Crinivirus (Closteroviridae) is transmitted in a semi-persistent manner by the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). Although several approaches exist for modeling insect population growth, modeling vector-born virus spread remains difficult because fundamental knowledge on the relationship between virus transmission and temperature is lacking for most vector transmitted viruses. To address this challenge, we initially developed a temperature-dependent phenology model for the whitefly vector using the Insect Life Cycle Modeling (ILCYM) software. In the present study, the effect of temperature on the efficiency of virus transmission by the whitefly was determined through controlled laboratory experiments at 8 constant temperatures in the range from 10 to 25 °C. The vector capacity to transmit the virus was highest at 15 °C (about 70 % probability of infection) but decreased radically as temperature deviated from this optimum temperature to <10 % at temperatures of 10 and 20 °C, respectively. The temperature-dependent probability of virus transmission by a single adult whitefly could be described by a nonlinear function, which was validated by transmission frequencies observed at fluctuating temperatures. This function combined with life table parameters calculated from previously established temperature-dependent phenology model for the vector provided a full temperature-responsive model for predicting PYVV spread potential and transmission probabilities. For spatial risk predictions, we devised two virus transmission risk indexes and tested their performance in correctly predicting virus presence/absence with field survey data. The best performing risk index was used to generate risk maps, which reflected well the current  occurrence of the virus but also predicted areas at high risk, where the virus has not previously been reported. One of them in western Panama was targeted for surveillance and resulted in identification of the virus in the country, where it was not previously known to occur. Simulated risk maps for the year 2050 revealed that climate change may significantly affect, the risk of distribution, generally reducing in tropical areas of the world, but increasing in the temperate regions.Management of viral plant diseases can be improved by using models to predict disease spread. Potato yellow vein virus (PYVV) of the genus  The crop is affected by various pest and disease, among which viruses are one of the most important due to their gradual accumulation over generations of clonal propagation, eventually leading to yield decreases of 50 % or more. Global trade and a changing climate affect the distribution and impact of pests and diseases leading to emergence in new areas. Potato yellow vein disease, caused by Potato yellow vein virus (PYVV), a member in genus Crinivirus (family Closteroviridae) is an important disease of the crop, in Colombia, Ecuador and northern Peru, causing up to 50 % yield reductions   can act as reservoirs for PYVV (PYVV is considered a quarantine pathogen by various agencies such as the European and Mediterranean Plant Protection Organization (EPPO) and the Animal and Plant Health Inspection Service (APHIS)  . The model, which was implemented in Insect Life Cycle Modeling (ILCYM) software  . A tempesoftware , 2017, psoftware . Also fosoftware , althouga) to assess the effect of temperature on the PYVV transmission rates by its vector, the greenhouse whitefly, b) establish a temperature-dependent model for PYVV transmission probability by its vector and c) combine this model with the previously established T. vaporariorum phenology model for predicting the risk of virus spread based on temperature on global, regional, or national scales. The virus transmission model was implemented as an additional module in the ILCYM software. Thus, we demonstrate an approach that extends the knowledge gained from insect phenology models to assess risks of diseases transmitted by insect species in cultivated crop.The objectives of this study were 22.1Virus–free potato plants (Clon W.A 077: CIP397077.16 and cultivar Canchan INIA: CIP380389.1) were obtained from the International Potato Center (CIP) germplasm bank in Lima, Peru.PYVV was obtained from the CIP potato virus collection and was propagated and maintained in potato. Plants grown from infected tubers served as source plants for the transmission experiments.For virus acquisition in the transmission experiments, two PYVV-infected source potato plants (grown from infected tubers) of the cultivar ‘Canchan INIA’ were planted in a temperature-controlled growth chamber per temperature tested. The temperature in the growth chamber was maintained at the same temperatures as used in the transmission experiments described below. Virus infection was confirmed approximately 25 days after planting by RT-PCR. In addition, properly sprouted healthy potatoes to be used as targets in inoculation experiments, were planted 10 days before virus transmission testing in growth chambers at the same temperature as used later in each transmission experiment.T. vaporariorum originating from Lantana camara, but maintained on potato plants for 5 years at the institutes own rearing facility (greenhouse) at a temperature between 20−23 °C, 70–95 % RH and a photoperiod of 12:12 h light (L): dark (D), was used in the experiments.A colony of Additional plant samples were collected from fields in Peru, Ecuador and western Panama and are described in section 2.2T. vaporariorum was determined at 8 constant temperatures of 10°,12°, 14°, 15°, 16°, 18°, 20°, and 25 °C (±0.5 °C). For each temperature, 50 single whitefly transmissions (described below) were performed per replication and replicated 2 times per temperature initially. Based on results, 2 more replications were performed to assess the variation in results at key temperatures . Whitefly transmissions were performed with an acquisition access period (AAP) to acquire the virus from the infected plants and inoculation access period (IAP) to transmit acquired virus to target plants . During the AAP, PYVV-infected potato plants (1 plant per 100 insects) were placed inside an acrylic box, covered with nylon mesh, in a growth chamber under each of the 8 predetermined temperatures. Adult whiteflies were caged with a clip-cage (17 × 13 × 13 cm) covered with nylon mesh to two leaves per plant  consisted of 50 + 2 plants; 50 plants were exposed individually to a single whitefly that had previously an AAP as described above and 2 plants were controls in which the AAP was performed on uninfected plants. After the 72-h IAP, the insects were removed from the cages and the plants treated with the insecticide Applaud (Buprofezin) (Bayer) at 0.1 % to ensure that no insects remained. The inoculated plants and were subsequently maintained in a greenhouse at a photoperiod of 12:12 h L: D and temperature of 18° and 23 °C during the light and dark period, respectively for 35 days. After this 35-d incubation period, virus presence was assessed taking systemic leaves (non-inoculated leaves) submitted to RT-PCR analysis section .Separately, to validate the predictions of the temperature dependent transmission function section  below we2.3Total RNA was extracted from sampled leaves using Trizol® reagent  according to the manufacturer's instructions. The cDNA for RT-qPCR was synthesized as follows: 7 μl of nuclease-free water (NFW), 2 μl of Random primers (250 ng/μl), 1 μl of dNTPs (10 mM) and 2 μl of total RNA (500 ng/μl) were mixed and denatured in a thermocycler  at 65 °C for 10 min and then cooled to 10 °C for 5 min.Subsequently, RT Mix  was added and then incubated at 37 °C for 50 min, followed by 70 °C for 15 min and finally cooled to 10 °C. The final volume obtained was a 20 μl cDNA, which was diluted with 80 μl of NFW to obtain a dilution of 1:5 for subsequent PCR.Relative PYVV concentrations in source leaves from each temperature were assessed to determine if temperature and leaf position had any effect on virus titres, that could influence transmission results. The PYVV primers  targeted the capsid region of the virus and the Cytochrome oxidase I (COX) gene was used as a reference gene (Supplementary Table 1).Ct values were determined using Applied Biosystem StepOne version 2.3 software. Samples were considered positive when they reached the Ct value before the 35th cycle. Each sample was performed in three technical replicates, including infected and healthy controls. Blanks were included in each separate PCR run. Data was analyzed using the 2(−ΔΔCt) method , 0.4μM of each primer, and 5μl PCR Master Mix Power SYBR Green .The following PCR program was used: PCR: 95 °C for 10 min, and then 35 cycles at 95 °C for 10 s, 50 °C for 45 s, 60 °C for 1 min, and finalized with a melting curve analysis. Fluorescence was measured after each amplification cycle. ) method  to deterTo verify successful virus-transmission by each individual whitefly to healthy potato plants as well as field samples from Panama (see section 2.6), regular RT-PCR was used, with primers PYVV CP1_F and PYVV CP2_R that amplified a 760 base pair fragment of the CP gene (Supplementary Table 1). The reaction conditions were as follows: 4 μL of PCR 5X Buffer (Promega), 1 μl of MgCl2 (25 mM), 0.5 μl of dNTPs (10 mM), 0.5 μL of Primers (CP1_F / CP2_R) both at 10 μM, 0.5 μl of the Taq polymerase (Promega), 8 μl of nuclease-free water (NFW) and 5 μl of diluted cDNA. The mixture was placed in a thermal cycler with the following conditions: one cycle at 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min followed by 10 min at 72 °C. Amplified bands were visualized in a 1% agarose gel stained with Gelred (Invitrogen) under UV light.Samples from field surveys in Peru and Ecuador were tested for PYVV by nucleic acid spot hybridization (NASH). Two ½ leaflets were macerated in 2 ml, of 5x SSC  buffer in 100 μm polyethylene plastic bags (4 × 6 cm) and 30 μl of crude leaf sap was blotted on a nylon membrane  dried and cross-linked by UV-irradiation (50 mJ) in a cross-linking oven (Stratagene). Each membrane also included extract from a positive PYVV infected and healthy control plant of cv. Canchan INIA. Digoxigenin (DIG)-labelled probes encompassing the coat protein gene region (CP) of PYVV were synthesized by PCR as described above using primers PYVV CP1 and PYVV CP2 , and DIG2.4jth temperature yij, with values of 1 for success and 0 for failure, for the Ith insect at the jth temperature to obtain the absolute frequency, or total number of insects that transmitted the virus for every temperature The transmission as evaluated for each adult in a We tested about 20 models available in ILCYM that were developed for describing insect development rates, and the best models were selected according to AICc. Parameters for each of these models are automatically estimated by ILCYM according to the Levenberg-Marquardt algorithm  using thAll candidate models were implemented as optional functions in a new tool within the ILCYM package for analyzing temperature-dependent vector virus transmission probability (efficiency) in insect vectors. All statistical calculations and coding for model implementation were made in R-3.4.1 .2.5T. vaporariorum temperature-dependent life table parameters–namely net reproduction rate (0R), mean generation time (T), intrinsic rate of natural increase (mr), finite rate of increase (λ) and doubling time (tD), based on monthly minimum and maximum temperature data. They provide useful information about the expected vector abundance in a given environment. The two indices were developed combining the information on virus transmission efficiency and the vectors life table parameters.We tested two alternative methods to establishing a PYVV risk index based on the temperature-dependent virus transmission efficiency combined with the phenology model established previously for the vector . The temperature based phenology model predicts λ, defines the average per-capita multiplication factor per time-step (in this study per month); it means, populations are expected to grow–assumed that the population has reached a stable age-stage distribution–by the factor λ each month. Thus, during a given time interval of x months a population of the size iN grows by the factor λx.The first index, “virus transmission index (VTI) 1″, is based on the virus transmission probability and the whitefly’s finite rate of population increase. The finite rate of increase, λ seems an adequate factor for projecting relative spatial or