The World Health Organization’s and Centers for Disease Control and Prevention’s definition of Zika infection are symptoms of fever, rash, joint pain, myalgia, headache and conjunctivitis. The diagnosis of Zika infection is based on the clinical history, physical examination and laboratory investigations which includes blood and urine Zika virus Polymerase Chain Reaction.Two patients presented with atypical presentation of Zika infection to Sengkang Health, Alexandra Hospital during the recent Zika outbreak in Singapore in August 2016.Madam A presented with isolated generalized rash with no fever, joint pain, myalgia, headache or conjunctivitis.Mr. B presented with isolated fever of 39.4 °C with no rash, joint pain, myalgia, headache or conjunctivitis.Both patients’ blood Zika Polymerase Chain Reactions were positive at the time of presentation.The described case reports illustrated the challenges that our community Family Physicians faced in diagnosing patients infected with Zika virus. Coupled with the knowledge that most patients are asymptomatic, Family Physicians need to have a high index of clinical suspicion for early identification of patients infected with Zika virus, so as to institute timely treatment and appropriate measures to mitigate the outbreak of Zika infection in the community. Appropriate epidemiological measures such as ensuring prompt and thorough contact tracing of the cases are instrumental in the control of this public health problem. With the recent surge in cases of Zika infection in South America and other countries like Cambodia and Thailand, WHO has declared that Zika virus is currently a global public health emergency. As Singapore is a major transport hub, it is inevitable that cases of Zika virus are imported to Singapore. There is also a high risk of local transmission due to the endemic presence of Aedes mosquitoes in Singapore.In Singapore, MOH has been closely monitoring the Zika virus situation and has introduced several measures to improve the surveillance of the disease and the protection of Singaporeans. Singaporeans are also advised to take precautionary measures to prevent mosquitoes breeding, thus emphasizing heavily on vector control as a critical component. To prevent Zika virus from becoming entrenched in our local population, MOH also undertook strict control measures. All confirmed symptomatic cases, as indicated by positive blood Zika PCR, were admitted to a public hospital until they recovered and were tested negative for the virus. The authorities in MOH also carried out active surveillance, looking for additional cases in high risk geographical regions with areas in relation to the confirmed cases. The first case of locally transmitted Zika virus infection was reported on 27th August 2016.Most patients with Zika virus infection were either asymptomatic or had mild symptoms. According to CDC, the most common symptoms of Zika infection are fever, rash, joint pain, conjunctivitis . Other eAND maculopapular rash AND any of the following:Arthralgia (Joint Pain)Myalgia (Muscle Pain)HeadacheNon-Purulent ConjunctivitisPoint to note:In Singapore, MOH case definition of Zika virus infection is as follows  :IndividAny individual with a recent travel history (within 2 weeks) to Zika-affected areas We propose that the criteria above serve only as a guide and further field studies need to be done to define the case definition of this disease.Madam A was a 53 years old lady, with previous history of total hysterectomy with bilateral salpingo-oophorectomy performed in 2013 for uterine fibroids. She presented with only generalized rash for one day. She did not complain of fever, joint pain, myalgia, headache or conjunctivitis. She also did not have other localizing symptoms suggestive of infection. She noticed a mosquito bite about three days prior to the onset of the rash. She stayed at the Eastern region of Singapore, which was identified as a Zika-affected area in Singapore. She did not have any recent overseas travel history.She came across information about Zika infection from an educational poster and hence sought medical consultation at Tan Tock Seng Hospital Accident and Emergency Department. On examination, she was well and clinically not dehydrated. Her temperature was 36.8 °C, blood pressure was 139/76 mmHg, pulse rate was regular at 82/min, and respiratory rate of 18/min with an oxygen saturation of 98% on room air. A generalized maculopapular rash was seen. Examination of her cardiorespiratory systems were unremarkable. Examination of her abdomen was unremarkable as well, with no evidence of hepatosplenomegaly. There were no lymphadenopathies detected during the physical examination.9/L)]; normal haemoglobin and platelet counts. Dengue NS1 antigen, Dengue IgM and IgG levels were not detected. Her Urea, Creatinine and Electrolytes test and Liver Function Test were normal. Blood and Urine Zika PCR were positive. All these tests were done one day after the appearance of the generalized rash. Diagnosis of Zika virus infection was confirmed and she was admitted to Sengkang Health @ Alexandra Hospital for further management.The initial laboratory investigations revealed a normal white blood cell counts with normal neutrophils and lymphocytes counts, slightly raised monocytes count at 1.14 [normal range: 0.20 - 0.70 ], slightly raised monocytes count at 1.02 [normal range: 0.20 - 0.70 (x109/L)]; normal haemoglobin and platelet counts. Dengue NS1 antigen, Dengue IgM and IgG levels are not detected. His Urea, Creatinine and Electrolytes test and Liver Function Test were normal. Blood and Urine Zika PCR were positive. All these tests were done on the second day of his fever presentation. Diagnosis of Zika virus infection was confirmed and he was admitted to Sengkang Health @ Alexandra Hospital for further management.The initial laboratory investigations revealed normal white blood cell counts with normal neutrophils, slight reduced lymphocytes counts at 0.75 [normal range: 0.90 - 3.30 (x10During his in-patient stay, he was given symptomatic treatment for his fever. He did not develop any rashes, myalgia, arthralgia, conjunctivitis or headache during the first two days of his hospitalization stay. He remained afebrile throughout his hospitalization stay. On the third day of his stay, he developed flushing of both his upper limbs which progressed to a generalised flushing affecting his trunk, limbs and face. His physical examination was otherwise unremarkable, with only the presence of generalised maculopapular rash. Daily Full Blood Counts were performed and his platelet counts remained stable. Daily serum Zika PCR were performed to trend for progression. On the fourth day of his stay, his serum Zika PCR was noted to be negative while urine Zika PCR remained positive. With a negative serum Zika PCR and clinical improvement, Mr. B was deemed fit to be discharged from the hospital. During his stay, he was educated about the Zika virus and its mode of transmission and vector control measures. Advice was also given to seek medical consult should he develop any additional symptoms.The described case reports illustrated the challenges that our community Family Physicians faced in diagnosing patients infected with Zika virus. Coupled with the knowledge that most patients are asymptomatic, Family Physicians need to have a high index of clinical suspicion for early identification of patients infected with Zika virus, institute treatment and measures to reduce the outbreak of Zika infection in the community. Family Physicians play an important role at the Primary Healthcare level in educating the public that vector control remains key to reducing the spread of the Zika virus. Residents should undertake vector control measures regularly to ensure proper housekeeping within their premises at all times and remove any potential mosquito breeding habitats. Residents also need to do their part to prevent mosquito breeding in their homes by doing the five-step Mozzie Wipeout .We believe that there may be more cases of patients that did not present typically and thus did not fit into the WHO and CDC case definitions, and thus might have been missed. Thus, more field reports of the presentation of the disease will be necessary to develop a more robust definition of this disease, especially within the South East Asia Region. The possibility that the Asian strain of the Zika virus may present differently from that of the South American strain needs to be further explored. There are public health implications especially for those couples planning to start families. It will be important to be aware of such atypical presentations of this disease as the child conceived by such unsuspecting mothers may develop serious developmental delays. Judicious allocations of resources can thus be made to uncover such cases who might be pregnant and presented atypically, for screening of birth developmental defects during their pregnancies.


