To reflect the initiative design and initiative of human security management and safety warning, ecological safety assessment is of great value. In the comprehensive evaluation of regional ecological security with the participation of experts, the expert’s individual judgment level, ability and the consistency of the expert’s overall opinion will have a very important influence on the evaluation result. This paper studies the consistency measure and consensus measure based on the multiplicative and additive consistency property of fuzzy preference relation (FPR). We firstly propose the optimization methods to obtain the optimal multiplicative consistent and additively consistent FPRs of individual and group judgments, respectively. Then, we put forward a consistency measure by computing the distance between the original individual judgment and the optimal individual estimation, along with a consensus measure by computing the distance between the original collective judgment and the optimal collective estimation. In the end, we make a case study on ecological security for five cities. Result shows that the optimal FPRs are helpful in measuring the consistency degree of individual judgment and the consensus degree of collective judgment. Ecological security refers to the overall level of ecosystem integrity and health, especially the minimum risk of survival and development, and the state of being not threatened. With the rapid growth of population and the development of social economy, the pressure of human activities on the environment is increasing, the contradiction between people and the earth is exacerbated, and the problem of excessive consumption of resources is becoming more and more serious. The threat of ecological damage and the environmental disasters caused by environmental degradation and ecological destruction to regional development, national security and social progress is increasing. Thus, the ecological security issue has been paid much attention on a global scale. The problem of ecological security has become a hot issue that needs to be solved urgently both in theory and practice ,4,5,6,7.The Yangtze River Delta urban agglomeration is the highest degree of urbanization, the most densely distributed towns and the highest level of economic development in China. Because the Yangtze River Delta urban agglomeration is located on the eastern coast of China and along the developed areas along the Yangtze River, the geographical advantages are prominent and the ecological status is very important. It plays an important role in maintaining the ecological balance of the middle and lower reaches of the Yangtze River and promoting the healthy development of the economy in the middle and lower reaches. In recent years, the economic development of the Yangtze River Delta region has shown a rapid growth trend, but, due to the degradation of the ecological environment and frequent ecological disasters, ecological security has become an important factor restricting the sustainable development of the Yangtze River Delta region. At present, the study on the ecological security of the Yangtze River Delta is still lacking. Therefore, it is urgent to carry out the comprehensive evaluation of the ecological security of the region, and then put forward the countermeasures to curb the deterioration of the ecological environment. Comprehensive evaluation of ecological security not only involves the natural environment, ecological and environmental disasters, environmental pollution, socio-economic and many other objective indicators, but also need to fully respect the subjective experience of experts and judgments. In the comprehensive evaluation of regional ecological security, the expert’s individual judgment level, ability and the consistency of the expert opinion (consensus level) have a very important influence on the evaluation result. This paper will study the consistency test method of expert opinion in ecological security assessment based on consensus decision theory.In group decision making (GDM) analysis, it is often required to establish a procedure to aggregate multiple individual subjective preferences into an optimal consensus and then select the most favorable alternative. Usually, preference relations are widely used to express the subjective preference in group decision. In each preference relation, the decision maker (DM) is expected to provide a rational judgment or estimation; in a collective preference relation, the whole group of DMs is expected to reach a full agreement . The firThe consistency framework of FPR was constructed in,  or Equation (3) is called the additively consistent condition of the FPR a et al.  gave a na et al. ,22,23,24a et al.  also derIn the following section, based on the indirect methods of Wang et al. , Ma et aLet However, Model (5) can be easily solved, leading to the unique solutionThat is,If Theorem 1. Iffor all, then the optimization Model (5) has a unique solution.(I).For all (II).For all (III).For all (IV).For all Let us now construct a new matrix Thus, we haveConsequently, the matrix Theorem 2. The matrixis an FPR which satisfies the multiplicative consistency, where,.We call the multiplicative consistent FPR Let We can readily see thatTheorem 3. The optimal Model (10) has a unique solution.Let us construct a new matrix (I).For all (II).For all We can readily see that(III).For all Thus, Meanwhile, we have Consequently, if Theorem 4. The matrixis an FPR and possesses the additive consistency if, where,.We also call the additively consistent FPR Let (I). Consistency degree associated with a pair of alternatives Definition 1. We callthe consistency degree associated with the pair of alternativeand, andthe consistency degree matrix associated with all the pair of alternativesand,.In fact, (II). Consistency degree associated with an alternative Definition 2. We callthe consistency degree associated with the alternative, andthe consistency degree vector associated with all the alternatives.The consistency degree (III). Consistency degree of the FPR Definition 3. Letbe an FPR, andthe corresponding optimal estimation matrix calculated using Equation (8) or Equation (11). We callthe consistency degree of the FPR.The bigger the value a et al. . Example 1. The FPRpresented by Chiclana [is given below:Chiclana  is givenFrom Equation (11), the optimal estimation matrix of The consistency measures of Here, we have two different consistency measures. The first consistency measure (our measure) is based on the optimal estimation matrix. That is, we regard this matrix as an objective matrix. The consistency degree of the FPR is based on the differences between the collective FPR  and its optimal estimation FPR. In this sense, the first consistency measure is constructed from the point of view of collective divergence. The second consistency (Chiclana’s measure ) is baseIn group decision making, the collective preference relation is very helpful in selecting the best alternative. Usually, when aggregating the individual preference relations to a collective preference relation, the consensus measure is desirable. The consensus index is a useful tool for measuring the degree of consensus between the individual preference estimations and collective preference estimation. Dong et al.  defined In group decision making, the weighted geometric average and weighted arithmetic average methods are often used to aggregate different individual preferences into a collective preference. In this section, we show by two theorems that the optimal estimation matrix for the aggregated FPR is equivalent to the aggregated optimal estimation matrix for different individual FPRs. Let Lemma 1. is a perfectly consistent preference relation if,, is multiplicative consistent.Let Lemma 2. is perfectly consistent if,, is multiplicative consistent.Lemmas 1 and 2 are obvious, the proof is omitted. Let us consider an FPR For Equation (17) is equivalent to(I).Suppose that Consequently, we define Theorem 5. Letbe an FPR,, then the weighted geometric average combinationis also an FPR, wheresatisfies Equation (17),. Moreover, if all the individual FPRs,, are multiplicative consistent, then the weighted geometric average combinationis also multiplicative consistent, wheresatisfies Equation (17),.