seasonal differences in field densities (abundance) of pest populations, assuming that the initial population size is equal among space or at the beginning of a season. The temperature-dependent vector transmission efficiency factor, p, as described in the previous section, defines the probability that a single virus-carrying vector transmits the virus to the plant after attacking the plant. Since knowledge about the biological processes for successful virus transmission and disease development in virus-inoculated potatoes is lacking, we favored a non-additive model, in which each vector that has acquired the virus has the same chance of causing an infection in the plant after feeding for a given time interval on the host plant. The attack (contact) rate is directly proportional to the density of the vector. If p is the vector’s probability of inducing a virus infection after a given feeding period, the probability that the plant will be not infected at vector densities of x vectors per plant is:P, isTherefore, the growth rate λ, as a useful indicator for a reasonably representation of relative differences in vector densities among different locations or between specific seasons in a given location in regard to environmental temperature. Accordingly, for our risk index we exchanged the parameter for vector densities in eq. [1] with the temperature-dependent life table parameter λ. The risk index for a given month in each location is calculated as follows:iVTI is the virus transmission risk index for the ith month, ip is the temperature-dependent virus transmission efficiency rate during the ith month (as described in the previous section), iλ is the temperature-dependent finite rate of increase of the vector population, and ω is a shape factor . The index values are bound between 0 and 1. The annual virus transmission risk index for given location is calculated as:We considered the vectors life table parameter, R0, multiplied with the temperature-specific virus transmission efficiency, p, over a year. The equation was:R0i and pi are the net reproduction rates and the virus transmission probability, respectively, predicted for the ith month.The second index VTI2 is a simple index, calculated by averaging the whitefly’s monthly net reproduction rate, To evaluate the predictive capacity of VTI1 and VTI2 we used a dataset containing 817 geo-referenced virus presence/absence data derived from a field surveys performed in Colombia, Ecuador and Peru during 2007–2010 (see section 2.6T. vaporariorum and PYVV in potato crops in Peru (1788 fields) and Ecuador (3 fields) between 2007–2010 . Depending on the size of the fields, a number of 10–20 plants were evaluated. The presence or absence of PYVV symptoms were recorded for each plant on fully expanded leaves. Collected leaves were stored in a Styrofoam box filled with ice and later (at the same day) plant sap blotted on nylon membranes and air dried for virus-detection tests using NASH as described in section Geo-referenced field surveys were performed to assess the presence of Subsequently, to evaluate the usefulness of the model to guide surveillance, we identified an area with significant risk, based on the selected VTI, for establishment and spread of PYVV in western Panama and surveyed the area for presence of whiteflies and symptoms corresponding to virus infection during 2016. Leaves from 26 potato plants were collected in 3 potato fields to verify the presence of PYVV, and identify the whitefly species from nymphs. The fields were located at Cerro Punta: ; Las Nuves  and La Gavidia . The samples were tested with RT-PCR using primers specific for PYVV as described above.2.7www.worldclim.org)and converted to .flt (float type) data format. For future predictions the temperature database with the RCP 6.0 scenario and the CCSM model were used. The extracted temperature data were organized in 12 × 2 matrices using the longitude as columns and latitude as rows representing 12 matrices each for the minimum and maximum temperatures. Thereafter, a point object was created for each geographical coordinate (longitude and latitude) in form of a two-column table (the first column included the minimum temperature and the second the maximum temperature for each point) that was used directly for spatial phenological simulation . These temperature data served as input data for calculating the Virus Transmission index (VTI) as, described in section Maps were generated using ILCYM, which works with monthly maximum and minimum temperature data sets for one year. Twelve months data sets with their respective geographical coordinates were obtained at a resolution of 10 min for world prediction and 2.5 min for the region between South America and Central America from the WorldClim database  at the optimum temperature (optT), and k is an empirical constant determining the shape of the function. The function explained >81 % of the variation in the data  causing 50 % reduction in the transmission rate (T½) can be written in terms of the decay constants as: T½ = lowk * ln (2) and T½ = lowk * ln (2), respectively.high1k into a state at which their activity (probability to be transmitted and cause infection in the plant) is reduced to γ, and 2) virus transmission of all states are reduced by a slower decay constant determined by high2k. Based on the conditions of the model and its application in terms of a function, the Sporleder 2 model can be defined as follows:where The Sporleder 2 function was 277 million times better than the modified Janish according to AICc evidence ratios To reveal if a relationship exists between virus transmission and virus titres in the source plantsat different temperatures, we performed qRT-PCR analysis from all source leaves used in the transmission experiments.The transmission efficiency had no correlation with virus concentration (correlation coefficient = -0.31) in the plant as determined by qRT-PCR, as virus titres did not vary much between 10° to 18 °C, but then increased significantly at temperatures above 20 °C , where transmission was inefficient . The relThe screenhouse experiment in La Molina for testing virus transmissions at naturally fluctuating temperatures during 3 seasons: February (summer), May (autumn), July (winter) were in agreement with the established virus transmission model. Only during July, that was the period in which temperature frequently fell below 17 °C at which efficient virus transmission was expected, plant exposure to vectors resulted in successful virus transmission. During the other season, temperature remained >17 °C, which was above the expected temperature limit for efficient virus transmission, and no transmission was observed (Supplementary Table 2).3.2Jand VTIS respectively, whereas VTI applies to the risk indices in general. The differences between the indices VTI1 and VTI2 is based on the expected temperature-dependent virus transmission efficiency and the vector’s two life table parameters, either the finite rate of population increase or the net reproduction rate are illustrated in J and VTIS indices was the narrower range of optimal temperatures in VTIS and a lower but broader secondary peak at higher temperatures in VTIS  of 0.71, while the highest AUC of 0.67 was obtained using VTIJ1 with ω = 35  vs specificity (true negative rate) plot for the field survey data for VTIJ2 and VTIS2 are illustrated in J2 and VTIS2 respectively and resulted in the best overall prediction accuracy of 70.4 % for VTIS2  and 72.1 % of the true absence (specificity) were correctly predicted, revealing a precision  of 29.0 %, while the negative predictive value was 93.9 %. The low positive predictive value is due to the unbalanced occurrence of the virus in the sampled locations (PYVV was detected in 10.2 % of the surveyed locations only).VTI2 performed better than VTI1 at any scale parameter setting when tested against the real virus presence/absence data sampled during the survey . VTIh ω = 35 d. Therefor VTIS2  whereas S2 threshold of 2.23. In contrast, VTI1 with variable parameter settings of ω revealed low F1 scores (not shown).The F1 score, as a measure of the threshold accuracy considering the tradeoff between precision and the true positive rate, (sensitivity) revealed a value of 0.516 for a VTI3.3J2 and year 2000 climate data (the most recent data available in 2015) and identified a region in western Panama that was predicted to have high VTIJ2 (red circle in T.vaporariorum was present in two of the three fields sampled (Station IDIAP and La Gavidia).To test the effectiveness of using the VTI2 maps for guiding surveillance, we used VTIS2 maps were generated. VTIJ2 and VTIS2 maps were very similar except that areas with higher annual average temperature (between 18−30 °C) increased in their risk predicted by VTIS2, but were still low, and areas of the highest risk were reduced .Subsequent to this effort the new Sporleder 2 function, which better fit the transmission data, was developed based on which new VTIS2 indices using current and future predicted annual climate data, revealing current and future areas at significant risk of PYVV epidemics , Colombia, Venezuela, Ecuador and Peru, that there are areas at risk of further spread and establishment in Costa Rica, Bolivia, Chile and Argentina as well  it can be a highly efficient vector with a transmission rate of 0.58−0.66 per individual insect. Strikingly, the transmission efficiency had no correlation with virus concentration in the plant as determined by qRT-PCR, as virus titres did not vary much between 10° to 18 °C, but then increased significantly at temperatures above 20 °C , where transmission was inefficient  for VTI1 and 2, always resulted in the simpler VTI2 function generating better predictions based on testing with the field survey data. VTI2 generates risk indices with values ranging from 0 to ∼11. The maximum accuracy of correctly predicting virus presence and absence using our field survey data was 70.4 % % using a VTIS2 of 2.23 as a cutoff  resulting risk maps may inform about potential establishment, but they do not provide information on the population growth and damage potential or temporal population change within a cropping season or year in a given region.T. vaporariorum using the RCP 6.0 climate scenario for 2050, reveals a general reduction of transmission risk in most tropical areas of the world , also showed a large temperature dependency with inoculation temperature having a larger effect than acquisition temperature  by its aphid vector perature . Similarperature , Barley perature  and Pea perature  by severT. vaporariorum and PYVV as an example. Risk maps generated can be used to guide surveillance, as demonstrated in this study, or pest risk analyses. Bases on the VTI2 maps for PYVV, the virus is predicted to have potential (VTIS2 > 2.23) to establish in southern regions of Peru, northern Bolivia, parts of Chile and Argentina, and this potential will increase at larger latitudes in the future, whereas it will decrease in tropical regions. The modeling described in this study was performed using ILCYM v4.0, which has a user-friendly “Shiny” (developed with Shiny software) interface making development of insect phenology models and virus transmission curves extremely easy once the data is entered. Generating maps is equally easy and it should thus provide a versatile tool for researchers to rapidly generate risk maps for insect pest or insect vectored diseases and could be particularly useful to prepare for changes in disease pressure during El Niño years for example. Whereas ILCYM is currently geared at generating risk maps based on monthly weather data, it should relatively easy be modified for local epidemiological simulation and forecasting to provide decision support, even miniaturizing it for smartphone use by farmers and extension workers alike.In Conclusion, our results show that the combination of insect life parameters and virus transmission functions can be used to effectively identify areas at risk for vector based spread, using H. Gamarra: Data curation, Investigation, Project administration, Supervision, Writing - original draft, Writing - review & editing. P. Carhuapoma: Data curation, Formal analysis, Software, Visualization. L. Cumapa: Investigation. G. González: Investigation. J. Muñoz: Investigation. M. Sporleder: Formal analysis, Methodology, Validation, Writing - review & editing. J. Kreuze: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - original draft, Writing - review & editing.