After the largest Ebola virus outbreak in history, experts have attempted to answer how the Zaire ebolavirus species emerged in West Africa and caused chains of human-to-human transmission. The widespread and untimely infection of Health Care Workers (HCW) in the affected countries accelerated spread of the virus within the community. Among the reasons attributed to this trend, it must be considered that HCW were exposed to the virus in their occupational environment. The contribution of environmental conditions to the spread of Ebola in West Africa was examined by investigating the effect of temperature/humidity on the virus’s environmental persistence and by modeling if saturation (liquid stress) allows for penetration of Ebola virus through personal protective equipment (PPE). Ebola-Makona virus persisted on PPE and materials found in outbreak settings for less than 72 hours at 27 °C and 80% relative humidity (RH). A difference in virus penetration was observed between dry  and saturated  samples of PPE. Infectious virus particles penetrated through saturated coupons of Tyvek Micro Clean, Tychem QC, whole surgical masks and N95 respirators. These findings suggest inclusion of saturation or similar liquid stress simulation in protective equipment testing standards. Filoviridae, has caused more than 20 sporadic and lethal outbreaks since its discovery in central Africa during 19762. From December 2013 to March 2016, the virus became an international concern due to the largest known outbreak in six West African nations including Liberia, Sierra Leone and Guinea3.The Ebola virus, a member of the 6. Contemporary studies suggest reconsideration of how the virus behaves and survives in the environment7. Infectious virus particles have been recovered from materials frequently used in clinical settings up to 172 hours8 and in outbreak conditions from 72–240 hours9. Epidemiological reports have cited a risk of acquiring Ebola from environmental sources such as the bodies of the deceased, excrement and contaminated medical equipment10. Climate also appears to heavily influence Ebola virus emergence, as most Ebola epidemics start with the end of the rainy season in December and January when temperature and humidity are both high11. On the front lines of Ebola outbreaks are HCW who volunteer to care for those sick with Ebola virus disease, a disturbingly high incidence of HCW infections was observed throughout the West African Ebola outbreak  which was highest during the record-setting temperatures of early 201412. The average rate of HCW infections peaked to 12.3% of the total during January, February and March, a greater incidence than any other three consecutive month period of the year13. The percentage of HCW infections per total infections decreased over the course of the outbreak suggesting the effectiveness of Ebola virus containment efforts including barrier protection, use of PPE, sanitary burial, epidemiological tracking and field diagnostics3.Until recently, little has been known about how the Ebola virus behaves in the environment outside of a host organism. Experts believed the virus was relatively weak with a short half-life and much environmental sensitivity16. In Ebola treatment units (ETU), personnel wear: rubber boots, a full length Tychem QC suit, a Tyvek Micro-Clean hood, googles, facial mask or respirator and two pairs of nitrile gloves18.The burden of this PPE, low gaseous permeability of the materials and, increased body temperature due to insulation and the retention of heat limit the shift length of HCW from half to a full hour19. Even short shifts do not prevent excessive sweating and heat exhaustion17. Perhaps temperature and humidity contributed to the high incidence of HCW infections during early 2014 as PPE may have become saturated by: perspiration, humidified air, condensation during respiration or bodily fluid exposure during use20. Saturation-thorough wetting- has been identified by the American Society for Testing and Materials (ASTM) as a stressor of protective materials capable of impacting the integrity of their protective barrier and performance21. Ebola virus may penetrate through liquid stressed material increasing the risk of infection.Besides dealing with high temperatures and humidity, aid workers combatting the Ebola outbreak in West Africa were challenged by the occupational risk of fluid-borne virus exposure (i.e. intubation), and having to routinely wear/remove standard Ebola virus PPEWe performed three experiments  to investigate how the Ebola-Makona virus persists outside a host in West African climatic conditions and if saturation with phosphate-buffered saline (PBS) affects the permeability of protective materials (i.e. PPE) worn by HCW responding to an outbreak.The ability for Ebola virus to remain viable on surfaces in average West African climatic conditions 27 °C and 80% RH has implications for fomite-driven transmission and the effectiveness of barrier protection. Ebola-Makona remained viable on all the tested materials between 24 to 72 hours post-inoculation with the exception of gloves <1 hour) and, cotton and goggles (<24 hours). No virus was detected at 72-hours on any of materials  log10 TCID50/mL for the hood and 1.1 (+/−1.7) log10 TCID50 units/mL for the suit  were inoculated with 6.8 (+/−0.4) loguit Fig. . Virus wuit Fig. .Figure 210 units/mL higher than the titre of infectious virus particles  were challenged with virus using a respiratory mannequin. Saturating the masks in PBS, affected the equipment’s permeability to Ebola virus particles. Regardless of mask type, saturation allowed for virus penetration in at least 1/3 biological replicates of the experiment. Virus particles were also detected on the face of the mannequin after removal of saturated KC 200 and KC 300 N95 respirators Fig. . In comples Fig. . The recles Fig. . In case10 units/mL higher than the titer of infectious virus particles , a potential surrogate for Zaire ebolavirus using the same conditions Fig. . Infecties Table . vRNA wa24. The ramifications of the outbreak cultivated our study and the findings suggest reconsideration of how to best protect HCW and others with the risk of Ebola virus exposure in outbreak environments. High temperature and humidity were demonstrated to shorten Ebola virus persistence on the challenged materials in comparison to conditions reflective of a clinical environment in resource wealthy nations7. Notably, virus persisted long enough (>24 hours) to allow for possible fomite driven transmission if contaminated surfaces are not properly identified and disinfected25. Other authors found that material and fluid matrix types complicate defining how temperature and humidity affect Ebola virus persistence. At high temperature and humidity, the virus survives longest in whole human blood9. The survivability of Ebola in whole blood - being longer than that of residual bodily fluids - could lengthen the window of opportunity for the virus to penetrate through material. Liquid stress/saturation of PPE, a consequence of hot humid conditions, perspiration and condensation from respiration, compromised the protection of: spun polyester attire, whole surgical masks and N95 respirators. Ebola virus penetrated through saturated materials at a greater incidence (n = 7/21) than the same materials when dry (n = 1/21) is greater than the perceived infectious dose27. In agreement with our previous work, recovery of Ebola virus RNA does not adequately represent infectious virus particles in stressful environmental conditions 25. Detection of vRNA in the mask penetration studies was more comparable to the titre of infectious virus particles, when they were detected together. Current standards for assessing PPE do not include saturation (i.e. perspiration) as a factor in their testing models. The passage of Ebola virus and the tested surrogate virus VSV-GFP through the examined materials should not be completely unexpected because, many items of PPE are certified to protect against penetration of particles ≥0.1–0.3 µm in size29. Ebola virus particles measure 80 nm (0.080 µm) in diameter and vary in length up to 14,000 nm (14 µm)30. VSV was chosen as an Ebola virus surrogate because it also possesses an envelope and measures similarly (70 nm (0.070 µm) in diameter)31. Both the Ebola and VSV particle sizes fall below the defined minimum pore size for each of the tested masks and the woven polyester fabric used to manufacture Tyvek and Tychem QC materials Tables  and S3. ls Table . Viral p32. Other limitations of our study include not measuring the surface tension of liquids on the material coupons or measuring the volume of PBS each mask absorbed after 30 minutes. We acknowledge that the use of PBS (pH 7) may not accurately represent perspiration of a human being, one of the pre-defined causes of saturation. However, it would be difficult to more closely model how human sweat directly affects the permeability of protective materials, because of the complex interactions between the solute, material and other environmental factors.The conditions applied in this study surpassed the recommendations for PPE use provided by the manufacturers and applied in current standards. Kimberly Clark recommends that neither the KC 200 or KC 300 N95 respirators be used in elevated temperature (>25 °C) or relative humidity (>60%). Dupont does not recommend the use of the Tyvek Micro Clean hood for biohazard protection but for use in controlled environments such as clean-rooms. The ASTM standards, condition fabric samples for a minimum of 24 hours to 21 +/− 5 °C and 30–80% relative humidity19. The use of powered air purifying respirators should be discussed as they provide air circulation and liquid impermeable shielding to the wearer’s face. The face is considered the most vulnerable region of the body to Ebola virus infection due to mucosal membranes of the eyes, nose and mouth30. Even the use of highly protective PPE does not guarantee safety of HCWs or others exposed to Ebola virus because user error (i.e. improper donning/doffing or use of the equipment) may lead to exposure or heighten exposure risk33.The possible penetration of Ebola virus through liquid stressed (saturated) PPE should impact