Breast cancer–related lymphedema is an adverse effect of breast cancer surgery affecting nearly 30% of US breast cancer survivors (BCS). Our previous analysis showed that, even 12 years after cancer treatment, out-of-pocket healthcare costs for BCS with lymphedema remained higher than for BCS without lymphedema; however, only half of the cost difference was lymphedema-related. This follow-up analysis examines what, above and beyond lymphedema, contributes to cost differences.d and multivariable analysis, we compared self-reported costs across 13 cost categories by lymphedema status. We elicited quotes about specific cost categories from in-person interviews with 40 survey participants.This mixed methods study included 129 BCS who completed 12 monthly cost diaries in 2015. Using Cohen’s Compared with BCS without lymphedema, BCS with lymphedema faced 122% higher mean overall monthly direct costs ($355 vs $160); had significantly higher co-pay, medication, and other out-of-pocket costs, lower lotion costs; and reported inadequate insurance coverage and higher costs that persisted over time. Lotion and medication expenditure differences were driven by BCS’ socioeconomic differences in ability to pay.Elevated patient costs for BCS with lymphedema are for more than lymphedema itself, suggesting that financial coverage for lymphedema treatment alone may not eliminate cost disparities.The economic challenges examined in this paper have long been a concern of BCS and advocates, with only recent attention by policy makers, researchers, and providers. BCS identified potential policy and programmatic solutions, including expanding insurance coverage and financial assistance for BCS across socioeconomic levels. The economic burden of cancer treatment and its consequences, termed financial toxicity , remainsOur previous analysis of costs for BCS in the US an average of 12 years after cancer surgery suggested significantly higher annual out-of-pocket health and wellness costs for BCS with BCRL compared with those without BCRL . That stn = 295) ). Despite an equal number of average monthly medical visits, costs were statistically higher for women with BCRL for the categories of co-pays , medications , and other out-of-pocket costs ; but lower for lotions . No other variables were statistically different by BCRL status based on effect size difference calculations.As shown in Table Table Table Women with BCRL reported ongoing costs that continued in the long-term, especially for co-pays and compression garments. For example, respondents with BCRL recounted how co-pay prices have increased over time and referred to comparative past and present experiences, while those without BCRL were more likely to discuss the impact of co-pay prices closer to the time of treatment. Additionally, for women with BCRL, other ongoing out-of-pocket direct costs included transportation to ongoing BCRL therapy or purchase of additional health-related foods and supplements to improve overall health and manage BCRL symptoms. Because BCRL is a chronic condition, these costs would be ongoing and life-long. Women with and without BCRL both suggested that medication costs were an ongoing challenge that compromised their ability to take multi-year oral anti-cancer adjuvant treatments.Both women with and without BCRL spoke on the inadequacy or high cost of insurance coverage that precluded getting care for BCRL or other comorbid conditions. Patients with BCRL reported BCRL-specific treatments or supplies that may not have been comprehensively covered by existing insurance plans. For example, insurance did not typically cover compression garments, bandages, or sleeves, resulting in several hundred dollars in out-of-pocket costs. One respondent without BCRL described how switching to public insurance that had a more limited formulary complicated her ability to take medications for comorbid conditions. She further described how costs for medications could change due to an insurance “donut hole”, referring to a point when, after having an initial amount of services covered, publicly insured  patients pay full price for medications until their out-of-pocket maximum is met and insurance coverage resumes. Cost for supplemental insurance contributed to higher out-of-pocket direct costs for BCS in both groups.BCS’ socioeconomic position amplified challenges with accessing health care for post-cancer care and BCRL, especially in the categories of medication and other out-of-pocket costs. One respondent without BCRL described having to stop work due to her cancer diagnosis and the resultant loss of income due to lost work made it challenging to afford medications. BCS with BCRL had to concurrently navigate costs for BCRL therapy on top of adjuvant treatments. As a result, BCS with BCRL had to trade off financial reserves initially intended to secure a future of financial stability, such as retirement funds, or to forgo taking medication as prescribed. For out-of-pocket costs, socioeconomic position emerged again as critical to understanding the patient’s experience. Another quote exemplified that patients at higher income levels still faced challenges with affording care, yet were often ineligible for assistance with their needs.This study is one of the few to examine the economic burden of breast cancer–related lymphedema, a chronic adverse effect of breast cancer surgery, and to explore specifically what contributes to higher costs for BCRL patients, above and beyond BCRL itself. Monthly average costs for breast cancer survivors (BCS) who have BCRL were 122% higher than BCS who do not have BCRL. Five types of costs vary significantly by BCRL status, and BCRL patients reported that higher costs persisted in the long-term: co-pays, lotions, compression garments, medications, and out-of-pocket costs. Study results reinforce previous findings that patients with BCRL continue to face higher costs, in some part, due to BCRL. This analysis extends this finding to show that divergent costs are exacerbated by underlying pre-morbid socioeconomic and insurance challenges.Our results point to a need to mitigate healthcare costs, including co-pays, medications, and other health needs that may be paid out-of-pocket by BCS across resource levels, especially for BCS with BCRL. Having BCRL was a contributor to higher costs for co-pays and items listed under other out-of-pocket costs. BCS with BCRL paid over double in co-pay costs despite having the same average number of medical visits compared with non-BCRL BCS. This disparity may suggest that BCS with BCRL have higher cost insurance plans, though this would need to be explicitly explored in further research. Lower expenditures on lotions and higher payments for medications were better explained by underlying differences in socioeconomic position than BCRL status. While high socioeconomic position patterned greater access to lotions and medications, interviewees suggested that high socioeconomic position did not fully protect against economic challenges and that women who do not quality for assistance may still need help. For both BCS with and without BCRL, patients reported a need to make trade-offs between healthcare spending and doing what was best for their health. Patients with limited financial resources had to choose between adherence to medically necessary treatments and managing other health and living needs, often resorting to creative ways to access medications. Taken together, we interpret these results to mean that intervening on BCRL costs alone may not be sufficient to mitigate cost differences or overall cost burden and that patients across the socioeconomic spectrum may need relief from economic challenges. Given the ongoing nature of economic challenges, relief may need to be extended well beyond the initial post-treatment stages.High medical non-cancer costs of nearly $15,000 for the first 2 years of BCRL treatment for working-aged BCS has been previously documented , with moWhile monthly out-of-pocket direct cost estimates found in our study are less than the previously estimated $393 to $561 for working-aged BCS , previouPrevious studies have suggested ways to reduce costs for BCS , 47–50, Inadequate insurance coverage for BCRL-related care emerged as a challenge for patients with BCRL and without BCRL. Costs for compression garments and bandages were present only for BCS with BCRL, which contribute to higher overall costs per month since, as the qualitative interviews report, they are not always covered by insurance. High costs will cause patients to use compression garments that no longer apply sufficient pressure to manage BCRL , 30. In This study contributes to the body of literature examining why costs for BCS who have BCRL may differ from those who do not have BCRL. Dollar amount estimates were based on the experiences of women in Southeastern Pennsylvania and New Jersey in the year 2015 and may not be generalizable to other regions of the United States; regional variations in insurance coverage may cause cost estimates to vary. This study may underestimate actual costs for the 3-month projected cost estimation. Underreporting was addressed by encouraging participants to supply receipts and medical visit bill summaries in lieu of writing them into the cost diaries themselves; and with monthly text-based, e-mail, and phone messaging reminders to complete the cost diaries, yielding a > 90% response rate in each month of data collected. This analysis did not have a comprehensive list of contributing factors; nevertheless, these findings suggest a starting point of potential contributing factors on which future studies could focus.Our results call for fresh consideration of enduring high out-of-pocket costs for breast cancer survivors (BCS) who experience adverse effects after cancer treatment. The study results reinforce the idea that there are significant long-term cost implications for BCS who have BCRL and especially BCS who are socioeconomically challenged. To better reduce cost disparities and provide patient relief from added cost burden, improved and sustained financial coverage  for BCS with BCRL should be considered. Patient-level interventions focused on mitigating or lowering costs for adverse treatment effects should consider both the patient’s socioeconomic context and their other healthcare needs. Given the long-term nature of economic challenges for BCS with BCRL, early financial interventions may be warranted to avert long-term economic burden. Upcoming studies should identify social and economic risk profiles of patients who would most benefit from such interventions.


