days 1–3; WNV: Days 1–4 |
Virus inocula are given as pfu per 0.1 mL BA-1 media.
Between days one and four post-inoculation when peak viremia was expected
Syringe samples were taken within 20 minutes of mosquitoes feeding on each animal. 0.1 mL blood was collected using a syringe attached to a 26 g ½-in SubQ needle. Blood was collected by jugular venipuncture of chicks on two occasions (one day apart) for each chick, and of sparrows on one or two occasions (three days apart), and by cardiac puncture of hamsters (post-mortem). Syringe samples were either expelled directly into Microtainer serum separators and centrifuged to isolate the serum for testing, or diluted in 450 µl BA-1 diluent (to achieve a 1∶10 dilution of serum). Blood samples were allowed to coagulate at room temperature for up to 30 minutes before placing them on ice.
Blood samples taken by syringe and mosquito were titrated simultaneously using the double-overlay Vero cell plaque assay
To ensure that any differences in titer between mosquito and syringe samples were not due to virus binding to mosquito tissues during blood feeding or grinding, six replicates each of 1 mL BA1+20% FBS containing approximately 102 pfu HJV or WNV were incubated at 28°C for 45 minutes with or without a homogenized mosquito. Virus titers of paired samples were compared by Vero cell plaque assay.
Three questions of interest concerning the association between virus log titer measurements obtained via syringe sampling and mosquito sampling of blood from a given animal were addressed: 1) as a function of the syringe-sampled log titer, what is the probability that the mosquito-sampled log titer would be positive, i.e., would detect virus? 2) among animals with positive log titer measurements using both mosquito and syringe sampling, at what syringe-sampled log titer would there be a 25%, 50%, or 75% probability that the corresponding mosquito-sampled log titer would be measurable, and what is the expected mosquito-sampled log titer as a function of the syringe-sampled log titer? 3) for each of the questions 1 and 2, do these associations vary by animal host (chicken, hamster, sparrow) or virus (HJV, WNV)?
A mixed discrete and continuous regression model was used to evaluate all three of these questions simultaneously. The discrete component of the model consisted of a logistic regression for log titer positivity, where the logit of the probability of a positive mosquito-measured log titer was modeled as a linear function of the syringe-measured log titer. The continuous component of the model was a linear model of the mosquito-measured log titer as a function of syringe-measured log titer, though no intercept was included in the model to reflect the fact that no mosquito-sampled values would be positive when the syringe-sampled values were 0. For both discrete and continuous components of the model, variables were included to permit different intercepts (discrete only) and slopes for animal and virus, including interactions. The models were fit using maximum likelihood, 95% profile confidence intervals (CI) for the model parameters were computed, and models were compared and selected using the likelihood ratio test and 5% significance. Inversion of the logistic model component provided estimates and 95% CIs of syringe-measured log titer values expected to yield positive mosquito-measured log titer values with probabilities of 25%, 50%, 75% and 90%. Standard regression diagnostics were used to evaluate model assumptions.
Analyses were carried out and graphs produced in the R statistical software package
For analysis of WNV and HJV viremia profiles (log titer over time), we took two approaches. For a direct comparison of mosquito-sampled versus syringe-sampled log ‘titers at each dpi separately, we used the paired t-test, which accounts for the paired observations on individual house sparrows. These comparisons by dpi within virus were adjusted for multiple comparisons using the Bonferroni adjustment. We then used mixed linear models to evaluate potential trends in the association between dpi and titer, including collection type (mosquito or syringe) as predictors; interaction of collection type with dpi was also included. Because titers were only measured at four time points, we interpret results of this modeling as indicative of general trends in the titers over time, rather than as definitive descriptions of the profiles. Estimation was done using ML when comparing fixed effect model parameters using the likelihood ratio tests, while final model parameters were estimated with restricted ML (REML). For these analyses, there were minimal 0-valued titers, so we did not model the probability of obtaining a 0 titer as a separate model component. Models were fit in R using the nlme package
Starved
All birds were >3 months old at the time of capture. Initial sampling indicated that 18% (13/71) of sparrows were positive for neutralizing antibodies to WNV, and 11% (8/71) were positive for alphavirus-neutralizing antibodies. Birds with prior exposure to WNV were excluded from experimental infection with WNV. Similarly, birds with alphavirus neutralizing antibodies were excluded from experimental infection with HJV.