Waterfowl and shorebirds are the primary hosts of influenza A virus (IAV), however, in most surveillance efforts, large populations of birds are not routinely examined; specifically marine ducks and other birds that reside predominately on or near the ocean. We conducted a long‐term study sampling sea ducks and gulls in coastal Maine for IAV and found a virus prevalence (1.7%) much lower than is typically found in freshwater duck populations. We found wide year‐to‐year variation in virus detection in sea ducks and that the ocean water temperature was an important factor affecting IAV prevalence. In particular, the ocean temperature that occurred 11 d prior to collecting virus positive samples was important while water temperature measured concurrently with host sampling had no explanatory power for viral detection. We also experimentally showed that IAV is relatively unstable in sea water at temperatures typically found during our sampling. This represents the first report of virus prevalence and actual environmental data that help explain the variation in marine IAV transmission dynamics. Environmental factors have large influences on populations and ecosystems. As the world's climate changes, these influences can be drastic and, to a very large extent, the consequences of climate change are unknown. This is particularly true regarding pathogens and disease transmission and prevalence. Any pathogen that is contact, aerosol, water‐borne, or fecally/orally transmitted, necessarily must interact with the physical environment and be able to withstand changes in temperature, humidity, wind, etc. to infect new hosts. Many viral pathogens such as influenza A viruses, are transmitted by these mechanisms and the stability of virions outside of infected hosts is critical for the generation of new infections, viral propagation, as well as the creation of potential human pandemics.The primary natural reservoir of influenza A viruses (IAV) is wild birds, mainly waterfowl, shorebirds and gulls. These hosts can be infected by all subtypes of IAV with the potential for very rapid genetic reassortment and viral evolution  are excreted through the digestive systems of infected wild birds and are spread via a fecal/oral transmission cycle. Whether new hosts become infected by directly contacting virus‐contaminated fecal matter or contaminated water, there are necessarily environmental factors involved in viral persistence and transmission. There have been several laboratory studies that showed a clear influence of water pH, salinity, and temperature on influenza virus stability. LPAIV were most stable in slightly alkaline (pH 7.4–7.8) conditions, freshwater vs. saline water, as well as cooler vs. warmer temperatures  of 2011–2013, 2015–2017. In addition, we opportunistically collected fecal samples from a variety of gull species. The geographic range of sampling locations extended from Frenchman's Bay  in the north to the Kennebec River estuary  in the south. Swab samples were taken using established methods  and real‐time polymerase chain reaction (RT‐PCR) analysis performed as described by Spackman et al. available online).We obtained ocean temperature data from the National Oceanic and Atmospheric Administration , and included the effects of categorical covariates of sampling season defined as the year that sampling was initiated during a sampling period, j , the species sampled based on taxonomic order Charadriiformes (gulls) relative to Anseriformes (ducks), and age category of the sampled bird defined as juvenile (hatch year) relative to after‐hatch‐year ageThe set of sample predictor variables, To examine the effect of ocean temperature we considered potential time lags and different measures of temperature variability to account for the timing of the transmission process and from daily fluctuations in temperature that could influence the environmental component of transmission, respectively. We hypothesized that ocean temperature would not have a direct effect on IAV detection at the same time as sample collection because there are three dynamic transmission components, each with a time period that must be completed sequentially for successful transmission and detection. First, an infected bird must excrete IAV into the environment and that the IAV must remain infective until a susceptible host encounters it. Second, the IAV must cause infection in the new host and undergo an incubation period before it begins to be excreted. Third, excretion persists until the host clears the infection and this step must align with our sampling process. We did not attempt to estimate or examine in detail the dynamics of all these processes; however, we hypothesized that water temperature will have the strongest effect on the initial process when IAV exists free in the environment and longer survival will lead to more transmission events. Hence, we created temperature covariates associated with time lags ranging from 0 (temperature on the day of sampling) to 14 d prior to sampling, as well as for temperature metrics summarized over 0–7, 3–10, and 7–14 d periods prior to sample collection. The choice of daily lag range and weekly periods as covariates was informed by the infectious period of IAV in sea ducks from laboratory infection trials  to examine which covariates had the strongest effect on model fit to the observed IAV detection data. We performed model selection in two stages because of the complexity of the temperature lag data. First, we fit a set of candidate models with all additive combinations of the sample covariate data in βx and used the AIC difference (ΔAICc) and AIC weights (wAICc) to select the best model , with one temperature‐metric–lag combination at a time to create a new candidate set of models , was diluted to 105 EID50/mL (where EID50 is the 50% embryo infectious dose) in Instant Ocean sea salt solution prepared according to the manufacturer's instructions . One‐milliliter aliquots of diluted virus were prepared and stored at −80°C. For each test temperature , we transferred 12 aliquots to a controlled temperature chamber  and held at constant temperature. At weekly intervals , three aliquots were removed and stored at −80°C until testing. Additional aliquots were stored at −80°C for the duration of the time course and thawed and frozen one additional time to replicate conditions of the test samples. These served as “day 0” controls. We determined virus titers in 10‐d‐old embryonated chicken eggs  according to the method of Reed and Muench (A LPAIV isolate (A/long‐tailed duck/Maine/295/2011 (H3N8)), collected from a sea duck, Long‐tailed Duck (d Muench .Somateria mollissima), with lesser numbers taken from Long‐tailed Ducks , three species of Scoters (Melanitta sp.), Bufflehead , Herring Gull (Larus argentatus), Common Goldeneye , and miscellaneous other species . On inspection of relative magnitude and explanatory ability of the fitted coefficients, we chose to only include the year covariate in a parsimonious model because it was the only covariate present in the top models and the only covariate with coefficients that had a significant effect on probability of detection as defined as 95% coefficient confidence intervals not overlapping zero , the effect of daily minimum sea temperature 11 d prior to the sample collection had the best fit to the data with wAICc = 0.45 and the next best model with ΔAICc = 3.8 and wAICc = 0.07 (Table Among the candidate set of models with the year covariate and sea temperature metric and lag combinations for each sample (07 Table :Table S2Phoca vitulina) with distinct risks of virus adaptation to mammalian hosts and potential to infect nearby human populations , a freshwater duck. In addition, water in bathing/swimming tubs provided for the challenged eiders contained significant amounts of IAV, indicating that similar fecal/oral transmission mechanisms are involved in both taxa of ducks (Hall et al. In a previous experimental challenge study, Common Eiders all became infected with LPAIV and cloacally excreted virus in amounts similar to infected Mallards (The interaction of all influenza viruses with the physical environment is a critical component of transmission and persistence and is no less important with IAV infections of sea ducks and other marine hosts. There are very few studies that have investigated environmental factors and influenza in marine habitats and this is the first that we are aware of that integrates actual real time environmental data with virus prevalence in a natural ecosystem. We present our findings in the hope that future studies will help to define the environmental/viral interactions across different host ecologies and aquatic environments. Click here for additional data file.


