At a fixed RH of 75%, infectivity was very sensitive to temperature, decreasing two orders of magnitude between 19°C and 25°C. We used random forest modeling to identify the best environmental predictors for modulating virus infectivity. The model explained 83% of variation in Phi6 infectivity and suggested that RH is the most important factor in controlling virus infectivity in droplets. This research provides novel information about the complex interplay between temperature, humidity, and the survival of viruses in droplets.Infectious diseases caused by enveloped viruses, such as influenza, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), cause thousands of deaths and billions of dollars of economic losses per year. Studies have found a relationship among temperature, humidity, and influenza virus incidence, transmission, or survival; however, there are contradictory claims about whether absolute humidity (AH) or relative humidity (RH) is most important in mediating virus infectivity. Using the enveloped bacteriophage Phi6, which has been suggested as a surrogate for influenza viruses and coronaviruses, we designed a study to discern whether AH, RH, or temperature is a better predictor of virus survival in droplets. Our results show that Phi6 survived best at high (>85%) and low (<60%) RHs, with a significant decrease in infectivity at mid-range RHs (∼60 to 85%). At an AH of less than 22 g · mIMPORTANCE Enveloped viruses are responsible for a number of infectious diseases resulting in thousands of deaths and billions of dollars of economic losses per year in the United States. There has been a lively debate in the literature over whether absolute humidity (AH) or relative humidity (RH) modulates virus infectivity. We designed a controlled study and used advanced statistical modeling techniques specifically to address this question. By providing an improved understanding of the relationship between environmental conditions and virus infectivity, our work will ultimately lead to improved strategies for predicting and controlling disease transmission. Infectious diseases caused by enveloped viruses, such as influenza viruses and the coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), cause thousands of deaths and billions of dollars of economic losses per year –3. Both –4–Although the root cause of influenza and SARS seasonal patterns remains elusive, a confluence of recent epidemiological, animal, and laboratory studies has found a relationship between environmental conditions—temperature, absolute humidity (AH), relative humidity (RH), and/or rainfall—and influenza virus incidence, transmission, or survival –18. The –21–There has been considerable debate over whether AH or RH modulates influenza virus survival and transmission. AH describes the mass of water vapor per volume of air , while RH describes the ratio of the actual concentration of water vapor to the maximum possible concentration, which varies strongly with temperature. Shaman and Kohn  have conDespite the agreement among these studies that AH is more important than RH for influenza virus survival and transmission, we contend that the debate is not settled. The relationship between survival and humidity is confounded by the fact that higher values of AH can only be achieved at higher temperatures, which are known to accelerate virus inactivation . EpidemiAlthough many factors affect disease transmission, we designed a study explicitly to discern whether AH or RH is a better predictor of virus survival in droplets, while also considering temperature in order to advance a mechanistic understanding of the relationship between virus inactivation and environmental conditions. We exposed an enveloped virus to seven environmentally relevant RHs and four temperatures to test the hypothesis that RH is more important than AH in modulating virus infectivity. Additionally, we hypothesize that over the range of temperatures typically found indoors (19 to 25°C), temperature does not have a significant effect on infectivity. The resulting data set is larger than has been obtained previously and enables a robust analysis that goes beyond simple linear regression. Due to the challenges and biosafety concerns of working with the influenza virus and coronavirus, this study employed the enveloped bacteriophage Phi6, which has been suggested as a surrogate for the influenza virus , 32 and P = 0.0002) influence of temperature on Phi6 infectivity; however, at 14°C and 19°C, virus infectivity remained high across all RHs tested. There was a significant effect of RH (P < 0.0001) on Phi6 infectivity at each temperature tested, except at 19°C (P = 0.0528). At 25°C and 37°C, we observed the same U-shaped RH dependence that Yang et al. .We tested the infectivity of Phi6 in droplets exposed to AHs ranging between 2.7 and 41.6 g · mP < 0.0001); however, at temperatures above 34°C, there was no effect of temperature on virus infectivity (P = 0.1293). For perspective, medical equipment meets the sterility assurance level when there is a 6-log reduction in the number of microorganisms.To examine the effect of temperature on Phi6 infectivity , we heldF statistics confirmed that the addition of RH to a model already containing AH did not improve the model. However, the overall fit of the multiple regression model was poor, with an R2 of 0.34. Using a multiple regression with the dependent variable of the log of infectivity improved the R2 to 0.64. For this model, both AH and RH were statistically significant, based on partial F statistics, but temperature was not.Multiple linear regressions of infectivity against temperature, RH, and AH revealed that the slopes for temperature and AH were statistically significant, but that for RH was not. Partial R2) was 0.83. The relative importance of the variables, measured as the increase in the mean squared error when the variable was deleted from the model, was RH > temperature > AH. The normalized variable importance was 1.00, 0.99, and 0.84 for RH, T, and AH, respectively. The random forest model applied to a log10 reduction in infectivity (instead of just relative infectivity) did not perform as well; the percentage of explained variance was 0.59.The random forest was the most successful model applied. P = 0.095).As some viral diseases are transmitted via the airborne route, the inevitable questions are whether virus inactivation is similar in stationary droplets and suspended aerosols and whether droplets, which are much easier to work with, can be used as a surrogate for aerosols in the study of virus inactivation. We measured virus stability in both droplets and aerosols under the same conditions, and the relationship between infectivity and RH was essentially the same in both . The corMany factors affect disease transmission, and we designed a study examining one piece of the puzzle to advance a mechanistic understanding of the relationship between virus inactivation and environmental conditions. The results of this study apply to transmission by droplets and aerosols that remain exposed to the environment on the time scale of many minutes to hours, or long enough for environmentally mediated inactivation to occur. This study was designed specifically to compare the importance of RH, AH, and temperature as predictors of enveloped virus survival in droplets. Ultimately, we hope this work can help explain the trends in diseases caused by enveloped viruses and lead to improved disease control strategies. Our results suggest that in determining virus infectivity, the interaction among RH, AH, and temperature is complex and not easily captured by linear or log-linear regressions. Shaman and Kohn  found thR2 = 0.83). The random forest model suggested that RH was the most important factor in controlling Phi6 infectivity in droplets. Random forests have the advantage that they do not need a prespecified model to evaluate as regressions do. Random forests develop the model based on the data and can thus provide improved results for models based on correlated interacting variables  –33. The –This study examined only one aspect, the effect of humidity and temperature on virus stability in droplets and aerosols, of many that control the transmission of infectious disease. Other environmental factors, such as sunlight, wind, and rainfall, are also known or suspected to affect transmission. Seasonal variations in contact rates between infected and susceptible hosts, such as an increased time spent indoors during the winter, have been suggested to explain the seasonality of influenza . Host suOur results provide insight into how RH, AH, and temperature affect enveloped virus droplet infectivity, and there are many directions for future work. First, as we have shown that the choice of statistical model has a significant effect on the conclusions, we recommend reanalyzing the data collected from previous studies of the effect of RH versus AH on enveloped virus infectivity using different statistical models beyond linear regression. Future efforts are also needed to study viruses that require more stringent biosafety protocols to assess whether the results shown here are generalizable to pathogenic viruses. Additionally, we recommend that future studies use more physiologically relevant media  to test the hypotheses that (i) medium composition has an effect on virus infectivity when exposed to different environmental conditions and (ii) glycoproteins provide a protective effect for virus in droplets against changes in RH and temperature. Finally, an interdisciplinary research effort is needed to define optimal indoor environmental conditions. Ultimately, these efforts will lead to improved strategies to control infectious disease transmission in the built environment.Pseudomonas syringae pv. phaseolicola, and we cultivated it on tryptic soy agar (TSA). We prepared stock solutions of virus by suspending propagated Phi6 in TSB at concentrations of approximately 108 to 1010 PFU · ml−1. We stored working stocks of Phi6 at 4°C and long-term stocks at −80°C.We used the bacteriophage Phi6  as a surrogate for influenza virus and other enveloped viruses in these experiments . The hosTo control humidity, we used a chamber as previously described . We placWe regulated temperature by incubating the chamber in temperature-controlled rooms (14°C and 37°C), an incubator (25°C), or on a benchtop (19°C). To isolate the effect of temperature at a single RH of 75%, we incubated the chamber at additional temperatures of 22°C, 28°C, 31°C, 34°C, and 40°C. We selected 75% RH for this experiment for the following reasons. First, there were distinct differences in infectivity versus temperature at 75% RH . Second,Before placing samples into the chamber, we allowed the chamber to reach equilibrium at the desired RH and temperature. When we briefly opened the chamber to place the samples inside, the temperature did not shift, but there was a small change in RH. However, the equilibrium was reestablished within approximately 5 to 10 min under all experimental conditions. Following every test, we washed the chamber with distilled water and ethanol to remove any salt residue.8 to 1010 PFU · ml−1 onto one well of a six-well, untreated, polystyrene cell culture dish (Greiner Bio-One CELLSTAR cell culture multiwell plate). We then placed the cell culture dish into the chamber, placed the lid on the chamber with a thin layer of vacuum grease to create a seal, and moved it to the appropriate location for incubation at the desired temperature. The entire process of diluting the working stock, spotting the droplets, and placing the cell culture dish into the RH chamber took place in a biological safety cabinet (BSC) at approximately 20°C.For each of three biological replicates under each experimental condition, we spotted 10 1-μl droplets of Phi6 at a concentration of 10Immediately following a 2-h incubation period, we returned the chamber to the BSC and removed the cell culture dish. We added 500 μl of TSB to each well containing droplets, which we resuspended via pipetting and then placed into separate cryovials for each well's solution. We placed the vials in a −20°C freezer for 20 min before moving them to a −80°C freezer for long-term storage. Prior to assaying these samples, we thawed them at room temperature before sampling and refreezing.Samples for each experimental condition included negative-control cultures containing 500 μl of Phi6 stock solution. We stored these controls in polyethylene tubes and did not spot them as droplets but otherwise incubated them in the RH chamber and stored and assayed them identically to the test cultures.