(II).Suppose there is at least one On the one hand, by Equation (7), the optimal estimation matrix By Theorem 2, On the other hand, the optimal estimation matrix Suppose that the weighted geometric average combination of Combining Equation (19) with Equation (21), we have that Theorem 6. The optimal estimation matrix of the weighted geometric average combination of individual FPRs is equivalent to the weighted geometric average combination of the optimal estimation matrix of the individual FPRs.Let Theorem 7. is an FPR ifis an FPR, for all;is additively consistent ifis additively consistent, for all, where,.According to Equation (11), the optimal estimation matrix for The optimal estimation matrix for Then, the arithmetic average of Thus, we have Theorem 8. The optimal estimation matrix of weighted arithmetic average combination of individual FPRs is equivalent to the weighted arithmetic average combination of the optimal estimation matrix of individual FPRs.Let According to Theorem 7, if Let (I). Consensus degree on a pair of alternatives Definition 4. The deviationis called the consensus degree on the pair of alternativesand, and the matrixis called consensus degree associated with all the pairs of alternativesand,.The smaller (II). Consensus degree on an alternative Definition 5. is called the consensus degree on the alternative, andthe consistency degree vector associated with all the alternatives.The number (III). Consensus degree on the FPR Definition 6. is the called consensus degree of the collective FPR.The bigger the value of a et al. . Example 2. The FPRs,, as presented by Chiclana [, are given as follows:Chiclana , are givSuppose that the relative weight of each DM is 0.25. Then, the arithmetic average combination of the FPRs According to The consensus measures of the collective FPR Here, we have two different consensus measures. The first consensus measure (our measure) is based on the optimal estimation matrix. That is, we regard this matrix as an objective matrix. The consensus degree of the FPR is based on the differences between the collective FPR  and its optimal estimation FPR. In this sense, the first consensus measure is constructed from the point of view of collective divergence. The second consensus (Chiclana’s measure ) is baseLet (I). Consensus degree on a pair of alternatives Definition 7. is called the consensus degree on the pair of alternativesand, and the matrixthe consensus degree associated with all pairs of alternativesand,.(II). Consensus degree on an alternative Definition 8. is called the consensus degree on the alternative,the consensus degree vector associated with all the alternatives.(III). Consensus degree on the FPR Definition 9. is called consensus degree of the collective FPR.Step 1. Construct the optimal estimation matrix of the FPRs by using Equation (8) or Equation (11). Step 2. Aggregate the individual FPRs into a collective FPR by using the weighted geometric average or the weighted arithmetic average method. Step 3. Compute the consensus degree matrix In the following, we use an example to show the process of measuring the consensus of the FPRs.Taking the ecological security evaluation of five important cities, Shanghai, Nanjing, Hangzhou, Suzhou and Wuxi, in the Yangtze River Delta as an example, with reference to the natural environment, ecological environment disaster, environmental pollution, social and economic indicators, experts Due to the limited rationality of experts and the incompleteness of knowledge, in the process of making judgments, it is often difficult for experts to ensure logical consistency. Based on the consistency measure and the consensus measure method proposed in this paper, the consistency of the logic and the validity of the judgment matrix are examined in the expert judgment process considering the consistency and multiplicative consistency of the FPR.Suppose that there are three FPRs Step 1. According to Equation (8), the optimal estimation matrices of For example, the  entry 0.6838 of Step 2. The weighted geometric average combination According to Equation (16), we also take the  entry 0.4797 of Step 3. The weighted geometric average combination According to Equation (16), we take the  entry 0.6007 of In fact, Step 4. The consensus degree matrix In particular, we look at how the  entry 0.7530 of Step 5. The consensus vector Step 6. The global consensus of The above research shows that the degree of consistency of the three experts’ comprehensive evaluation of ecological security for five cities is 0.8780, which has a high degree of consensus. It also shows that the overall judgment of three experts have a certain degree of reliability.(1)Every entry in an inconsistent FPR is useful for the reason that it reflects the overall thinking of a DM. In this paper, we use the method of optimization to obtain the optimal additively consistent or the optimal multiplicative consistent FPR. The optimization method is in fact either the geometric average or the arithmetic average. It comprehensively considers each entry in the inconsistent FPR. Consequently, all information in the inconsistent FPR is fully used.(2)Every FPR in GDM is useful for the reason that it reflects the influence of each individual in the group. In this paper, we use the weighted geometric average or the weighted arithmetic average to aggregate individual FPRs into a collective FPR. We also show by two theorems that the optimal estimation matrix of the aggregated FPR is equivalent to the aggregated optimal estimation matrix of different individual FPRs. The theorems indicate additionally that there is no information loss that occurs during the aggregation of the individual FPRs and reconstruction of the optimal collective FPR.(3)The optimal FPRs are helpful in measuring the consistency degree of individual judgment and the consensus degree of collective judgment. Being similar to the consensus model as proposed by Chiclana , a new c(4)Additionally, we want to mention that our consensus measure is based on the deviation between the original collective estimation and its optimal estimation, while Chiclana’s consensus measure  on the dThe evaluation of regional ecological security risk by group decision-making method can not only make full use of the subjective experience of expert groups, but also overcomes the difficulties of complex objective structure and incomplete objective data to a certain extent. Based on the theory of consensus decision making of fuzzy preference relation, this paper studies the consistency test method of expert opinion in ecological security assessment. In this paper, we proposed our consistency measure model by computing the distance between the original individual FPR and its optimal estimation matrix, and consensus measure model by computing the distance between the original collective FPR and its optimal estimation matrix. The contribution of this work can be seen from several different angles.