Duck aviadenovirus B. The pathogenicity of the new isolate DAdV-3 FJGT01 was investigated using challenge experiments. The gross lesions in the animal experiment were similar to the clinical lesions observed in the diseased ducks. TEM examination of liver sample showed that virions accumulated and arranged in crystal lattice formations in the nuclei of hepatocytes. The present study provides new information about the epidemiology and characteristics of duck adenovirus associated with Muscovy ducklings.Recently, infectious disease outbreaks characterized by swelling and hemorrhagic liver and kidneys occurred in Muscovy ducklings in China. Four viruses were isolated and identified as adenoviruses by transmission electron microscopy (TEM) and polymerase chain reaction (PCR). Sequence analysis identified the new isolates as duck adenovirus 3 (DAdV-3), species The online version of this article (10.1007/s00705-018-4105-2) contains supplementary material, which is available to authorized users. Adenoviridae includes five genera: Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus and Siadenovirus [Aviadenovirus includes 14 species: Fowl aviadenovirus A-E and Goose aviadenovirus A, Duck aviadenovirus B, Pigeon aviadenovirus A-B, Falcon aviadenovirus A, Psittacine aviadenovirus B, and Turkey aviadenovirus B-D [Atadenovirus and DAdV-2 in the genus Aviadenovirus. Waterfowl are known to be natural hosts of DAdV-1, and infections are usually asymptomatic [et al. published the complete genomic sequence of DAdV-2 GR isolated from Muscovy ducklings in France. Based on its genetic characteristics, DAdV-2 has been suggested to belong to the genus Aviadenovirus and the species Duck aviadenovirus B [According to the International Committee on Taxonomy of Viruses (ICTV), the family enovirus . The genirus B-D . So far,ptomatic . In chicptomatic . To our ovirus B . Anotherovirus B .et al. [g for 15 min at 4 °C. The supernatants were then used for polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) assays to detect avian influenza virus [Recently, several emerging outbreaks, characterized by swelling and hemorrhagic liver and kidney, have been threatening Muscovy duckling farms in Fujian, Zhejiang, Anhui and Guangdong provinces in China Fig. . The moret al. . None ofza virus , avian pza virus , 11, Musza virus , novel dza virus , avian Tza virus , duck asza virus , goose pza virus , duck enza virus  and aviaza virus . The sam5.5 TCID50/ml, 103.5 TCID50/ml, 104.3 TCID50/ml, 103.0 TCID50/ml, respectively. For transmission electron microscopy (TEM) examination, the cell culture fluid of infected DEFs was harvested and then clarified by centrifugation at 8,000g for 30 min using a Ti19 rotor  at 4 °C. The supernatant was collected, overlaid onto a 20% sucrose solution, and then centrifuged at 100,000g for 3 h at 4 °C in a Ti70 rotor . The pellet was resuspended in TE buffer . The virus preparations were stained with phosphotungstic acid  and examined using TEM. Spherical particles ranging from 60 nm to 80 nm in diameter were observed . These results indicated that the newly isolated viruses were AdVs.Duck embryo fibroblasts (DEFs) were freshly prepared from 11-day-old Muscovy duck embryos and maintained in DMEM supplemented with 5% fetal calf serum  for ~ 16 hours. AdV-positive samples were inoculated onto DEFs, and four virus isolates, designated as FJGT01, AHAQ13, ZJJH07 and GDMM10, were isolated successfully. Compared to normal DEFs, the infected cells became round and clustered like grapes , which is a classical characteristic of the cytopathic effect in adenovirus-infected cells –22. The TM Thermal Cycler, Bio-Rad, Hercules, CA, USA). The PCR products were purified using a QIAquick Gel Extraction Kit  and then cloned into pGEM®-T Easy Vector  according to the manufacturer’s instructions. After transformation of competent Escherichia coli strain DH5α  with these constructs, three positive clones for each PCR product were sequenced in both directions using an ABI 3730XL DNA Analyzer  at BioSune Biotech Co., Shanghai, China. The genomic sequences of the AdV isolates were assembled using the Lasergene sequence analysis software package . The complete genomic sequences of FJGT01, AHAQ13, ZJJH07 and GDMM10 isolates were obtained and deposited in the GenBank database with the accession numbers MH777395, MH777396, MH777397 and MH777398, respectively. The full-length genomes of all four isolates were 43842 bp in length. The G+C content of 47.13% and the predicted ORFs were similar to those reported for other adenoviruses.The complete genomes of the identified were amplified using a set of specific primers (Table S1). PCR was carried out in a PCR system , suggesting that the new isolates DAdV-3 FJGT01, GDMM10, AHAQ13, and ZJJH07 are members of the species Duck aviadenovirus B . Briefly, a total of 30 ten-day-old Muscovy ducklings were randomly divided into two groups (15 ducklings per group). The ducklings in the infection group were inoculated with 300 μl of FJGT01 containing 10Tissues of the heart, liver, spleen, lung, kidney, brain, and pancreas of dead Muscovy ducklings were collected for histological examination. The tissues were fixed, cut into sections, and stained with hematoxylin and eosin Fig. . HistopaLiver samples were also collected for examination by TEM. The liver samples were embedded, and ultrathin sections were stained . TEM demDuck aviadenovirus B in the genus Aviadenovirus.In summary, in the present study, we isolated and identified DAdV-3 that was circulating in China. The newly isolated DAdV-3 strains could be classified as numbers of the species Supplementary material 1 (TIFF 18138 kb)Supplementary material 2 (TIFF 18548 kb)Supplementary material 3 (TIFF 20500 kb)Supplementary material 4 (TIFF 19954 kb)Supplementary material 5 (TIFF 17893 kb)Supplementary material 6 (DOCX 15 kb)Below is the link to the electronic supplementary material.


There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015–2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne , aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control. In the near past blueberry production was primarily concentrated in North America, with over 80% of world production in 2003 . There wBlueberry, Cranberry, and Lingonberry Compendium lists 13 viruses known to infect highbush blueberry [The movement of large volumes of vegetative plant material has inherent risks in terms of biosecurity ,7. Symptlueberry . Blueberlueberry ) can caulueberry ) can be lueberry ), the vilueberry ,13 whereThere is an effort in the U.S. to harmonize blueberry certification programs (for more information visit ncpnberries.org), which has been the impetus for this study. For effective certification programs, understanding the viruses that pose the greatest risk for reinfection in nurseries can be used to develop best management practices. The objectives of this work were to identify the high risk blueberry viruses by production region in the North America and make this information available to regulators, and nursery managers. The outcome of such actions will be improved certification systems with higher quality plants that benefit fruit producers and consumers alike.Samples were collected from the field randomly. They were brought back or shipped to the USDA-ARS Horticultural Crops Research Laboratory in Corvallis, Ore. or the Department of Plant Pathology at the University of Arkansas between 2015 and 2017. Areas of collection were the Pacific Northwest , the Midwest , the Southeast , and the Northeast . All samples consisted of the youngest but fully expanded leavesTotal nucleic acids (TNA) were extracted from leaf samples using the buffers described previously ,15. Tiss405 values were determined using the ELx808 plate reader . Samples were considered positive if the absorbance values were three times or greater than that obtained in wells loaded with buffer rather than leaf sap.For ELISA, leaf samples were homogenized using a Pollähne roller press with buffer added to the rollers using a peristaltic pump. The grinding buffer used was that described in Martin and Bristow . For BLMBlueberry fruit drop associated virus (BFDaV), blueberry latent virus (BlLV), blueberry mosaic associated virus (BlMaV), blueberry necrotic ring blotch virus (BNRBV), blueberry red ringspot virus (BRRV), blueberry virus A (BVA), and as well as an internal control standard  were tesNADH dehydrogenase ND-2 subunit as described [Reverse transcription reactions (50 μL) consisted of 50 mM Tris acetate (pH 8.4), 75 mM potassium acetate, 8 mM magnesium acetate, 20 mM DTT, 0.4 mM dNTPs, 0.3 μg random primers, and 60 U Maxima reverse transcriptase . The solution was vortexed, centrifuged briefly, incubated at room temperature for 2 min, and then 50 °C for 60 min. The reactions were terminated by 5 min incubation at 85 °C. The cDNA constituted 10% of the total PCR volume. Before virus detection, all samples were evaluated for quality of the RNA by carrying out amplification of the escribed . Virus-sOlpidium virulentus [The viruses detected in blueberry in the major production areas in the U.S. are shown in rulentus  very sloThe majority of the samples obtained from California were from nurseries, yet the BlShV and BlMaV positive samples were from production fields or breeding plots.In the Pacific Northwest, most samples were collected in Oregon and Washington with the exception of 184 samples collected in British Columbia, Canada primarily because BFDaV was observed in two fields there . BFDaV wIn the southeast U.S.A., there were two viruses that were common, BRRV and BNRBV with the latter detected only in this region. BRRV has been reported sporadically in other regions but it is considered an important pathogen in New Jersey where it was first identified . The absIn the northeast BLMoV, BlScV, BVA, and TRSV were the most common viruses. BlScV is aphid-transmitted as is probably the case of BVA, a closterovirus . TRSV waIn the upper Midwest, the aphid or hypothesized aphid-transmitted BlSSV and BVA were the most common viruses suggesting that there may be high aphid populations in this region, or an efficient aphid vector of these two viruses. All three nematode transmitted viruses, PRMV, ToRSV, and TRSV, were also present in this region.There is little information on virus resistance in blueberries. “Jersey” is reported to be immune to BRRV . “BluecrThe information from this survey highlights the most prevalent viruses in blueberry by production region in the North America. Several viruses were very limited in distribution, the extreme example being BFDaV which was detected in only one field in northwest Washington State and in several fields in adjoining British Columbia, Canada. In the U.S. efforts are underway to eradicate this virus. It was only detected in the cultivar “Bluecrop” in the field, but not in over 300 samples tested from other cultivars planted adjacent to fields with the infected material. The presence of the virus in “Aron” at the National Clonal Germplasm Repository in Corvallis, Oregon is of concern but no other cultivar in the block tested positive for the virus. BNRBV was only detected in the southeast U.S.A. where it was quite common. BRRV was primarily a problem in the southeast U.S.A., though there was a single positive from the northeast U.S., where it was originally described . BRRV haBlShV was widespread in the Pacific Northwest, with over 40% of the samples testing positive for this virus. This was clearly the most widespread of any of the viruses within a production region. This virus is pollen-borne; plants generally lose one full year’s crop but then recovers to full production . For thiThe information on high risk viruses for blueberries in the U.S. and British Columbia, Canada can be used by growers to manage virus diseases in production fields, by State Departments of Agriculture to manage more efficiently certification programs and by nurseries to inform the development of their pest management plan. Certification programs are based on best management practices (BMPs) to minimize risk of pathogen infection, combined with testing to ensure effectiveness . TestingNematodes are one of the more challenging vectors to manage in a nursery system since fumigation is only effective in the upper layers of soil and nematodes below the depth of treatment escape and can repopulate the site. Many blueberry nurseries are in canyards, potted plants maintained on a bed of course gravel >5 cm thick, or on plastic laid over the soil. Also, the blueberries are grown in soilless media. This growing system avoids the risk of nematode transmitted viruses. From a total of 79 samples no parasitic nematodes were detected in either the potting media or below the plastic barrier . This apFor some commodities such as potato, where the growers only have the crop in the field for one year, there tolerances for virus incidence built into certification schemes. With long term crops such as blueberry, even a very low level of infection is problematic for fruit producers since it takes 5–8 years for plant to come into full production. No certification system will be 100% effective, even if every plant was fully tested prior to planting. Thus, it is important for producers to monitor their fields for infection and rogue infected plants promptly to increase the productive life of a field. With good certification programs, there should be few plants that will need to be removed, but if we look at plant production as a system, the final check is to monitor what growers planted in the production fields.