−1) for stock, control, and test cultures. For the plaque assays, we first performed serial dilutions of our working stock in TSB medium. We then mixed 50 μl of the diluted Phi6 with 250 μl of overnight cultured P. syringae, with the coculture inoculated into 6.5 ml of TSB soft agar prewarmed to 50°C. We quickly, but fully, hand-shook the mixture and poured it onto TSA plates, which we rotated by hand to allow the top agar to form a smooth even layer on the TSA. After drying, we sealed the plates in Parafilm and incubated them at 25°C for 24 h. To determine the concentration of infectious Phi6 in a sample, we multiplied the number of observed plaques by the appropriate dilution coefficient. We calculated the relative infectious ratio after aging Phi6 droplets at a given temperature, RH, and AH as relative infectious ratio (%) = EN/CN, where EN and CN are the measured Phi6 concentrations (PFU · ml−1) for the exposed and control samples, respectively.We performed plaque assays to quantify infectious Phi6 in the form of PFU  in Phi6 infectivity between different temperatures, RH, and AH in JMP Pro 13.We determined the average Phi6 infectivity and the standard error from triplicate samples for each RH-temperature combination tested. We calculated all statistics in R version 3.4.2  and JMP


In 2015 and 2016, we observed 15 malformed calves that were exposed tointrauterine infection with Shamonda virus, a Simbu serogroup orthobunyavirus,in Japan. Characteristic manifestations were arthrogryposis and gross lesions inthe central nervous system. Our results indicate that this arbovirus should beconsidered a teratogenic virus in ruminants. Two viruses (KS-1/P/15 and KS-2/P/15) were isolatedfrom cattle blood collected during August and September 2015, and another virus(KSB-1/C/15) was isolated from a pool of C. tainanus midges sampledduring September 2015.To obtain data on arboviruses circulating in 2015, we attempted to isolate viruses onBHK-21 and HmLu-1 cells inoculated with blood samples obtained from 60 sentinelcattle maintained on 30 farms and from pools of We performed reverse transcription PCR (RT-PCR) with primer pairs  specific for small RNA segment of Simbuserogroup viruses and the One Step RT-PCR Kit . Wegenerated a product of the expected size from RNA samples of the isolated viruses.Preliminary sequence analysis for the RT-PCR product (443-nt) showed that theviruses were highly similar to SHAV. We sequenced and analyzed complete small andmedium RNA segments and a partial region of the large RNA segment by using primersspecific for SHAV  on virus-infected HmLu-1 cells byusing an established method . Serum sAlthough 2 calves ingested colostrum substitute containing immunoglobulins, allcalves tested were deprived of colostrum produced by their mothers. SHAV had notbeen detected in mainland Japan for ≈10 years until we identified new casesof infection in 2015. It is highly improbable that the colostrum substitutecontained antibodies against SHAV. Malformed calves were delivered at or beyond term(gestation periods range 275–299 days), but 6 calves were born dead.The small RNA–specific RT-PCR showed positive results for samples from thecentral nervous system  of 7 deformed calves.Sequences obtained were identical (except for 1 of 2 nt substitutions) with relevantsequence of SHAVs isolated in 2015.Torticollis (10/15) or arthrogryposis (12/15) were often observed among affectedcalves . Head deHistopathologic analysis identified calcification of nerve cells (11/15),perivascular infiltration of mononuclear cells (6/15), and gliosis (5/15), whichwere often observed in the cerebrum and brainstem . Severe >1:256).VNTs did not detect neutralizing antibodies against teratogenic arboviruses, such asChuzan virus, Akabane virus, Aino virus, or Peaton virus in serum samples fromaffected calves. All tested dams of affected newborns were positive for SHAVantibodies in VNTs (titer range 1:16–,Our findings support an association between SHAV and congenital deformities in calvesinfected in utero. Manifestations and macroscopic and microscopic observations inthe malformed calves were similar to those of calves infected with Schmallenbergvirus (–Twelve of the dams of the malformed calves in this report were vaccinated with atrivalent inactivated vaccine containing Akabane, Aino, and Chuzan viruses. To ourknowledge, no effective preventive measure for infection with SHAV is available.Previous surveillance in Africa, the Middle East, and Asia (


To determine and compare hospitalization costs of bacterial community-acquired pneumonia cases via different costing methods under the Brazilian Public Unified Health System perspective. Cost-of-illness study based on primary data collected from a sample of 59 children aged between 28 days and 35 months and hospitalized due to bacterial pneumonia. Direct medical and non-medical costs were considered and three costing methods employed: micro-costing based on medical record review, micro-costing based on therapeutic guidelines and gross-costing based on the Brazilian Public Unified Health System reimbursement rates. Costs estimates obtained via different methods were compared using the Friedman test. Cost estimates of inpatient cases of severe pneumonia amounted to R$ 780,70/$Int. 858.7 , R$ 641,90/$Int. 706.90 (therapeutic guidelines) and R$ 594,80/$Int. 654.28 . Costs estimated via micro-costing  did not differ significantly (p=0.405), while estimates based on reimbursement rates were significantly lower compared to estimates based on therapeutic guidelines (p<0.001) or record review (p=0.006). Brazilian Public Unified Health System costs estimated via different costing methods differ significantly, with gross-costing yielding lower cost estimates. Given costs estimated by different micro-costing methods are similar and costing methods based on therapeutic guidelines are easier to apply and less expensive, this method may be a valuable alternative for estimation of hospitalization costs of bacterial community-acquired pneumonia in children. In 2010, an estimated 120 million new episodes of the disease affected children aged under 5 years, with 935 thousand cases progressing to death worldwide.6 Several costing methods with different levels of accuracy and quality may be applied to estimate disease costs, bottom-up micro-costing, top-down micro-costing and gross-costing being the most common.7Estimated costs of inpatient treatment of pneumonia in children aged less than 5 years in Latin America range from US$ 804.46 to US$ 1,076.89.8 The Brazilian Public Unified Health System (SUS - Sistema Único de Saúde) covers about 75% of the population; therefore, estimating pediatric pneumonia costs under the SUS perspective is important. Given the variability of results across costing methods, estimation of Brazilian costs of inpatient cases of severe pneumonia in children via different methods is of particular relevance.Pneumonia represents a high economic burden to society and the health care system.Sistema Único de Saúde hospital costs to treat bacterial community-acquired pneumonia cases estimated via different costing methods.To determine and compare An observational descriptive partial economic evaluation study under the SUS perspective. Costs of the disease were estimated using different methods. Costing studies were carried out in Goiânia (GO) between October and December 2011.8 This costing study was based on a sample of children admitted to two of these hospitals, totaling up 111 clinical beds and 38 pediatric intensive care unit (ICU) beds, accounting to approximately 30% of children hospitalized due to CAP, in a prospective population-based design. Selected cases corresponded to children hospitalized due to suspected pneumonia (cough and/or dyspnea) with clinical or radiological confirmation and receiving antimicrobial therapy during the period of admission. Cases with a discharge diagnosis of viral pneumonia, bronchiolitis, asthma or atypical pneumonia, as well as those involving patients admitted through health insurance plans were excluded.The study population comprised children aged between 28 days and 35 months hospitalized due to community-acquired pneumonia (CAP), and enrolled in a prospective population-based pneumonia surveillance study conducted in 17 child hospitals in the city of Goiânia (GO).Over the course of the study period, 1,520 children with suspected pneumonia were identified via the surveillance study, 330 of which met CAP definition criteria. Of these, 106 (32%) were admitted to the two hospitals included in this study, of which 11 (10%) were excluded due to missing records. Out of 95 children whose medical records were evaluated, 11 (12%) were excluded due to lack of antimicrobial therapy over the course of hospital stay, 5 (5%) due to diagnosis upon discharge of viral pneumonia, bronchiolitis, asthma or atypical pneumonia, and 31 (31%) due to admission costs paid by a health insurance plan. The final sample comprised 59 cases of children with pneumonia and admitted through the SUS, in that, 52 children considered severe cases and treated in inpatient units, and 7 graded as very severe and treated at ICU.10 direct costs were considered, including medical and non-medical costs used in previous published studies on pneumonia costs.12 Medical costs included hospital services (admission to ward and/or ICU), professional services , drugs, radiological examinations and laboratory tests, and respiratory physical therapy. Non-medical costs comprised daily companion charge rates. Data concerning healthcare resource utilization were analyzed from date of admission to date of discharge.In compliance with international recommendations,reals  and converted to international dollars ($Int.) at the rate of R$  1,00=$Int. 1.10; 2011 exchange rates).13Costs were calculated in Three costing methods were used in this study: (1) bottom-up micro-costing ; (2) top-down micro-costing, (cost estimation based on therapeutic guidelines); and (3) top-down gross-costing .e.g., suction, nebulization