future concepts and strategies of infection prevention and control in tropical environments. The negative impacts of saturation on the protection afforded by PPE should be considered and mitigated. PPE could either be re-designed for increased temperature transfer/gaseous exchange to reduce perspiration or to offer more complete protection13. The contributions of the West African environment also deserve consideration. Teams deployed to the Ebola crisis in Guinea reported that excessive deforestation around ETU made daily temperatures very hot34. Such extreme environmental conditions paired with the physical exertion of work within ETU likely caused HCW to perspire and the build-up of condensation on the inner surface of their equipment. These sources of saturation may have increased the risk of Ebola virus exposure. Although our understanding of Ebola is evolving, many questions remain. Specifically, can the PPE used in Ebola outbreaks be made more safe? And do current testing standards accommodate for the stress of outbreak scenarios?Continued investigation into the high incidence of HCW infections during the Ebola-Makona outbreak in West Africa has attributed numerous factors including lack of suitable PPE and its effective use, virus exposure in the community, reporting bias in epidemiological numbers and propensity for colleagues to treat each other forgoing personal safety36. Masks are evaluated by three ASTM standards  which test penetration of horizontal projection of synthetic blood, bacterial filtration efficiency and particulate penetration using latex spheres39. None of these standard tests factor for saturation/perspiration or challenge the material with infectious Ebola virus. In fact, the ASTM reference document F1671 states that protection afforded by protective material must be assessed on a pathogen-specific basis21. Reviews on the usage of PPE in Ebola virus outbreaks call for more empirical evidence to aid in the selection of appropriate materials40. As a solution they suggest that items of PPE should undergo testing in a BSL-4 facility following ISO or ASTM methodology and challenged with Ebola instead of a surrogate virus17. The bacteriophage Phi-X174 functions as a surrogate for blood-borne viruses in the ISO 16604 standard. We were unable to perform the standard using Ebola virus, because it requires a positive pressure apparatus not currently approved for use in high containment laboratories. Future efforts should aim to complete the aforementioned standards including saturation as a preconditioning factor with the Ebola virus or VSV as a surrogate. The methods of this work follow an amended QCT-2 method published as ASTM E2197 – 11 and by the US Environmental Protection Agency42. Generating knowledge of Ebola in environmental systems particularly the West African climate, will allow for better policy on hemorrhagic fever virus infection prevention control and outbreak management.We propose a new model for testing the protection that Ebola virus PPE affords to the wearer which includes saturation (liquid stressing) of the tested materials in PBS or a like simulant of perspiration or condensation. Spun polyester fabrics used to manufacture PPE such as Tychem, Tyvek are subjected to the ISO 16604, ASTM F1670 and ASTM F1671 as benchmarks of liquid permeability and virus penetrationWe wish to highlight that real world use of PPE may involve complex potentially risk amplifying conditions not recommended by the manufacturer or tested in standards. The experimental model we devised- liquid stress of PPE and challenge with high titre infectious Ebola virus- was intended to demonstrate a worst case scenario. This scenario did not incorporate available strategies to decrease the examined risks such as disinfection and removal of affected equipment. We hope that this work helps policy makers define acceptable levels of liquid stress such as perspiration or condensation during use of PPE.Ebola virus Makona variant stocks (Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05) were propagated in African green monkey kidney (VeroE6) cells (ATCC CRL-1586) within the Containment Level 4 (CL-4) laboratory at the National Microbiology Laboratory (NML) of the Canadian Science Centre for Human and Animal Health (CSCHAH) located in Winnipeg, Manitoba, Canada. When approximately 80–90% CPE was observed (10–12 days post-infection), virus was harvested from supernatants by centrifugation: 5000 × g for 10 min and 108,000 × g for 2 hr with a 20% sucrose cushion. Stocks were prepared by suspending pelleted virus from the first centrifugation step (un-concentrated stock) or second centrifugation step (concentrated stock) in in Dulbecco’s modified eagle medium (DMEM)(HyClone) containing 2% Fetal Bovine Serum (FBS)(Gibco) and 1% antibiotics (Penicillin-Streptomycin) (Gibco) and stored at −80 °C. The concentrated stocks were used for the environmental persistence and PPE penetration experiments; the non-concentrated stocks were used for the mask protection experiment.Carriers measuring 1.5 inches in diameter were prepared from PPE and environmental materials, they were gamma-irradiated (1 MRad) and placed inside 12 well tissue culture plates (Corning) prior to inoculation. The PPE materials included: Tyvek Micro-Clean hood and mask (Dupont) the mask was removed, Tychem QC suit (Dupont), Sol-Knit 39–124 green nitrile-coated gloves (Ansell), Flex-seal goggles (Uvex), KC 300 Fluidshield N95 particulate filter respirator including the fluid-shield layer (Kimberley-Clark) and Hazmax Kneeboot boots (Onguard). The environmental materials included: cotton-polyester pillow case, maple wood with an epoxy (CSCHAH), plastic and stainless steel (CSCHAH).et al. 20157) to accomplish this an organic soil load composed of 12.5 μL 5% bovine serum albumin (Sigma), 17.5 μL 5% tryptone (Becton Dickinson) and 50 μL 0.4% bovine mucin (Sigma) was mixed with 125 µl of Ebola-Makona. Using a positive displacement pipette, 10 µl of the virus-soil load mixture was added to each material in triplicate and an additional 10 µl added to 990 µl DMEM (2%FBS/1% antibiotics) as a positive control. The carriers were incubated at 27 °C with 80% relative humidity supplied by an electronic humidifier (Cigar Oasis) and measured daily using a digital hygrometer (Xikar). At the desired collection time point, virus was eluted from each carrier type by repeated scraping and washing with 990 µl DMEM (2% FBS/1% antibiotics) and stored at −80 °C for processing for TCID50 and qRT-PCR quantification.The virus inoculum was designed to mimic virus contained within residual bodily fluids from an infected Ebola patient (summarized previously Cook 50 assay (500 µl) and qRT-PCR (140 µl).Carriers 2–3 inches in diameter were prepared from Tyvek Micro-Clean hood (Dupont) with the mask removed, Tychem QC coveralls (Dupont) and KC 300 Fluidshield N95 particulate filter respirator including the fluid-shield (Kimberley-Clark). The carriers were gamma-irradiated (1 MRad) and placed inside of 6 well tissue culture plates (Corning). The saturated samples were soaked in sterile PBS (pH 7) for 30–45 minutes prior to testing. Ten µl of the soil load-virus mixture was added with a positive displacement pipette onto each PPE type, in quadruplicate, independent experiments. As an positive (inoculation) control, 10 µl of Ebola-Makona was added to 990 µl of DMEM (2% FBS/1% antibiotics). After 30 minutes of incubation, each sample-type  was grouped and processed on the lid of a 6 well tissue culture plate. Using sterile forceps all the samples were moved to clean 6-well plates and swabbed on either the inoculated surface-as a control (recovery control)- or the unexposed side. Swabbing was performed with dry rectangular foam swabs , both sides of the swabs were used to maximize virus recovery. Virus was eluted from each swab in 990 µl of DMEM (2% FBS/1% antibiotics) within a 14 mL Falcon tube (Corning). The tips of the swabs were expunged on the inside of the tube three times before removal. The swab supernatants were processed for TCIDThree types of facial protection were challenged with Ebola-Makona virus in dry and saturated conditions. A flat surgical mask  and two duck-bill N95 respirators KC 200 and KC 300 (Kimberly Clark) were included in the study, the KC 300 respirator was actively used during the Makona outbreak, the other two masks or similar designs may have been available in resource limited areas. Six masks of each type were packaged and gamma-irradiated at 1 MRad, before use in CL-4. Dry masks were fitted directly onto the face of a mannequin  capable of simulating human respiratory action. Saturated masks were placed in 1 L of sterile PBS and left to absorb liquid for 30 minutes. Four 200 μl inoculations of Ebola-Makona stock were added to the outside surface of the dry or saturated masks in the four quadrants of a cross centering on the nose. The same volume of virus 4 × 200 μl was titrated to serve as a positive (inoculation) control. The mannequin was programmed to breath at a rate of 20 breaths per minute for 30 minutes. Each inhale/exhale moved 0.5 L of air, approximately the average tidal volume of human adult lungs. At the end of the breathing cycle, the masks were removed from the face of the mannequin. The inside surface of the mask and face of the mannequin were dry swabbed with sterile foam swabs . The swabs were treated identically to those used in the PPE penetration experiments. The experiment was conducted in three biological replicates for each item of facial protection in the dry and saturated test conditions.Dry and saturated surgical masks were also challenged in CL-2 using Vesicular stomatitis virus genetically modified to express green fluorescent protein as an Ebola virus surrogate. VeroE6 Cell tissue culture positive for VSV-infection were visualized using a confocal microscope (Zeiss Axiovert 40 CFL fitted with a HB0 50 light source).2. After virus absorption, 150 µl of DMEM (2% FBS/1% antibiotics) was added and the cells were incubated at 37 °C/5% CO2. Monolayers were scored for CPE on 7 and 14 days post-infection for Ebola and 1 and 2 days for VSV-GFP, titres reported as Log 10 TCID50 Units/mL were determined using the Reed and Muench formula8.Virus supernatants from the environmental persistence, PPE penetration and mask protection experiments were 10-fold serially diluted and 50 µl of each dilution in triplicate was used to infect 80% confluent VeroE6 monolayers in 96-well culture plates for 1 hour at 37 °C/5% COZaire ebolavirus species. The conditions of the PCR reaction were programmed to: 61 °C for 3 min, 95 °C for 30 sec, 40 cycles of 95 °C for 15 sec and 60 °C for 40 sec. VSV-GFP RNA was detected by using a primer probe set targeting the nucleoprotein region . The same thermo-cycling conditions were used.RNA was extracted from viral supernatant using commercial reagents  and quantified by qRT-PCR . Ebola virus RNA was detected by use of a 2:1 primer probe mix  specific for et al. (C = Q × VDNA/VPCR× 1/VEX× 1000)43. A paired T-test was used to analyze the results of the mask protection experiment in Fig. Genomic equivalents per millilitre were calculated using the formula published by Chiu Supplementary Information