The main objective of this study was to develop various models using North American Animal Disease Spread Model (NAADSM) to simulate the transmission of Porcine reproductive and respiratory syndrome (PRRS) virus between farms in Nghe An Province in Vietnam in order to inform the prevention and control of this important disease. Using real data from the household survey, credible parameters for direct/indirect mean contact rates between different farms were estimated. A total of eleven models were developed, including immunization scenarios. In addition, we conducted sensitive analysis on how the mean contact rates influenced the results. The immunization scenarios showed that a high proportion of pigs in medium size farms needs to be vaccinated in order to reduce the transmission to pigs in small farms under the Vietnamese pig production system. In order to promote the use of vaccinations, incentives (such as a vaccine subsidy) for medium size farms may be needed. It could be the most cost‐effective control and prevention strategy for pig diseases in Vietnam. Our study provides insights on how pig diseases can be spread between pig farms via direct and indirect contact in Nghe An under the various hypothetical scenarios. Our results suggest that medium/large farms may play an important role in the transmission of pig diseases. In Vietnam, PRRS is endemic and has a major impact on production. It is a viral disease in pigs caused by a single‐stranded and small enveloped RNA virus of the family Previous studies suggest that the main risk factors for PRRS are the movement of infected animals between farms and the introduction of infected semen , Vietnam. All methods were performed in accordance with the relevant guidelines and regulations.2.2NAADSM requires three key parameters: (a) mean contact rates (estimated number of direct/indirect contacts between farms per week); (b) probabilities of infection transfer with each contact; and (c) contact distance between farms  “How often did vehicles enter your farm over the last 6 months?”; (2) “How often did veterinarians/ animal health workers visit your farm over the last 6 months?; (3) How often did other farmers and traders visit your farms over the last 6 months?”; and (4) “How often did you share any equipment with other farms”. A Poisson distribution was fitted for the different model parameters, with the average number of contacts used to define λ. The distributions for mean direct and indirect contact rates were computed on a weekly basis. This period was selected as the virus can be infective for a week at 21°C  were not considered. In addition, we assumed that a continuous flow (CF) system was used in all farms in our models instead of all‐in‐all‐out system (AIAO) which is not common in Vietnam.2.3In Vietnam, medium farms are mainly responsible for supplying piglets and weaners to small farms Figure . HoweverOur scenarios were simulated on a weekly basis for 52 weeks over 1,000 iterations, which was sufficient to capture the pig production life cycle (6–8 months) in Vietnam. The number of median infected pig farms and the time it took to reach peak epidemic levels were calculated for each scenario. We assumed that infected farms became infectious for long periods of time, with new susceptible animals consistently replaced during the study period. Previous studies suggest that a PRRS virus can survive in the infected farms for long periods of time  was decreased from the baseline model (contact rate:1) to 0.75, 0.5 and 0.25, respectively, while the indirect contact transmission probability (ICTP) was modified from the baseline model (contact rate: 0.1) to 0.5, 0.25 and 0.05. The number of median infected farms from each scenario was compared to the baseline model (scenario B2). In addition, we assessed the impact of the mean contact rate of indirect contact towards large farms by varying values from 3.5 to 2, 1, 0.75 and 0.5 in the model.3A total of 60 farmers [Female (16): male (44)] were interviewed in two districts (30 farmers/district). The mean and range of age were 51 and 25–90 years old, respectively. The survey captured data of 46 small, 11 medium and 3 large farms (See Table S1).Of the farms enrolled in the survey, none reported that boars or equipment were shared with other farms. Table The modelled number of infected farms  by each production type is presented in Table The number of median infected farms for scenario B1 was much lower than scenarios A2 and B2, where a single outbreak occurred in a small farm . For each scenario, the median number of infected small farms was 14 (scenario B1), 51 (scenario A2) and 51 (scenario B2). In addition, the median number of infected medium farms was 0 (scenario B1), 139 (A2) and 138 (B3), respectively.The sensitivity analysis showed that the number of median infected farms was very sensitive to the DCTP Table , suggest4To our understanding, this was the first study to evaluate the spread of PRRS virus between farms via direct and indirect contacts in Vietnam. Using real data from the household survey, credible parameters for mean contact rates between different farms were estimated. Furthermore, the number of pig farms by production types used in our models was based on local data, adding to the robustness of the model.Scenario A2 included both direct and indirect contact while scenario A1 only had direct contact. As a result, scenario A2 showed that the number of median infected farms was the highest and quickest to reach the peak epidemic. In addition, scenarios A2 and B2 had relatively higher number of median infected farms. Our findings suggest that medium and large farms may play an important role in the transmission of PRRS virus in Nghe An. In general, large farms are isolated and mostly managed by big commercial companies in Vietnam. For this reason, certain direct contact routes were not taken into account as in practice, and there is no animal movement towards the large farms. There is a hierarchical structure of animal movement from medium to small farms. Therefore, no animal movement from small to medium farms was considered as the small farms are the last stage of pig's life cycle in Vietnam, which are brought to the slaughterhouses. Our survey showed that the indirect contact rate in large farms was considerably higher than small and medium farms, likely reflecting the fact that there are frequent vehicle and human movements for pig sales and farm managements in comparison with small and medium scale farms.Our models showed that the transmission route to large farms via indirect contact could have a significant impact on our results. Given the strict restrictions in place, however, it is unlikely that the large farms allow the sharing of vehicles and human movement with other farms. Therefore, the medium farms need to be targeted to efficiently reduce/prevent the transmission of the PRRS virus to small‐scale farms, which account for 70% of the total production in Vietnam (Lapar, Binh, & Ehui, 0 of PRRS virus was higher than 1 (Charpin et al., The model assumed that the whole farm became infectious when one pig was infected from the farm, which was reasonable as it is unlikely that the PRRS virus would die out without onward transmission of diseases to other pigs in the farms. Similarly, many studies have suggested that RThe main limitation of our study was that our direct contact transmission probability was considered to be 1 which may have led to an over‐estimation as some of farms may use AIAO production system, so virus transmission rate might be less than 1. The importance of the transmission probability was also confirmed in the sensitivity analysis. Values used were based on studies by others (Neumann et al., This study is the first attempt to quantify the indirect contact rates from the survey. Our estimates are mainly based on vehicle and human movements and may have underestimated the number of infected farms in practice. Some studies have suggested that the virus could be disseminated by aerosol and fomite transmissions (Pirtle & Beran, A few simulation studies on the spread of PRRS virus have been conducted in New Zealand and Canada using similar software (Neumann et al., It is essential to educate farmers to have an isolation area for newly pig arrivals and an AIAO system in order to prevent the spread of disease on farms. Based on our observations, these are not properly implemented in small farms due to the low level of awareness in biosecurity and for economic reasons. In addition, it is important to control the movement of infected or high‐risk farms as this would help stop the spread of PRRS virus. The PRRS virus vaccine has been introduced to farmers in Vietnam, but is mainly used in large‐scale pig farms. They are not in use, in small‐scale farms due to economic constraints and the absence of incentives to promote the use of PRRS vaccination, since pigs can be traded without restrictions.Despite the discussed limitations and difficulties to validate our model, it provides valuable insight into how PRRS virus can be spread between farms via direct and indirect contact. Our model could aid decision makers in directing resources towards the prevention and reduction of transmission of PRRS virus in farms. The model can be easily adjusted to other provinces or countries in the region with modified parameters.The authors declare that they have no competing interests.H.S.L, K.K.T, and B.W. designed research: H.S.L, V.B.N, N.A. and D.M. performed research; H.S.L and K.K.T. analysed data; H.S.L, K.K.T. and B.W. wrote the paper. Click here for additional data file.