C. neoformans. In the Northwestern United States, C. gattii has received considerable attention secondary to increased virulence resulting in significant morbidity and mortality. There are no cases in the extant literature describing a patient with C. gattii requiring neurosurgical intervention in Alabama. A middle-aged immunocompetent male with no recent travel or identifiable exposure presented with meningitis secondary to C. gattii. The patient underwent 12 lumbar punctures and a ventriculoperitoneal shunt and required 83 days of inpatient therapy with 5-flucytosine and amphotericin B. The patient was found to have multiple intracranial lesions and a large intramedullary spinal cryptococcoma within his conus. Following an almost 3-month hospitalization the patient required treatment with oral voriconazole for one year. In the United States meningitis caused by C. gattii infection is not isolated to the Northwestern region. Cryptococcal infections are seen throughout the United States in both immunocompromised and immunocompetent patients. The most common form is Cryptococcus neoformans, which is most commonly seen in immunocompromised individuals. Cryptococcosis frequently presents as pneumonia but also has the capability to spread hematogenously to the central nervous system (CNS) causing meningitis and accumulations of yeast known as cryptococcomas. Cryptococcal disease is most often caused by two species complexes: C. neoformans species complex and the C. gattii species complex [ C. neoformans/C. gattii species complex was recently updated in 2015. What were genotypes within these species complexes now have all been given a different species name. Cryptococcus neoformans, Cryptococcus deneoformans, Cryptococcus gattii, Cryptococcus bacillisporus, Cryptococcus deuterogattii, Cryptococcus tetragattii, and Cryptococcus decagattii are the new names [ C. neoformans var. grubii, is now known as C. neoformans. The serotype D isolates, previously named C. neoformans var. neoformans, are now members of the species C. deneoformans.Cryptococcal disease in the United States has generally been attributed to complex . The taxC. neoformans is the most common cryptococcal pathogen with a global distribution and predominantly infects immunosuppressed hosts. There is a greater predilection of C. gattii and C. deuterogattii to infect immunocompetent individuals than that of C. neoformans [ C. bacillisporus, Cryptococcus tetragattii, and Cryptococcus decagattii are also nearly exclusively involved in infections among HIV-positive subjects. The ability of these species to spread is dependent on the yeasts' extracellular proteolytic activity, on melanin production, capsule formation, inositol, urease-activity, and ability to replicate inside macrophages. A deficiency in the protease produced by C. gattii as well as other properties limits its ability to cause cryptococcemia [ C. gattii forms larger inflammatory cryptococcomas that may require long-term antifungal therapy and neurosurgical interventions [oformans . C. bacicoccemia . Insteadventions . C. gattii species complex  [ C. gattii and C. deuterogattii are frequently seen in immunocompetent patients but differ in their source: C. gattii is endemic to Australia and is a common source of infection in Aboriginal groups, while C. deuterogattii is the predominant form found in the recent Pacific Northwest outbreak [ C. bacillisporus, C. tetragattii, and C. decagattii are more frequently seen in immunocompromised patients [ C. bacillisporus occur more frequently and was found to be the major type in Californian HIV patients, while C. tetragattii is endemic to sub-Saharan Africa and mainly seen in HIV-infected individuals [ C. decagattii and C. tetragattii are unable to be distinguished by conventional PCR-fingerprinting and URA5-RFLP. Thus it is difficult to distinguish the two for specific distribution data [There are 5 known species of theagattii) . As mentoutbreak , 5. C. bpatients . Infectiividuals . C. decaion data . C. gattii species complex disease emerged on Vancouver Island in 1999 and has since spread to the Pacific Northwest region of the United States [ C. gattii species complex cryptococcosis reported in the Southeastern United States, revealing the need to further define the populations at risk. For the first time, we report a case of C. gattii infection in an immunocompetent Alabama native with no recent travel or known exposure who developed severe meningoencephalitis requiring long-term antifungal therapy and neurosurgical intervention. The patient was previously in the military, however, and had extensive travel to Hawaii, Philippines, Thailand, Australia, Okinawa, and Hong Kong. His military experience was over a 5-year period ending in 1987. As this disease is associated with increase morbidity and mortality, this case highlights that the C. gattii species complex should be on the minds of those who treat cryptococcal disease in the United States.Previously uncommon in North America, thed States , 8. Ther Aspergillus antigens were all negative. Four weeks prior to admission, the patient began to experience fevers, chills, headache, back pain, and vomiting. He was admitted to an outside hospital and a complete workup revealed cryptococcal meningitis via India ink stain of his cerebrospinal fluid (CSF). The patient was subsequently started on amphotericin B and 5-flucytosine for the cryptococcal meningitis, as well as vancomycin and ceftriaxone for possible concomitant bacterial meningitis.A middle-aged male native to northern Alabama without a recent history of travel  was transferred to our hospital with the diagnosis of cryptococcal meningitis. The patient had a low complement levels (CH50) but was noted to have normal C5, MBL ELISA, C3, C4, and AH50. He was admitted with a history of cocaine use, but no injectable drug use. HIV testing including a viral load, gonorrhea, chlamydia, serum CMV antigen, urine histoantigens, and serumThe patient was transferred to our institution for treatment of cryptococcal meningitis, ultimately requiring 83 days of IV amphotericin B (Abelcet) and oral flucytosine therapy. Initial imaging revealed a peripheral right upper lobe lung nodule on chest CT, suggestive of cryptococcoma with negative acid-fast stain on bronchial lavage. MRI of the brain shown in  Cryptococcus gattii by CDC) determined the patient was infected with a member of the C. gattii species complex and further analysis was performed. The patient had a ventriculoperitoneal shunt placed for intractable headaches and elevated pressures on serial lumbar punctures.Workup (+India ink stain, + cryptococcal antigen 1 : 512, and molecular typing ofInterferon-gamma therapy was initiated on day twenty for refractory infection and was administered three times per week. After 7 weeks of hospitalization, repeat imaging suggested worsening of intracranial cryptococcal disease, prompting an increase in amphotericin B from 5 to 6 mg/kg/day. During the following week, the patient complained of back pain radiating down his lower extremities that was exacerbated by amphotericin infusions. MRI of the spine shown in  C. gattii meningitis.It was decided to initiate corticosteroid therapy to treat cord