and oxygen supply) were not taken into account.For costing purposes, inpatient ward and ICU length of stay were taken into account in cases of very severe pneumonia treated at both levels. Procedures not listed in SUS reimbursement tables and which therefore did not have a predefined reimbursement rate .Banco de Preços em Saúde (BPS),14Câmara de Regulação do Mercado de Medicamentos (CMED)15 and Revista ABC Farma.16 Prices of similar drugs manufactured by different pharmaceutical industries vary; therefore, drug prices in this study were based on the formulary of one of the hospitals considered, and the 17% Goiás State tax (Imposto sobre Circulação de Mercadorias e Serviços - ICMS) was calculated.Unitary prices of individual resources consumed were multiplied by the estimated amount of resources. Drug costs per child were calculated according to prescribed doses administered over the curse of hospital stay. Drug prices were based on average prices listed in Sistema de Gerenciamento da Tabela de Procedimentos, Medicamentos, Órteses, Próteses e Materiais Especiais do SUS (SIGTAP),17 or “SUS table”.Costs of professional and hospital services, respiratory physical therapy, radiological examinations and laboratory tests were estimated based on Daily hospital bed rates and medical fees were estimated by dividing respective SUS reimbursement rates by patient average length of stay.Sociedade Brasileira de Pneumologia e Tisiologia and Sociedade Brasileira de Pediatria for treatment of children hospitalized due to CAP18  were used.16 The remaining components were priced according to SIGTAP.17 Respiratory physical therapy was not included in therapeutic guidelines. As mentioned, oxygen supply, although recommended for hypoxemic children, was not listed in SUS tables and was therefore not considered.Drug prices listed by the national agencies BPS,19 This amount is paid whenever a minimum of 50% of expected length of stay is actually completed . Additional R$ 20,00 are paid for longer hospitalization time (from 9 days on).19Hospitalization rates practiced by SUS hospitals, or by private hospitals with contract with the SUS, are reimbursed via a package of professional and hospital services including meals, hospital room rates, hospital materials, drugs and ancillary diagnostic tests. This package has a fixed value defined according to diagnosis of each admission (based on ICD code indicated as diagnosis upon discharge of each patient). Reimbursement rates correspond to R$ 504,07/$Int. 554.47  and R$ 78,40/$Int. 86.24 . A standard value reflecting minimum length of stay for a given diagnosis is paid.Autorizações de Internação Hospitalar) were obtained from the Hospital Information System  of SUS Information Technology Department (DATASUS - Departamento de Informática do Sistema Único de Saúde) via the Municipal Health Department of Goiânia. Sistema Único de Saúde reimbursement rates per patient, including hospital services, daily companion charge rates, professional services and respiratory physical therapy were identified and analyzed for hospitalization cost estimation on a per patient basis.Copies of authorizations for hospital admission  and standard deviation (SD) were calculated for severe and very severe pneumonia. Given the low numbers of very severe cases, only severe inpatient cases treated at wards were considered in comparative analysis of costs estimates. The Friedman test was used to investigate cost differences in the methods, via one-to-one comparisons, at a significance level of 0.05. Data were entered into Excel spreadsheets and statistical analyses were performed using the Statistical Package Social Sciences (SPSS) software version 23.Hospital das Clínicas da Universidade Federal de Goiás, Research Ethics Committee (CEP) protocol MHA/HC/UFG 174/09.This study was approved by the Ethics Committee of The clinical and demographic features of the 59 CAP inpatients are presented in Health resources and respective amounts utilized for treatment of 52 patients affected with severe pneumonia in this study are detailed in Very severe pneumonia treatment costs were significantly (p<0.001) higher compared to severe pneumonia, regardless of the costing method employed. Severe pneumonia treatment costs varied across costing methods, with mean costs corresponding to R$ 780,70 , R$ 641,90  and R$ 594,80  .Based on medical record review, daily cost estimates for the mean length of stay in this study (4 days) amounted to R$ 126,00/$Int. 138.60 and R$ 19,58/$Int. 21.53 . Mean overall costs corresponded to R$ 780,70 /$Int. 858.77, with R$ 750,10/$Int. 825) and R$ 30,60/$Int. 33.6 reflecting medical and non-medical services respectively. Ward hospitalization costs amounted to R$ 507,20 /$Int. 557.90, while drugs costs amounted to R$ 159,00 / $Int. 174.90. Antibacterial and ancillary drug costs accounted for 40% and 60% (R$ 64/$Int. 70.4 and R$ 95/$Int. 104.5) of total drug costs respectively.Therapeutic guideline based costing yielded mean overall cost estimates of R$ 641,90 /$Int. 706, with R$ 609,90/$Int. 670.90 and R$ 32,00/$Int. 35.20 reflecting medical and non-medical costs respectively. Mean values estimated via reimbursement based costing corresponded to R$ 594,80 /$Int. 654.28, with R$ 565,50/$Int. 622 and R$ 29,30/$Int. 32.23 reflecting medical and non-medical costs respectively .Estimated very severe pneumonia treatment costs varied across costing methods. Cost estimates obtained via micro-costing methods  did not differ significantly (p=0.405), while the estimated cost for repayment was significantly lower than the costs estimated by therapeutic guideline (p<0.001) or medical record review (p=0.006) basead cost estimates.20 including pneumonia or pneumococcal disease treatment.22Mean length of stay in this study (4 days) is within expected SUS time frame for pneumonia treatment17 and is consistent with WHO  recommendations of 5 days, and with data from a recent global systematic review addressing treatment costs of pneumonia in children (mean of 5.8 days).23There is evidence to suggest that length of stay has a significant impact on overall hospital costs,24and up to R$ 1.373,5325 or R$ 2.957,34,26 combined bottom-up/top-down costing) following adjustment for inflation in the country of origin and conversion to American dollars (PPP 2010)6 to reals (2011). As expected, costs estimates based on reimbursement rates were significantly lower compared to costs estimates based on medical record review.The hospital costs of severe pneumonia in children estimated in this study reflect data from Latin American studies .28 investigating the value of combined gross-costing and micro-costing to assess variations in utilization of essential hospital resources failed to reveal differences between top-down and bottom-up approaches. Wordsworth et al.,29 compared top-down and bottom-up cost estimates but did not explore the gross-costing method. Tan et al.,20 compared the reliability of overall hospital service cost estimates based on bottom-up/micro-costing, top-down/microcosting or gross-costing analysis in the Netherlands. This study is the first to assess inpatient costs of a single patient sample via three different costing methods.Studies comparing costing methods are scarce. A study by Swindle et al.,i.e., costing estimates based on medical record review) is thought to be the most accurate costing method and the gold standard.7 However, this methodology is difficult to apply, labor intensive, time consuming and expensive. The top-down approach combined with micro-costing or gross-costing may represent an easier and less expensive alternative.9 Still, evidence suggests that bottom-up micro-costing is the method of choice to estimate the cost of components with greater impact on overall daily hospital stay costs.29Bottom-up micro-costing  or bottom-up  micro-costing did not differ significantly in this study, suggesting that top-down micro-costing may be a more practical and less expensive costing alternative.This study has several limitations. First, the low number (n=7) of very severe pneumonia cases in the sample limited comparing the three methods in this group of patients. Further costing studies with an adequate number of pneumonia cases requiring ICU hospitalization are therefore warranted. Also, the average daily hospital stay cost of R$ 126,00 adopted in this study may have translated into increased therapeutic guideline and medical record based cost estimates.SUS cost estimates obtained via top-down (based on therapeutic guidelines) or bottom-up  micro-costing did not differ significantly in this study, suggesting that top-down micro-costing may be an alternative to micro-costing based on medical record review for severe pneumonia treatment cost estimation. Studies comparing different costing methods and focusing on different diseases are warranted to support our findings. These results may subsidize economic evaluation of interventions for prevention and control.


Parapoxvirus genus of the family Poxviridae and infects sheep and goats, often around the mouth, resulting in acute pustular skin lesions. ORFV encodes several secreted immunomodulators including a broad-spectrum chemokine binding protein (CBP). Chemokines are a large family of secreted chemotactic proteins that activate and regulate inflammation induced leukocyte recruitment to sites of infection. In this study we investigated the role of CBP in vivo in the context of ORFV infection of sheep. The CBP gene was deleted from ORFV strain NZ7 and infections of sheep used to investigate the effect of CBP on pathogenesis. Animals were either infected with the wild type (wt) virus, CBP-knockout virus or revertant strains. Sheep were infected by scarification on the wool-less area of the hind legs at various doses of virus. The deletion of the CBP gene severely attenuated the virus, as only few papules formed when animals were infected with the CBP-knock-out virus at the highest dose (107 p.f.u). In contrast, large pustular lesions formed on almost all animals infected with the wt and revertant strains at 107 p.f.u. The lesions for the CBP-knock-out virus resolved approximately 5–6 days p.i, at a dose of 107 pfu whereas in animals infected with the wt and revertants at this dose, lesions began to resolve at approximately 10 days p.i. Few pustules developed at the lowest dose of 103 p.f.u. for all viruses. Immunohistochemistry of biopsy skin-tissue from pustules showed that the CBP-knockout virus replicated in all animals at the highest dose and was localized to the skin epithelium while haematoxylin and eosin staining showed histological features of the CBP-knockout virus typical of the parent virus with acanthosis, elongated rete ridges and orthokeratotic hyperkeratosis. MHC-II immunohistochemistry analysis for monocytes and dendritic cells showed greater staining within the papillary dermis of the CBP-knock-out virus compared with the revertant viruses, however this was not the case with the wt where staining was similar. Our results show that the CBP gene encodes a secreted immunodulator that has a critical role in virulence and pathogenesis.Orf virus (ORFV) is the type species of the  Orf is a debilitating skin disease of sheep and goats and is transmissible to man  produced by the NZ7) by ELISA and its effects on monocyte