Concerns regarding outbreaks of human monkeypox or the potential reintroduction of smallpox into an immunological naïve population have prompted the development of animal models and countermeasures. Here we present a marmoset model of monkeypox and smallpox disease utilizing a relevant poxvirus via a natural exposure route. We found that 1000 plaque forming units (PFU) of Monkeypox virus was sufficient to recapitulate smallpox disease, to include an incubation period of approximately 13 days, followed by the onset of rash, and death between 15 and 17 days. Temporally accurate manifestation of viremia and oral shedding were also features. The number of lesions ranged from no lesions to 299, the most reported in a marmoset exposed to a poxvirus. To both evaluate the efficacy of our antibodies and the applicability of the model system, marmosets were prophylactically treated with two monoclonal antibodies, c7D11 and c8A. Of three marmosets, two were completely free of disease and a single marmoset died 8 days after the mock (n = 1) or PBS control(s) (n = 2). Evaluation of the serum levels of the three animals provided a possible explanation to the animal succumbing to disease. Interestingly, more females had lesions (and a greater number of lesions) and lower viral burden  than males in our studies, suggesting a possible gender effect. Poxviruses are large viruses capable of causing disease in humans. Of the known poxviruses, variola virus and monkeypox virus cause the most severe disease, smallpox and human monkeypox, respectively. Models utilizing these viruses are valuable for the study of host-virus interaction and for evaluating countermeasures. Arguably the best models utilizing variola virus or monkeypox virus involve the intravenous inoculation of nonhuman primates. Because these models have an unnatural route of infection and bypass the incubation of the disease, more genuine models are sought. Here we tested whether marmosets could be infected with monkeypox virus via a natural route and, if so, whether the model could be applied to study countermeasures. We found that the animals were susceptible to monekypox virus at a low dose via a natural route, uncannily mirrored human disease onset, and could be protected by prophylactic treatment of two novel monoclonal antibodies. Furthermore, we found potential disease disparity between males and females that, if proven true, could stimulate the exploration of a mechanistic explanation to describe the disparity and deepen our understanding of poxvirus pathogenesis, immunology and/or marmoset biology. Family Poxviridae. All share a double stranded DNA genome and replicate in the cytoplasm. Members of the genus Orthopoxvirus are arguably the most recognizable poxviruses as they include variola virus, monkeypox virus, and vaccinia virus, the causative agent of smallpox, monkeypox, and the virus converted to a vaccine to eradicate smallpox from nature, respectively. Because of the lack of, or waning, immunity due to the cessation of vaccination, there are concerns about our vulnerability to natural (zoonotic) or man-made poxviral threats.Poxviruses are large and complex viruses and belong to the For the most part, human monkeypox is an endemic disease in Africa. In 2003, multiple cases of monkeypox were reported within the U.S., representing the first confirmed incidence of monkeypox breaching the African continent . Within Unlike monkeypox virus, variola virus is host restricted to humans and is not zoonotic. Because of this, the eradication of smallpox disease was possible through intense survellience and vaccination efforts. Variola virus, was not eradicated and still resides in at least two locations in the world, the Centers for Disease Control and Prevention, Atlanta, Gerorgia, United States and State Research Center of Virology and Biotechnology, Novosibirsk, Russia. Whether accidental, such as the potential harm from the 6 unaccounted vials of variola virus found at a U.S. Food and Drug Administration facility, or throi.e., fever, headache, malaise). Unlike smallpox, lymphadenopathy is commonly reported for monkeypox during this prodromal phase. A progressive rash ensues. Again, the extent and severity of the rash tends to be attenuated in monkeypox cases. Overall, monkeypox is likely to have a more favorable outcome, but, the extent and presentation of disease will likely depend on many factors Both monkeypox and smallpox have a similar disease presentation in humans. After an approximate one to two week incubation period, both diseases present with general symptoms  licensed product with an indication for smallpox disease, the ACAM2000 vaccine. The benefit-to-risk ratio with ACAM2000 is very small and was a major factor for cessation of vaccinating after the eradication of smallpox disease. In fact, vaccinia immune globulin (VIG) was licensed to counteract the adverse events associated with the smallpox vaccine. Being that a significant portion of the United States is contraindicated for our only line of defense against monkeypox or smallpox, safer vaccines –9 and thCallithrix jacchus) model for the treatment of poxvirus disease. More specifically, we used a cocktail of two human-chimeric monoclonal antibodies, c7D11 [Here, we describe a model for the study of human monkeypox  and present the first application of any marmoset  was propagated and titrated as previously described , 20. Theet al. . BS-C-1 StableFast platform and the single cholesterol selection strategy as described by Sampey, et. al. [The discovery and characterization of the antibodies are reported elsewhere , 14. For et. al. . BrieflyAntibodies were combined 1:1 prior to subcutaneous injection into NHPs. Isotype control antibody contained identical constant human heavy and light chains as the c7D11 mAb, but with different murine heavy and light chain variable domains that bind an unrelated filovirus glycoprotein.et al. [Samples  were subjected to three freeze-thaw and sonication cycles and plaque titrated as reported by Golden, et al. . For assVirus from animal #9, D25 oral swab was used to perform two types of assays. Briefly, virus was diluted and incubated with a constant concentration of c7D11 and adsorbed. Samples incubated with c7D11 were incubated for 1h at 37°C, adsorbed for 1h, and overlayed with Minimal Eagle Medium  containing 5% heat inactivated fetal bovine serum, FBS (Gibco).Whole blood samples were collected in Sarstedt K3E Micro Tubes and analyzed by either the AcT Diff 3 (Coulter) or a Hemavet HV950 (Drew Scientific). Plasma was collected by centrifugation at 1800xg for 10 minutes.Callithrix jacchus) weighing between 233–437 grams, were exposed via the intranasal route by instilling (pipet) 50μL of virus into each nare  while animal was inhaling. Physicals, lesion counts, weights, rectal temperatures, oral swabs, and venous blood collections were performed previous to, and every three days following, exposure. Animals were anesthetized with isoflurane for all animal manipulations, and prior to euthanasia were anesthetized with Telazol before administration of an intravenous barbiturate overdose.Both male and female adult marmosets  24 hours prior to exposure. Two control animals received an equivalent volume of PBS, while a third received 40mg/kg of a human chimeric, non-poxvirus targeted monoclonal antibody (BioFactura). Survival curve comparsions (Log-rank (Mantel-Cox) test) were performed usng Graphpad Prism.Plates were coated with poxvirus antigen B5, L1 or diluent  and incubated at 4°C until assay was performed (1–3 days). Recombinant 6xHis-tagged soluble poxvirus antigens B5 and L1 were transiently expressed in HEK-293T cells and purified by cobalt affinity chromatography . Antigen B5 is the product of the vaccinia virus gene B5R and is the target of monoclonal antibody c8A and antigen L1 is the product of the vaccinia virus gene L1R and is the target of monoclonal antibody c7D11. On the day of the assay, wells of the plate were aspirated and washed with PBS with 0.05% sodium dodecyl sulfate (SDS) and subsequently blocked for two hours with blocking buffer (PBS with 5% skim milk and 0.05%SDS). Wells were washed with PBS with SDS before samples were added. Samples were first diluted into 5% skim milk with SDS and serially diluted 1:5. The standard antibody was diluted 1:10 into marmoset sera (Bioreclamation) and serially diluted 1:5, leaving a single tube that contained only marmoset serum. The spiked and control marmoset sera were diluted in blocking buffer analogous to the samples. Samples were added to the plate in triplicate (two on wells containing antigen and one with no antigen). Plates were sealed and incubated at 37°C for one hour before being mechanically washed. Horseradish peroxidase labeled, anti-human IgG detection antibody  diluted in blocking buffer at 1:8000 was then added to each well and incubated at 37°C. After washing,3,3’,5,5’- tetramethylbenzidine (TMB) Sureblue Reserve (KPL) was added, followed by TMB Stop solution (KPL). Absorption readings were performed on a Tecan plate reader (450nm). The data was normalized and values interpolated using curve fit  on Graphpad Prism. Antigens and monoclonal antibodies c8A and c7D11 were provided by Biofactura.Research was conducted in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. All animal experiments were approved by USAMRIID’s Institutional Animal Care and Use Committee.Animals were housed in individual metal cages meeting current standards for the duration of the housing period in animal biocontainment level 3. Room environment was centrally controlled by an HVAC system that maintained room humidity and temperature. Animals were provided pelleted commercially available feed and