Corona viruses cause common cold, and infections caused by corona viruses are generally self-resolving. During the last 4 years, corona viruses have become the most important viruses worldwide because of the occurrence of several recent deaths caused by corona viruses in Saudi Arabia. Spread of the infection occurred worldwide; however, most cases of mortality have occurred in the Middle East. Owing to the predominance of outbreaks in the Middle Eastern countries, the virus was renamed a Middle East respiratory syndrome corona virus (MERS-CoV) by the Corona virus Study Group. The Center for Diseases Control and Prevention and World Health Organization maintain a website that is updated frequently with new cases of MERS-CoV infection. In this review, we describe the history and epidemiology of this novel virus. Studies of the genetics and molecular mechanisms of this virus are expected to facilitate the development of vaccines in the future. The first cases of corona virus infection in Saudi Arabia, specifically Jeddah, were reported on June 13, 2012; after this outbreak, corona virus continued to spread overseas to many countries in Asia, Africa, Europe, and America . During The corona virus responsible for this outbreak was a novel virus that mainly affected adults. The transmission mechanism and potential treatment strategies were still unclear. Notably, although this virus initially appeared to only affect adults, cases have also been observed in pediatric patients. Thus, this novel, potentially fatal virus represented a substantial public health risk. In this review, we discuss the history, epidemiology, and molecular mechanisms of this novel virus, called the Middle East respiratory syndrome corona virus (MERS-CoV).This study was scheduled and directed to be adhered to PRISMA standards of quality for reporting meta-analysis. We used PRISMA 2009 checklist in writing all parts of the article. It was a meta- analysis.Article discussed the corona virus history and epidemiology.Case reports or studies involving patients who were exposed to corona virus.All age group.Article published within last 4 years.Suspected case with no proves.The two authors independently screened all titles and abstracts, evaluated both the full-text of eligible publications and the risk of bias of included studies, and extracted data Table .Table 1PSearch in Google.Search in PubMedEvaluated the papers, divided them depending on the inclusion and exclusion criteria.Evaluated the articles and analyzed them.The authors searched the topic through the following stepsWe searched through Google, Pub Med, Pub Med Central, CAS, Citebase, DOAJ, Embase, Embiology, MEDLINE, OAIster, SCImago, Scopus, SOCOLAR and Zetoc by using the keywords.www.robis-tool.info) and (www.jclinepi.com). The risk of bias was low . Until 2002, corona virus was considered a relatively simple, nonfatal virus; however, an outbreak in 2002–2003 in Guangdong province in China, which resulted in spread to many other countries, including Thailand, Vietnam, Taiwan, Hong Kong Singapore, and the United States of America, caused severe acute respiratory syndrome (SARS) and high mortality rates in over 1000 patients. After this outbreak, microbiologists and infectious disease experts focused on the understanding the pathogenesis of the disease and discovered that this infection was caused by a new form of corona virus. A total of 8096 individuals were infected with this virus, resulting in 774 deaths; thus, in 2004, the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) declared a state of emergency [Corona virus is a single-stranded, enveloped RNA virus 1 that is spherical or pleomorphic in shape with bear’s club-shaped glycoprotein projections. There are subtypes of corona virus, alpha corona virus, beta corona virus, gamma corona virus, and delta corona virus, and each subtype has many serotypes. For example, OC43-like and 229E–like have been shown to affect humans, whereas the other types mainly affect animals , and multi-organ failure, which can result in death. The CDC recommends collecting multiple specimens at different times from different sites; for example, samples should be collected from oropharyngeal and nasopharyngeal swabs, sputum, blood, and stool and rectal swabs, as well as the lower respiratory tract, which is most frequently positive for the virus.Typically, samples will be processed by real-time reverse transcription (rRT)-PCR to analyze the following sequences: upstream of the E protein gene (upE), open reading frame 1b (ORF 1b), and open reading frame (ORF 1a), which is the most sensitive sequence for identification of the specific virus. RNA dependent RNA polymerase (RdRp) and N genes are also target sites in the MERS-CoV genome. To confirm the diagnosis, one of the following criteria must be present: (1) a positive PCR result for at least two different specific targets in the MERS-CoV genome or (2) one positive PCR result for a specific target on the MERS-CoV genome and an additional different PCR product confirming a known sequence of MERS-CoV. The algorithm for diagnosis is as follows: (A) upE-specific rRT-PCR – if positive, confirm with ORF 1a rRT-PCR analysis, and, if positive, the case is confirmed; (B) upE-specific rRT-PCR – if positive, confirm with sequencing of one of the two target sites (RdRp or N assay), and, if positive, the case is confirmed. False negative results can occur due to issues with specimen collection, including early or late collection or shipping/handling problems –23.In June 13, 2012, the first reported case of MERS-CoV 1 occurred in Jeddah, Saudi Arabia. This outbreak resulted in many cases of infection, particularly among health care workers who were in direct contact with the patients, indicating that the virus could be transmitted from human to human via air droplets. This prompted strict contact precautions, such as isolation and the use of personal protective equipment, i.e., gloves, N 95-typemasks, and gowns. In Saudi Arabia, many cases of MERS-CoV were reported in almost all provinces, with Jeddah, Makka, Riyadh, and Al-Hassa being the most commonly affected cities. Additionally, many outbreaks were reported in hospitals, causing affected hospitals to close down and not to accept any infected patients. Most of the patients who died were immunocompromised, having conditions such as chronic renal failure, congestive heart failure, and diabetes or having recently received organ transplants . After the initial outbreak in Saudi Arabia, MERS-CoV was reported in several other countries, including Qatar, Bahrain, Kuwait, Jordan, and Tunisia. According to the Minister of Health in Saudi Arabia, from June 13, 2012 until December 2015, a total of 1227 cases of MERS-CoV have been reported, with 728 recovered, one still under treatment, and 549 expired due to MERS-CoV-related symptoms. The Minister of Health, primary healthcare facilities, and public health officials immediately sought to educate the population in Saudi Arabia through internet, lectures, and brochures and continued to carefully record each new case in order to improve knowledge and therapeutic strategies for this virus. The latest report from the WHO on December 7, 2015 showed that MERS-COV has been identified in 26 countries, with 1621 confirmed cases and 584 deaths globally. Notably, the highest numbers of both reported cases and deaths have been in the Saudi Arabia , 25.Pipistrelluspipistrellus, P. nathusii, P. pygmaeus, Nycteris, and Neoromiciazuluensis, and 5.3–24.9% were found to be positive for MERS-CoV, with most positive results (> 70%) being identified in fecal samples with high viral loads [P. kuhlii, Eptesicusbottae, Eidolon helvum, and Rosettusaegyptiacus. Thus, although there was a positive association between bats and corona virus infection, there was no association between bats and MERS-CoV. Therefore, these data have suggested that MERS-CoV is not transmitted through bats [Corona virus as known to be a zoonotic virus; however, the MERS-CoV is a novel virus, and whether zoonotic transmission occurs is not clear yet. International studies carried out from 2012 to 2014 in Mexico, European countries , Ghana, and South Africa have examined whether bats may be carriers of MERS-CoV. These studies have tested bats mainly for the 329-bp fragment of RdRp using blood, fecal, and oral samples , and treatment with antibiotics in the case of bacterial super infections. However, such treatments may not be sufficient for MERS-CoV infections, which may be more severe. Ribavirin and interferon alpha have been shown to have synergistic effects and are more beneficial when started early. Additionally, mycophenolic acid has been shown to be efficacious and can be used as a monotherapy; however, initial clinical trials included few patients, and further studies are necessary –49. AlthMERS-CoV is a lethal zoonotic virus that originated in the Middle East. The main source of transmission, as has been shown in several studies, is through camels. Therapies are still under development and include ribavirin a, interferon alpha, and mycophenolic acid. Further studies are underway to develop an effective vaccine for MERS-CoV aiming to reduce the incidence and mortality rate of infection with this virus.