migration in response to specific chemokines in a chemotaxis assay and inflammatory cell trafficking using a murine skin inflammation model. To examine the in vivo effects of this factor on pathogenesis and host response we constructed a knock-out recombinant in which the CBP gene was deleted. Infection studies were carried out in sheep using the recombinant virus and the clinical pathology and host response compared with the parent strain.We hypothesized that the ORFV CBP disrupts chemokine gradients within the skin tissue of its host during infection and thereby sets up a blockade to inhibit the recruitment of leukocytes to the infected site. For these studies we used ORFV strain NZ7. The NZ7 strain induces large pustular lesions in sheep and has been used to characterize other ORFV virulence factors ] intradermal injection. The optimal levels of LPS to inject into the dermis of the shaved abdomen of C57BL/6 mice and the time at which maximal monocyte and DC recruitment was evident, has been described previously  and Gr-1  for monocytes or CD11c  and MHC-II (rat anti-mouse I-A/I-E PE-conjugated from clone M5 (BD Pharmingen) at 4°C on ice for 2 h. Isotype control was IgG2b) for DC. Flow cytometric analysis was performed using a FACSCalibur (Becton Dickinson). All flow cytometric data were analyzed using CellQuest and FlowJo softwares. All samples were analyzed using flow cytometry counts of 10,000 events and were normalized to haemocytometer counts and weight of individual skin biopsies.The mouse experiments described in this study were approved by the University of Otago Animal Ethics Committee. Specific Pathogen Free C57BL/6 mice and enhanced green fluorescent protein (eGFP) transgenic C57BL/6 mice were obtained from the University of Otago Animal Facility and used with institutional ethical approval. Monocyte and DC recruitment from the blood was assessed using adoptive transfer of eGFP bone marrow transgenic cells into sex-matched 6–8 week old C57BL/6 recipients as described previously , was PCR amplified and cloned into the Hind-III and EcoRI restriction sites of pSP70 to generate pSP70/left arm. A fragment which spans 556 bp downstream of the CBP coding region and which includes 18 bp of the CBP gene (right arm), was PCR amplified and cloned into the vector pSP70/left arm at the EcoRI and BgI-II restriction sites. Finally the Eschericia coli gene (lacZ), placed under the control of an ORFV late promoter PF1 . In a further construct to produce a CBP-FLAG fusion protein, the FLAG sequence was incorporated at the C-terminus of the CBP coding sequence, that is a FLAG sequence was also incorporated into a second primer to make the left arm by amplification as described above. A fragment which spans 556 bp downstream of the CBP coding region and which includes 18 bp of the CBP gene (right arm used to make the knock-out construct), was cloned into the vector pSP70/left arm at the EcoRI and BgI-II restriction sites. The reporter gene ß-glucuronidase (GUS) running off an early/late promoter was then inserted into the KpnI and EcoRI site, that is between the left and right arms of the construct. This placed the GUS gene downstream of CBP in an intergenic region in the ORFV-CBP revertant. In addition an early transcription termination sequence, TTTTTCT, was engineered immediately downstream of the GUS gene. The final plasmid construct was called pCBP-rev. The recombinant virus was generated from the ORFV CBP-ko recombinant by methods described above. Putative ORFV-CBP revertant plaques were identified by their blue plaque phenotype in the presence of 5-bromo-4-chloro-3-indolyl B-D-glucuronide cyclohexylammonium salt and lack of blue color in the presence of X-gal. The revertant virus was named CBP-rev. A second revertant was made that incorporated a FLAG sequence at the C-terminus of CBP and named CBP32P-labeled dsDNA probe. The size of mRNA was determined using a BRL RNA ladder. Autoradiography was performed for 72 h at −70°C.To analyse mRNA from virus infected cells by Northern blotting, total RNA was isolated by methods described previously . Virus-induced lesions were photographed daily over a 3 week period and the clinical scores determined. Sheep were examined for erythema, papule and pustule formation and firmly attached scab associated with the scarified/infected areas of the skin. The clinical score was determined by counting the discrete round raised papules with a creamy appearance that formed on the skin surface along the lines of scarification using magnified photographic images and the width of the lesion (1–10 mm) that formed when papules coalesced (days 5–7). Clinical scores ranged from 1 to 25. Once the papules/lesions lost their creamy appearance, that is were in the process of forming scab, the clinical score was recorded as zero. The clinical scores for all animals at all-time points were scored by two researches. Punch biopsies (5 mm) were taken at days 4, 6, and 8 from the right leg. Blood was obtained from the jugular vein of each animal before infection and then at 13 and 18 days pi when all animals were euthanized.All animal experiments described in this study were approved by the University of Otago Animal Ethics Committee. The animals were housed in a PC2 containment facility at AgResearch, Invermay, Otago. Twenty-four ORFV sero-negative merino-cross lambs (ewes) were randomly divided into four groups of six and inoculated on the wool-free region of the inner hind legs. Group 1 was infected with 490 nm.Antibodies produced against ORFV antigen and ORFV-075 scaffold protein were analyzed in sheep serum by ELISA. ORFV antigen was prepared from ORFV-infected LT cells as described previously  from sheep were treated with zinc salt fixative  in 10 mg/ml BSA/0.05% Triton X/TBS, and incubated O/N at 4°C. DAPI 1 was added for 30 min before sections were washed three times in 0.05% Tween20 TBS, then rinsed in TBS only. Sections were mounted with SlowFade Gold Antifade Mountant (Invitrogen S36936). The stained sections were viewed by fluorescent microscopy using an Olympus BX51 and then photographed using an Olympus digital camera. Cell counts were performed using Image J Fiji software by subtracting background, increasing contrast, creating a RGB stack, selecting either the epidermis, or the papillary dermis, determined by the smooth collagen and elastic fibers then measuring percentage area that stained positive in the green field.For immunohistochemical staining of the ORFV F1L envelope protein, tissue sections were de-paraffinated and rehydrated as described above. Sections were then blocked in TBS + 5% FCS for 30 min and reacted with anti-ORFV F1L monoclonal antibody (8D7) . The sheep MHC-II immunofluorescence data was analyzed by One-Way ANOVA with means comparison using Sidak's test (GraphPad Prism 6).The mouse skin inflammation data was analyzed by paired Student's CBPNZ7 making the gene 27 nts longer than CBPNZ2 with a predicted polypeptide of 32.193 kDa compared with 31.182 kDa. It was therefore necessary to first examine the in vitro properties of CBPNZ7 and compare these with the published data of CBPNZ2. To examine the chemokine binding properties of CBPNZ7, the CBPNZ7 gene was cloned into the pAPEX eukaryotic expression vector and expressed as a C-terminal FLAG tagged fusion protein following transfection of HEK293 cells. The protein was affinity purified using anti-FLAG beads and tested for its ability to bind murine chemokines using an ELISA assay. The chemokine binding specificity of CBPNZ7 was compared with CBPNZ2 over a broad range of chemokines that are produced during inflammation in skin and the constitutive chemokine CCL19. The ELISA carried out is an indirect assay and measures free chemokine (not in complex with CBP) following CBP incubation with chemokine. Therefore, the stronger the binding the less free chemokine that remains. The assay showed that CBPNZ7 bound the murine inflammatory chemokines CCL2, CCL3, CCL5, and CXCL2  into the dermis of the shaved abdomen of the recipients. Twenty-four hours later mice were euthanized and the skin around the injection site excised and cells isolated and stained. The number of monocytes and DC in skin was determined by flow cytometric analysis. The results showed that CBPNZ7 potently inhibited the recruitment of blood derived eGFP monocytes and eGFP DC at 1 and 100 ng of CBPNZ7  with the greatest effect seen at 100 ng CBPNZ7 . Infected skin was photographed to provide a record of the clinical pathology from which the clinical score could be determined from the number, appearance and size of papules and pustules that developed along the scratch line. The clinical score for each animal infected with a dose of 107 pfu virus is shown in Figure wt virus or CBP revertant viruses. By day 4 p.i., 4 of the animals in the ko group had developed small pustules. Sheep infected with wt and the CBP revertants developed lesions typically observed for the NZ7 strain of ORFV and papules and pustules were visible by day 4 p.i. By day 5 p.i., large pustular lesions had developed in the wt and CBP revertant groups and the clinical scores were significantly greater than sheep infected with the CBP ko virus (P < 0.05). In contrast, lesions in the CBP knockout group were beginning to resolve by day 5 p.i. By day 6 p.i., lesions in the wt and CBP revertants groups had reached their maximum clinical pathology , CBP rev 20.33 (SE 2.26), CBPFLAG rev 19.50 (SE 2.80). In contrast, lesions had resolved in 3 of the animals in the CBP knock-out group by this time and the group had a mean clinical score of 4.83 (SE 2.46). The clinical scores for the wt and CBP revertant groups were significantly different to the CBP knockout group (P < 0.02) at day 6 p.i. By day 7 p.i., lesions had resolved in all but 2 animals in the CBP knock-out group. By day 8 p.i. the clinical pathology in almost all animals was less apparent and lesions were beginning to form scabs. By day 10 p.i., scabs had formed on all animals except for 3 in the CBP revertant group. By day 18 p.i., lesions had resolved on all animals and scab had detached. Throughout the course of the experiment, the clinical scores for the wt and revertant groups were not significantly different to each other.Experimental infection of sheep with 5 virus per inoculum, the trend in clinical pathology at day 6 was similar to that for animals infected with a virus dose of 107  and for the CBP rev and CBPFLAG rev groups 4.50 (SE 2.29) and 5.33 (SE 1.96) respectively. In contrast the clinical score for the CBP ko virus group was 0.50 (SE 0.50). The clinical scores for the wt and the CBP revertant groups were significantly different to the CBP knock-out group (P < 0.05). At 105 virus per inoculum the lesions resolved in less time than for the 107 dose administered. At a dose of 103 virus per inoculum few animals developed lesions and these were only seen in the wt and revertant groups  where contiguous sections were stained by H & E and for F1L , elongated rete ridges and orthokeratotic hyperkeratosis (thickening of stratum corneum) typical of the s Figure . The sams Figure . Again twt, CBP-ko and CBP revertant viruses during the early stages of infection (day 4) when clinical signs of infection were first becoming apparent. At a dose of 107 virus per inoculum, the numbers of inflammatory cells for the wt, CBP-ko