potable water was provided at libitum from an automatic watering system. In addition, animals received supplemental foods, treats and fruits daily. Animals were provided manipulanda , foraging devices, treats and fruits as enrichment. Treats and extra fruits were increased while in biocontainment. Euthanasia was performed when the animal(s) met the criteria for euthanasia using a score sheet for intervention or when found moribund . The scoOur goal for these initial experiments was to determine the dose of intranasally administered monkeypox virus required to cause disease in marmosets. We previously showed that marmosets exposed to low titer monkeypox virus administered intravenously was able to initiate severe disease . Based oBecause there were no indications of disease, we chose to re-expose the three marmosets at a higher dose (1000 PFU). A fourth marmoset, #4, was included and exposed at an even higher dose (5000 PFU). All animals displayed signs of illness and were either euthanized or succumbed to disease 15 days post exposure . In geneSlight changes in behavior were observed on Day 12, but animals did not meet our criteria for disease scoring until Day 13. The higher dosed animal #4 was the first to score using the criteria , based on observed unresponsiveness and dyspnea . The thrSkin lesions were present on two of the four animals . On animAnalysis of whole blood samples revealed increases in white blood cells and decreased platelet counts . Both lyVirermia and oral shedding were assessed by plaque titration of whole blood and oral swabs. Using whole blood samples, all animals were viremic by Day 15 . AnimalsAfter determining a dose of virus that causes severe disease from the previous experiments, we next wanted to explore whether or not we could protect the marmosets from such a disease. A group of three marmosets  were treated subcutaneously with a 1:1 mixture of monoclonal antibodies c7D11 and c8A at 20 mg/kg (per antibody) 24 hours previous to monkeypox virus exposure. As controls, one marmoset was treated with an equivalent dose of isotype matched  monoclonal antibody (#5) and two marmosets were treated with an equivalent volume of PBS (#6 and #7).The disease course in the control animals was similar to our initial experiments. There were no significant increases in temperature, but animal’s had slight decreases on Day 15 . Body weAnimals prophylactically treated with c7d11 human-mouse chimera and c8a human-chimpanzee chimera antibodies did not exhibit signs of any illness throughout the critical period, that is, when the untreated animals were showing signs of disease or died (Days 13–18). With the exception of animal #9, all treated animals survived the exposure . Animal We further explored the implications of the circulating and shed virus by assessing the virus susceptibility to monoclonal antibodies. To do this, we performed a plaque reduction assay  with c7D11and virus collected using the oral swab from animal #9 on Day 25. We found that 10μg/mL of c7D11 greatly reduced the virus as follows: >1250 PFU/mL reduced by greater than 89%; 210 PFU/mL was reduced by 96% and 55 PFU/mL was reduced by 100%. These experiments allowed us to conclude that the virus was indeed monkeypox virus and also that the shed virus was itself not resistant to our most potent therapeutic component (c7D11). With limited plasma sample, we also looked at levels of c7D11 and c8A on Days 0, 12, and 21 by ELISA. Animal #9 had approximately 1/3 less c7D11 in the plasma as the other two animals and, 15% less c8A. Differences between animal #8 and #10 were slight in levels of c7D11 and c8A . Differences became apparent (or more apparent) on Days 12 and 21. For instance, animals #8 and #9 had c7D11 levels amounting to 36% and 8% on Day 12 as compared with animal #10, and 19% and 2.3% on Day 21. For c8A, this equated to 85% and 33% for Day 12 and 5%, and 41% for Day 21  before succumbing to the disease . Unlike All control animals were viremic at Day 12 post exposure . Animal Infectious virus was detected in oral swabs on Day 12  and Day 15 . Low levels of virus were detected (less than 100 PFU) at these time points and, interestingly, there was not an appreciable increase between Day 12 and Day 15 for the animals that were positive on Day 12 .To assess the relative levels of monoclonal antibodies c7D11 and c8A at the time of exposure and persistence over time in uninfected animals, we recapitulated the efficacy experiment by injecting 20mg/Kg  into a single marmoset . RepreseHere we present for the first time the successful treatment of a monkeypox virus exposed marmoset. The antibodies tested are known to target both morphogenic forms of the virus: the MV, in the case of c7D11, and the EV, in the case of c8A. Previously, 7D11 was studied as a full mouse monoclonal antibody , 23 and Using a small number of marmosets, we found that a relevant dose of 1000 PFU was sufficient to cause uniformly severe disease. Exposure with 100 PFU was not sufficient to cause disease. Other than a single hematological analyte (platelets) from a single animal, all parameters measured were normal. This includes a lack of detectable virus in the samples. From our data, the most likely scenario is that monkeypox virus failed to infect and instead was mechanically  cleared. The evidence for this is that there have been no reported experimental survivors that have exhibited clinical disease or presented with hematological/physiological manifestations  . Also, tDuring the incubation  for naturally acquired smallpox, it is thought that a primary viremia occurs from the local lymph tissue which ultimately seeds the hematopoietic system . A seconOf the six mock or un-treated marmosets exposed to 1000 PFU of monkeypox virus, the sequence and temporal onset of disease events were analogous to those delineated above for human disease . For insSince animal #4 was exposed to 5000 PFU, we will have to consider it separately from 1000 PFU, and, given it is the only animal exposed at the dose, not infer too much from the data. The onset of viremia and detection of infectious virus on oral samples were earlier than the 1000 PFU animals. Sequentially opposed to the 1000 PFU animals, oral swabs were positive for infectious virus on Day 6 and viremia was not detected until Day 9. This could be potentially be explained by the sensitivity of the titration assays or that oral tissues are seeded prior to, not by, the viremia we detected in the blood. There were no real differences in magnitude in any of the parameters measured .Unlike the monkeypox virus intravenous marmoset model, a visible cutaneous rash was not a consistent feature of the disease in the intranasal marmoset model . In factWhether adaptive and/or innate response(s) is insufficient in marmosets and leads to more severe disease is unknown and will require more experimentation. Severe disease in mice infected with ectromelia virus has been linked to Natural Killer cells  and low Interestingly, there appears to be a gender effect related to cutaneous lesion burden . FemalesHow this applies to the outcome of smallpox and human monkeypox is difficult to conclude. It is well known that in humans, pregnant females were significantly more likely to develop more severe form of the disease (hemorrhagic disease), but in the absence of pregnancy there was no clear gender effect on severity or outcome. Others have reported differences in immune response after smallpox vaccination. For instance, it has been reported that in humans, females generate a higher neutralizing response to Dryvax . The oppBoth human monkeypox and smallpox have similar disease manifestations and were difficult to decipher in the age of smallpox. In the eradication era, this was not an issue from the clinical perspective as the vaccine provided protection from both monkeypox and smallpox. Now that the global population is becoming more immunologically susceptible to monkeypox, there are more monkeypox outbreaks with more associated cases of monkeypox. Therefore, a global predicament is more likely than before and an adequate level of preparedness should be sought. Concerns for monkeypox should not completely overshadow the threat of accidental or purposeful reintroduction of smallpox. Because of the relatedness of the monkeypox and variola viruses, therapeutic and vaccine strategies can be bundled for both indications. With the advent of the intranasal monkeypox virus model in marmosets, the entire disease course of monkeypox and smallpox can be accounted for by nonhuman primate models utilizing a virus known to cause severe disease in humans . For sma