Background: Incidental findings of virus-like particles were identified following electron microscopy of tissue-engineered tendon constructs (TETC) derived from equine tenocytes. We set out to determine the nature of these particles, as there are few studies which identify virus in tendonsper se, and their presence could have implications for tissue-engineering using allogenic grafts.Methods: Virus particles were identified in electron microscopy of TETCs. Virion morphology was used to initially hypothesise the virus identity.  Next generation sequencing was implemented to identify the virus. A pan herpesvirus PCR was used to validate the RNASeq findings using an independent platform. Histological analysis and biochemical analysis was undertaken on the TETCs.Results: Morphological features suggested the virus to be either a retrovirus or herpesvirus. Subsequent next generation sequencing mapped reads to Equid herpesvirus 2 (EHV2). Histological examination and biochemical testing for collagen content revealed no significant differences between virally affected TETCs and non-affected TETCs. An independent set of equine superficial digital flexor tendon tissue (n=10) examined using designed primers for specific EHV2 contigs identified at sequencing were negative. These data suggest that EHV is resident in some equine tendon.Conclusions: EHV2 was demonstrated in equine tenocytes for the first time; likely fromin vivo infection. The presence of EHV2 could have implications to both tissue-engineering and tendinopathy. Sparsely distributed fibroblasts (tenocytes) produce ECM. Tenocytes are utilised in the production of tissue-engineered tendon constructs (TETCs), which are used within tendon research as a model forin vivo tendons, and have been explored as a potential therapeutic modality for tendinopathy3.Tendons transfer force from muscle to bone, in addition to playing a key role in positioning and reducing locomotion expenditure, such as in the equine superficial digital flexor tendon (SDFT). Their structure is hierarchical4. This study in broiler hens suggests that a primary viral infection coupled with subsequent bacterial infection leads to catastrophic musculoskeletal infection and death.There are few causes of musculoskeletal disease which have been attributed to viral infection, with no previous literature citing viral causes of tendinopathy in the horse. Previous viruses implicated in tendinopathy of other species include adenovirus and reovirus in tenosynovitis of broiler henset al.5 describe NGS as a high throughput, highly sensitive method for transcriptome analysis. We have previously used it to characterise transcriptome-wide gene expression in numerous studies, including tendon ageing and disease6. Additionally, it has infrequently been used to identify viral isolates within tissues7 with a high sensitivity.Next generation sequencing (NGS) is frequently used as a research tool to characterise the transcriptome of eukaryotes, providing superior profiling to previously used methods such as microarrays. WangIn this study equine TETCs were produced to report changing protein profiles with ageing. Following electron microscopy, two samples were found to contain virus-like structures. The aim of this study was to identify these particles using NGS and investigate their functional consequences on TETCs.All reagents were supplied by Sigma unless otherwise stated.Equine SDFT was collected from young  and old  donors from the equine hospital and abattoir as a by-product of the agricultural industry. The Animal (Scientific Procedures) Act 1986, Schedule 2 does not define abattoir collection as a scientific procedure and hence ethical approval was not required. Samples collected from an equine hospital were subject to the University of Liverpool ethical approval and consent (VREC462).8. Constructs were engineered as previously described by Kharazet al.9. In brief, tenocytes were seeded at 600,000 cells per well then scored every other day until full contraction of the construct. Constructs were harvested at 28 days and either snap frozen, collected for standard histology or transmission electron microscopy (TEM).Tenocytes were digested from equine SDFT using standard collagenase protocolTEM of tendon constructs was performed following fixation in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 8 hours, followed by buffer washing procedures and second fixation and contrast stain with 0.1% osmium tetroxide for 90 minutes. Samples were stained with 8% uranyl acetate in 0.69% maleic acid for 90 minutes, dehydrated in ascending ethanol concentrations and embedded in epoxy resin. Ultrathin cross-sections (60–90 nm) were cut with a Reichert-Jung Ultracut on an ultramicrotome  using a diamond knife. Sections were then mounted on 200 mesh copper grids and stained with ‘Reynold’s Lead citrate’ stain for 4 minutes. Images were viewed in Philips EM208S Transmission Electron Microscope  at 80K.10 and counts made of virus with:On examination of TEM images, two TETCs were identified as containing virus-like particles . These particles were morphologically assessed for virion identification. Particle diameter was estimated from electron micrograph images. Particle morphological characteristics were observed for virion diameter, presence of a viral envelope, nucleocapsid shape and surface projection presence. The number of full capsids, nucleocapsids and empty capsids were counted in 22–26 TEM images from each of the infected donors. For each sample, resin sections were mounted onto copper grids and viewed in the TEM at X 44000 magnification. 25–30 successive grid squares were viewed and the first area in each grid square which was found to contain virus was photographed. The images were loaded into ImageJ (version 1.51n)i) envelope + nucleocapsid + DNA (full)ii) envelope + nucleocapsid – DNA (nucleocapsid only),iii) envelope only (empty).TEM images were compared to current literature in order to provide a morphological reference .11, and shown inTETCs were fixed in 4% paraformaldehyde and paraffin embedded. Sections were cut at 4µm onto polylysine slides and subsequently stained with haematoxylin and eosin (H&E) and Masson’s Trichrome. Histology was assessed using a scoring system developed in an unpublished report by Charterset al.6. Analysis was undertaken by the Centre for Genomic Research, University of Liverpool. One µg of total RNA was ribosome depleted with the RiboZero Magnetic kit . NGS libraries were prepared using the ScriptSeq v2 NGS Library Preparation Kit . All of the enriched material was used as input material and following 15 cycles of amplification, libraries were purified using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser . Libraries were pooled in equimolar amounts and quantity and quality of each pool assessed by using Bioanalyzer  and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa (KK4854) on a Roche Light Cycler LC480II according to manufacturer’s instructions. The template DNA was loaded at 300 pM. The sequencing was carried out on one lane of an Illumina HiSeq4000 at 2×150 bp paired-end sequencing with v1 chemistry.Two samples of TETCs were submitted for RNASeq, one virally affected (V: Y1) and one control (NV). RNA was extracted from constructs as previously described, and NGS was performed in accordance with the method described by Peffers12. Coverage plots were created using R. Initially a reference sequence file was created using known herpesvirus genomes. Reads were then aligned to the reference sequence files using Bowtie2 global alignment. PCR and optical duplicate reads were subsequently removed using Picard. Resulting alignment files were then used as input to Bedtools, (version 2.16.2) which was used to calculate coverage across the genomes. The coverage data output of this was used as input for a custom R script. Once the reads were mapped the number of reads mapped to each transcript was undertaken. These counts were calculated using HTSeq-count (version 0.6.1p1)13, accepting only hits of quality 10+, and excluding ambiguous hits and hits on the opposite strand.Initial processing and quality assessment was undertaken as previously described14.Illumina sequence reads were adapter- and quality-trimmed using Cutadapt version 1.2.1  and Sickle version 1.200. Host sequences were removed by aligning trimmed reads to the Equus caballus genome  with HiSat (version 2.0.3b)15) search of the assembled transcripts against the NT database from NCBI was carried out and full taxa information  was derived based on the best hit, using the NCBI taxonomy database (ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdump.tar.gz). The search was carried out with an e-value cut off of 1e-5. The results were filtered to identify all hits to the virus superkingdom.To taxonomically assign contigs, a BLASTN All raw read data produced in this study has been submitted to the EBI ENA, primary accession PRJEB20552, secondary accession ERP022713.16.Freeze-dried samples (V (n=2) and NV (n=5) of TETC derived from the young donors were hydrolysed overnight at 60°C using papain reagent. Digests were stored at -20°C and subsequently assayed for collagen content by hydroxyproline assayP≤0.05. All normality testing and statistical analysis was undertaken using Excel  and GraphPad prism .Data for the hydroxyproline assay were normality tested prior to statistical analysis. Data was considered statistically significant atAs RNAseq is not definitive, we used a pan-herpes PCR to confirm that the virus in question was indeed a herpes virus and there were no other herpesviruses present.et al.17. This protocol uses dI-substituted primers that offer improved sensitivity and specificity than previous protocols18.In