and CBP revertants varied from medium to heavy. At a dose of 105 virus per inoculum, there was a similar influx of inflammatory cells but again this was variable for infected animals within groups. In the PBS controls there were few inflammatory cells seen . Interestingly the wt and both revertant viruses were not significantly different to the mock-infected animals whereas the CBP-knock-out-virus was significantly higher than the mock-infected control group (P = 0.0479). The percent area MHC-II staining in the papillary dermis revealed slightly higher staining for MHC-II for the wt virus compared with the CBP-ko virus, however the difference was not significant. Interestingly the percent area staining of MHC-II cell in the papillary dermis was significantly less for both revertant viruses compared with the CBP-ko virus and the wt virus (P < 0.01 and P < 0.05 respectively). The percent area MHC-II staining in the papillary dermis for the revertant viruses was again not significantly different to the mock-infected animals.The quantification of MHC-II staining is shown in Figure de novo synthesis. ORFV-075 is a scaffold protein that is produced late in infection but is not incorporated into the viral particle. Figure wt and CBP ko virus (P = 0.061) however there was a significant difference between the wt and the revertant (P = 0.0465). Detection of antibodies against ORFV-075 showed that the ORFV-CBP-knock-out virus had replicated in all infected animals  with the wt producing a higher antibody response. Further there was a significant difference in antibody levels for the wt compared with the revertant virus (P = 0.0465) but not between the other groups.We were interested to determine whether animals produced an immune response against antigen produced by the virus in infected cells by s Figure . A signiNZ7 prior to investigations in sheep with recombinant virus. Chemokine binding assays showed that it displayed the equivalent biological properties as CBPNZ2 despite differences in their primary structure. In addition, CBPNZ7 potently blocked the inflammatory cell infiltrate when injected intradermally at ng levels in a mouse skin model, suggesting that it could have a role in pathogenesis.We firstly characterized the biological properties of the purified ORFV-NZ7 CBP protein and then investigated the effects of deleting the CBP gene from ORFV on replication, virulence, pathogenesis, and host response. As described above, the CBP gene shows surprising variability within the ORFV species with the NZ7 strain showing 78% identity to the NZ2 strain  (Graham et al., The only other study in which a poxvirus CBP knock-out virus has been tested in the context of wt virus was observed.Our studies clearly showed that the CBP gene is critical in ORFV pathogenesis and virulence and although ORFV encodes a number of other secreted anti-inflammatory factors, the deletion of CBP gene severely attenuated the virus. The secreted CBP appears to induce its effects early during infection, since there were few signs of clinical orf in almost all CBP knock-out virus infected animals. At this time we do not know which cells or factors were involved in the faster control of the ORFV CBP-knock-out virus infection. The deletion of the CBP gene may however be advantageous for recombinant ORFV vaccine development. Although its deletion severely attenuated the virus, a primary antibody response typical of the SF, ZL, and LW designed and conceived the experiments. SF, NR, CM, ZL, and AD performed the experiments. SF wrote the paper. LW and AM assisted with the manuscript preparation and proof reading.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Beauveria bassiana has a wide host range and is used as a biocontrol agent against arthropod pests. Mycoviruses have been described in phytopathogenic fungi while in entomopathogenic fungi their presence has been reported only rarely. Here we show that 21.3% of a collection of B. bassiana isolates sourced from worldwide locations, harbor dsRNA elements. Molecular characterization of these elements revealed the prevalence of mycoviruses belonging to the Partitiviridae and Totiviridae families, the smallest reported virus to date, belonging to the family Narnaviridae, and viruses unassigned to a family or genus. Of particular importance is the discovery of members of a newly proposed family Polymycoviridae in B. bassiana. Polymycoviruses, previously designated as tetramycoviruses, consist of four non-conventionally encapsidated capped dsRNAs. The presence of additional non-homologous genomic segments in B. bassiana polymycoviruses and other fungi illustrates the unprecedented dynamic nature of the viral genome. Finally, a comparison of virus-free and virus-infected isogenic lines derived from an exemplar B. bassiana isolate revealed a mild hypervirulent effect of mycoviruses on the growth of their host isolate and on its pathogenicity against the greater wax moth Galleria mellonella, highlighting for the first time the potential of mycoviruses as enhancers of biocontrol agents.The entomopathogenic fungus  Naranaviridae which comprise the simplest unencapsidated RNA viruses. Finally, we illustrate for the first time the potential of mycoviruses as enhancers of the biological control agent Beauveria bassiana, an entomopathogenic fungus utilised as an environmentally friendly alternative to chemical insecticides.Virus diversity is constantly increasing and the findings often challenge our perceptions of the virosphere, enhance our understanding of the global ecology and evolution of viruses and virus-like elements and offer a range of new tools. Here we describe a fungal viral family, provisionally designated as Polymycoviridae, with a variable number of genomic segments. Polymycoviruses contain at least four and up to seven genomic segments; the three smallest ones are individual and non-homologous among different members of the family, demonstrating an unprecedented dynamic genomic organisation in terms of both segment number and sequence. Additionally we report the smallest known virus to date, belonging to the family  Beauveria bassiana  Vuillemin is an entomopathogenic ascomycete belonging to the family Clavicipitaceae, order Hypocreales. It has a wide host range of approximately 750 arthropod species and a widespread geographical distribution. B. bassiana isolates have also been recovered from soil and several plant species as endophytes [B. bassiana strains, such as GHA and ATCC 74040, are available commercially as biocontrol agents against a variety of arthropod pests [dophytes . Specifiod pests . Althougod pests .Narnaviridae and Hypoviridae, while the latter includes the families Totiviridae and Partitiviridae. Recently an increasing number of novel mycoviruses have been reported including a negative-strand RNA mycovirus [B. bassiana the presence of dsRNA elements, virus-like particles and associated hypovirulence have been reported [Victorivirus, family Totiviridae have been sequenced [Mycoviruses have been described in a wide range of fungi and are classified into eleven major families, six accommodating single-stranded (ss) and five accommodating double-stranded (ds) RNA genomes. The former group includes the families ycovirus , a geminycovirus , and a nycovirus . In B. breported ,8; howevequenced ,10.B. bassiana isolates were characterised. These findings include a description of the smallest virus reported up to date, new members of the established families Partitiviridae and Totiviridae and the proposed novel family Tetramycoviridae now renamed Polymycoviridae which appear to have an unprecedented dynamic nature in terms of genomic element number and sequence. Additionally, the potential of mycoviruses as enhancers of the biocontrol agent B. bassiana is demonstrated.In the present study all mycoviruses and other dsRNA elements found in a large collection of B. bassiana, we screened a well-characterized panel of isolates sourced from worldwide locations [B. bassiana isolates harbor unique nucleic acid elements following electrophoretic separation on agarose gels -1 and -2, respectively. Analysis of the BbPV-1 and BbPV-2 RdRP aa sequences revealed typical motifs  confirmed the presence of victoriviruses in EABb 00/23Su, and EABb 00/88Su and in EABb 01/12Su and EABb 01/103Su , 704 aa (75 kDa), 610 aa (67 kDa) and 306 aa (32 kDa) in size, flanked by 5’- and 3’-UTRs. The 5’-UTRs of the four segments are respectively 26, 71, 31 and 72 bp long while the 3’-UTRs are 71, 74, 57 and 380 bp long. The 5’- and 3’-terminal sequences of all four dsRNAs are very similar . PartialCladosporium cladosporioides and Botryosphearia dothidea [poly = ‘many’ in Greek, in contrast to tetra = ‘four’). Therefore, the viruses derived from isolates EABb 92/11-Dm, IMI 391043 and ATHUM 4946 were designated as Beauveria bassiana polymycovirus (BbPmV)-1, -2 and -3 respectively. Following a search of the public databases, all complete or partial sequences of putative polymycoviruses were collected (C. cladosporioides (Cc) PmV-1 to 63% for AfuPmV-1. The RdRPs, encoded by the largest dsRNA1 segments of all the proposed members of the Polymycoviridae family, contain three partially conserved motifs , BdPmV-1 from B. dothidea (class Dothideomycetes) and BbPmV-2 (class Sordariomycetes) group together, while the two B. bassiana polymycoviruses do not. This may indicate that host shifts are quite frequent for polymycoviruses.BLAST searches of the viral proteins revealed that the dsRNA segments constitute the genome of viruses belonging to a newly proposed family, the Tetramycoviridae, the prototype member of which is Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) . Howeverdothidea  each witollected  and anald motifs  found inoviridae  which arThe proteins encoded by polymycovirus dsRNA2 segments contain a cysteine-rich, zinc finger-like motif  and, unlThe proteins encoded by the dsRNA3 segments contain a conserved catalytic methyltransferase motif  and a N-The proteins encoded by the dsRNA4 segments are proline-alanine-serine (PAS) rich  and in tAlternaria sp. FA0703. Further analysis revealed that their 5’- and 3’- termini are very similar to those of the larger segments from the same isolate  and appears to be distantly related to fungal transcription factors with helix-loop-helix domains . Additionally the AltPmV-1 dsRNA5 product belongs to the nucleotide kinase protein superfamily . The function and the biological role of these polypeptides are unknown, but since they are not present in all polymycoviruses they are probably not essential for virus replication and maintenance. In some mycovirus families e. g. “chryso-like” viruses, at least two divergent virus clades with either 3 or up to 5 genome segments have been described [None of these polypeptides have significant homology with sequences deposited in the public databases. The protein encoded by CcPmV-1 dsRNA5 contains a viral transcriptional regulator domain , with no ORFs of significant length in either strand and no statistically significant similarity with other known sequences. Their termini are very similar to those of BbSNLV , suggestitovirus  and the itovirus  predicteitovirus .In order to assess the effects of mycoviruses on the growth and virulence of the fungal host, isogenic lines of virus-free and virus-infected EABb 