Marsupenaeus japonicus shrimp) miRNA (shrimp miR-1000) was characterized. The miRNA target gene prediction showed that only two virus genes (wsv191 and wsv407) might be the targets of miR-1000. The results of insect cell transfection assays revealed that shrimp miR-1000 could target multiple virus genes (wsv191 and wsv407). The mRNA degradation analysis and RNA FISH (fluorescence in situ hybridization) analysis indicated that miR-1000 triggered the mRNA degradation of target genes through 5′-3′ exonucleolytic digestion in vivo and thereby inhibited the virus infection in shrimp. The miRNA-mediated 5′-3′ exonucleolytic digestion of target mRNAs stopped near the 3′UTR (3′untranslated region) sequence complementary to the seed sequence of miR-1000. Therefore, our study provided novel insights into how a host miRNA targeted multiple viral genes and prevented host from virus infection.MicroRNAs (miRNAs) function as crucial suppressors of gene expression via translational repression or direct mRNA degradation. However, the mechanism of multi-gene regulation by a host miRNA in antiviral immunity has not been extensively explored. In this study, the regulation of two white spot syndrome virus (WSSV) genes by its host ( Drosha and Dicer, two genes required for miRNA biogenesis, leads to a decreased mature miRNAs production and increases host sensitivity to virus infection (wsv477 to inhibit virus infection (p53 gene (It is well known that virus invasion of its host and the host immune response against the virus depend on virus-host interactions . During nfection . These fnfection . Similarp53 gene . On the p53 gene . In thisMcl-1 gene (BCL2 family apoptosis regulator)  in length, regulates its target gene expression through the binding of the miRNA seed sequence to the 3′UTR of mRNA, resulting in the translation repression or the direct mRNA degradation of target gene . The miRgulator) . It has gulator) . All theMarsupenaeus japonicus shrimp. The white spot syndrome virus (WSSV), a member of the Nimaviridae family and Whispovirths genus, has become one of the most dangerous and devastating marine pathogens that affect crustaceans (wsv191 and wsv407), which triggered mRNAs degradations through 5′-3′ exonucleolytic digestion, leading to the inhibition of virus infection. Therefore, the present data provides novel insights into how host miRNA targets multiple viral genes thus preventing host cells from the virus infection.In this study, we sought to explore how a single miRNA could target and regulate multiple genes, by studying miR-1000, a host miRNA, which is upregulated during WSSV infection in staceans . As an estaceans , 16. It staceans . The resMarsupenaeus japonicus) of ~15 g each were raised in tanks (60 cm × 40 cm × 30 cm) filled with aerated seawater at 20°C. The seawater salinity was 21–26%0. For each treatment, 20 individuals were used. To ensure that the shrimp were virus-free before experiments, three shrimp of each treatment were randomly selected and subjected to PCR with WSSV-specific primers (5′-TATTGTCTCTCCTGACGTAC-3′ and 5′-CACATTCTT CACGAGTCT AC-3′). At the same time, the shrimp were subjected to the detection of pathogenic microorganisms using a commercial kit for shrimp pathogen detection , which could detect acute hepatopancreatic necrosis disease, yellow head virus, shrimp iridovirus, enterocytozoon hepatopenaei, covert mortality nodavirus, Taura syndrome virus, infectious hypodermol and hematopoietic necrosis virus, hepatopancreatic parvovirus, infectious myonecrosis virus, Vibrio cholera and Vibrio parahemolyticus. The detection indicated that the shrimp were pathogen-free. To infect shrimp with WSSV, 100 μl of WSSV (104 copies/ml) prepared in sterile phosphate buffered saline (PBS) was intramuscularly injected into each virus-free shrimp at the lateral area of the fourth abdominal segment using a syringe with a 29-gauge needle. At different time points post-infection, shrimp were collected and stored in liquid nitrogen for later use. At the same time, the cumulative mortality of shrimp was examined daily. In shrimp mortality assays, shrimp without any treatment and shrimp injected with PBS were used as negative controls. The WSSV used was obtained as described previously  and miR-1000 (5′-ATAT TGTCCCGTCACAGCAGT-3′) were modified with 2′-O-methyl (OME) (bold letters) and phosphorothioate (the remaining nucleotides). As controls, the sequences of miR-1000 and AMO-miR-1000 were randomly scrambled, generating miR-1000-scrambled (5′-AACTTGCGTCCGATAGTACCT-3′) and AMO-miR-1000- scrambled (5′-TAAGGGTAGTGAACCCAGCTT-3′), respectively. All oligonucleotides were synthesized by Sangon Biotech . AMO-miR-1000 or miR-1000, at a concentration of 15 μg/shrimp, was intramuscularly injected into shrimp at the lateral area of the fourth abdominal segment. At different time points after the last injection, shrimp hemocytes were collected for later use. The experiments silencing or overexpressing miR-1000 in shrimp were conducted independently for three times.To silence or overexpress miR-1000 in shrimp, anti-miR-1000 oligonucleotide (AMO-miR-1000) or miR-1000 was delivered into shrimp by injection according to our previous study . AMO-miRTotal genomic DNA was extracted from WSSV-infected shrimp with a DNA isolation kit  according to the manufacturer's manual. The WSSV copies were then quantified using WSSV-specific primers (5′-TTGGTTTCATGCCC GAGATT-3′ and 5′-CCTTGGTCAGCCCCTTGA-3′) and TaqMan probe (5′-FAM- TGCTGCCGTCTCCAA-TAMRA-3′) according to previous studies , 19. A DIn order to predict the viral genes targeted by miR-1000, two softwares including Targetscan  and miRawsv191, wsv407 and wsv024, the wsv191 3′UTR, wsv407 3′UTR, and wsv024 3′UTR were cloned into the pIZ/V5-His vector  which carried the sequence of enhanced green fluorescent protein (EGFP) gene. As a control, the sequence of wsv191 3′UTR and wsv407 3′UTR matching the seed sequence of miR-1000 was mutated to yield the EGFP-Δwsv191-3′UTR and EGFP-Δwsv407-3′UTR construct. All the recombinant plasmids were confirmed by sequencing.To evaluate the interactions between miR-1000 and the genes Cell culture, transfection and fluorescence assays were carried out as described previously . Insect wsv191 and wsv407, quantitative real-time PCR was performed with the wsv191-specific primers (5′-TTGACGAGGAGGATTGTAA AGG-3′ and 5′-ATACCAGGGTTTATTTTGTTGCG-3′) or wsv407-specific primers (5′-AACCCATTCCACCCCAATATC-3′ and 5′-ATATCTTTGTCGGCCAACTTGTC-3′) as described before . Quantitative real-time PCR reaction mixture contained 5 μl of SYBR® Premix Ex Taq, 0.5 μl of 10 μM forward and reverse primers and 100 ng of cDNA template. The PCR reaction conditions were: 95°C for 1 min, followed by 45 cycles of 30 s at 95°C, 30 s at 52°C, and 30 s at 72°C. For each treatment, quantitative real-time PCR was independently carried out for three times to quantify mRNAs.To determine the mRNA level of Western blot analysis was carried out according to our previous study . Shrimp wsv191 or wsv407 gene were designed, which were designated wsv191-siRNA (5′-CAGTGACGGTAAATTGTGTACCGTT-3′) and wsv407-siRNA (5′-GGGTGG AGAGTTAGAAAGTTGTACA-3′), respectively, and synthesized by the in vitro transcription T7 kit  according to the manufacturer's instructions. To knock down the expression of virus genes, shrimp were infected with WSSV, followed by injection of 15 μg/shrimp synthesized siRNA at 48 h post-infection. The siRNAs were diluted into the 100 μl siRNA buffer . Twelve hours later, shrimp were re-injected with the same amount of siRNA per shrimp. The duration of siRNA was 72 h in shrimp. At different time points after the last siRNA injection, three shrimps were randomly selected per each treatment group and stored in liquid nitrogen for later use. The RNAi assay was biologically repeated for three times.RNAi was conducted according to the procedure described before , 19. To in vitro T7 transcription kit  according to the manufacturer's instructions. After incubation, the mixture was subjected to electrophoresis on 1% agarose gel at 120 V for 30 min. Next, the RNA was transferred onto Hybond-N+ nylon membrane. After ultraviolet cross-linking, membranes were pre-hybridized in DIG Easy Hyb granule buffer  for 0.5 h at 42°C and then hybridized with the DIG-labeled probe  at 42 °C overnight. The detection was done with the DIG High Prime DNA labeling and detection starter kit II (Roche).The mRNA degradation analysis was conducted as described before in our laboratory . To deteThe remaining 3′UTR of the target mRNA after miR-1000-mediated degradation was separated using 1% agarose gel electrophoresis . The remin situ hybridization assay was carried out according to our previous study  to obtain the mean values and standard deviation of biological repeats.To explore the role and the relevant mechanism of miR-1000 during virus infection of shrimp, shrimp were challenged with WSSV, followed by the content detection of miR-1000. As in our previously study .To further assess the relationship between miR-1000 and virus infection, miR-1000 was overexpressed and/or silenced in shrimp, followed by evaluation of the virus infection. The results indicated that when miR-1000 was overexpressed in shrimp Figure , the WSSin vivo.All these data indicates that miR-1000 could reduce WSSV infection in shrimp, suggesting its antiviral activity wsv191 and wsv407) might be the target genes of miR-1000 , but was downregulated in shrimp co-injected with AMO-miR-1000 and wsv191-siRNA or wsv407-siRNA  and synchronously trigger the mRNA degradation of its targets in vivo, leading to the inhibition of virus infection. The results of the present investigation indicated that miR-1000 silencing led to significant increases in WSSV copies and shrimp mortality compared with shrimp injected with only WSSV, while our previous study revealed that the mortality of miR-1000-silencd shrimp was slightly increased (4 copies/shrimp) was used to trigger virus infection of shrimp in the present study. Our findings demonstrated that wsv191 and wsv407 had positive effects on virus infection in shrimp. Given that wsv191 encodes a non-specific nuclease, which can hydrolyze to both DNA and RNA  and virus genes (wsv191 and wsv407). As shown in Figure Our previous investigation indicated that miR-1000, a conserved miRNA in invertebrates, could target the TrCPβ E3 ubiquitin ligase, is suppressed by miR-183 through direct mRNA degradation -specific ribonuclease (PARN) (RNA interference (RNAi) mediated by miRNAs can function in antiviral immune responses via post-transcriptional regulation of gene expression . By bindradation . In HCT1e (PARN) . In the XZ and YG designed the research and conceived the study. YG and CJ performed the experiments. XZ and YG wrote the manuscript.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.