During the coronavirus pandemic (COVID-19), health care workers are innovating patient care and safety measures. Unfortunately, many of these are not properly tested for efficacy. The objective of this study was to determine the efficacy of the novel COVID-19 Airway Management Isolation Chamber (CAMIC) to contain and evacuate particulate.Multi-institutional proof-of-concept study.Two academic institutions: Walter Reed National Military Medical Center (WRNMMC) and Madigan Army Medical Center (MAMC).Smoke, saline nebulizer, and simulated working port models were developed to assess the efficacy of the CAMIC to contain and remove ultrafine particles. Particulate counts were collected at set time intervals inside and outside the system.r(18) = −0.88, P < .001, WRNMMC; r(18) = −0.91, P < .001, MAMC. Similarly, saline nebulizer particulate counts inside the chamber significantly decreased over time: r(23) = −0.82, P < .001, WRNMMC; r(23) = −0.70, P < .001, MAMC. In the working port model, particulate counts inside the chamber significantly decreased over time: r(23) = −0.95, P < .001, WRNMMC; r(23) = −0.85, P < .001, MAMC. No significant leak was detected in the smoke, saline nebulizer, or working port model when the CAMIC was turned on.With the CAMIC on, smoke particulate counts inside the chamber significantly decreased over time: The CAMIC system appears to provide a barrier that actively removes particles from within the chamber and limits egress. Further studies are necessary to determine clinical applicability. The CAMIC may serve as an adjunct to improve health care worker safety and patient outcomes. Health care systems have been overwhelmed due to the highly communicable nature of SARS-CoV-2 and the high morbidity of COVID-19 infection. Although it is primarily transmitted through droplets , evolving evidence suggests that it also has aerosol and airborne tendencies.9 Increased viral concentrations within the nasopharynx10 place otolaryngologists at a high risk for exposure11 during commonly performed aerosol-generating procedures. Endonasal procedures have been shown to generate significant particulate, which places the provider at risk.12 Guidelines to prevent viral propagation during otolaryngologic clinical and surgical treatment recommend donning appropriate personal protective equipment (PPE) based on the procedure being performed.13 Health care workers are at high risk for exposure, despite wearing appropriate PPE, as viral particles have been identified in the ventilation systems of hospitals treating patients with COVID-19.14 Furthermore, airway interventions present an even higher risk of transmission due to viral aerosolization during intubation3 and noninvasive respiratory support mechanisms, including continuous positive airway pressure (CPAP), bilevel positive airway pressure (BiPAP), and high-flow nasal cannula.2Mitigation strategies for noninvasive airway management have been proposed, but aerosolization concerns limit their use.In this proof-of-concept study, we introduce the COVID-19 Airway Management Isolation Chamber (CAMIC) system. This functions as a physical barrier and a containment chamber with suction to isolate an infected patient. We hypothesize that the CAMIC will contain and evacuate particles within the system, with limited egress into the surrounding environment. The primary outcome will examine the reduction in particulates over time through the CAMIC system. The secondary outcome will evaluate particulate escape from the CAMIC system.Supplement 1 (available online). Suction is attached and distributed via the frame. Oxygen is delivered through the contralateral port. The CAMIC system is inserted into a clear surgical bag  and placed around the head, neck, and shoulders of the patient. At WRNMMC, the barrier covering included a drawstring that was cinched around the mannequin’s torso, whereas at MAMC, the barrier had no drawstring and was tucked tightly around the mannequin’s torso.The CAMIC system is a polyvinyl chloride hollow frame with fenestrations. It is placed at the head of a hospital or surgical bed  negative-pressure rooms. For the smoke and nebulizer models, particle readings were taken inside (center of the chamber) and outside (3 in from the bag on the torso of the mannequin) of the CAMIC system at set intervals. The particle detector remained in a constant position on the mannequin’s torso during testing. An internal sample line was suspended in the middle of the CAMIC above the mannequin’s face and run externally, which allowed internal measures without placement of the particle detector within the CAMIC. The sample line was capped to prevent undue particle egress. For the working port model, particle readings were taken 1 in from a 4-in slit in the superior panel of the CAMIC. An internal sample line suspended above the mannequin’s head allowed for internal measurements.15 A smoke model approximated ultrafine particles (0.5 µm), while a saline nebulizer model approximated microdroplets/aerosol particles (0.5-5 µm). A working port model assessed the effectiveness of the CAMIC with a deliberate fenestration into the barrier. Each scenario was tested with the suction and oxygen on within the CAMIC and without any suction or oxygen within the CAMIC. Five iterations of each scenario were conducted at both institutions. Background readings were taken inside and outside the CAMIC prior to each trial for reference. The particle detector was kept in the same location for each testing scenario. Negative controls were recorded to establish particle dissipation for each test room via the same testing methods as in the smoke model with a CAMIC without the polyethylene barrier, suction, or airflow  collected particle counts with 10-second means. The 2 particle meters show good correlation on comparative evaluations.Institutional Review Board approval was obtained at both institutions .A ventilation smoke tube (MSA Safety Incorporated) containing ethylenediamine and acetic acid delivered 6 puffs of smoke within the CAMIC directly above the mannequin’s face. Six puffs of smoke were used to produce a replicable high particulate count within the CAMIC system. A 2-minute period was used to allow for adequate smoke distribution within the CAMIC. Initial particle counts were recorded. Suction (120 mm Hg) and oxygen (10 L/min) were started after 2 minutes of equilibration. Internal and external particle counts were recorded every 2 minutes over an 8-minute period, which was chosen after initial testing where 8 minutes were required to evacuate particulate near external environment baseline.Power Nebulizer 2  or AeroEclipse II Breath Actuated  nebulized normal saline within the CAMIC for 1 minute to allow for adequate particle distribution. At WRNMMC the nebulized normal saline was delivered via a face mask attached to the mannequin, whereas at MAMC the mouthpiece was secured to the mannequin’s mouth. An initial particulate count was recorded. Suction (120 mm Hg) and oxygen (10 L/min) were started. The nebulizer was run for 4 additional minutes to simulate a nebulizer treatment. Internal and external particle counts were recorded every 2 minutes over a total of 12 minutes, which was 8 minutes after cessation of nebulization.Our smoke model methodology was modified to simulate a working port or a 4-in vertical slit (where a provider managing the airway would stand) with the CAMIC. After application of 6 puffs of smoke within the CAMIC, a peak reading was taken at 90 seconds before creation of a working port. Wall suction (120 mm Hg) and oxygen (10 L/min) were started at 2 minutes. Internal and external particle counts were recorded every 2 minutes over an 8-minute period.t testing and Pearson’s correlation coefficient assessed the relationship between particle counts within trials over time. For the smoke and working port models, the 2-minute and 90-second readings were used as the reference for intratest comparisons. For the nebulizer model, intratest comparisons were performed over the first 5 minutes when the nebulizer was running and for the subsequent 8 minutes after discontinuation of nebulizer therapy. Student’s t testing was performed to compare treatment versus control at each time point. A power analysis was performed; 95% CIs were reported; and statistical significance was determined to be P < .05. Background readings were removed for all statistical analyses.Particulate counts were analyzed with Microsoft Excel. Descriptive statistics were reported. Paired r(18) = −0.88, P < .001, WRNMMC; r(18) = −0.91, P < .001, MAMC. There was no significant decrease in internal particle count over time in the CAMIC-Off trials. At both institutions, the internal particle count was significantly lower with CAMIC-On versus CAMIC-Off at 4, 6, and 8 minutes.Internal particle counts decreased at each time point at both institutions with CAMIC-On . No evidence of external particle leak was detected for the CAMIC-On or CAMIC-Off scenarios at either institution  = −0.82, P < .001, WRNMMC; r(23) = −0.70, P < .001, MAMC. There was a significant decrease in internal particle count over time in the CAMIC-Off trials at the 11- and 13-minute time points at MAMC and the 9-, 11-, and 13-minute time points at WRNMMC. At both institutions, the internal particle count was significantly lower with CAMIC-On versus CAMIC-Off at all time points following the cessation of nebulizer treatment.During the nebulizer treatment, internal particle counts significantly decreased at MAMC at 3 and 5 minutes. After the cessation of nebulizer treatment, internal particle counts decreased at all time points  at both institutions with CAMIC-On . No evidence of external particle leak was detected for the CAMIC-On scenarios at either institution  = −0.95, P < .001, WRNMMC; r(23) = −0.85, P < .001, MAMC. Internal particle counts significantly decreased over time after creation of the working port in the CAMIC-Off scenarios at each institution. CAMIC-Off particle counts negatively correlated to time at both institutions: r(23) = −0.96, P < .001, WRNMMC; r(23) = −0.7, P < .001, MAMC. Internal particle counts were significantly lower with CAMIC-On as compared with CAMIC-Off at 4, 6, and 8 minutes at WRNMMC and 6 and 8 minutes at MAMC.After the working port was created, internal particle counts significantly decreased at each institution at all time points with CAMIC-On and CAMIC-Off . Elevated external particle counts (CAMIC-Off vs CAMIC-On) were detected at 4, 6, and 8 minutes at MAMC.15 asymptomatic carriers,16 viral droplets that can remain on surfaces for several days,17 and inaccurate patient testing18 all contribute to its high transmission rates. Critically ill patients requiring mechanical ventilation often succumb to a deadly pulmonary illness1921 with features atypical to acute respiratory distress syndrome.22 Initial concerns regarding aerosolization of the virus prompted deviation from standard critical care airway algorithms, limiting the use of noninvasive therapy such as CPAP and BiPAP.24 These guidelines recommend early intubation for definitive airway management,26 which has downstream effects on the availability of ventilators. Retrospective data from China and Italy suggest that hypoxemic patients with COVID-19 respond well to positive pressure, indicating a crucial role for noninvasive respiratory treatment as an alternative to intubation.27 Our aim in development of the CAMIC was to create a multifunctional adjunct PPE barrier to protect health care workers intervening on the airway in various capacities. In this proof-of-concept study, we designed and tested a novel isolation chamber, the CAMIC, which has the potential to serve 3 vital functions: (1) it provides a resilient barrier between patients and health care workers; (2) it facilitates a safer environment for noninvasive airway management; and (3) it is assembled with readily available equipment.The COVID-19 pandemic has tested the limits of health care systems around the world. Airborne spread,28 However, intubation is not possible through this system, and clearance of particulates has not been adequately assessed. Another barrier, the rigid aerosol box, may decrease droplet exposure, though droplets may escape the working ports.9 In addition, the open end of the box allows respiratory particles to escape into the room.29 In the otolaryngology literature, endonasal drilling and transnasal cauterization have been shown to