order to validate the RNASeq findings with a different platform, a pan-herpesvirus PCR was undertaken. Samples V; Y1 and Y6  and a negative control (O3) were tested using a pan-herpesvirus PCR using a modified methodology described by EhlersModifications of the components included using 12.5ul of 2x Qiagen multiplex PCR master mix containing HotStar Taq DNA polymerase . The list of primers used in pan herpesvirus PCR is available inIn order to determine the presence of EHV-2 in SDFT of a larger equine population, PCR primers were designed to amplify a region within a 37kb contig demonstrating an excellent depth of coverage (mean 4919x coverage) and 99% homology to EHV-2. The primer sequence was; V1 forward GGGCGGAGAATGTAGAGACG, V1 reverse GGTGGACTTTAACGGGGAGG (product size 443). DNA was extracted from 10 (mean ±SD age: 12.8±7.1) grossly normal equine SDFT collected from an abattoir. A QIAmp DNA extraction kit  following the manufacturer’s protocol was used. A sample of EHV-2 positive DNA  was used as a positive control.PCR was performed in a final volume of 50ul consisting of 12.5ul ThermoPrime 2x ReddyMix PCR Master Mix, 1.5ul forward primer, 1.5ul reverse primer, 24.5ul water and 10ul extracted DNA template. Following an initial denaturation at 94°C for 9 min, products were amplified by 5 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and elongation at 72°C for 1 minute. This was immediately followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for one min and elongation at 72°C for one minute. Amplification was followed by a final extension at 72°C for 7 min. 15µl of product was electrophoresed on a 1.5% 1× Tris-acetate-EDTA agarose gel containing 10ul of PeqGreen. TrackIT 1Kb Plus DNA ladder was used as the standard.All phylogenetic analysis was carried out within MEGA (version 5). DNA sequences were aligned using Clustal W . Neighbour-joining trees constructed. Bootstrap analysis  was used to provide support for individual nodes.Two out of 13 samples contained virus-like particles. The virus-like particles identified were approximately 100–150nm in diameter, and had an indistinct outer border with surface protrusions and a cylindrical nucleocapsid . Viral pThere were no significant differences in histological score between V and NV based upon the characteristics measured .Contrasting V and NV TETCs showed that there was no significant difference in collagen content .NGS analysis: Transcript assembly and removal of host genomic reads. Illumina sequence reads were adapter- and quality-trimmed using Cutadapt version 1.2.1  and Sickle version 1.200. A summary of raw and trimmed sequence data is shown in19 for both samples. V resulted in 913,443 transcripts (including isoforms) and NV resulted in 788,076 transcripts (including isoforms).NGS analysis: Viral sequence determination. Y6 had 131 transcripts that hit viral sequences, 129 were assigned to Equid herpesvirus 2, a single read was assigned Equine Infectious Anaemia (EIA) virus and a single read to Bovine viral diarrhea virus (BVDV). For Y6 84.25% reads were mapped to the Equus caballus genome and 1.32% to Equid herpesvirus 2 strain G9/92 complete genome ; the strain that most reads mapped to in this sample. Identified gene transcripts are inNGS analysis: Latency associated transcripts. Two different types of mapping were undertaken in an attempt to identify latency associated transcripts. Firstly a megablast run against the entire nt database to get taxonomic information, and secondly a blastx run against a database created of all know ERV genes in animals from NCBI (these sequences only appeared for human in NCBI). We assessed the two sequences in NCBI that result from a search for 'latency associated transcript and herpes' and ran a blastx for both samples against these. Neither resulted in any significant hits.20 search against all animal ERV sequences from the NCBI database was carried out to check that the contigs suggested to be herpesvirus were not ERVs. No significant hits to known ERVS were found for contigs assigned as EHV2 in the previous search.O3 had 7 transcripts aligned to virus sequences, all assigned Equid herpesvirus 2. A further BLASTNNGS: Genome coverage plots of discovered viral sequence against EHV2. Coverage plots of the samples were checked against the following genomes; Bovine herpesvirus type 1.1 , Equid herpesvirus 1 strain T953 (KM593996.1), Equid herpesvirus 2 strain G9/92 (KM924294.1), Equid herpesvirus 3 strain AR/2007/C3A (NC_024771.1), Equid herpesvirus 4 (NC_001844.1), Equid herpesvirus 5 strain 2–141/67 (NC_026421.1). From these plots for samples NV (O3) (NV (O3)  and V (YNV (O3) , it is cThe coverage plot shows that the genomic coverage of the TETC virus of EHV2 strain G9/92 was 86.26%. The virus was mapped to 3,875.605 contigs to EHV-2, compared with 2–339 from the rest of the herpesvirus panel .Pan herpesvirus PCR. Pan herpesvirus PCR was found to be positive for EHV2 for the two V samples and negative for NV samples  demonstrated the close relationship between the virus in this study and the currently sequenced strains.et al.26 studied the presence of feline herpesvirus-1 (FHV1) within the feline tendon following experimental inoculation. It has previously been described that tissue-engineered tendons can be used as allographic transplant agents27. They implied that the presence of FHV1 for application of TETCs in allogenic transplantation could lead to immune rejection of the engineered tissue and hence transplant failure. Whilst the histological structure and collagen content of the TETCs was not altered by the presence of EHV2, the ability of EHV2 within TETCs here to survive culture and multiple passages whilst retaining the ability to reproduce, may have implications for their use as a therapeutic option in terms of transplant rejection.Swensonet al.28 describe the persistent infection of equine dermal fibroblasts with EIA. Further virus isolated from fibroblasts include herpes simplex virus within avian fibroblast cells. Stulberget al.29 discuss the growth of herpes simplex virus within cultured fibroblasts, illustrating a cytopathic effect upon cells cultured using a non-plasma technique. Such areas of focal necrosis were not evident in the TETCs here.In the literature, isolation of primary virus within tenocytes is limited, though fibroblasts from other anatomical locations have demonstrated the ability for viral replication. Klevjer-Anderson4. The study investigated outbreaks of tenosynovitis in commercial broilers using virus isolation. Tendon from affected flocks yielded isolation of reoviruses and adenoviruses. To our knowledge, viral agents have not previously been isolated from tendons in horses. Zenget al.30 used TEM to visualise adenovirus particles. Their TEM images suggest that adenoviruses are much smaller than the particles isolated in this experiment, with the approximate diameter being 70–90nm. A similar result was evident with reoviruses’, which have an average diameter of 60–80mm.Viral causes of tenosynovitis have previously been described in chickens32. Membrane filtration removes many contaminants, however these may be an ineffective against viruses due to their small size33.Whilst all culturing was undertaken in sterile conditions the identified virions could have originated from a culture contaminant. Likely causes include cross-contamination between TETCs and other tissue within the laboratory or use of contaminated reagents during production. Previous studies have found viral contamination in commercially available foetal calf serum such as that used in TETC production here. However, as the virus was limited to a subset of cultured TETC this is unlikely. Viral isolates include BVDV, bovine parainfluenza virus-3 and bovine herpesvirus-1 (BoHV1)34. The single read to BoHV1 when compared with EHV2 (129) suggest that BoHV-1 was not the virus isolated here. Moreover, the BoHV1 read could be a consequence of cross mapping. One read was found to EIA. This is a notifiable disease within the UK. The single read was of only 52bp hence it was suspected due to cross-mapping and further investigation was not pursued.It is important to consider host-range and tissue specificity of viruses, which could be present as a contaminant of tissue culture reagents. Foetal calf serum was used in the tissue-engineered culture system. As a result, we compared the viral reads obtained to a variety of bovine virus’ with suggestive morphology. One read to BoHV1 was identified in virally affected TETC. As an alpha-herpesvirus BoHV1 has previously shown limited scope for crossing species-barriers35. One might predict that it is not surprising to identify EHV-2 in tendon but perhaps more surprising that it was identified in only two of 13 samples. A number of studies have described equine gammaherpesviruses such as EHV-2 in peripheral blood mononuclear cells (PBMCs)36. EHV-2 is latent in B lymphocytes and direct cell to cell contact between epithelial cells and B lymphocytes is required for the production of infection37. In a study of Icelandic horses of EHV-2 in PBMCs and a selection of tissues using a co-cultivation technique 80% of horses examined demonstrated infectious replicating virus38. These studies suggest that EHV-2 or cells containing the viral genes could have been present in a high proportion of tendon taken. Whilst every effort was made to remove tendon from the lower limbs during dissection in a aseptic manner cross contamination by other infected or latently-infected cells cannot be ruled out. Thus it is possible the tendon derived EHV-2 came from cell to cell contact of either PBMC or other donor cells and that this was the route of infection. However of the two infected samples (both from young donors) one was from the abattoir collection and one from the equine hospital. It would have been expected that more than one abattoir sample would be affected if this was the case as these were harvested, handled and processed at the same time.One additional potential explanation for the presence of EHV-2 in the samples is the ubiquitous nature of EHV-2 in both foals to adult horsesWhilst the PCR for EHV-2 on tendon did not find any viral DNA this test cannot confirm lack of presence of latency associated transcript RNA. We did not identify the presence of latency associated transcripts in the RNAseq data. When the two sequences in NCBI that result from a search for 'latency associated transcript and herpes' were used neither resulted in any significant hits. Thus whilst latency associated transcripts cannot be ruled out with data currently available this is unlikely. However in order to confirm this a more in-depth analysis than this would require further research in latency associated transcripts linked to herpes in other animals which is beyond the scope of this study.39. Moreover, the equid herpesvirus most associated with myeloencephalopathy is EHV140. NGS data in this study revealed minimal coverage of the EHV1 genome when compared with EHV-2. There was no clinical history of the TETC donor as this was derived from an abattoir and hence further conclusions cannot be drawn. Since a small population of other horses were assessed for the presence of EHV2 RJW 248419 it would appear that the presence of this virus in equine tendon is uncommon.Unfortunately the EHV status of the donor horses was unknown. The clinical history for one of the TETC donors containing virus was available. The donor was presented at the hospital with right hind lameness and proprioceptive deficits. The horse was euthanased on humane grounds. EHV myeloencephalopathy was not suspected on presentation due to the absence of cauda equina signsFinally, an interesting point to consider is the potential role of an equid herpes virus in tendinopathy. EHV2 has not previously been isolated in cases of musculoskeletal disease in the horse. Whether this is because it has not been investigated or because it has no significant role in disease has not been established. Due to the unknown status of horse from the abattoir it is difficult to draw conclusions from the single case in which hind limb pathology was described. Whilst histological examination and limited biochemical tests within this study implies that EHV2 has no significant affect upon the structure of TETCs the mechanical properties of the TETCs were not assessed. Further work is required to determine if EHV-2 has a role in tendinopathy.This study identifies EHV2 in equine tendons for the first time, and describes NGS as a useful tool for virus identification. The implications of the presence of EHV2 in tendon to both tissue-engineering and tendinopathy requires further work. However, there are potential implications for the use of TETCs as allogenic transplant agents, as the presence of virus could result in transplant failure.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Wardle R et al.10.17605/OSF.IO/WYPKQ41.All raw read data produced in this study has been submitted to the EBI ENA, primary accession PRJEB20552, secondary accession ERP022713. The data underlying this work has been uploaded to the Open Science Framework Database, and can be accessed via DOI, The authors have responded to comments from both reviewers and adjusted the manuscript accordingly such that a decision of approve can be made.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have taken attention of both reviewers’ comments. The paper status satisfies my recommendation of being accepted.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. "This study suggests that a primary viral infection coupled with subsequent bacterial infection leads to catastrophic musculoskeletal infection and death" Please clarify that this is the study on broiler hens and not the current work. It is not clear from the current work what statistical tests were carried out in the programmes described in the materials and methods. Were any of the RNA seq hits for the latency associated transcript RNA? The specific PCR for EHV2 on tendon tissue did not find any viral DNA however can this test confirm lack of presence of latency associated transcript RNA? Was there any RNA from the virus infected tenocytes left over for analysis? Did the authors collect these tenocytes from tissue that was known to be septic antemortem? I wonder if this statement is a jump too far?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. ReviewerThis study suggests that a primary viral infection coupled with subsequent bacterial infection leads to catastrophic musculoskeletal infection and death" Please clarify that this is the study on broiler hens and not the current work.ResponseWe have amended the manuscript to clarify that this was indeed in broiler hens.ReviewerIt is not clear from the current work what statistical tests were carried out in the programmes described in the materials and methods.ResponseWe have amended the manuscript to clarify that these statistical tests relate to the hydroxyproline assay.ReviewerWas there any RNA from the virus infected tenocytes left over for analysis?ResponseAll the RNA samples were used in the studies pertaining to this manuscript.ReviewerDid the authors collect these tenocytes from tissue that was known to be septic antemortem? I wonder if this statement is a jump too far.ResponseAll samples were grossly normal on collection. Hospital derived samples showed no signs of sepsis.ReviewerWere any of the RNA seq hits for the latency associated transcript RNA?ResponseTwo different types of mapping were undertaken. Firstly a megablast run against the entire nt database to get taxonomic information, and  secondly a blastx run against a database  created of all know ERV genes in animals from NCBI. Therefore the’ latency associated transcripts’ (only appear to be sequences for these in humans in NCBI) would not have been included. We assessed the two sequences in NCBI that result from a search for 'latency associated transcript and herpes' and ran a blastx for both samples against these. Neither resulted in any significant hits.  Thus whilst latency associated transcripts cannot be ruled out with data currently available this is unlikely. A more in-depth analysis than this would require further research in latency associated transcripts linked to herpes in other animals and is beyond the scope of this paper.  We have amended the manuscript accordingly.ReviewerThe specific PCR for EHV-2 on tendon tissue did not find any viral DNA however can this test confirm lack of presence of latency associated transcript RNA?ResponseThe reviewer is correct in their assumption. We have amended the manuscript. 1 described detection of EHV-2 and EHV-5 in PBMC and a selection of tissues in about 80% of the animals they examined using co-cultivation methods to demonstrate infectious replicating virus. EHV-2 is common in foals but these observations were also made in older horses on this occasion. As with other gammaherpes viruses, EHV-2 is known to be latent in B lymphocytes  and these latent viruses were re-activated and identified by co-cultivation methods. These and other findings strongly suggest that EHV-2 virus or cells containing EHV-2 genes would have been present in a high proportion of tissue samples taken from horses for this study. Many samples were from slaughterhouse materials but even so, it would have been very difficult to remove tendon tissues from horses without significant contamination by infected (or latently-infected) cells. This is a very well executed study carried out with high level of rigour to define and describe the findings of EHV-2 in equine tendon cells. There is one important aspect which potentially confounds the interpretation of the data. The authors refer to the possibilities of some contamination of samples and tissue culture reagents as a possible source of EHV-2 virus and PCR products in tendon cells and provide a good argument to discount these. However, one aspect is not fully considered in their justification of this potentially novel finding. There have been many published papers describing equine gammaherpes viruses in PBMC and several tissues/organs in both foals and adult horses. EHV-2 is ubiquitous in horses and virus and viral genes are commonly found. For example, Torfason et al. These possibilities should be discussed fully in the paper. It is possible also that cell-to-cell contact of infected PBMC or other donor cells with tenocytes is the route of infection of the latter in vivo. This would be perfectly acceptable as a method by which EHV-2 genes and virus were detected in cultured tenocytes. The fact that the authors detected virus in only 2 of the 13 samples might also be worthy of note, considering that this virus is so ubiquitous.   Do the authors know if the positive samples were from old or young horses? The considerations above do not detract from the findings that equine tendon cells may contain EHV-2 and that this might have implications for use of these cells therapeutically to treat tendinopathy. The contention is that the paper should discuss the findings in relation to published papers such as those indicated above and the potential likelihood of this finding not being unexpected.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors appreciate the comments made by the reviewer. We have taken on board their concerns and amended the manuscript accordingly to include discussion of other possible reasons for the findings of EHV-2 in equine tenocytes.