Human challenge trials (HCTs) deliberately infect participants in order to test vaccines and treatments in a controlled setting, rather than enrolling individuals with natural exposure to a disease. HCTs are therefore potentially powerful tools to prepare for future outbreaks of emerging infectious diseases. Yet when an infectious disease is emerging, there is often substantial risk and uncertainty about its complications, and few available interventions, making an HCT ethically complex. In light of the need to consider ethical issues proactively as a part of epidemic preparedness, we use the case of a Zika virus HCT to explore whether and when HCTs might be ethically justified to combat emerging infectious diseases. We conclude that emerging infectious diseases could be appropriate candidates for HCTs and we identify relevant considerations and provide a case example to illustrate when they might be ethically acceptable. Human challenge trials (HCTs), or controlled human infection models (CHIMs), involve deliberately exposing human participants to diseases to learn more about the early stages or transmission of a disease or to accelerate testing of interventions. One prominent example was the yellow fever experiments conducted by Walter Reed and colleagues in the early 1900s that proved that yellow fever was transmitted by mosquitoes , 3. DespAt first blush, HCTs seem ideal to prepare for future infectious diseases epidemics. Consider that the Coalition for Epidemic Preparedness Innovations (CEPI) was recently created in order to “finance and coordinate the development of new vaccines to prevent and contain infectious disease epidemics” . For epiHigh priority diseases for epidemic preparedness include Nipah virus, MERS-CoV, Lassa virus, Ebola virus disease, Marburg virus disease, Zika virus, and Rift Valley Fever . These aIn this paper, we consider the case of a Zika virus HCT as a springboard to determine whether and when an HCT of an emerging infectious disease could be permissible. Through this analysis, we isolate several conditions that might make it ethically acceptable to conduct an HCT on an emerging infectious disease. Recognizing that, for many high priority diseases, an HCT would expose participants to such high risk that it might not be ethically acceptable, we conclude by providing an illustrative case involving a potential outbreak of a Disease X and considering how an HCT might be useful in this scenario.In 2015, Zika virus emerged as a major public health crisis in Brazil and other South American and Caribbean countries. Although the WHO declared the state of emergency surrounding Zika virus over in November 2016, it indicated that Zika is likely to be a persistent and unpredictable public health threat for years to come . Some esIn 2016, researchers proposed conducting an HCT in which healthy volunteers would be intentionally exposed to Zika virus, which was a newly emerging public health threat at the time. Although the protocol for this trial is not publicly available, some of the details are widely known. The Zika virus HCT was proposed to learn more about the early stages of Zika infection and efficiently test whether vaccines can protect against Zika infection. The trial was to be conducted in the United States and enroll healthy volunteers who would not otherwise be exposed to Zika virus. Previous exposure to other viruses in the same family  would have been an exclusion criterion for the study since there is a theoretical possibility of enhancement of disease in patients with antibodies from other flaviviruses. Participants would be confined in clinics for approximately two weeks , 18.As the potential funders of such a trial, the National Institute of Allergy and Infectious Diseases and the Walter Reed Army Institute of Research were concerned that Zika virus HCTs were ethically complex. They therefore assembled an independent, multidisciplinary expert panel to address the question of whether a Zika virus HCT could be ethically justified, and if so, under what conditions. The panel included ethicists with expertise in several subfields of research ethics , a neurologist, two obstetrician/gynecologists, and an infectious disease physician. Panel members were vetted to ensure there were no conflicts of interest. The panel was charged with making recommendations to NIH about the ethical considerations for Zika HCTs in general. Unlike standard ethics review committee or institutional review board processes, the panel members did not review a study protocol. The panel’s deliberations were not meant to supplant existing review processes, but to supplement them. Accordingly, the panel considered the latest scientific and epidemiological information about Zika virus, existing ethical frameworks for HCTs, and ongoing research into Zika vaccines.The panel held several teleconferences and one in-person meeting with presentations from experts from a variety of backgrounds. After extensive deliberation, the panel drafted a report that was released in February 2017. The Zika HCT ethics expert panel offered a preliminary ethical framework for Zika HCTs and concluded that Zika virus HCTs could be ethically justified in principle but would be premature at the time . The twoAlthough these were the most critical and unresolved issues facing Zika HCTs at the time, the panel also noted several other important ethical considerations, including ensuring that there was a robust informed consent process, sufficient but not undue compensation for research participation, a plan to respect the right to withdraw, a system for compensation for research-related injury, and consultation with the community in which a Zika HCT would be conducted—considerations which may be helpful for HCTs more generally .It is important to note that external conditions, the evidence available about Zika virus, and the epidemiology of Zika virus have changed since the panel submitted its recommendations. The period of infectivity for those infected with Zika virus is much better known; Zika virus can be transmitted for roughly 30 days in individuals whose viral loads are not high   crowded field of interventions and ongoing studies and uncertain social value; and (2) potentially high and uncertain risk to participants and third parties if the trial was conducted in the United States.First, as was the case with Zika virus, many stakeholders are likely to be interested in addressing an emerging infectious disease in the midst of an outbreak, making it difficult to determine how much value an HCT would add. Within a year of the panel’s report, it became clear that an efficacy trial would be extremely difficult to conduct giving the declining numbers and unpredictability of Zika outbreaks, and the initial uncertainty about the value of a Zika HCT was resolved . One posSecond, as previously mentioned, emerging infectious diseases that are prioritized have potentially serious complications and are often characterized by high uncertainty. For example, the modes and duration of transmission of Zika virus were not known at the time of the review of the ethics of Zika HCTs, so the risks of transmission to people outside of the study were difficult to determine, let alone mitigate. As has been described elsewhere, the Zika HCT ethics panel struggled with the question of how to justify uncertain risks to bystanders. Third parties outside of the research may not know or give consent to being exposed to risk of infection with Zika virus, and there is limited guidance on how to address risk to research bystanders , 22. We There is limited regulatory and ethical guidance on whether there is an upper limit of allowable risk in research. The Nuremberg Code was one of the earliest codes of research ethics and addresses this issue directly, stating that “No experiment should be conducted where there is an a priori reason to believe that death or disabling injury will occur; except, perhaps, in those experiments where the experimental physicians also serve as subjects” . RelevanFor example, the U.S. Federal regulations merely state that risks must be minimized and justified by the benefits to society, suggesting that any level of risk could be permissible as long as there was sufficient social value and informed consent by the participants . The mos[V] olunteers for high-risk public-health research may be subject to substantial background risk from public-health threats, such as potentially lethal infectious diseases for which effective treatment is lacking. It is thus the incremental net risk from the research that must be assessed against the prospect of public-health benefits from the study results .In contrast, others have not ruled out HCTs on emerging infectious diseases and noted that the way risks should be calculated in the context of an ongoing outbreak might be relevant. Joffe and Miller argue that:Joffe and Miller give the example of an epidemic of avian influenza and note that, just as firefighters or rescue workers facing a large-scale disaster take on higher risks than they would in the ordinary course, so too could research participants .Accordingly, another possible way an HCT on an emerging infectious disease could be ethically acceptable is if the HCT was conducted in a setting where an outbreak was occurring. In the context of HCTs, this is typically referred to as an “endemic setting,” but perhaps a clearer way to describe it is that the participants should be drawn from a group already facing some background risk of infection. This may be a way of reducing the net risk to which research volunteers are exposed, since they are already at some level of risk of having the disease, and the difference is that they now will have near-certainty of being infected.There are potential drawbacks to conducting HCTs in endemic settings, however, but these can be addressed in some cases . First, In sum, if a population could be identified who could give consent and had a likelihood of being largely unaffected by the disease, researchers could be confident that the disease would not spread outside of the research participants, and if the research could be conducted in a setting with a relatively high background risk of infection and careful monitoring throughout the study, then an HCT on an emerging infectious disease could be relatively straightforward to justify ethically (provided of course that standard research ethics criteria were satisfied as well).Although Zika virus was discovered in Uganda in 1947, it did not raise international concern until relatively recently. In 1956, Bearcroft inoculated himself with Zika virus and noted only very mild, transient symptoms . Due to We use a case of a hypothetical neurovirulent non-polio enterovirus to illustrate a possible use of HCT with an emerging infectious disease; this is one way to flesh out the WHO’s description of a “Disease X” that might also be ethically acceptable to study in an HCT Fig.  Additio.Fig. 1EProvided that the general conditions for ethical research are met , this exAs with the example of the Zika HCT, there are several other issues that should be addressed for an HCT on Disease X to be ethically acceptable. Although we lack the space to address them fully, we will touch on two critical issues here. First, the level of payment would have to be sufficient to compensate participants for the amount of time they would contribute to the study and could be relatively high if participants had to be confined to avoid transmission to others for an extended period of time. This may raise concerns about undue inducement—more specifically, that participants might ignore the risks involved —or lie oSecond, there might be justice concerns that the data from an HCT of Disease X would not be used to generate benefits that would be valuable to those who are most affected by Disease X. For an HCT on Disease X to have sufficient social value to justify exposing individuals to risk, it is unlikely that producing a vaccine aimed at travelers rather than those in endemic regions would be sufficient to motivate the study. To the extent that the potential global social value is what justifies conducting the research, researchers and sponsors should endeavor to make the benefits of their research broadly available .Even if the conditions outlined above were not able to be met initially, an HCT might still be an ethically acceptable response down the line. As more information was gathered about the disease and it became difficult to conduct research in the field to license a vaccine, the social value of an HCT would increase.Finally, it is worth noting that the scientific production during a public health emergency is overwhelming. The number of articles on Zika virus registered in the National Center for Biotechnology Information’s PubMed database was 81 between 1952 and 2013, 26 in 2014, 38 in 2015, 1735 in 2016, 1855 in 2017, and 5480 by the beginning of April 2018. Although this can be a potential challenge for ethics review processes to sort the wheat from the chaff, it is also another reason that waiting to conduct an HCT for a relatively short period could result in much more information being available to conduct the HCT safely and in a more targeted fashion.Examining the case of a potential Zika virus HCT reveals that HCTs on emerging infectious diseases could be ethically acceptable under the right conditions. A more comprehensive framework and rigorous review processes are urgently needed , particuAdditional file 1. Reviewers Report