The mixed discrete and continuous regression model fits and model comparisons using the likelihood ratio test resulted in a final model with no statistically significant differences in the logistic component’s intercept and slope by either animal or virus. In contrast, a statistically significant difference was found in the slopes of the linear component by virus, though not by animal (
Model | Logistic | Linear | Comparator Model | Total df | Deviance | Chi-sq | df | p-value |
1 | AVi | AVi | – | 19 | 221.4 | |||
2 | AV | AVi | 1 | 14 | 223.3 | 1.9 | 4 | 0.76 |
3 | AVi | AV | 1 | 17 | 225.0 | 3.6 | 2 | 0.16 |
1 | 13 | 226.9 | 5.5 | 6 | 0.48 | |||
4 | AV | AV | 2 | 3.6 | 2 | 0.16 | ||
3 | 1.9 | 4 | 0.76 | |||||
5 | AV | A | 4 | 12 | 231.4 | 4.5 | 1 | |
6 | AV | V | 4 | 11 | 229.8 | 2.9 | 2 | 0.23 |
7 | A | AV | 4 | 11 | 230.0 | 3.1 | 2 | 0.21 |
8 | V | AV | 4 | 9 | 232.9 | 6.0 | 4 | 0.20 |
9 | A | A | 5 | 10 | 234.5 | 3.1 | 2 | 0.21 |
7 | 4.5 | 1 | ||||||
10 | A | V | 6 | 9 | 233.0 | 3.1 | 2 | 0.21 |
7 | 2.9 | 2 | 0.23 | |||||
11 | V | V | 6 | 7 | 235.8 | 6.0 | 4 | 0.20 |
8 | 2.9 | 2 | 0.23 | |||||
12 | V | A | 5 | 8 | 237.4 | 6.0 | 4 | 0.20 |
8 | 4.5 | 1 | ||||||
13 | A | 1 | 9 | 8 | 239.5 | 5.0 | 2 | 0.08 |
10 | 6.5 | 1 | ||||||
14 | 1 | A | 9 | 6 | 239.1 | 4.6 | 4 | 0.33 |
12 | 1.8 | 2 | 0.42 | |||||
15 | V | 1 | 12 | 6 | 242.3 | 5.0 | 2 | 0.08 |
11 | 6.5 | 1 | ||||||
16 | 1 | V | 10 | 5 | 237.6 | 4.6 | 4 | 0.33 |
11 | 1.8 | 2 | 0.42 | |||||
13 | 4 | 244.1 | 4.6 | 4 | 0.33 | |||
14 | 5.0 | 2 | 0.08 | |||||
17 | 1 | 1 | 15 | 1.8 | 2 | 0.42 | ||
16 | 6.5 | 1 |
Models are specified by their logistic and linear components, with “A” indicating a term for animal was in the model component, “V” indicating a term for virus was in the model component, and “I” the interaction between animal and virus was in the model component. For convenience, model labels are given in the first column (Model), and the nested/reduced model being compared to that is given as the Comparator Model, referenced by the Model label of column one. P-values <0.05 are in boldface.
Logistic component | Estimate | 95% CI |
Intercept | −4.37 | −6.16, −2.94 |
Slope | 1.20 | 0.83, 1.67 |
Slope – HJ | 1.03 | 0.97, 1.09 |
Slope – WNV | 0.93 | 0.89, 0.97 |
Variance | 0.59 | 0.42, 0.85 |
Recall there was no intercept included in the linear component of the model.
Chicken, hamster, and house sparrow all developed detectable viremia for both WNV and HJV infections. The probability of detecting virus in a mosquito blood meal increased with virus titer (as syringe-measured;
Data for viruses (a) or animals (b). Panel (c) shows the data by virus with graphical representations of the linear (scale on left margin) and logistic (scale on right margin) components of the final fitted model. Also shown below the horizontal 0 in panel (c) are estimates and 95% confidence intervals for the syringe-measured titers that have 25% (o), 50% (+), 75% (×), and 90% (▪) probabilities of obtaining a positive mosquito-measured titer.