produce significant particulate, which places providers at risk for exposure in the absence of using N95 respirators.12 Other novel systems have been used for otolaryngologic aerosol-generating procedures, including a flexible barrier for otologic procedures30 and a negative-pressure system for performing tracheostomy.31 While these reports demonstrate the clinical feasibility of these systems, all are based on anecdotal evidence. In contrast, we have demonstrated that the CAMIC contains and removes particulate with reproducible efficacy.Previous barrier methods appear to have significant limitations. In Italy, a self-contained helmet was used with the intent to reduce intubation rates and extend the benefits of noninvasive ventilation.32 Our data demonstrate that the CAMIC contained and removed >99% of smoke and nebulized saline particles introduced within the system, with minimal leaks into the environment. This is particularly notable in the nebulizer model, where particle counts decreased despite active nebulization within the closed CAMIC system. When a working port was introduced, the CAMIC prevented a detectable leak when turned on. While dynamic testing is beyond the scope of this proof-of-concept study, these findings encourage future investigations to use the CAMIC as a device for particulate or droplet containment and evacuation during active respiratory interventions. Furthermore, the CAMIC could function as an additional PPE barrier during otolaryngologic interventions, such as flexible and direct laryngoscopy, nasal endoscopy, and sinus surgery, as well as otologic surgery.The CAMIC provides a droplet barrier but also uses an internal vacuum and oxygen system, which may create laminar flow that facilitates particulate removal.The 2 institutions performed testing by using different nebulizers and polyethylene bags to confirm efficacy with commonly available hospital resources. This variability demonstrated that results are reproducible. The simple design and easily accessible materials allow the CAMIC to be assembled without specialized equipment. This device could be useful in a variety of clinical settings and has the potential for mass production.Supplemental Table S1). Additionally, we were able to conduct particle measurements only at set time points and locations.This was a small-sample study to demonstrate proof of concept. Other limitations include variability in test room conditions, such as differences in air turnover in the MAMC and WRNMMC procedure rooms, as well as the use of a mannequin. Control testing of the procedure rooms at MAMC and WRNMMC demonstrated different rates of smoke dissipation, suggesting differences in the HVAC systems  at each institution  creation, and then immediate capture of the potential leak. The additional 30 seconds allowed for performance of the additional tasks and reading capture.Supplemental Table S2 to the HVAC air turnover within the procedure rooms at MAMC and WRNMMC.The decrease in particle counts in the nebulizer model at the 11- and 13-minute time points at MAMC and the 9-, 11-, and 13-minute time points at WRNMMC may be due to settling of the larger droplets to the bottom of the chamber. Both institutions had similar results, but significance may be due to using 2 different nebulizers and particle counters. Similar to our smoke external data as compared with the no-CAMIC control model, we attribute the differences seen in Future studies should incorporate continuous measurements from multiple locations when assessing dynamic treatments and aerosol-generating procedures, such as laryngoscopy, endoscopic sinus surgery, mastoidectomy, and tracheostomy. These should examine the applicability of intubation, patient transport, and surgical intervention. While we demonstrated that the CAMIC significantly reduced particulate count, clinical effectiveness warrants future study.The CAMIC appears to provide a barrier that actively removes particles from within the chamber and limits egress into the treatment room. This could allow for safer noninvasive airway management, such as high-flow oxygen, nebulizers, and CPAP/BiPAP. The device evacuates internal particulate, even with a working port, suggesting procedural utility during invasive airway management. Furthermore, the CAMIC is easily assembled with readily available inexpensive materials. The CAMIC does not replace the need for individual PPE but may serve as an adjunct to improve health care worker safety and patient outcomes.Click here for additional data file.Supplemental material, CAMIC_appendix_build_design_final05032020 for COVID-19 Airway Management Isolation Chamber by Timothy C. Blood, Jonathan N. Perkins, Paul R. Wistermayer, Joseph S. Krivda, Nathan T. Fisher, Charles A. Riley, Douglas S. Ruhl and Steven S. Hong in Otolaryngology–Head and Neck SurgeryClick here for additional data file.Supplemental material, E-Tables_Revised for COVID-19 Airway Management Isolation Chamber by Timothy C. Blood, Jonathan N. Perkins, Paul R. Wistermayer, Joseph S. Krivda, Nathan T. Fisher, Charles A. Riley, Douglas S. Ruhl and Steven S. Hong in Otolaryngology–Head and Neck Surgery


Phys. Rev. Lett. 2019, 123, 138101). In this short commentary, we call into question the relevance of these results because of key model assumptions. First, the model of plant hosts assumes virus transmission only occurs between adjacent plants. This assumption overlooks the basic but imperative fact that most multipartite viruses are transmitted over variable distances by mobile animal vectors, rendering the model results irrelevant to differences between plant and animal hosts. Second, when not all genome segments of a multipartite virus are transmitted to a host, the model assumes an incessant latent infection occurs. This is a bold assumption for which there is no evidence to date, making the relevance of these results to understanding multipartitism questionable.Multipartite viruses have segmented genomes and package each of their genome segments individually into distinct virus particles. Multipartitism is common among plant viruses, but why this apparently costly genome organization and packaging has evolved remains unclear. Recently Zhang and colleagues developed network epidemiology models to study the epidemic spread of multipartite viruses and their distribution over plant and animal hosts ( Multipartite viruses have segmented genomes and package these genome segments into multiple virus particles . VirusesTraditionally, the advantages of multipartitism have been sought in their molecular structure. Early proposals regarded reassortment—that is the exchange of entire genome segments between two distinct viral genotypes—as the possible key advantage for multipartite viruses , which wThe observation that multipartite viruses preferentially infect plants has prompted the investigation of how the network of contacts between hosts affects their propagation . In a reIn In the framework of the SLIR model, with the assumptions stated, the authors find that a multipartite virus is more likely to persist under a static contact network than an annealed network if the average number of contacts per individual (the average degree of the network) is above a threshold that they estimate. This result is to be expected given the model assumptions. An infectious host is more likely to transmit multiple viral segments to the same susceptible host in a static rather than an annealed interaction structure, since in the static structure the transmission of various segments between a specific S–I or L–I pair can happen at multiple time points. In an annealed network, the transmission of segments is affected by a dilution process that prevents the formation of clusters of infected hosts, which are the only ones that effectively produce and transmit segments to other hosts in susceptible or latent states. As the authors assume the static interaction structure is representative for the transmission of plant diseases, they conclude that this difference may explain the distribution of multipartite viruses over hosts. This result is based on an untenable assertion about plant–virus transmission and on the existence of an L state that, to date, does not have empirical support. Since these two assumptions qualitatively affect the results attained, it is unclear how this article enhances our understanding of the between-host spread of multipartite viruses. Finally, as the study concerns the distribution of multipartite viruses over plants and animals, it does not suffice to study the epidemic behavior of different viruses in isolation. Below we detail each of these three important criticisms.First, the assumption that virus-transmission patterns between plants will be captured by a static network  cannot bSecond, the study results are based on an assumption that is insufficiently substantiated and not discussed: the existence of L states. The authors propose that transmission of one virus segment changes the host state, so that it can become infectious if the other virus segment(s) is transmitted later on . EpidemiIs there any evidence L states might exist? It was recently shown that nanoviruses have distributed replication at the cellular level . Within Third, the study focuses on which contact network, static or annealed, is more amenable to multipartite virus epidemics . As statThe intriguing phenomenology of the model, explosive transitions to endemicity  to M.P.Z.Conflict of interest: None declared.


N-terminal moiety. In addition, these two molecules offered protection against several other viruses, such as herpes simplex virus, yellow fever virus, Zika virus, and human coronavirus, classifying these glycopeptides as broad antiviral molecules with a favorable therapeutic index.Influenza A and B viruses are a global threat to human health and increasing resistance to the existing antiviral drugs necessitates new concepts to expand the therapeutic options. Glycopeptide derivatives have emerged as a promising new class of antiviral agents. To avoid potential antibiotic resistance, these antiviral glycopeptides are preferably devoid of antibiotic activity. We prepared six vancomycin aglycone hexapeptide derivatives with the aim of obtaining compounds having anti-influenza virus but no antibacterial activity. Two of them exerted strong and selective inhibition of influenza A and B virus replication, while antibacterial activity was successfully eliminated by removing the critical  Seasonal infections by influenza A and B viruses are each year responsible for significant morbidity and mortality . BesidesSeveral recent investigations demonstrated that some antibiotics from bacterial origin display interesting antiviral properties  represenRecently, we prepared a series of teicoplanin pseudoaglycone (TC) derivatives by coupling one or two lipophilic side chains to the N-terminus of the glycopeptide core, using triazole, sulfonamide or maleimide linking elements . Some ofN-terminal N-methyl-d-leucine moiety can be easily removed by Edman-degradation +: 1064.16. Found: 1064.129.Synthesis of azido vancomycin aglycone hexapeptide (terature . Sodium 6: 132 mg (0.127 mmol) of 2 was dissolved in 2.0 mL of DMF:H2O 3:1 mixture. Next, 72 mg  of alkyne 4 was added, followed by 6 mg CuSO4 · 5H2O. The reaction mixture was stirred at room temperature for 12 h. By this time TLC  indicated good conversion. The solvents were evaporated until a syrupy residue was obtained. Ether was added, and the product was filtered off after precipitation and washed with additional ether to remove the excess alkyne. Purification was carried out by C18 reverse phase column chromatography (H2O:MeCN = 70:30→60:40→55:45) followed by gel chromatography using Sephadex LH-20 in MeOH. The title compound was obtained as a white powder in 69 mg yield (37%). NMR: see m/z calcd. for C70H80Cl2N12O21 + Na+ [M + Na]+: 1517.48. Found: 1517.68.Synthesis of compound 7: 93 mg (0.09 mmol) of compound 3 was dissolved in 2.0 mL of DMF:H2O 3:1 mixture. 