Many plant viruses express their proteins through a polyprotein strategy, requiring the acquisition of protease domains to regulate the release of functional mature proteins and/or intermediate polyproteins. Positive-strand RNA viruses constitute the vast majority of plant viruses and they are diverse in their genomic organization and protein expression strategies. Until recently, proteases encoded by positive-strand RNA viruses were described as belonging to two categories: (1) chymotrypsin-like cysteine and serine proteases and (2) papain-like cysteine protease. However, the functional characterization of plant virus cysteine and serine proteases has highlighted their diversity in terms of biological activities, cleavage site specificities, regulatory mechanisms, and three-dimensional structures. The recent discovery of a plant picorna-like virus glutamic protease with possible structural similarities with fungal and bacterial glutamic proteases also revealed new unexpected sources of protease domains. We discuss the variety of plant positive-strand RNA virus protease domains. We also highlight possible evolution scenarios of these viral proteases, including evidence for the exchange of protease domains amongst unrelated viruses. Eukaryotic RNA viruses have a long evolution history, which is driven by their necessary adaptation to their hosts . VirusesPositive-strand [(+)-strand] RNA viruses have a constrained genome size and have adapted to this limitation by encoding multifunctional proteins and/or by expressing different forms of their proteins using sophisticated expression strategies ,15. One Positive-strand RNA viruses have been divided into two large groups, alpha-like or picorna-like based on the phylogeny of their RdRps . PicornaViral proteases are often multi-functional, playing key roles in multiple steps of the infection cycle. Indeed, they have been described to counteract antiviral defense responses by suppressing RNA silencing or having deubiquitinase activity, facilitate symplastic and systemic long distance movement, aid in viral replication and virion maturity, and promote insect host transmission and retention. For an in-depth discussion of the multifunctional activities of plant virus proteases, we refer the readers to a recent review . In manywww.ebi.ac.uk/merops) for protease clans and families . . 158 resactivity , and theactivity .Aphtovirus, family Picornaviridae) revealed a papain-like fold with Cys51, His148, and Asp163 (replacing the Asn of papain) forming the catalytic cleft [in cis at a single KLK↓GAG cleavage site to release itself  is the main protease and it cleaves the nonstructural polyprotein at four sites -strand RNA virus proteases were identified in the early 1990s and they were based on sequence alignments that confirmed the presence of conserved cysteine and histidine residues ,119. Eluic cleft  -strand RNA viruses also encode cysteine proteases. A putative papain-like protease was initially identified in the polyprotein of potyviruses . Since tin cis at a single site [Potyviridae encode an HC-Pro domain [Bymovirus have a bipartite genome and also encode a related cysteine protease, which is termed P2-1. The P2-1 protease is located in the N-terminal region of the RNA2 polyprotein and it cleaves at a single related cleavage site with the consensus sequence of xG↓ [Like the P1 protease, the helper component protease (HC-Pro) of potyvirids is a minor protease that cleaves the polyprotein gle site ,123    is shorter than that observed in papain or in the leader proteases of animal and human picornaviruses [cis-cleavage mechanism [The multi-functional HC-Pro plays roles in vector transmission and in the suppression of antiviral plant defense responses, notably RNA silencing . HC-Pro aviruses . The cryaviruses  -strand RNA genome (between 15 and 20 kb) amongst plant viruses [Closterovirus) was first characterized in 1994 [509 and His569 to cleave at the single G↓G cleavage site [Viruses in the family  viruses ,127. The in 1994 . It was age site ,129. Simage site ,131. AlsClosteroviridae encode two L-Pros, notably citrus tristeza virus (CTV) and grapevine leafroll-associated virus 2 (GLRaV-2). The tandem proteases probably arose by domain duplication [It is interesting to note that some members of the family lication ,133,134.lication ,134. In lication . It has lication ,134.Tymovirales, Nidovirales and Bunyavirales (an order encompassing a diverse group of negative-strand RNA viruses). It is now well-established that many OTU-like cysteine proteases have deubiquitinase (DUB) activity and cleave lysine-bound ubiquitin units at GG↓K cleavage sites [A new superfamily of cysteine proteases was proposed in 2000 that shares the catalytic Cys and His residues with papain, but has sequence relationships closer to the Ovarian Tumor Domain (OTU) proteins than to papain . Similarge sites .Tymovirus, family Tymoviridae, order Tymovirales) was initially referred to as papain-like, following the identification of the catalytic Cys783 and His869 residues in the mid-1990s [783 was followed by Leu rather than by the aromatic residue normally found in papain-like proteases. Determination of the TYMV protease structure confirmed a fold with more similarities to the yeast OTU DUB than to papain [Alphaarterivirus, family Arteriviridae, order Nidovirales) and the OTU-DUB of Crimean-Congo hemorrhagic fever virus  are strikingly similar, although they also display interesting differences [The cysteine protease of turnip yellow mosaic virus  in the early 1990s with only the C-terminal dyad implicated in polyprotein cleavage [Tymovirales will be necessary to gain a better understanding of their evolution history.The order  modules . The acqe motifs A. Indeedcleavage . The N-tcleavage   were only known to encode a single main protease E IWRF and GWEYQ, respectively. Mutation of two other conserved residues (Gln1180 and Gln1322) reduced the protease activity.Until recently, members of the family 1192, Glu1274, and Gln1180 are brought in close proximity in this model. Concanavalin A-like lectin/glucanases are a superfamily of proteins that share very low sequence identity and are found in a wide range of species, including microorganisms, insects, plants, and animals [Scytalidium lignicolum [136) and glutamine (Gln53) residues that are arranged on opposing beta-sheets [Secoviridae, suggest that it was relatively recently acquired.Homology-based modeling of the SMoV Pro2-Glu catalytic region implied a beta-sandwich structure that is typical of a concanavalin A-like lectin/glucanase fold  Figure B. Althou animals . Most megnicolum . Structua-sheets ,152,153.a-sheets . High-reClosteroviridae) [Closteroviridae incorporate the CPm in the short tail of the virion filamentous particle and the CPm is required for virus transmission by its aphid vector [A putative Pro2-Glu domain was detected in the minor coat protein (CPm) of several criniviruses and velariviruses (family viridae) . Sequencd vector ,155,156.d vector . Competid vector . These oOrtervirales encode aspartic proteases that are related to the cellular pepsin and they are often referred to as retropepsin [25 and Asp25’) [Caulimoviridae  is a family of plant-infecting pararetroviruses. Pararetroviruses share genomic organization, replication strategy, and phylogenetic relatedness with ancestral retroviruses but lack an integrase gene and package DNA instead of RNA into virions [Caulimovirus) was the first pararetrovirus identified to encode an aspartic protease, which has also been implicated in virion maturation and regulation of the viral coat protein nuclear targeting [Reverse-transcribing viruses belonging to the order ropepsin . Retroviropepsin . The aspropepsin . The HIV Asp25’) ,163. The virions . Cauliflargeting ,165,166.Ortervirales. Indeed, a retropepsin-like aspartic protease domain with a conserved DTG sequence was found to be encoded by a plant negative-strand RNA virus [Ophiovirus, family Aspiviridae) is a tripartite virus that contains a protease domain within its movement protein. Mutation of the catalytic Asp residue prevented not only the maturation cleavage of the MP, but also the formation of tubular structures that are necessary for cell-to-cell movement of the virus. Whether the CPsV 20K protease was acquired from a plant reverse-transcribing element or from a cellular protease by parallel evolution is not known. To date, aspartic protease domains have not been identified with certainty in association with positive-strand RNA viruses. Although an early study identified a possible retropepsin-like DSG motif in the polyprotein of a closterovirus (BYV) [Recent findings revealed that aspartic proteases are not restricted to members of the order NA virus . Citrus us (BYV) , a proteus (BYV) , the posSecoviridae. Understanding these protease specificities is critical not only for accurate prediction of viral polyprotein cleavage sites in emerging viruses, but also to identify host protein targets of viral proteases, an area that remains unexplored for plant viruses.Detailed functional and structural analyses of viral proteases have provided new insights into the recurrent acquisition of proteolytic enzymes by viruses and into the adaptation of these protease domains to accommodate and facilitate virus infection cycles. The origin of some viral proteases are ancient, as exemplified by the main chymotrypsin-like serine and cysteine proteases of animal, plant, and lower eukaryotes picorna-like viruses that have limited sequence identities but share conserved catalytic residues and a similar general fold. As detailed in While the large family of chymotrypsin-like viral proteases share a common and ancient origin, other viral proteases have more complex evolutionary histories, which is reflected by the sequential acquisition of diverse protein domains, duplication and/or deletion of protein domains, and in some cases adaptation of non-proteolytic enzymes to attain protease activities . It is cThe recent discovery of an atypical glutamic protease encoded by a plant picorna-like virus  implies