edema as the patient was experiencing constipation and urinary urgency. Dexamethasone 4 mg every 6 hours was initiated, as it was believed that the patient had paradoxical antifungal-induced immune reconstitution inflammatory syndrome in setting ofAfter ten days of dexamethasone therapy, repeat MRI of the brain and spine showed improving white matter lesions with no evident spinal lesion, although meningeal enhancement was still present. Concurrent analysis of the CSF showed increased glucose and decreased protein, suggestive of an appropriate response to therapy. Final CSF cultures demonstrated no growth but India ink stain remained positive. After 83 days of therapy, amphotericin and flucytosine were discontinued and the patient was started on voriconazole 300 mg bid. The patient was monitored for three days and was subsequently discharged on the same voriconazole regiment with a dexamethasone taper.C. neoformans species complex and C. gattii species complex share some common virulent attributes but are also distinct in their pathogenicity, ecology, and epidemiology. C. neoformans has a global distribution and certain members of the C. neoformans/C. gattii species complex commonly infect predominantly immunocompromised individuals. In contrast, other members of the C. gattii species complex are more geographically restricted and commonly infect immunocompetent patients. Following inhalation of aerosolized yeast, these pathogens usually cause pulmonary disease with the capability of hematogenous dissemination to the CNS resulting in meningitis, cryptococcomas, and neurologic deficits [deficits . Cryptocdeficits . Accumul C. gattii species complex in the United States have been in the Pacific Northwest and usually produce pulmonary disease [ C. gattii species complex in the Southeastern United States are few and are frequently associated with travel to endemic areas. No member of the C. gattii species complex is considered to be endemic to the Southeastern United States and has never been isolated from environmental sources in the area [ C. gattii species complex-members can be dormant for decades until the host's immune system becomes suppressed, and this patient may have contracted the pathogen during his time overseas in the military. Here we have reported for the first time a case of C. gattii species complex in an immunocompetent Alabama native that had no recent travel in over three decades, which required long-term antifungal therapy and neurosurgical intervention.Most cases of disease . The numthe area . C. gatt


Aerosolized pathogens are a leading cause of respiratory infection and transmission. Currently used protective measures pose potential risk of primary/secondary infection and transmission. Here, we report the development of a universal, reusable virus deactivation system by functionalization of the main fibrous filtration unit of surgical mask with sodium chloride salt. The salt coating on the fiber surface dissolves upon exposure to virus aerosols and recrystallizes during drying, destroying the pathogens. When tested with tightly sealed sides, salt-coated filters showed remarkably higher filtration efficiency than conventional mask filtration layer, and 100% survival rate was observed in mice infected with virus penetrated through salt-coated filters. Viruses captured on salt-coated filters exhibited rapid infectivity loss compared to gradual decrease on bare filters. Salt-coated filters proved highly effective in deactivating influenza viruses regardless of subtypes and following storage in harsh environmental conditions. Our results can be applied in obtaining a broad-spectrum, airborne pathogen prevention device in preparation for epidemic and pandemic of respiratory diseases. Droplets with aerodynamic size  salt is locally dissolved by the viral aerosols and ii) supersaturation is followed by evaporation-induced salt recrystallization. Consequently, viruses are exposed to increasingly higher concentrations of saline solution during drying and physically damaged by recrystallization.bare) were pre-wet to contain about 600 μL of coating solution (abbreviated as Filterwet). The amount of NaCl salt  coated on the filter per unit area, considering that the filters thickness is constant, was easily controlled by changing the coating solution volume  during drying of pre-wet filter  (c = 133.0 ± 4.7°) to completely hydrophilic   microfiber filter, was coated with NaCl salt as an active virus negation unit  . Scannin, n = 7) . Both th n = 10) . Hydrophe images .bare did not exhibit any significant level of resistance against penetration of virus under our experimental conditions . Conversely, salt-coated filters showed substantially increasing filtration efficiency with pressure and amount of coated salt. In particular, in the case of Filterwet+600μL, filtration efficiency varied from 43 to 70%, with increasing pressure from 3 to 17 kPa, and Filterwet+1200μL exhibited persistent, high-level efficiency (~85%) .Filtration efficiency of salt-coated filters was tested against aerosols with volumetric mean diameter (VMD) of 2.5–4 μm containing H1N1 pandemic influenza virus  at different pressure conditions  infected with penetrated dosages of H1N1 virus under breathing pressure (~10 kPa)P < 0.005). This is consistent with lower levels of inflammatory cytokines, interferon-γ (IFN-γ), from salt-coated filter groups compared to negative control and bare filter groups   .bare, salt-coated filters exhibited almost complete HA activity loss within 5 min of incubation  (P < 0.001). TEM analysis showed that influenza virus on Filterbare experiences morphological change into non-spherical shape during aerosol drying (30P < 0.001)  . It is al drying . Notably< 0.001) . In parahe virus  comparedus  and A/Vietnam/1203/2004 (VN/04 H5N1). Similarly to CA/09 H1N1, 100% of mice survived viral infection (PR/34 and VN/04), with no evidence of weight loss, due to higher filtration efficiency of salt-coated filter than that of bare filter   for 1 day did not cause any significant difference in filtration efficiency  . As a re= 0.718) . Even af= 0.718) , despite= 0.718) .Staphylococcus aureus aerosols, by employing S. aureus ATCC 6538S. aureus by one order of magnitude. Additionally, whereas during BFE evaluation all three layers of surgical masks are used, in this work filtration efficiency refers to mask filters (middle layer). It is worth noting that the conditions for BFE standard evaluation (such as flow rate and time of application of flow) do not coincide with the experimental procedure we used for measurement of the filtration efficiency, which may further contribute to the different result. The enhanced filtration efficiency of salt-coated filters against influenza virus aerosols as compared to bare filters can be explained by the observed wetting of aerosols, favoring greater adhesion to salt-coated filters. Furthermore, the significant improvement in filtration efficiency resulted in complete protection of mice against lethal influenza aerosols, which demonstrates the high level of protection provided by salt-coated filters, outperforming currently used bare filters.Development of a universally applicable, low-cost, and efficient