92/11-Dm isolate were generated. Initial attempts to cure EABb 92/11-Dm using protein synthesis inhibitors as described  and throGalleria mellonella, as described before [G. mellonella larvae infected with three virus-free strains in comparison to EABb 92/11-Dm was noted  derived from isolates IMI 331273, IMI 392612, EABb 01/103Su and EABb 92/11-Dm were inoculated into 10 mL of Czapek-Dox CM broth and incubated on a rotary shaker (150 rpm) over a period of 7 days. The mycelium from individual cultures was harvested daily by filtration through Miracloth and the pellets were lyophilized before been weighed. In order to assess the viral genome levels, dsRNA was extracted and quantified on an agarose gel using ImageJ [8) of the two isolates were inoculated into 10 mL of Czapek-Dox CM broth as described above. G. mellonella infectivity assays were performed as described [G. mellonella larvae were dipped into 2 mL of 105 spore suspensions for 10 sec. Mortality was recorded every 24 h over a period of 8 days. Log-Rank and Wilcoxon tests as implemented by GraphPad Prism 6 were used to analyse the data for significant differences in survival.Fungal spores . LiCl fractionation of dsRNA was carried out as described . Isolatiin vitro transcription of template DNA in the presence of digoxigenin-UTP, using T7 RNA polymerase , followed by immunological detection using alkaline phosphatase-conjugated, anti-digoxigenin antibody (Roche).Gel electrophoresis, denaturation, neutralization and electrophoretic blotting of dsRNA or ssRNA were carried out according to standard protocols . Blots wB. bassiana ITS sequence that served as an endogenous control were amplified using the target-specific primer pairs 5’-TTC CGC CTC TCG AAT AGA AA-3’ and 5’-CGA ACA GAG TGG CAA GAT GA-3’; 5’-GAT CTC TTG GCT CTG GCA TC-3’ and 5’-TTG AAA TGA CGC TCG AAC AG-3’, respectively, after cDNA synthesis using the same target-specific primers as appropriate.After electrophoretic separation on agarose gels dsRNAs were used, either collectively or individually, as templates for cDNA synthesis and PCR amplification of products using random priming, sequence-specific priming and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) which were subsequently cloned and sequenced as described ,35. At lSequence similarity searches of the GenBank, Swissprot and EMBL databases were conducted using the BLAST program . SearchePurification of virus-like particles was performed as described . PurifieB. bassiana strains were generated from hyphae using a similar procedure to that described previously [w/v) Trichoderma harzianum lysing enzyme , the protoplasts were washed repeatedly in isotonic buffer and the viruses (10 μl at 100 ng/μl × 107 protoplasts) were introduced via polyethylene glycol-mediated transfection. The transfected protoplasts were then rescued and regenerated on 1% (w/v) D-glucose-enriched Czapek-Dox CM agar where they formed a lawn. Pooled transfected fungal colonies were collected and the mycelia was subcultured at least three times. To verify that the mycelia were transfected with BbPmV-1 and BbNV-1, total RNA extracts were prepared from the cultures using the RNeasy Plant Mini Kit (Qiagen) and RT-PCR amplification of viral amplicons 838 and 502 bp in size using gene-specific primer pairs that were designed based on the sequences of BbPmV-1 and BbNV-1, respectively, was performed.Protoplasts of eviously . BrieflyData deposition: The sequences reported in this paper have been deposited in the GenBank database: Beauveria bassiana partitivirus-1 and -2 (BbPV-1 and -2) accession numbers LN896303-LN896306; Beauveria bassiana victorivirus-1 (BbVV-1) strains EABb 01/103Su, EABb 01/12Su, EABb 00/88Su, EABb 00/23Su accession numbers LN896314-LN896317; Beauveria bassiana polymycovirus-1, -2 and -3  accession numbers LN896307-LN896313, LN896318-LN896320; Beauveria bassiana small Narna-like virus (BbSNLV) and satellite RNAs accession numbers LT627647, LN896321- LN896322.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Fig(a) Schematic representation of the electrophoretic profiles of dsRNA elements extracted from 17 B. bassiana isolates and their relative sizes. (b) Geographical distribution of mycoviruses found in B. bassiana.(PDF)Click here for additional data file.S2 Fig(a) 1% (w/v) agarose gel electrophoresis of viral dsRNA extracted from B. bassiana isolates IMI 331273 (lane 2) and IMI 392612 (lane 3), harboring BbPV-1 and BbPV-2, respectively. Lane 1 contains the DNA marker Hyperladder I (Bioline), the sizes of which are shown to the left of the gel. (b) TEM images of BbPV-1 in fungal mycelia (left) and negative stained, purified BbPV-2 (right). Both are visualized as icosahedral particles approximately 50 nm in diameter. Virus particles are indicated by arrows. (c) Schematic representation of the genomic organisation of BbPV-1 and BbPV-2. The BbPV-1 and BbPV-2 genomes consist of two dsRNAs each containing one ORF (grey boxes) flanked by 5’- and 3’-UTRs (black boxes). (d) A comparison of the conserved motifs of the RdRP in BbPV-1, BbPV-2 and other partitiviruses ((e) Maximum likelihood phylogenetic tree created based on the alignment of RdRP and CP sequences of members of the Partitiviridae family ((f) Electrophoresis and northern blot hybridization of B. bassiana partitiviruses. DsRNA extracted from seven B. bassiana isolates, including IMI 331273 that harbors BbPV-1 and IMI 392612 that harbors BbPV-2, was electrophoresed in 1% (w/v) agarose gels and blotted onto nylon membranes. Hybridization was carried out using probes specific for the RdRP and the CP of BbPV-1 and BbPV-2.iviruses . Numberse family  using th(PDF)Click here for additional data file.S3 Fig(a) 1% (w/v) agarose gel electrophoresis of dsRNA extracted from B. bassiana isolate EABb 01/103Su harboring BbVV-3 using virus purification (lane 3). Lane 1 contains the DNA marker Hyperladder I (Bioline), the sizes of which are shown to the left of the gel. (b) Schematic representation of the genomic organisation of BbVV-1. The BbVV-1 genome consists of a single dsRNA that contains two overlapping ORFs encoding a CP (light grey box) and an RdRP (dark grey box) flanked by 5’- and 3’-UTRs (black boxes). In the alignment of partial RdRP sequences of members of the genus Victorivirus, family Totiviridae isolated from B. bassiana strains, including BbVV-1  and BbVV-2 , asterisks signify identical aa residues, colons signify highly conserved residues and single dots signify less conserved but related residues.(PDF)Click here for additional data file.S4 Fig(a) BdPmV-1 and (b) CcPmV-1. Each genome consist of five dsRNAs, each containing one ORF (grey boxes) flanked by 5’- and 3’-UTRs (black boxes). A comparative alignment of the 5’- and 3’-terminal sequences of (c) BdPmV-1 dsRNAs 1–5, (d) CcPmV-1 dsRNAs 1–5, (e) AltPmV-1 dsRNAs 2, 5 and 6, (f) BbPmV-1 dsRNAs 1–4 and (g) BbPmV-1 dsRNAs 1, 6 and 7. Asterisks signify identical nucleotides. AltPmV-1 dsRNA 6 does not share a similar 3’-terminal sequence with dsRNAs 2 or 5; however there is an internal sequence in its 3’-UTR with significant similarity to the 3’-terminus of dsRNAs 2 and 5, indicating a possible sequencing error. Virus names, acronyms, and GenBank accession numbers are listed in Schematic representation of the genomic organisation of (PDF)Click here for additional data file.S5 Fig(a) Comparison of the conserved motifs of the RdRP in polymycoviruses. Numbers within the brackets indicate the number of aa not shown. In the RdRP motifs, the symbol ‘#’ signifies S or T and the symbol ‘&’ signifies bulky hydrophobic residues . In all sequence alignments, asterisks signify identical aa residues, colons signify highly conserved residues and single dots signify less conserved but related residues. (b) Comparison of the cysteine-rich zinc finger-like found in the proteins encoded by dsRNA 2 of polymycoviruses. (c) Comparison of the catalytic methyltransferase motifs found in the proteins encoded by dsRNA 3 of polymycoviruses and 5’-RLM RACE oligo-cap analysis of BbPmV-1 dsRNA 1. Viral dsRNA was (i) treated with CIP and TAP and ligated to an oligoribonucleotide adaptor at the 5’-terminus, (ii) treated with CIP only and ligated, (iii) left untreated and ligated and (iv) left untreated and unligated. Subsequently, RT-PCR was performed with a primer homologous to the oligonucleotide adaptor and a sequence-specific primer, and the products were electrophoresed on a 4.5% (w/v) native polyacrylamide gel. The expected product size is 310 bp, indicated by an arrow, and the positive reaction in lane 1 indicates that the positive-strand of dsRNA 1 is capped. (d) Proline-alanine-serine (PAS) content of the proteins encoded by RNA 4 of polymycoviruses. (e) PAS content of the proteins encoded by ORF-1 of unirnaviruses. (f) AFM image of the non-conventionally encapsidated viruses from the EABb 92/11-Dm isolate. BbPmV-1 and BbNV-1 are visualized as chain-like linear nucleic acids of different lengths corresponding to those predicted from the size of the genomic dsRNAs. (g) PAS content of the proteins encoded by the extra dsRNAs of polymycoviruses. Virus names, acronyms, and GenBank accession numbers are listed in (PDF)Click here for additional data file.S6 FigMaximum likelihood phylogenetic tree created based on the alignment of RdRP sequences of polymycoviruses and related viruses belonging to the Superfamily 1  Tables u(PDF)Click here for additional data file.S7 Fig(a) 1% (w/v) agarose gel electrophoresis and northern blot hybridization of dsRNA extracted from the EABb 92/11-Dm and EABb 92/11-DmC1 isogenic lines. Hybridization was carried out using a probe specific for BbPmV-1 dsRNA1. Attempted RT-PCR amplification of (b) a 838 bp BbPmV-1 dsRNA1 fragment and (c) a 502 bp BbNV-1 dsRNA fragment from the EABb 92/11-Dm, EABb 92/11-DmC1, EABb 92/11-DmC2, EABb 92/11-DmC3 and EABb 92/11-DmT isogenic lines. In all gels, lane 1 contains the DNA marker Hyperladder I (Bioline), the sizes of which are shown to the left of the gel. Comparison of the fungal growth in solid and liquid medium; (d) radial growth and (e) biomass production of EABb 92/11-Dm, EABb 92/11-DmC1, EABb 92/11-DmC2 and EABb 92/11-DmC3. At least three independent repetitions were performed in triplicate  or duplicate (biomass production) and error bars represent standard deviation. A yellow background indicates the time points when the difference in growth is statistically significant . (f) Mean survival curves of G. mellonella larvae infected with the EABb 92/11-Dm, EABb 92/11-DmC1, EABb 92/11-DmC2 and EABb 92/11-DmC3 isogenic lines. At least three independent repetitions were performed in triplicate.(PDF)Click here for additional data file.S8 Fig(a) IMI 331273, (b) IMI 392612, (c) EABb 01/103Su and (d) EABb 92/11-Dm in liquid Czapek-Dox CM was assessed daily for 7 days (grey shading). (a) BbPV-1, (b) BbPV-2, (c) BbVV-3, (d) BbNV-1 and BbPmV-1 dsRNAs extracted from equal amounts of dry mycelia were electrophoresed in 1% (w/v) agarose gels, dsRNA levels were quantified by ImageJ and the results are presented in graphical form (black lines). At least three independent repetitions were performed in duplicate and error bars represent standard deviation. Representative agarose gel analyses of the dsRNAs are shown below each graph.Biomass production of isolates (PDF)Click here for additional data file.


Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location, and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus.We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge, mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a possible mechanism through which the species may not experience severe influenza virus infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza virus neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection. Influenza A virus (IAV) is an important respiratory pathogen and is the major cause of ongoing global morbidity and mortality. Innate immunity is critical in the early stages of IAV infection, limiting virus spread in host tissues before the induction of the adaptive response. There are several innate immune mechanisms that participate in the early containment of IAV, including a variety of soluble innate inhibitors that are present in respiratory secretions. They include collectins, defensins, pentraxins, mucins, and salivary scavenger cysteine-rich glycoprotein 340 (gp340) –8. Under–4.5 PFU are nonlethal and infectious virus is rarely recovered from the lungs. The difference in viral progression to the lower respiratory tract (LRT) between the Udorn and PR8 viruses was due to the relative sensitivity of the viruses to a previously undescribed innate inhibitor of influenza virus present in mouse saliva (Vibrio cholerae or Arthrobacter ureafaciens receptor-destroying enzyme (RDE), suggesting that sialic acid, to which the viral hemagglutinin (HA) and neuraminidase (NA) bind, was not the primary determinant of sensitivity to the inhibitor.In the mouse model of IAV infection, it has been reported that when the viral inoculum is initially restricted to the nose during intranasal delivery to the upper respiratory tract (URT), the A/Udorn/307/72  strain of influenza A virus causes a localized infection that gradually progresses to the trachea and lungs, whereas the A/Puerto Rico/8/34  virus causes an infection that remains almost entirely in the nasal passages , 10. In e saliva . Saliva e saliva . Inhibitin vitro resulted in potent virus neutralization . Inhibition was also seen when either the M  or HA  gene of Udorn virus was expressed on the PR8 backbone, referred to as PR8(Ud-M) and PR8(Ud-HA), respectively. However, expression of the Udorn NA gene on a PR8 backbone in PR8(Ud-NA) virus resulted in a low level of neutralization comparable to that of parental Udorn virus .To determine the target of the murine salivary inhibitor, we used reverse genetics to create hybrid viruses expressing either the HA, NA, or M gene of the inhibitor-resistant Udorn virus on the inhibitor-sensitive PR8 virus backbone. Parental PR8 and Udorn viruses were also constructed from plasmids to serve as controls. An y saliva , was they saliva . At a doP < 0.0001 compared to Udorn), although this was not as potent as that observed against the PR8 parent virus (P < 0.001 compared to PR8). Together, these data confirmed that the salivary inhibitor was indeed targeting the NA of PR8 virus to exert its effect.These data indicated that the viral NA was the critical determinant of sensitivity to the neutralizing inhibitor in mouse saliva. To support this, a hybrid Udorn virus bearing PR8 NA, referred to as Ud(PR8-NA) virus, was created. In the inhibition assay , this viV. cholerae RDE to remove sialic acid residues (P < 0.0001 compared to Udorn). Hybrid viruses containing PR8 NA, i.e., PR8(Ud-HA), PR8(Ud-M), and Ud(PR8-NA), were also sensitive to neutralization by RDE-treated saliva, while PR8(Ud-NA) virus containing the Udorn NA showed less inhibition once the sialic acid had been removed from the saliva . In contrast, viral progression and subsequent replication in the lungs differed dramatically between these five viruses (P < 0.0001 compared to Udorn). Of the hybrid viruses on a PR8 backbone, only PR8(Ud-NA) virus consistently progressed to the lungs and replicated to high titers (P > 0.05 compared to Udorn). PR8(Ud-M) virus was not detected in the lungs following URT inoculation, and PR8(Ud-HA) virus showed only low or undetectable titers (both P < 0.0001 compared to Udorn). When mice were infected with Ud(PR8-NA) virus, again, no statistically significant difference was observed in replication in the nasal turbinates compared to that of the parental viruses (P < 0.0001 compared to Udorn), with virus detected at low titers in only 1/5 mice  , sialylation may not be required. Unfortunately, we found that saliva itself contained intrinsic sialidase activity that cleaved sialic acid from the larger NA substrate fetuin (48.4 kDa) independently of virus, precluding experiments to test the activity of the salivary inhibitor against larger substrates. The sialidase activity of saliva was not evident against the MUNANA substrate.Although the viral NA appeared to be the key determinant of susceptibility to the salivary inhibitor, it was unclear whether binding of the inhibitor to the NA also affected its sialidase function. To examine this, reverse-genetics-derived parent and hybrid viruses were preincubated with untreated or RDE-treated saliva, or with phosphate-buffered saline (PBS) as a control, and assayed for the ability to cleave the NA substrate 4-methyl-umbelliferyl-(MUNANA) . When pr(MUNANA) . In cont(MUNANA) . Surpris(MUNANA) . This waus ratio . This suAn alternative hypothesis, that the binding of the salivary inhibitor to NA sterically affects the binding of HA to its receptor, was ruled out in experiments examining the ability of saliva to prevent the hemagglutination of chicken red blood cells. Untreated saliva had very low levels of hemagglutination inhibition (HI) activity against Udorn, PR8, and hybrid viruses (titers ≤ 16), and this did not correlate with the inhibitor sensitivity of the tested viruses (data not shown). Furthermore, this weak HI activity was completely lost when the saliva was RDE treated, effectively ruling out HA as an additional viral target of the inhibitor.To further investigate whether the inhibitor in saliva affects NA activity against large substrates, we took advantage of the fact that the sialidase function of NA prevents self-aggregation of newly formed virus particles and their reattachment to infected cells after budding, thereby aiding efficient virus release , 13. ThiTo support this conclusion, MDCK cells were infected with PR8 virus and cultured in the presence of medium alone, saliva, or zanamivir, the commercially available NA inhibitor drug, and then stained and visualized by transmission electron microscopy (TEM)  and scanSimilar results were observed by TEM of cells infected with the Ud(PR8-NA) virus . In the P > 0.05). Neutralization of the Ud(BJx109-NA) virus was statistically significantly higher than that of Ud(BJx109-HA) (P < 0.05) despite not reaching the level of the donor virus.To further investigate the target of the inhibitor and to define the precise residues involved, we created a panel of hybrid viruses by engineering the HA or NA genes of other closely related H3N2 strains with differing sensitivities to the salivary inhibitor on a Udorn backbone. The susceptibilities of Udorn viruses expressing either the HA or NA of HKx31, A/Memphis/1/71 (Mem71), BJx109, or A/Bangkok/1/79 (BK79) donor virus were tested in the saliva neutralization assay  and compin vitro susceptibility and resistance of these viruses predicted their ability to progress to the lungs in vivo, mice were inoculated by the URT route, and virus titers were determined in the nose and the lungs at day 6 postinfection. Despite growing to high titers in the nasal turbinates (P < 0.0001). This was also apparent for mice infected with the Udorn virus expressing the NA of HKx31 (P < 0.0001). Conversely, both the Udorn strain and Ud(HKx31-HA) virus consistently traveled to the lungs and grew to high titers in all the mice examined (P > 0.05). Viruses with Mem71 HA and NA on a Udorn backbone showed the same pattern of tropism as the HKx31 hybrids . Similarly, introduction of the E368K (KDS) mutation into Udorn NA increased neutralization of the resulting virus to 74.0% ± 7.1% (P < 0.0001).To determine whether residues 368 to 370 were involved in binding the salivary inhibitor, we generated Udorn viruses containing D369E or E368K mutations in the viral NA, changing the consensus sequence from the putative resistance motif EDS to EES  or KDS (in Mem71), respectively, which are found in inhibitor-sensitive strains. When tested for inhibition by mouse saliva in the on assay , introduin vitro, Udorn(Mem71-NA) showed 62.1% ± 8.4% neutralization, similar to Mem71 virus . However, when Udorn virus expressed the NA of Mem71 bearing an EDS motif instead of KDS, the virus was now largely resistant to the salivary inhibitor , confirming the importance of residues 368 to 370 in determining in vitro sensitivity to the salivary inhibitor.To support these findings, Udorn virus was engineered to contain the NA of Mem71 in which the introduction of a K368E mutation changed the KDS motif to EDS of the resistant Udorn strain. When tested for neutralization by saliva in vitro susceptibility motif at N2 residues 368 to 370 on progression of the virus down the respiratory tract, groups of 6 to 10 mice were inoculated via the URT route with 104.5 PFU of the reverse-engineered mutant and parent viruses, and titers in the nose (P < 0.0001). Introduction of the KDS motif into the Udorn NA also reduced the ability of the virus to progress to the lungs, but the effect was not as pronounced as the change to EES, showing 40% (4/10) of the mice with undetectable pulmonary virus and a 30-fold reduction in the mean viral titer compared to unmodified Udorn virus (P < 0.05). In addition, the progression of Mem71 virus to the lungs showed the virus to be highly impeded (P < 0.001). However, introduction of the EDS motif in Ud(Mem71-NA EDS) virus resulted in the virus progressing to the lungs in 100% of the mice (6/6). These data show that, despite breakthrough pulmonary replication in some individual animals, assessment of the overall ability of engineered viruses to be halted in their progression to the lungs is a reflection of their capacity to be neutralized by the salivary inhibitor in vitro.To determine the effect of mutations in the the nose  and lungthe nose  were exathe nose . The PR8the nose , with PR impeded , consistThis work identified a site on influenza virus neuraminidase that confers sensitivity to a previously undescribed natural inhibitor of the virus in mouse saliva, and to our knowledge, this is the first description of any innate inhibitor of influenza virus specifically targeting this viral protein. This is significant in providing insight into a novel antiviral defense mechanism that may have evolved to inhibit the spread of virus from the upper respiratory tract to protect the trachea and lungs. Although laboratory mice are routinely used to study influenza virus pathogenesis, as far as we know, they do not naturally succumb to influenza in the wild. Our work shows that mice already produce their own natural form of NA inhibitor, with a