In general, good correlation was seen between mosquito and syringe samples at titers ≥5.0 log10 pfu/mL serum as compared with titers <5.0 log10 pfu/mL serum (
Virus | DPI | Mean difference in log-titer | 95% CI | p-value |
HJV | 1 | 0.30 | −0.04–0.62 | 0.08 |
2 | −1.56 | −2.75–−0.37 | 0.02 | |
3 | −1.46 | −3.67–0.76 | 0.11 | |
4 | no observations | – | – | |
WNV | 1 | −0.80 | −2.25–0.65 | 0.23 |
2 | −0.19 | −0.65–0.28 | 0.37 | |
3 | −0.15 | −0.84–0.53 | 0.61 | |
4 | −1.02 | −2.33–0.30 | 0.11 |
There were 38 instances where multiple mosquitoes fed on the same viremic sparrow (HJV or WNV) and each mosquito was analyzed separately. When sparrow virus titers were >5.0 log10 pfu/mL serum, mosquito titers varied on average by 0.44 log pfu/mL serum from each other (range 0–1.5 log difference, n = 22). When sparrow titers were <5.0 pfu/mL serum, mosquitoes fed on the same animal had titers that varied on average by 2.7 log pfu/mL serum (range 0.2–4.7 log difference, n = 16).
Viremia profiles (mean daily viremia) were generated for house sparrow infected with HJV (
Mean virus titers were similar for WNV and HJV incubated with and without a homogenized
We have evaluated a novel technique for viremia determination in vertebrates which uses mosquitoes in place of syringes for drawing blood. This technique can be applied to a variety of virus - vertebrate systems, and although demonstrated with a mosquito-borne flavivirus and alphavirus, is not limited to the study of arboviruses. Single engorged mosquitoes fed on viremic animals were successfully used to approximate mean daily virus titer and describe viremia profiles. Mosquito-derived blood also informed the day of maximum viremia, and the duration of detectable viremia. Importantly, fewer animals are required since individuals can be repeatedly sampled on consecutive days and blood samples can be obtained in a less invasive manner.
Mean daily titers determined from mosquito-drawn blood were usually slightly lower than titers determined by syringe (
In looking at variability among virus titers in mosquito blood meals from the same sparrow, most mosquitoes that fed on a sparrow with a titer >5.0 log pfu/mL serum had blood meal titers within 0.5 log of each other. However in one case a few mosquitoes had very different titers from the rest. For example, 10 mosquitoes fed on a WNV-infected sparrow with a syringe titer of 6.0 log10 pfu/mL serum. For reasons that remain unclear, seven mosquitoes had titers ranging from 6.5 to 6.9 log10 pfu/mL serum, and three had titers of 9.7, 9.7 and 9.5 log10 pfu/mL serum. These results were re-confirmed by Vero cell plaque assay. Concentration of blood in the midgut post-feeding was considered, however mosquitoes were not given enough time to undergo diuresis prior to freezing, and no such droplets were ever seen at the bottom of the cup. Alternatively the location on the animal where those aberrant blood meals were obtained and uneven distribution of virus particles within the blood stream could possibly have played a role.
A viremia profile for house sparrows infected with HJV has not been previously reported. The sparrows reached a peak viremia within 24 hours post inoculation, after which viremia dropped off sharply (
The viremia profile generated for house sparrows infected with WNV (NY99-4132) was different from that previously reported
We have developed a novel technique for arbovirus viremia determination in small vertebrates. By testing the blood meals of single engorged mosquitoes fed on viremic animals, we have closely approximated the viremia profile, duration of detectable viremia, and day of peak viremia in chicks, hamsters, and sparrows infected with HJV and WNV. Additionally, we have applied the technique to generate a novel HJV viremia profile for house sparrow, and an updated WNV viremia profile in a Colorado population of house sparrows. While we have demonstrated the proof of concept by testing mosquitoes individually for comparison against syringe titer, future work should seek to compare the precision of pooling engorged mosquitoes fed on a viremic host against testing them individually for approximating virus titer. Additionally, the minimum number of mosquitoes necessary to obtain an accurate estimate of viremia will vary by titer, and has yet to be modeled. Still, this technique creates the opportunity to investigate the competence of other small vertebrates such as reptiles, rodents, and nestling birds as arbovirus amplifying hosts, which until this point has been impossible due limitations in serial blood sampling capabilities.
We would like to thank the late Dr. Andrew Spielman for inspiring this work. Animal care was provided by Verna O’Brien, Andrea Peterson, and Dr. Paul Spurlock. Insectary support was provided by Andrea Peterson and Erin Borland. Permission for sparrow capture was provided by Cozy Cow Dairy, Windsor, CO. This project was financially supported by the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors only, and do not necessarily reflect the views of the United States Government.