mechanism for virus negation is regarded as a major challenge in public health against general airborne biological threats. This led us to propose a new concept of personal/public preventive and control measures using salt-recrystallization against pathogenic aerosols based on two hypotheses. The salt-coating can enhance adsorption of virus on the filter fibers and inactivate virus by the increase of osmotic pressure followed by the crystallization of salts. As shown in in vivo. According to previous reports, hyperosmotic stress (>541 mOsm) and crystallization induce membrane perturbation with irreversible deformation of the viral envelope and structural virus damage, respectively, resulting in infectivity loss of virus30Rapid loss of HA activity and viral infectivity on salt-coated filters can be explained by physical destruction of virus during recrystallization of coated salts. When the salt-coated filter is exposed to virus aerosols, salt crystals below the aerosol droplet dissolve to increase osmotic pressure to virus. Due to evaporation, the salt concentration of the droplet significantly increases and reaches the solubility limit, leading to recrystallization of salt. As a consequence, virus particles are exposed to increasing osmotic pressure during the drying process and are physically damaged by crystallization. As shown in in vivo due to higher filtration efficiency of salt-coated filters compared to bare filters and deactivation of virus on salt-coated filters were observed following exposure to PR/34 H1N1 and VN/04 H5N1 . The coating solution was prepared by dissolving sodium chloride  in filtered DI water  under stirring at 400 rpm and 90 °C, followed by the addition of Tween 20 (Fisher Scientific) to a final concentration of 29.03 w/v% of NaCl and 1 v/v% of Tween 20. To obtain the salt-coated filters, the mask bare PP filters were pre-wet to contain approximately 600 μL of coating solution by incubating overnight at room temperature. Any remaining dry areas were removed by applying gentle strokes with tweezers to the filters while immersed in the coating solution. Subsequently, the filters were deposited in the desired volume of coating solution  on petri dishes  to control the amount of NaCl per unit area and dried in an oven  at 37 °C for 1 day.The commercial surgical masks had a three-ply structure. The middle layer is the filter media, whereas the inner and outer layers provide support and protect the filter against wear and tear. The metal nose clips and elastic ear loops were removed and circular samples (radius: 3 cm) were cut from the masks. The PP filters (middle layer) were isolated by removing the inner and outer protective layers , A/Puerto Rico/8/34  and A/Vietnam/1203/2004  were grown in 10-day old embryonated hen eggs, in which H5N1 virus was derived by reverse genetics from HPAI A/Vietnam/1203/2004For experiments involving aerosols exposure, an aerosol chamber  was used . It has For all assays and analysis, suspensions of the filters were prepared as follows, unless otherwise indicated. To reconstitute virus adsorbed onto filters, virus-laden filters were immersed in 400 μL of sterilized DI water for about 5 min, and then removed after vortexing from the suspension. The virus suspension was centrifuged at 19,800 g and 4 °C for 10 min , followed by resuspension of pellets in 70 μL of DI water to eliminate any interference from materials in supernatant during assays.The filters were exposed to the virus aerosols at 3, 10 and 17 kPa and suspensions of the filters were obtained, as described above. The filtration efficiency was calculated as the ratio of the amount of virus  reconstituted from the filter to that from the virus in the exposure aerosols. The concentration of virus in aerosols was determined by generating viral aerosols into a 15 mL centrifuge tube, containing 1 mL of DI water. After vortexing, virus concentrations  were measured with bicinchoninic acid assay  with bovine serum albumin as a standard. In the case of virus reconstituted from salt-coated filters, virus-laden filter suspension was replaced with DI water prior to BCA assay.bare, Filterwet, Filterwet+600μL, and Filterwet+1200μL) was calculated from the filtration efficiency at 10 kPa (near breathing pressure) using the relationship: penetration dosage = virus dosage in lethal aerosol × penetration efficiency (%)/100, where penetration efficiency (%) = 100 − filtration efficiency (%). To examine the effects of the aerosolization process on the viral infectivity change, two mice groups were infected with a lethal dose of virus before and after aerosol formation, which served as negative control groups. Body weight changes and survival rate of mice were monitored daily for 15 days. Mice with body weight loss greater than 25% were euthanized. All animal protocols were approved by the Kyung Hee University (KHU) Institutional Animal Care and Use Committee (IACUC). All animal experiments and husbandry involved in this work were conducted under the approved protocols and guidelines of KHU IACUC. KHU IACUC operates under National Veterinary Research and Quarantine Service (NVRQS), and animal welfare law and regulations of the WOAH-OIE .Lethal infectivity of influenza viruses (CA/09 H1N1) was examined in 8 week old female inbred BALB/c mice  by using the intranasal route. For bare and salt-coated filters, 12 mice per group were infected with individual penetration dosage of influenza virus through each filter. The penetration dosage of the virus through the filters  were infected with the penetrated dosage of viral aerosols (PR/34 H1N1 and VN/04 H5N1 viruses) through FilterOn day 4 after infection 6 mice of each group were sacrificed for the collection of lung samples. Lung virus titers were measured on six-well plates containing confluent MDCK cell monolayers. Inflammatory cytokines (IFN-γ) were determined using BD OptEIA mouse IFN-γ ELISA kit  following the manufacturer’s procedure.bare, Filterwet, Filterwet+600μL, and Filterwet+1200μL). Since Filterbare exhibited almost complete penetration upon pressure application, aerosols were exposed to the bare filter in the absence of pressure and samples were carefully handled to prevent mechanical agitation. To measure time-dependent stability change of virus, virus-laden filters were incubated at ambient conditions for 0, 5, 15, and 60 min after aerosol exposure, and suspended in DI water to reconstitute virus at each time point. In vitro stability of virus was characterized by measuring hemagglutinin activity (HA) and virus titers at the same concentration as lethal doseTo investigate the effects of salt-coating on viral infectivity loss, lethal influenza aerosols were exposed to four different types of filters  after incubation for 60 min at RT . 