function similar to that which humans have produced synthetically in the form of zanamivir and oseltamivir to reduce the burden of influenza virus infection. This would render virus naturally transmitted to the URT inefficient in causing pulmonary infection in these animals, despite the virus being able to grow to high titers in the lungs if delivered directly to that site, as when infected experimentally. Whether similar inhibitors are also naturally produced in other species is not known. We were unable to demonstrate an inhibitor with equivalent virus strain specificity in ferret saliva and saliva taken from human infants before any exposure to influenza virus (unpublished findings).The presence of inhibitors in saliva would appear to be a useful protective mechanism against respiratory pathogens, and there have been various reports of inhibition of influenza virus , 16 and in vitro but also significantly reducing the ability of the Udorn EES virus to progress to the lungs after URT infection compared to the wild-type strain. Although D and E residues have similar properties and substitution of these residues maintains the same overall charge, glutamic acid has a longer side group than aspartic acid, potentially altering binding of the salivary inhibitor. The other amino acid substitutions resulted in changes to charge, polarity, hydrophobicity, or acidity, which may also affect potential binding.The activity of the inhibitor against N2 virus strains was shown to be critically dependent on amino acid residues 368 to 370 of the NA, so that NAs bearing EDS at these positions were resistant and those with EES/EEL/KDS were sensitive. Although change of an aspartic acid (D) residue at position 369 to a glutamic acid (E) residue (EDS to EES) was the most conservative change, it had the most pronounced effect, increasing susceptibility to saliva in vitro and to cause aggregation of virus at the budding sites of infected cells, even after RDE treatment, indicates that the action of the inhibitor is sialic acid independent and therefore unlikely to act by binding within the active site. We therefore propose that the salivary inhibitor binds near the active site and occludes entry of the viral receptor in a similar way to neuraminidase-inhibiting (NI) antibody. Residues 368 to 370 are located on the surface of the NA near the outer rim of the active site, making them ideal candidates to be involved in such binding , the mechanism of action of the salivary inhibitor appears to be different from that of the synthetic NA inhibitor antiviral drugs. These compounds are sialic acid analogs that function by tightly binding to the NA active site, blocking interaction with receptor-bound sialic acid . This in binding  and B. TIt was noted that swapping of residues 368 to 370 from the NA of inhibitor-sensitive virus strains onto a Udorn backbone did not result in complete inhibition of progression of virus to the lungs of all individual mice given a URT infection. It may be that to help facilitate binding of the salivary inhibitor to the surface of the NA, additional residues, in conjunction with 368 to 370, are involved. We investigated a number of candidate residues both within the active site (Q220K and I290V) and distal to the site (H155Y and N328K) that may have either affected the interaction of highly conserved catalytic residues with sialic acid  , 22 or aIt is interesting that residues 368 to 370 form one of three loop structures that in avian NAs form a secondary sialic acid binding site . Initiall-glutamine (Sigma-Aldrich), 2 mM sodium pyruvate , 24 μg/ml gentamicin , 50 μg/ml streptomycin (Life Technologies), and 50 IU/ml penicillin (Life Technologies). Medium as described above, without serum, was used for dilutions, and the same medium containing 1 mg/ml bovine serum albumin (BSA) (RPMI plus BSA) was used as a control for virus neutralization assays. All media were sterilized by 0.45-μm-pore-size membrane filtration and stored at 4°C. All the cells were maintained at 37°C in 5% CO2.MDCK cells and 293T human embryonic kidney (293T) cells were maintained in RPMI 1640  and Dulbecco's modified Eagle's medium (DMEM) , respectively, supplemented with 10% heat-inactivated fetal calf serum (FCS) (Life Technologies), 2 mM Influenza viruses were propagated in 10-day-old embryonated hen's eggs at 35°C for 2 days before allantoic fluid was harvested and stored at −80°C. HKx31 (H3N2) influenza virus is a laboratory-derived reassortant consisting of the internal proteins of PR8 (H1N1) with the surface proteins of A/Aichi/2/68 (H3N2) , 28. BJxTotal RNA was isolated from virus-infected allantoic fluid using an RNeasy kit (Qiagen) according to the manufacturer's instructions. The cDNA of PR8 and Udorn viruses was synthesized by reverse transcription of viral RNA (vRNA) with an oligonucleotide (Uni12 primer) complementary to the conserved 3′ end of the vRNA. The cDNA was amplified by PCR with segment-specific primers containing a BsmBI, BsaI, or AarI restriction site , and theMutations were introduced into the Udorn NA gene using custom primers designed to incorporate the desired amino acid substitutions, E368K (EDS to KDS) and D369E (EDS to EES). Primers were also used to incorporate a K368E (KDS-to-EDS) mutation into the Memphis NA. Two complementary products were amplified by PCR and then used as templates in another round of PCR with the universal primers to generate the full-length NA gene encoding the mutations. The custom primer sequences are available upon request.6 cells of each were added to 36 ml of Opti-MEM (Life Technologies) without FCS, supplemented with penicillin and streptomycin (termed Easy Flu medium). Each well of a 6-well tissue culture (TC6) plate was then seeded with 3 ml of this cell suspension (1 × 105 to 3 × 105 cells/well). FuGene6 transfection reagent  was used to transfect the cells according to the manufacturer's instructions. Briefly, 2 μl of FuGene6/μg of each plasmid was mixed in Opti-MEM, incubated at room temperature for 45 min, and added to subconfluent 293T and MDCK cell cocultures. The transfection medium was carefully removed after 6 h of incubation at 37°C and replaced with 1 ml of Easy Flu medium for a further 24 h. Another 1 ml of Easy Flu medium containing 2 μg of l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-trypsin was then added to each well. The cells were incubated at 37°C until 72 h posttransfection, when cytopathic effects (CPE) were observed. Supernatants were then harvested and clarified by centrifugation, and rescued virus was amplified in 10-day-old embryonated hens' eggs. The identities of the reverse-engineered viruses were confirmed by restriction digestion and full-length sequencing of RT-PCR products for the HA, NP, NA, M , and NS genes.An 8-plasmid reverse-genetics system described previously  was usedd) were bred in the Animal Facility of the Department of Microbiology and Immunology, University of Melbourne, under specific-pathogen-free conditions and used with the approval of the University of Melbourne Animal Ethics Committee.Inbred 6- to 8-week-old BALB/c mice  were determined by plaque assay on MDCK cell monolayers  was treated with 1 volume of V. cholerae RDE  for 30 min at 37°C. The final concentration of RDE was 50 mU/ml. The RDE was then inactivated by incubation at 56°C for 1 h. Samples were transferred to 1,000-MW-cutoff Spectrapor membrane tubing  and dialyzed against 50 mM NH4HCO3 at 4°C overnight. Following freeze-drying, samples were reconstituted to their original volumes with PBS and stored at −20°C. Control saliva was untreated, as we had previously shown that the inhibitor is resistant to heat treatment and lyophilization  with 200 μl of 20 μg/ml Carbachol  in PBS, and saliva was collected. Where specified, the saliva was treated with RDE. One volume of saliva in 8 volumes of Ca-Mg saline  virus/saliva ratio. Mixtures of virus with RPMI plus BSA (1 mg/ml) at the same ratio were used as a control. The mixtures were then incubated at 37°C for 30 min before being added to MDCK cells, which were then overlaid, and infectious virus was enumerated by the ability to form plaques as described previously . Virus nMDCK cells were exposed to PR8 or Udorn virus at a multiplicity of infection (MOI) of 10 for 1 h at 37°C. The cells were then washed thoroughly and cultured in the presence of untreated or RDE-treated saliva , medium alone, or 50 ng/ml of zanamivir for a further 10 h at 37°C.For TEM analysis, cells were pelleted and resuspended in 2.5% glutaraldehyde in PBS for 2 h at room temperature before postfixing in 1% osmium tetroxide in buffer for 1 h. The cells were rinsed three times in fresh buffer for 10 min between treatments. The cells were then dehydrated in increasing concentrations of 10%, 30%, 50%, 70%, 90%, and two changes of 100% anhydrous ethanol for 15 min each. Following dehydration, the cells were infiltrated with increasing concentrations of 25%, 50%, 75%, and 100% LR white resin  in ethanol for 6 h each. After a second change of 100% resin, the cells were embedded in fresh resin in gelatin capsules and allowed to gently sink to the bottom to form a loose pellet. The gelatin capsules were capped to exclude air, and the resin was polymerized in an oven at 60°C for 24 h. Embedded cells in blocks were sectioned with a diamond knife on a Leica Ultracut R microtome, and ultrathin (90-nm) sections were collected onto pioloform-coated 100-mesh hexagonal copper grids. The sections on grids were sequentially stained with saturated uranyl acetate for 10 min and Triple Lead Stain for 5 min  and view2 for 10 h.For SEM analysis, 13- mm coverslips (Nunc) were gold coated in an Edwards S150B sputter coater  for 5 min, giving a coating of approximately 30- to 40-nm gold thickness. MDCK cells treated as described above were placed onto the coverslips in 24-well plates and incubated at 37°C and 5% COThe coverslips were removed and dehydrated in increasing concentrations of ethanol as for TEM . The cov2CO3. The plates were read in a PerkinElmer fluorimeter with an excitation wavelength of 365 nm and an emission wavelength of 450 nm.NA activity was measured using the MUNANA fluorescence-based assay as described previously . The assTests were performed in round-bottom 96-well microtiter plates at room temperature using 1% (vol/vol) chicken erythrocytes. Hemagglutination titers were determined by titration of virus samples in PBS, followed by addition of an equivalent volume of chicken erythrocytes. For hemagglutination inhibition tests, dilutions of untreated or RDE-treated saliva were prepared in PBS, and 4 hemagglutinating units (HAU) of virus was added. Following 30 min of incubation, chicken erythrocytes were added, and the ability of the saliva to inhibit virus-induced hemagglutination was assessed after 30 min.P value of <0.05.All statistical analyses were performed with GraphPad Prism version 6.0e using one-way analysis of variance (ANOVA) or, for the neuraminidase inhibition titration, two-way ANOVA, both with Sidak's multiple-comparison tests. Statistical significance was demonstrated by a