54 mg  of alkyne 5 was added followed 5 mg CuSO4 × 5H2O. After 12 h stirring at room temperature, TLC indicated good conversion. The reaction mixture was worked up and purified by C18 reverse phase column chromatography as described above. The title compound was obtained as a yellow powder in 52 mg yield (38%). NMR: see m/z calcd. for C69H74Cl2N10O22S2 + Na+ [M + Na]+: 1551.369. Found: 1551.367.Synthesis of compound 8: 78 mg (0.077 mmol) of 2 was dissolved in 3 mL dry pyridine and 0.5 mL dry DMF, then 19 μL  of n-hexanesulfonyl chloride was added. After stirring 3 h at room temperature, ethyl acetate and ether was added, the resulting precipitate was filtered and washed with ether. The crude product was purified by flash column chromatography using Toluene: MeOH = 7:3→6:4 as eluent, followed by gel chromatography using Sephadex LH-20 with MeOH: H2O = 6:4 as eluent. The yield was 20 mg (22%). NMR: see m/z calcd. for C52H51Cl2N7O18S + Na+ [M + Na]+: 1186.228. Found: 1186.276.Synthesis of compound 9): 207 mg of 4 +: 562.37. Found: 562.37.10): 208 mg (0.43 mmol) of compound 5  and 44 mg  of azidoethylamine were dissolved in 2 mL dry DMF under argon. 66 μL  Et3N was added followed by 16 mg (20 mol%) Cu(I)I. The reaction mixture was stirred for 2 h at room temperature. After evaporation the crude product was purified by flash column chromatography using DCM: MeOH = 100:0→93:7 (+0.1% v/v NH4OH) as eluent. The yield was 154 mg (55%), yellow syrup.Synthesis of 1-(1-(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11-tetraoxatridecan-13-yl)-3,4-bis(butylthio)-1H-pyrrole-2,5-dione  δ 7.77 , 4.69 , 4.44 , 3.73–3.57 , 3.28 , 3.21 , 1.99 , 1.63 , 1.45 , 0.93 . 13C NMR  δ 166.42 , 144.81 (triazole Cq), 135.61 , 123.27 (triazole CH), 70.30, 69.80, 69.54, 67.75, 64.47 , 53.01 (N-CH2), 41.79, 37.64 (2 x CH2), 32.32, 31.36, 21.48 , 13.43 . MALDI-MS m/z calcd. for C25H43N5O6S2 + Na+ [M + Na]+: 596.25. Found: 596.238.11: Vancomycin aglycone hexapeptide 2  was dissolved in 1.0 mL dry DMF. 95 mg  of compound 9 was added followed by 25 μL  Et3N and 55 mg  of PyBOP. The reaction mixture was stirred for 2 h at room temperature, after which TLC  indicated complete conversion. The product was precipitated by the addition of 100 mL of cold EtOAc:Et2O = 1:1 mixture, filtered off and washed thoroughly with diethyl ether. The crude product was purified by gel column chromatography using Sephadex LH-20 in MeCN:H2O = 8:2 mixture, followed by flash column chromatography in MeCN:H2O = 9:1 mixture. The product was obtained as a white powder in 52 mg yield (38%). NMR: see m/z calcd. for C72H86Cl2N14O20 + Na+ [M + Na]+: 1559.54. Found: 1559.76.Synthesis of compound 12: Vancomycin aglycone hexapeptide 2  was dissolved in 1.0 mL dry DMF, then 101 mg  of compound 10 was added followed by 25 μL  of Et3N and 55 mg  of PyBOP. The reaction was stirred for 2 h at room temperature, after which TLC  indicated complete conversion. The product was worked up and purified as described for compound 12. The title compound was obtained as a yellow powder in 44 mg yield (31%). NMR: see m/z calcd. for C71H80Cl2N12O21S2 + Na+ [M + Na]+: 1593.43. Found: 1593.44.Synthesis of compound 13: Step 1: 837 μL (12.5 mmol) ethylenediamine was dissolved in 5 mL dry DCM and stirred at room temperature, while 115 mg (0.63 mmol) n-hexanesulfonyl chloride in 0.5 mL dry DCM was added via syringe over the course of about 30 min. The reaction mixture was stirred for 4 h, after which it was thoroughly evaporated. The crude product was purified by flash column chromatography using hexanes: acetone = 6:4 (+0.2% v/v Et3N) as eluent. 95 mg of N-(2-aminoethyl)hexane-1-sulfonamide was obtained as a slightly yellow syrup with acceptable purity (based on 1H and 13C NMR spectra), which was suitable for the amide coupling in Step 2: Vancomycin aglycone hexapeptide 2  was dissolved in 1.0 mL of dry DMF:DMSO mixture. 25 μL Et3N  was added followed by 70 mg N-(2-aminoethyl)hexane-1-sulfonamide  obtained in step 1, followed by 54 mg  PyBOP. The reaction was stirred at room temperature for 3 h, then the product was precipitated by the addition of ethyl acetate, filtered out and washed with diethyl ether. The product was purified by flash column chromatography using step gradient elution (toluene: MeOH = 7:3→1:1) then by gel column chromatography . The title compound was obtained as an off-white powder in 36 mg yield (34%). NMR: see m/z calcd. for C54H57Cl2N9O17S + Na+ [M + Na]+: 1228.29. Found: 1228.27.Synthesis of compound For antiviral testing, 25 mM compound stocks were prepared in 100% DMSO and stored at 4 °C. The compounds were fully soluble under these conditions. In the antiviral tests, the highest concentration tested was 100 µM, corresponding to a non-toxic DMSO content of 0.4%.N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin). One day later, virus (MOI: 0.001) was added together with 1:5 serial compound dilutions, to reach a total volume of 200 µL per well. Besides the test compounds, two references were included, i.e., zanamivir and ribavirin (positive controls) plus a condition receiving medium instead of compound (negative control). In parallel, the compound dilutions were also added to a mock-infected plate (in which medium was added instead of virus), to determine compound cytotoxicity. Each plate contained two wells in which all reagents yet no cells were added, to serve as blanks in the MTS calculations. After three days incubation at 35 °C, viral CPE was first monitored by microscopy. Then, the supernatants were replaced by MTS reagent (CellTiter 96® AQueous MTS Reagent from Promega) diluted 1:10 in PBS, and 4 h later, absorbance at 490 nm was measured in a plate reader. From all OD values, the blank OD was subtracted. The % protection against virus was defined as: [(ODCpd)virus−(ODContr)virus)]/[(ODContr)mock−(ODContr)virus] × 100, where (ODCpd)virus is the OD for a given concentration of the compound in virus-infected cells; (ODContr)virus is the OD for the untreated virus control; and (ODContr)mock is the OD for the untreated mock-infected control. The % cytotoxicity was defined as: [1−(ODCpd)mock/[(ODContr)mock] × 100, where (ODCpd)mock is the OD for a given concentration of the compound in mock-infected wells. The values for EC50  and CC50 (50% cytotoxic concentration) were calculated by interpolation based on semi-log dose response.The virus strains  were propagated in embryonated hen eggs. The antiviral procedure was published elsewhere ,39. Madi99 values were calculated by interpolation and defined as the compound concentration causing 100-fold reduction in vRNA copy number, as compared to the virus control receiving no compound. It was ascertained that the cell controls showed no detectable qPCR signal.To monitor the inhibitory effect of the compounds on virus replication, MDCK cells were seeded, infected  and treated with 1:2 serial compound dilutions. The plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). At day 3 p.i., supernatants were collected and frozen at –80 °C, to quantify the virus yield by one-step qRT-PCR analysis of viral copy number . Two µl - mutant), herpes simplex virus type 2  and vaccinia virus (strain Lederle); human cervixcarcinoma HeLa cells, used for respiratory syncytial virus , and African Green Monkey kidney Vero cells, used for yellow fever virus (vaccine strain 17D) and Zika virus (strain MR766). The medium used for virus infection was Dulbecco’s Modified Eagle’s Medium supplemented with 2% fetal calf serum. To prepare virus stocks, confluent cell cultures in 75-cm2 flasks were infected with the virus and frozen after 3 to 5 days incubation at 37 °C (or 35 °C in case of human coronavirus 229E), when full-blown CPE was visible. After freeze-thawing and centrifugation, the clarified lysates were stored at −80 °C. For the antiviral experiments, the cells were grown in 96-well plates until confluent. Virus was added (MOI: 0.001) together with 1:5 serial dilutions of the compounds. For each virus, appropriate reference compounds were included. The compound dilutions were also added to a mock-infected plate, to determine compound cytotoxicity. When manifest CPE was reached, i.e., after 3 to 5 days incubation at 37 °C (or 35 °C in case of human coronavirus 229E), CPE and compound cytotoxicity were quantified by the MTS assay, and EC50 and CC50 values were calculated as explained above for influenza virus.The viruses were propagated and evaluated in the following cell lines: Human embryonic lung (HEL) fibroblast cells, used for human coronavirus 229E , herpes 99 value, i.e., the concentration causing 100-fold reduction in vRNA copy number, as compared to the virus control receiving no compound. It was ascertained that the cell controls showed no detectable qPCR signal.To assess inhibition of human coronavirus 229E replication, HEL cells were infected and treated with 1:2 serial compound dilutions. The plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). At day 4 p.i., supernatants were frozen at −80 °C to determine the virus yield by one-step RT-qPCR assay. Two microliters supernatant was mixed with 11 µl lysis mix containing lysis enhancer and resuspension buffer at a 1:10 ratio , and heated for 10 min at 75 °C. Five microliters of lysate was transferred to a PCR plate containing 9.75 µl RT-qPCR mix (CellsDirect One-Step RT-qPCR) and 0.25 µl Superscript III RT/Platinum Taq enzyme, and coronavirus-229E N-gene specific primers and probe . The RT-qPCR program consisted of 15 min at 50 °C, 2 min at 95 °C, and 50 cycles of 15 s at 95 °C and 45 s at 60 °C. An N-gene plasmid standard was included for absolute quantification. Compound activity was expressed as the EC6 and 7 having the same antiviral potency as the analogous teicoplanin pseudoaglycon derivatives [Starting from vancomycin, we have successfully prepared two derivatives with strong activity against influenza virus. The modifications that we introduced were based on our previous work on the glycopeptide antibiotic teicoplanin. Interestingly, some of these modifications yielded compounds ivatives ,30 but l


Dear Editor,The African swine fever (ASF) is a highly contagious hemorrhagic and lethal disease in domestic pigs. The ASF outbreaks of in many Asia countries, including China, Vietnam, Mongolia, and South Korea, have posed a huge threat to the pig industry. In the attempt to stop ASF from spreading further in Mainland China, more than 10 million pigs have been culled since August 2018. The causative agent for ASF is a DNA virus, African swine fever virus (ASFV). Although this deadly disease has been reported in Kenya nearly one hundred years ago, there has been no vaccine for protection from ASFV infection or effective treatments to cure ASF until now.Asfarviridae, is a large, enveloped virus with a regular structure. The DNA genome of ASFV vary in length from approximately 170 to 193 kbp and encodes 151 to 167 proteins depending on the different viral isolates   and size exclusion chromatography (SEC). The SEC result shows that ASFV p35 may exist multiple forms in solution, with the monomer as predominant peak  and explore its potential role in core shell assembly. The polyprotein pp62 produces three mature structural proteins by the cleavage of viral protease pS273R, give rise to mature product p15, p35 and p8  by a mercury (II) derivative and refined to 2.1  Å with a Rfactor and Rfree of 20.3% and 23.0% (Table S1). The rod-like crystal grows in relative higher pH solution belongs to space group P21 with one p35 molecule in one asymmetric unit, in contrast to the cube-like crystals with larger unit cell and containing two p35 molecules in one asymmetric unit. In the final model, residues D1-Y11, D128-D138, G265-I273 and E299-N304 of Molecular A and B are not visible due to the lack of interpretable electron density data, suggesting their high structural flexibility.Two different shapes of crystal are obtained by the vapour-diffusion method. The rode-like crystals appear in the condition containing 0.2 mol/L ammonium acetate, 0.01 mol/L magnesium acetate tetrahydrate, 0.05 mol/L sodium cacodylate trihydrate pH 6.5, 30% PEG 8000, which is subject to heavy atom screening and soaking. While the high-resolution data were collected by the cube-like crystal grows in 0.1 mol/L BIS-TRIS pH 5.0, 25% 10-helices  matrix protein, which is the only virus that the full-length matrix structure has ever been reported in orthomyxovirus to date. The polymerized matrix proteins M1 of ISAV are underneath the viral envelope to enforce the shape and structural integrity  analysis software. The result shows that the accessible surface area buried in the interface is 485.2 ÅNumerous studies on virus matrix protein have indicated its critical role for membrane association primarily mediated by a positively charged patch from the protein surface Below is the link to the electronic supplementary material.