12 mice per group were infected with a lethal dose of virus collected from each type of filter. Body weight change and lung virus titers were measured as described above.To test infectivity difference observed from wet, Filterwet+600μL, and Filterwet+1200μL) were stored at 37 °C, 70% RH in an incubator  for 15 days. Every day, the filters were collected and incubated at ambient conditions for 5 min. At 1-day incubation, filtration efficiency was measured at 10 kPa from Filterwet+600μL, followed by in vivo infection test. Lethal infectivity between two different filter groups  was compared by measuring body weight change and survival rate of mice after exposure to lethal CA/09 H1N1 aerosols. XRD analysis was performed to salt-coated filters incubated for 1 and 15 days, and SEM/EDX mapping analysis for 15-day incubated samples.Salt-coated filters  to a metal, flat substrate and 3 μL of DI water were added on the surface of the filters. The contact angles were measured from images collected with an optical microscope  at RT. Images of aerosols on filter fibers were obtained using a dispersive Raman microscope .The bare and salt-coated filters were fixed with carbon tape to a metal, flat substrate and exposed to aerosols generated from 5 μL of Sulforhodamine B Dye solution . Aerosol drying time was determined with timer by observation with optical microscope.For virus stability tests, bare and salt-coated filters were exposed to CA/09 H1N1 aerosols and, after 5 and 60 min incubation, virus was recovered by suspension of the filters, as described above. To study the effects of the coating formulation during aerosol drying independently from crystal growth, bare and salt-coated filters were immersed in DI water and removed after 60 min. Subsequently, virus was incubated in the obtained suspension for 60 min. Additionally, the virus suspension was centrifuged at 19,800 g and 4 °C for 10 min to collect the samples and suspend them in DI water. For TEM analysis , samples were deposited on copper grid  and negatively stained with solution comprised of phosphotungstic acid hydrate .To identify the morphology of salt-coated filters and recrystallized salts, SEM/EDX analysis was performed for bare and salt-coated filters after coating with 10 nm thick gold layer. Scanning electron microscopy analysis  was operated in secondary electron mode at 20 kV and EDX analysis was obtained with EDX detector .To confirm the formation of crystalline NaCl coating during drying process and its stability during storage at 37 °C and 70% RH, XRD analysis  was performed at different coating conditions. Filters (1 × 1 cm) were mounted on a slide glass for XRD analysis (θ–2θ mode) using a CuKα radiation.P value of less than 0.05 was considered to be significant.To compare multiple conditions, Student’s t-test, One-way analysis of variance (ANOVA), and general linear model were used . How to cite this article: Quan, F.-S. et al. Universal and reusable virus deactivation system for respiratory protection. Sci. Rep.7, 39956; doi: 10.1038/srep39956 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Picornaviridae, genus: Enterovirus) sequences were successfully enriched using foot-and-mouth disease virus  oligonucleotide. The enrichment factor relative to the background nucleic acid was about 1,000-fold. As hybridization has less stringent oligonucleotide match requirements than PCR, few oligonucleotides are sufficient to cover the potential sequence variation in the whole genus and may even enrich nucleic acids of viruses of other related genera. Efficient enrichment of viral sequences makes its use in diagnostics cost-efficient.Despite recent advances, our knowledge of potential and rare human pathogens is far from exhaustive. Current molecular diagnostic tools mainly rely on the specific amplification of marker sequences and may overlook infections caused by unknown and rare pathogens. Using high-throughput sequencing (HTS) can solve this problem; but, due to the extremely low fraction of pathogen genetic material in clinical samples, its application is only cost-effective in special, rather than routine, cases. In this study, we present a method for the semi-specific enrichment of viral conservative sequences in a HTS library by hybridization in solution with genus-specific degenerate biotinylated oligonucleotides. Nucleic acids of the test viruses  were enriched by solution hybrid selection using pan-flavivirus oligonucleotides. Moreover, enterovirus (family:  Novel pathogens are among the major challenges to public health2. Fast and accurate identification of infectious agents is a critical step in any surveillance strategy.The majority of emerging infectious diseases (EIDs) among humans are viral zoonoses. This is exemplified by recent outbreaks of zoonotic diseases with high lethality rates 4. However, all these methods have one serious limitation: diagnostic systems efficiently detect specific NA markers only if the pathogen genome was previously identified, sequenced and annotated5.Most of the current diagnostic systems, such as PCR, NASBA and LAMP, are based on the specific amplification of nucleic acids (NAs)6. Some of these viruses can pose a real threat to human health. HTS techniques  can directly identify genetic markers of unknown infectious agents in a pool of NAs from biological samples without using classical time-consuming virological or microbiological methods. However, total NA sequencing is not used in routine infectious disease diagnostics because of certain limitations. The genetic markers of a pathogen usually account for only a few reads among a huge number of background host-related sequences 7. For example, when HTS was used to identify the Schmallenberg virus, only192 out of 94,722 reads (0.2%) were mapped to its genome8.According to some estimates, there are hundreds of thousands viruses awaiting discovery12. Some tools for viral NA enrichment are based on PCR with non-specific or genus-specific degenerate oligonucleotide primers. The preferential amplification of pathogenic sequence (PATHseq) is based on using a set of 88 specific 8-mer oligonucleotides  as primers for the synthesis of secondary cDNA strands13. Alternatively, the VIDISCA method based on the presence of virus-specific endonuclease restriction sites was suggested14. Advanced VIDISCA uses non-rRNA binding hexamers in a reverse transcription (RT) reaction15. It is also possible to enrich viral sequences by a broad-range RT-PCR using a set of degenerate oligonucleotides17.The following strategies have been developed to increase the viral/host NA ratio before or after NA extraction: physical enrichment methods ; biological methods (exonuclease treatment); host NA depletion18. The authors described target selection of genome sequences of known viruses by hybridization in a solution using 1,993,200 biotinylated oligonucleotides representing 342,438 viral coding sequences. However, as this approach used specific (non-degenerate) oligonucleotides, theoretically, it can only detect viruses that have regions with > 80% sequence identity to known viruses.The recently developed virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) technique increases the sensitivity of sequence-based virus detection by HTSIn this study, we describe a cost-effective method for the significant enrichment of conservative fragments of viral genomes by hybridization with degenerate genus-specific biotinylated oligonucleotides. We analyze the utility of the technique for NA library enrichment in order to increase HTS efficiency and decrease the cost per sample.19, sequences sharing more than 90% identity were discarded. The data set was refined manually to exclude pegiviruses and incomplete sequences. Foot-and-mouth disease virus alignment was prepared similarly. Hybridization oligonucleotides matching the most conserved genome fragments were designed according to the following principles:All full-genome flavivirus sequences available in Genbank in October 2014 were extracted. After alignment with MAFFT-length between 50–90 nt- 90% match with all target sequences (degenerate nucleotides were used where necessary)20.