S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10−5. A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach.Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae  MHC) locus (S) loci as in the Brassicaceae S-locus, have a large number of highly divergent alleles maintained by negative frequency-dependent selection over long periods of time (DNA sequencing has come a long way since Sanger’s “chain termination” technique first improved our ability to sequence DNA . In the C) locus  or plant of time . Their g of time .S-locus would rely on polymerase chain-reaction (PCR) amplification of specific regions of interest (often the S-locus receptor kinase gene SRK) in combination with either Sanger sequencing  containing the S-locus  errors and indel errors. By comparison of two assemblies of independent SMRT sequencing one of the two S-locus BACs, we find that this approach is efficient and highly accurate with regard to structural errors and single nucleotide polymorphisms. Mapping short-read data to the an SMRT assembly of the second S-locus BAC for error correction, we find that correction for indel errors is necessary, especially for studies aiming to identify functional polymorphisms. This method can thus be valuable for a wide range of genomic studies of complex genomic regions, where reference-based approaches for studying genetic variation are not feasible.In contrast to short-read sequencing technologies, SMRT sequencing should provide a better basis for reliably assembling repetitive regions, due to mean read lengths >20 kb and maximum reads >60 kb . EventuaCapsella grandiflora  with 10% bleach and 70% ethanol. Seeds were stratified at 2-4° in the dark on plates with 0.8% agar and half-strength MS medium . After two weeks, we moved the plates to climate controlled growth chambers  to allow the seeds to germinate. After approximately 1 week, we transplanted seedlings to pots with soil in the climate-controlled chambers. We kept the plants under dark conditions for 4 days prior to sampling young leaves for BAC library construction.We surface-sterilized seeds of four accessions of the self-incompatible crucifer S-locus haplotypes, we followed a strategy similar to HindIII and ligated to pCC1BAC cloning vector , after several size selection steps. BAC libraries were screened for flanking regions of the S-locus by hybridization with DNA probes and PCR amplification with specific primers and further selected based on mean insert size. Clones of colonies that tested positive for U-box and ARK3 flanking genes of the S-locus were selected for sequencing. All BAC library production and screening was performed by the French Plant Genomic Resource Centre (CNRGV) at INRA.To sequence full-length S-locus sequences in two BAC clones at the Uppsala Genome Center, National Genomics Infrastructure Sweden. DNA fragments over 10 kbp were selected using BluePippin Size selection  and the SMRTbell Template Prep Kit 1.0  was used for library preparation, with an insert size of 500 bp to 20 kb. SMRT sequencing was done on the RSII system, using P5-C3 chemistry.We conducted SMRT sequencing  of two different Brassicaceae S-locus BAC (CgrS-BAC2). The sequencing library  was prepared from 1 μg of DNA, following the manufacturers’ guidelines. We generated 1.1 million paired-end 250 bp reads on the MiSeq using v2 sequencing chemistry .To assess sequencing and assembly errors, we generated two independent libraries of one BAC clone (CgrS-BAC1), which was then subjected to independent SMRT sequencing and assembly, whereas the second BAC, (CgrS-BAC2), was sequenced once with SMRT sequencing. To assess indel errors, we also generated short-read sequencing  data for the second de novo assembly with Celera Assembler 8.3rc2 (S-locus), as well as several contigs containing E. coli sequences. As HGAP.3 does not split reads, assembling a circular molecule results in overlapping ends of reduced coverage, and we therefore conducted circularisation and removed overlapping ends using minimus2 v3.1.0 of the AMOS suite  , and the numbers and base-pair locations of indels and SNPs, that represent sequencing errors.To estimate assembly and sequencing error rates, we compared the https://github.com/douglasgscofield/PacBio-utilities). Indel error rate was calculated as number of insertions and deletions, divided by assembly length to get a per base-pair error rate.To generate an additional estimate of sequencing error rates, we used Illumina MiSeq data for CgrS-BAC2. We mapped short reads to the polished assembly of the BAC clone using bwa-mem v 0.7.8 . FinallySRK exon 1 sequences from the S-haplotype assemblies by searching for BLAST hits to general SRK exon 1 forward (SLGF) and reverse (SLGR) primers (SRK exon 1 sequences or conversely rejected them based on stronger sequence homology to known ARK3 (Aly8) sequences using BLAST (v2.5.0+). Exact parameters for sequence homology varied between candidate sequences due to high divergence in SRK alleles, but were always above 90%.To assess the utility of using SMRT sequencing of BAC clones to reconstruct complex loci, we extracted Capsella SRK-like sequences, and known SRK and ARK3 alleles, we bulk downloaded 722 publicly available Brassicaceae SRK and ARK3 sequences of >500 bp length from GenBank (Table S1) and retained only those under 2000 bp of length. Duplicates were removed using dedupe.sh from BBMAP v34.56 (Joint Genome Institute), and we made an initial alignment between our SRK sequences and the publically available SRK and ARK3 using MAFFT v7.245 with the E-INS-I algorithm .In order to characterize the relationship between our l v8.2.3 , generatS-locus, we annotated our S-locus assemblies with Augustus v3.2.3 (http://www.repeatmasker.org) via Maker v2.31.9 .To assess patterns of sequence conservation across the entire v1.03.54  and the v1.03.54  , and 153563 bp (for CgrS-BAC2), see We obtained one large contig containing the S-locus into one contig, in contrast to assemblies of the S-locus based on short-read data, which resulted in several contigs , and an average of 0.02 substitutions errors per bp (range 0-0.1) based on 454 sequencing of SRK amplicons  already work well at removing assembly errors if, as in our case, the coverage of long reads is high enough  validation, which enables a faster and simpler workflow, once the assembly and error correction is complete. SMRT sequencing is still relatively costly, adding to the costs of BAC library production (∼1700 € at time of publishing), but for certain studies long reads are indispensable, for instance to assemble regions of high repeat content and to accurately assemble intergenic regions . Using ae.g., PBcR from ∼85% up to 99.9% (de novo assembled with higher confidence. Hybrid assemblies however are computationally intensive, especially early programs (High quality assemblies are necessary for many genetic studies, by the alignment of short read data directly to SMRT long reads, hybrid software are able to improve the accuracy of SMRT sequencing long reads , e.g., Pprograms , as theyS-locus, a locus that has been difficult to study due to its high content of repeats and high divergence among alleles. Independent SMRT sequencing runs of the same BAC clone allow us to estimate an error rate of 5.7 × 10−5 indels per bp. These errors can efficiently be corrected using short reads, and such correction is important especially in the context of highly accurate studies of functional gene variants.We show that SMRT sequencing of BACs is an efficient way to obtain high-quality assemblies of the Brassicaceae This approach can be useful for studies of other genomic regions characterized by high divergence and repetitive content, such as other loci under long-term balancing selection , where rwww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300467/-/DC1.Supplemental material is available online at Click here for additional data file.


Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008–2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008–2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region. Foot-and-mouth disease virus (FMDV) causes a severe and highly contagious acute vesicular illness that affects livestock and wildlife. The wide genetic and antigenic variation of this virus is evidenced by the existence of 7 serotypes  and multiple subtypes . Interestingly, there is evidence of FMDV tropism at sites of persistent infection with favorable conditions for long term survival of infected cells  and withAn investigation of serotype O persistently infected cattle reported up to 63 nt changes and 24 aa substitutions in the ORF in persistent infections but none of the aa was consistently found in all the isolates . In seroOur analysis of Asia-1 genomes detected few positively selected codons. Four out of five isolates from the fourth sampling displayed positively selected codons in P1, consistent with the clustering pattern observed during the phylogenetic analysis where these isolates formed a monophyletic group. Positive selection has been reported on 1 to 7% of codon positions in FMDV capsid genes for other serotypes in field isolates . More stPhylogenetic analyses indicated that persistent viruses from this study clustered closely with viruses from acute infections circulating in Pakistan during 2011 , and witDifferent methods have been proposed to classify FMDV strains, subtypes, and sublineages , 58. We h epitope in VP4 that was conserved across most of the Asia-1 sequences. Knowledge of signature amino acid sequences, receptor binding motifs, and key antigenic sites could be applied to epitope based or chimeric vaccine research [Antigenic variation is a key obstacle for FMD control; it leads to the lack of cross-protection between serotypes and sometimes between strains , 14, 59.research .The association between viral genetic sequence and antigenicity is not well defined; consequently, vaccine matching studies using serology are necessary to monitor cross-protection of current vaccines. A better understanding of mechanisms driving virus evolution, particularly understanding selective forces exerted under subclinical and persistent infections might help inform the development of more effective FMD vaccines.S1 Fig(TIF)Click here for additional data file.