The main goal of the paper is to calculate the heat conductivity for three experimental hemp–lime composites used for structural construction purposes with the use of the experimental stand inside two compartments. Due to current construction trends, we are constantly searching for eco-friendly materials that have a low carbon footprint. This is the case of the analyzed material, and additional thermographic heat distribution inside the material during a fire resistance test proves that it is also a perfect insulation material, which could be applied in addition of popular isolating materials. This paper presents the results of certain hemp–lime composite studies and the potential for using hemp–lime composite for the structural construction industry. Hemp–lime composite heat transfer coefficient, fire resistance, and bulk density properties are compared to those of other commonly used construction materials. The obtained results show that the material together with supporting beams made of other biodegradable materials can be the perfect alternative for other commonly used construction materials. The idea of sustainable development in construction, i.e., one that limits the negative impact of buildings on the environment and is user-friendly, is gaining in popularity . BuildinCannabis sativa L.) has a long history with human civilization and was often found near early nomadic settlements close to streams in well-manured areas [Cannabis sativa L. is a quick-growing, annual herb with a multitude of uses covering a range of products derived from fiber or oilseed that have been known throughout history [The objective of the work is the experimental identification of heat conductivity for three experimental mixtures of hemp–lime composite used for construction purposes analyzed for the use for construction of single-family buildings. Industrial hemp . ArCADia-TERMOCAD is computer software available in several versions, it is one of the most popular programs on the Polish market designed for preparing energy performance certificates required for construction, modernization, and lease and sale transactions of buildings or premises, as well as for calculating the demand for heat and cooling of rooms and facilities. In ArCADia-TERMOCAD PRO version it is possible to perform energy audits, overhauls, and energy efficiency audits, e.g., for the purpose of obtaining modernization bonus. It can also be used for BREEAM certified calculations. Thanks to a rich database, the user of the program develop the necessary documents in accordance with the legal requirements applicable in Poland. ArCADia-TERMOCAD program in all versions has a built-in, fully functional graphics editor allowing to model the body of the building. The TERMOCADIA editor enables import of drawings in DWG format and import and export of ArCADia BIM system projects. Its main purpose is to perform building designs according to Building Information Modeling (BIM) technology assumptions. In addition to traditional architectural documentation, the program also performs a digital building model . The hom2. Building energy performance certificates were issued based on the brick home design, while for our simulation we used the hemp–lime composite home that had been modified by changing its partition structure, i.e., using hemp–lime composite and other natural materials instead. The partitions were fabricated taking into account designers’ and home contractors’ new technology recommendations and guidelines. For our comparative analysis, we selected a new building made of commonly used construction materials as shown in The calculations were performed for a single-family two-story home with a loft and a total floor area of ~220.0 mTesting was performed while the box was tightly closed. During the tests, the obtained value of the heat transfer coefficient was comparable to that found in the literature and reported by manufacturers. The calculated value of the coefficient was also used later on in the study to determine the power demand of a single-family home constructed with hemp–lime composite. 2 × year of nonrenewable primary energy. The energy characteristics provide a lot of vital information about the material and its environmental impact. Our calculations indicate a traditionally constructed building requires 85.35 kWh/m2 × year).A hemp–lime composite building requires nonrenewable primary energy amounting to 44.81  were entered into the library of ArCADia-TERMOCAD program, which enabled to calculate the coefficients of penetration of individual building partitions  and, as a result, to determine the energy demand of the whole analyzed building. Next, the obtained results were compared with a building made of traditional building materials used in Poland. The obtained results are presented in 3. The parameters give hemp–lime composite very good thermal insulation properties with the thermal conductivity coefficient ranging from 0.038–0.055 (W/m*K), depending on how much the shiv has been compacted and what mixing method was applied. The heat transfer coefficient obtained during the calculations had the value of UC = 0.11 (W/m2*K) for external wall and 0.20 (W/m2*K) for inside wall with the addition of the hemp–lime composite. The above value makes it possible to erect walls without any additional insulation needed: the hemp–lime composite is a technology that eliminates thermal bridges in the building. The material was also tested for its flame resistance; the sample did not ignite following a 10 min-long exposure to an open 1800 °C flame, because the material contains lime which not only improves its flame resistance but it also protects it from biological degradation corrosion or fungal deterioration [Further development of conventional building materials is not critical for the construction industry since nature offers the best choices it is up to us to use them properlyKnowledge of hemp–lime as a building material is still at the beginning of the process. In spite of the new research undertaken in this area, there are still no unified standards to ensure the appropriate parameters of a given compositeA great variability of parameters, such as the conductivity, fire, weather, and biological resistance, of the hemp–lime composite is related to so many factors such as morphology of the fillers, the orientation of the filler particles, porosity, the method and the ratio of compaction, the distribution of the filers and many othersThe authors acknowledge the importance of these factors in terms of hemp–lime structure-related issues. This is a very wide range of interdisciplinary research the authors are in the process of preparing samples of the composites for further investigations.The study was designed to analyze the potential of using natural construction materials for single-family buildings. To perform such an analysis, researchers developed their own natural material to take advantage of the hemp life cycle potential for sustainable construction industry. The most significant benefits of using hemp–lime composite include its potential for use as a non-combustible, renewable and natural raw material with a zero-carbon footprint, and good thermal insulation properties. The lime–hemp composite is very light thanks to its high porosity and low bulk density, which ranges from 300 to 400 kg/mioration . The tes


Aedes aegypti mosquitoes. These viruses have become endemic in large parts of North, Central, and South America. Arboviruses persistently infect mosquitoes throughout their life span and become infectious  after a period of time called the extrinsic incubation period (EIP). The duration of this infectiousness, however, is not well characterized. This is an important shortcoming because many epidemiological models assume that mosquitoes continue to be infectious for the duration of their life span. To define the duration of infectiousness for CHIKV and ZIKV, mosquitoes were infected orally with these viruses. Every 2 days, legs/wings, midguts, salivary glands, and saliva were collected from 30 to 60 mosquitoes and viral load measured. In CHIKV-infected mosquitoes, infectious virus in saliva peaked early (2–4 dpi), and then decreased rapidly and was rarely observed after 10 dpi. Viral RNA in infected tissues also decreased after the initial peak (4–8 dpi) but did so much less drastically. In ZIKV-infected mosquitoes, the infectious virus in saliva peaked at 12–14 dpi and dropped off only slightly after 14 dpi. In infected tissues, viral RNA increased early during infection, and then plateaued after 6–10 days. Our findings suggest that significant variation exists in the duration of the infectious period for arboviruses that is in part influenced by virus clearance from expectorated saliva.Chikungunya virus (CHIKV) and Zika virus (ZIKV) are arthropod-borne viruses transmitted mainly by  Alphavirus) and Zika virus  are arthropod-borne viruses transmitted by Aedes aegypti and Aedes albopictus mosquitoes. Both viruses have spread across the world over the last two decades and have recently become endemic in large parts of North, Central, and South America.3 Clinical symptoms of Chikungunya fever and Zika fever are very similar: fever, arthralgia, myalgia, rash, and conjunctivitis.4 However, both viruses can cause more serious diseases including persistent arthralgia (CHIKV) or neurological symptoms (CHIKV and ZIKV), although neurological complications are extremely rare in CHIKV infections.5 Zika virus has also been known to cause Zika congenital syndrome,6 which is a collective term for neurodevelopmental disorders in infants as a result of ZIKV infection of the developing fetus in pregnant women.Chikungunya virus . The duration of the EIP is a complex phenotype that is determined by specific virus–mosquito pairings and influenced strongly by environmental conditions.12 Formulas to quantitatively estimate the basic reproductive rate (R0) for a vector-borne pathogen 13 include only a term for the duration of the EIP but not for loss of transmissibility. Therefore, epidemiological models tend to implicitly assume that mosquitoes are infectious for the duration of their life span.Arboviruses persistently infect mosquitoes throughout their life span of approximately 2–4 weeks in nature,14 This was not observed in mosquitoes infected with ZIKV. However, we did not evaluate infectious virus present in the saliva, and only two time points were taken in the study.14 We therefore sought to determine whether virus replication and transmission dynamics differ between these two viruses. We thus investigated the dynamics of replication, dissemination, and estimated transmission of CHIKV and ZIKV in a