- whenever possible, degenerate positions were simplified to G or K (T/G), given that mismatches involving T and G result in a minimal penalty to hybridization efficiency21.The potential ability of oligonucleotides to hybridize with selected target sequences was checked using the Mfold web serverFinally, three pan-flavivirus biotinylated oligonucleotides were chosen: Flavi-8469 ATGACNGAYACYACNVCNTTTGGRCARCARMGRGTKTTYAARGARAARGTKGACAC ; Flavi-8844 GCNAARGGNAGNMGNGCYATNTGGTWYATGTGGCTNGGNDSNMGNTTYYTNGARTTYGARGCYYTNGGNTTYYTNAAYGARGAYCAYTGG ; and Flavi-9834 GGNGMNTGGATGACNACWGANGACATGYTNNNNGTNTGGAAYVRRGTNTGGAT .The hybridization probe aimed at all FMDV sequences was designed similarly: ggTggKWTgCCKTCTggKTgTKCTggKACYTSTgTgTTKAATTCAATKWTKAA .Donor whole blood samples were collected with written informed consent from volunteers in the CRIE . All experiments were performed in accordance with relevant guidelines and regulations and were approved by the CRIE ethics committee. Total human RNA was extracted from the human mononuclear cell fraction of the samples using the RNeasy Mini Kit (Qiagen).The Gagar Japanese encephalitis virus (JEV) strain was cultured in pig embryo kidney cells. The 17D yellow fever virus (YFV) strain was passaged in Vero cells. The Gregory Echovirus 11 (E11) strain, which was originally obtained from ATCC, was cultured in human rhabdomyosarcoma cells. Viral RNA was extracted from cell culture medium using the Viral RNA Mini Kit (Qiagen).The relative concentrations of isolated viral RNA and host RNA were estimated using in-house qPCR assays. Then, total human RNA (100 ng) was mixed with either 20 pg of YFV, 30 pg of JEV or 100 pg of E11. The total volume of each sample was adjusted to 10 µL with water.First-strand cDNA was synthesized from RNA using Reverta-L RT kit (AmpliSens) according to the manufacturer’s instructions. The kit uses MMLV reverse transcriptase and random hexamer primers.Second-strand cDNA synthesis was performed by adding 10 µL of RT mixture, 8 µL of 5x buffer C (AmpliSens), 5 µL of dNTP (2.5 mM) (AmpliSens), 1 µL of random hexamer oligonucleotide (168 pmol/µL) (in-house) and 1 µL of RNAse H (New England Biolabs), made up to 50 µL with milli-Q water. The mixture was incubated at 37 °C for 10 min. The enzymes were inactivated by heating at 95 °C for 2 min. After cooling on wet ice, 1 µL of Bst polymerase (New England Biolabs) and 0.5 µL of Klenow exo- (New England Biolabs) were added. Incubation conditions were as follows: 4 °C for 45 secs, 20 °C for 5 min, 37 °C for 5 min, 45 °C for 5 min, 50 °C for 5 min and 68 °C for 5 min. The reaction product was purified with Agencourt AMPure XP beads (Beckman Coulter) using the manufacturer’s protocol, with the exception of an altered bead-to-DNA ratio, where we used a 1.5:1 beads-to-DNA solution ratio, instead of a 1.8:1 ratio. DNA was eluted to 17 µL of milli-Q water.22. Total reaction volume was adjusted with milli-Q water up to 25 µL. The mixture was incubated at 55 °C for 5 min. The reaction was stopped by adding 1 µL of 200 mM EDTA. The reaction product was purified with Agencourt AMPure XP beads (Beckman Coulter) using the manufacturer’s protocol. DNA was eluted to 22 µL of milli-Q water.To incorporate sequencing adapters, purified cDNA libraries (15 µL) were mixed with 5 µL of 5x transposase buffer (in-house) and 0.5 µL of the transposase and transposon complex, termed transposome (in-house)The cDNA libraries were amplified by real-time PCR. PCR reactions (50 µL) included 200 nM of NTS1 primer (5′ TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG 3′), 200 nM of NTS2 primer (5′ GTC TCG TGG GCT CGG A GAT GTG TAT AAG AGA CAG 3′), 0.25 mM of dNTP, 14 µL of PCR-mix-2-FL (AmpliSens), 1.2X of EvaGreen (Biotium Inc.), and a 20 µL purified cDNA sample.23.The oligonucleotides  were separated from other parts of the PCR mixture  by a wax layer. Amplification conditions were as follows: 30 °C for 1 min; 37 °C for 1 min; 72 °C for 1 min; denaturation at 95 °C for 5 min; 18 cycles at 95 °C for 20 s, 65 °C for 30 s and 72 °C for 60 s. Real-time PCR amplification was performed using a Rotor Gene 6000 instrument (Qiagen). The amplification was monitored to stop the reaction during the exponential phase and before the reaction reached the plateauAliquots of PCR product were stored at −20 °C for further analysis.The cDNA libraries containing human and YFV or human and JEV genetic material were hybridized with three pan-flavivirus biotinylated oligonucleotides . The library containing human and E11 genetic material was hybridized with FMDV-specific biotinylated oligonucleotide.24 with minor modifications.Hybrid selection was performed similarly to a previously published methodBriefly, a 20 µL mix containing 1X hybridization buffer , biotinylated oligonucleotide(s) , 1 µg Cot-1 DNA (Invitrogen), 50 uM oligonucleotide Nts1_SpC3 (5′ CTG TCT CTT ATA CAC ATC TGA CGC TGC CGA CGA spC3 3′), 50 uM oligonucleotide Nts2_SpC3 (5′ CTG TCT CTT ATA CAC ATC TCC GAG CCC ACG AGA C spC3 3′) and 3 µL of an amplified cDNA library was heated at 97 °C for 5 min. Next, the mixture was slowly cooled to 47 °C (3 °C every 10 min). After 15 h at 47 °C, the hybridization mixture was added to 100 µg of M-270 streptavidin Dynabeads (Invitrogen), which had been initially processed as follows: the beads were washed twice in 150 µL of a buffer containing 2 M NaCl, 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, then incubated in 14 µL of a solution containing 1X hybridization buffer, 2.2% casein (Stereospecific Detection Technologies GmbH) and 100 mg/L of Cot-1 DNA (Invitrogen) for 60 min at 37 °C, and then resuspended in 80 µL of a buffer containing 0.4 M NaCl and 6.25 mM Tris-HCl (pH 7.5).After 15 min at 37 °C, the beads were pulled down by the magnetic rack and washed once at 47 °C for 5 min with 1 mL of prewarmed 1xSSC /0.1% SDS (sodium dodecyl sulfate). Next, the beads were washed twice at 47 °C for 5 min with 1 mL of prewarmed 0.2xSSC/0.1%SDS, once at room temperature for 5 min with 1 mL 0.2xSSC/0.1%SDS, and once at room temperature for 5 min with 1 mL of 0.2xSSC/0.1% Tween 20. The beads were resuspended once after each washing step and collected by the magnetic rack. Hybrid-selected DNA, which was attached to the beads, was resuspended with 20 µL of 0.1% Tween 20.The cDNA libraries captured on the beads were reamplified by PCR. PCR reactions (50 µL) consisted of 200 nM NTS1 primer (5′ TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG 3′), 200 nM NTS2 primer (5′ GTC TCG TGG GCT CGG A GAT GTG TAT AAG AGA CAG 3′), 0.25 mM dNTP, 14 µL of PCR-mix-2-FL (AmpliSens), 3 µL of 20X EvaGreen (Biotium Inc.) and 20 µL of bead suspension.The oligonucleotides and dNTP  were separated from other components of the PCR mixture  by