Mexican colony of Ae. aegypti mosquitoes to see when virus in the saliva peaks and whether transmission drops off over time. We exposed Ae. aegypti mosquitoes to a blood meal containing CHIKV or ZIKV, sorted blood-fed mosquitoes into individual tubes, and then collected the midguts, legs/wings, salivary glands, and saliva every 2 days from 2 to 20 days post-infection (dpi). We used infectious virus in collected saliva as a proxy for transmission efficiency. Transmission experiments using live animals may provide results that differ from ours, but saliva collection is the most feasible method to estimate transmission in vector competence studies. We found that in mosquitoes infected with CHIKV, viral RNA copies and amount of infectious virus in saliva peaked early and gradually decreased over time. In ZIKV-infected mosquitoes, viral RNA and infectious virus peaked later and persisted in saliva throughout the experiment. Overall, our study provides evidence that virus transmission may not always be maintained throughout the mosquito’s life span, and that the rate of virus clearance from saliva varies in a virus–mosquito–specific manner. This finding has clear implications for modeling the epidemiology and transmission of arboviruses.During a previous study, we observed that the proportion of CHIKV RNA-positive saliva was higher at an earlier time point than a later time point.Aedes aegypti mosquitoes from Poza Rica, Mexico, were colonized in 2012.15 Mosquito husbandry and insectary practices are as described elsewhere.3 Briefly, larvae were raised on a diet of powdered fish food, and adult mosquitoes were maintained at 28°C with a 12:12 (L:D) photoperiod and 70–80% relative humidity. Water and sugar were provided ad libitum.® CCL-81™, Manassas, VA) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 50 µg/mL gentamycin, and 100 µg/mL penicillin/streptomycin at 37°C in a 5% CO2-containing humidified atmosphere.Vero cells (ATCC7 PFU/mL and 3 × 107 plaque forming units (PFU)/mL for CHIKV and ZIKV, respectively.Chikungunya virus 99659  and ZIKV PRVABC59  were propagated in Vero cells, and single use aliquots were prepared and frozen at −80°C. Stocks were titrated on Vero cells by the standard plaque assay. Briefly, cells were infected at ∼90% confluency using 10-fold serial dilutions of virus in DMEM (without FBS). After 1 hour of incubation, an overlay was added, consisting of 6 g/L Tragacanth gum  in Eagle’s Minimum Essential Medium (EMEM) supplemented with 4% FBS, 100 µg/mL gentamycin, 100 µg/mL penicillin/streptomycin, and 5 µg/mL amphotericin B. The overlay was removed 3 dpi (CHIKV) or 5 dpi (ZIKV), and cells were fixed in 20% ethanol containing 1 g/L crystal violet. Stock titers were 1.8 × 106 PFU/mL of CHIKV or 1.5 × 107 PFU/mL of ZIKV in 20% DMEM (1:1 mix of blood:virus stock). The artificial blood meal contents were added to water-jacketed glass feeders sealed with hogs gut and connected to a 37°C water bath. Mosquitoes were allowed to feed for approximately 45 minutes, after which they were anesthetized at 4°C and sorted. Engorged female mosquitoes were transferred to individual 50-mL conical tubes with a small piece of paper towel at the bottom. The tops of the conical tubes were covered in mesh and secured with a rubber band. After females began to recover, water was added directly to the conical tube until a small pool collected at the bottom after absorption into the paper towel. Two filter cards were then placed on top of each tube as a sugar source for the duration of the experiment. One card was soaked in 20% sucrose solution, whereas the other was soaked in a solution of 50% honey and 20% sucrose.Female mosquitoes, 5–7 days old, were fed an infectious blood meal of defibrinated calf blood containing 9 × 10g for 5 minutes at 4°C to expel saliva-containing oil into mosquito diluent before freezing. All samples were stored at −80°C until sample processing. Experiments were performed twice for CHIKV and ZIKV infections.Mosquitoes were then held for up to 20 days in a BSL-3/ACL-3 insectary under the same conditions as described earlier. Every 2 days following the infectious blood meal, 30 mosquitoes were cold-anesthetized. Legs/wings were collected first, and then saliva was collected by inserting the mosquito proboscis into a capillary tube containing immersion oil for 30 minutes. After salivation, the midguts and salivary glands were dissected. Legs/wings, midguts, and salivary glands were placed directly into lysis buffer . Capillary tubes containing saliva were placed in microcentrifuge tubes with 100 µL mosquito diluent  and centrifuged at 15,000 × 16 with minor modifications. Vero cells were seeded in 12-well plates and allowed to reach ∼90% confluency. Saliva samples were thawed, briefly vortexed, and centrifuged for 3 minutes at 15,000 × g. In the meantime, Vero cell culture media was removed and replaced with 200 µL of DMEM without additives. Then, 30 µL of saliva sample were added to each well. Plates were rocked for 10 minutes at room temperature to allow spread of virus over the monolayer, returned to the 37°C CO2 incubator, and briefly rocked every 15 minutes for 1 hour. After 1 hour, 1 mL of Tragacanth/media overlay was added to each well, and plates were incubated at 37°C. After 3 days (for CHIKV) or 5 days (for ZIKV), the plates were fixed with a staining solution . Plaques were visualized on a light box, counted, and recorded. For samples with high viral titers, this process was repeated with 10-fold dilutions of input sample to permit accurate plaque quantification. The detection limit for plaque assays was 3.33 PFU/saliva sample.Saliva samples were titrated as previously describedLegs/wings, midgut, and salivary gland samples were thawed, vortexed thoroughly, and briefly centrifuged. Viral RNA was extracted from individual samples using the MagMax total RNA isolation kit (Thermo Fisher Scientific Cat #AM1830), following the manufacturer’s protocol, and the KingFisher Flex System. Samples were eluted in 50 µL of nuclease-free water, of which 5 µL were placed directly into quantitative real-time reverse transcriptase–PCR (qRT-PCR), and the rest was collected into 8-strip tubes and stored at −80°C.3 Positive RNA standards were generated for both viruses3 and included in each qRT-PCR plate, as well as negative controls. Saliva samples were thawed, vortexed, and briefly centrifuged, and 5 µL were used directly for qRT-PCR. The limit of reliable detection for qRT-PCR was 335 viral copies/sample.One-step qRT-PCR was performed on individual samples of legs/wings, midguts, salivary glands, and saliva with specific probes and primers for CHIKV and ZIKV in a multiplex reaction, as previously described.Ae. aegypti mosquitoes continues to be present throughout the mosquito’s lifetime . Chikungunya virus infection rates were high (94–100%) for all time points, as measured by viral RNA in the midgut , as measured by viral RNA in the midgut  and declined thereafter, ZIKV transmission peaked later (14 dpi) and remained more consistent over time. Chikungunya virus RNA levels in saliva also decreased after 6 dpi, but numerous mosquito saliva samples remained CHIKV RNA positive up to 18 dpi. This highlights the importance to measure infectious virus in vector competence studies.Here, we compared CHIKV and ZIKV replication and transmission in a colony of 14 However, although the same virus isolate and mosquito colony were used, the experimental setup was quite different. Fewer time points were used, infectious virus was not determined, and female mosquitoes were sequentially blood-fed in various chronological orders with uninfected blood, blood containing ZIKV or blood containing CHIKV. In the latter study,14 all mosquitoes tested had thus received at least two blood meals, which may explain overall higher numbers of viral RNA-positive saliva samples of these mosquitoes.17 It has recently been shown that a second noninfectious blood meal  can enhance CHIKV dissemination and transmission.17 It may thus be possible that a second blood meal could have resulted in renewed or prolonged transmission of CHIKV at later times after infection. Aedes aegypti mosquitoes tend to take multiple smaller blood meals (“sipping” behavior), due to interruptions in feeding and necessary resources for oviposition.18 Although multiple smaller blood meals thus represent what happens in nature, using one infectious blood meal for vector competence experiments is common practice and makes our data relevant to researchers selecting time points to determine vector competence for CHIKV. Many studies evaluate transmission at late time points, such as 14 dpi, which may not reflect peak transmission for CHIKV.We had observed a similar, albeit less dramatic, decrease in CHIKV RNA in saliva between time points in a previous study.19 Both Rückert et al.3 and Magalhaes et al.14 focused on the study of viral coinfections and, thus, measured only viral RNA in saliva because of the inherent difficulty of plaque-titrating two viruses from the same sample. The estimated transmission rates thus cannot be directly compared. In Sanchez-Vargas et al.Ae. aegypti and Ae. albopictus saliva and salivary glands infected with CHIKV. The authors detected infectious virus in 50% of saliva samples at 9 dpi for this particular virus–mosquito combination (Ae. aegypti from Poza Rica and CHIKV strain 99659), which is significantly higher than our estimated transmission rate of 10% at 8 dpi. However, saliva sampling and virus titration methods also varied slightly between our study and Sanchez-Vargas et al.19 study, and infectious virus titers of saliva samples 9 dpi were also low in the latter study , despite a higher proportion of positive saliva samples.We were surprised to observe lower overall transmission levels for CHIKV than some previous studies using this virus strain and the same mosquito colony.In conclusion, the results from our study suggest that once a mosquito is infected with CHIKV and able to transmit virus, it may not necessarily continue to do so for the remainder of its life. This contrasts to the general assumption that the EIP of a mosquito presents an end point after which the mosquito will transmit virus with each subsequent blood meal. Our results could impact further considerations for the EIP of CHIKV-infected mosquitoes and how vectorial capacity is calculated, and they may provide insight into saliva sampling for vector competence experiments. The observed discrepancy between CHIK RNA and infectious virus in mosquito saliva also further highlights the importance of sampling infectious virus whenever possible because RNA copies may not correlate directly to infectious virus.