a wax layer. Amplification conditions were as follows: denaturation at 95 °C for 5 min; 19 cycles at 95 °C for 20 s, 65 °C for 40 s and 72 °C for 60 s. Real-time PCR amplification was performed using the Rotor Gene 6000 instrument (Qiagen).The relative concentration of viral cDNA before and after hybridization was measured by a specific qPCR using GAPDH  as the housekeeping gene. The cycle threshold  was chosen as being at the middle of the exponential phase of the amplification curve. The ratio between the host and viral NAs was assessed in terms of the Ct value difference. Oligonucleotide primers and probes for viral cDNA quantification Table  were choFor HTS, the initial and enriched cDNA libraries were indexed by PCR using Nextera-compatible oligonucleotide primers. The libraries were pooled at equal ratios. Paired-end 250-base sequencing by synthesis was performed on the MiSeq System (Illumina) using protocols provided by the manufacturer. Samples were de-multiplexed using the Illumina software, with FASTQ files generated.25. The Bowtie 226 tool was used for aligning sequencing reads to references. Alignments were processed using SAMtools27 and pysamstats software, and figures were generated in the R environment.Reads in the FASTQ format were filtered by quality (Q30), and the adapter sequences were removed by TrimmomaticFirst, enrichment efficiency was estimated by comparison of the host:viral cDNA ratio in libraries before and after hybridization Table .Table 2EIn unprocessed samples, delta Ct, indicating the host:pathogen ratio, was 6.2, 5.0 and 9.3 for human/YFV, human/JEV and human/E11 libraries, respectively. As a result of hybridization, this ratio changed to −5.1, −7.5 and 2.3 for human/YFV, human/JEV and human/E11 libraries, respectively. According to qPCR data, the ratio of target sequences to host sequences increased by approximately 2^11, or 2,000 times, in the case of the human/YFV library, approximately 2^12, or 4,000 times, in the case of the human/JEV library after hybridization with three pan-flavivirus oligonucleotides, and about 2^7, or 100 times, for the human/E11 library after hybridization with the FMDV-specific probe. Due to the use of the semi-quantitative PCR, the enrichment efficiency estimates should be considered as approximate; however, they at least indicate the order of magnitude of enrichment efficiency.HTS was also performed to validate the hybridization-induced change in the host:virus cDNA ratio. Significant enrichment of target viral sequences was observed in samples after hybridization Table . The numThe pattern of HTS reads coverage along the YFV, JEV and ECHOV genomes corresponded to an expected enrichment in the genome regions, which were complementary to the biotinylated oligonucleotides Figs , S1. IntRapidly evolving methods in molecular diagnostics face limitations associated with extremely high genetic diversity of viruses. Furthermore, most of the global virome remains undiscovered, resulting in a lack of reference sequences for novel pathogen identification. One way to overcome this situation is by developing a system, which allows specific enrichment of unknown virus content in processed biological material based on known homologous sequences.Enterovirus and Aphthovirus genera are very distantly related within the Picornaviridae family. Therefore, the specificity breadth of the method goes beyond a genus and could cover distantly related genera within a highly diversified virus family.We were able to increase viral cDNA content for two distinct flaviviruses (YFV and JEV) by hybridization of HTS-ready libraries with pan-flavivirus oligonucleotides. Comparison of the genomes of these viruses  using BLASTn shows a 66% identity level at 27% of the genome coverage. Oligonucleotide probes were designed based on the most conserved genome fragments and had 96.4–100.0% sequence identity (considering degenerate positions as matches) with the viruses . The hybridization oligonucleotides showed no preference towards YFV and JEV in the enrichment experiment. The data set for the oligonucleotide design included over 100 highly diversified flavivirus sequences, therefore, comparable enrichment efficiency may be expected for any known flavivirus. To further test the specificity range of the method, we tested whether it was possible to enrich the cDNA of one virus genus with hybridization probes designed for another genus of the same family. Indeed, it was possible to enrich the content of enterovirus cDNA using an oligonucleotide designed to to the FMDV virus (79.2% sequence identity between the FMDV oligonucleotide and the enterovirus). The 12 and 246, thereby greatly exceeding the maximum degeneracy of 28, which is acceptable in a PCR primer.Importantly, cDNA enrichment by hybridization lacks theoretical specificity limitations typical of PCR, such as the strict requirement of the precise match of the three 3′-end nucleotides of the PCR primer. Any small number of additional mismatches in any part of the hybridization oligonucleotide will only reduce hybridization efficiency, but not completely prevent it. Therefore, this approach is theoretically more robust in terms of sequence variation than genus-specific PCR with degenerate primers. Moreover, the degeneracy of the probes (the number of possible unique oligonucleotides within a degenerate oligonucleotide preparation) was between 228. As expected, this bias was further deepened in our protocol. Therefore, it is advisable to use several hybridization oligonucleotides per taxon. On the other hand, using specifically designed degenerate probes, which are complementary to conserved genome regions, is much more affordable than synthesizing multiple specific oligonucleotides to the whole genome, as suggested earlier18.Enrichment of cDNA libraries by hybridization involves four steps of NA copying and amplification: reverse transcription, second-strand synthesis and two rounds of library amplification before and after hybridization. Even a standard library preparation procedure results in the uneven amplification of genome fragments29. Moreover, the correctness of assumption about a pathogen’s presence in a sample completely depends on the qualification of a medical specialist.At the present time, molecular diagnostic tools mostly rely on the specific amplification of marker sequences. In other words, while a standard molecular test answers the question about whether pathogen X is present in the sample, this does help investigators to answer the more important question, “which pathogen is present in the sample?”To date, many tools for unbiased HTS-based pathogen detection exist. However, due to the extremely low fraction of a pathogen’s genetic material in clinical samples, its usage is only cost-effective in very special cases. The proposed technique can enrich the share of pathogen genome fragments in a library, even if the NA sequence of a pathogen is currently unknown. It can also significantly enhance the utility of HTS for diagnostics.Supplementary information


Prunus spp. are being discovered and characterized, especially in the family Betaflexiviridae. Double-stranded RNAs from symptomatic leaves of a Japanese apricot (Prunusmume) tree from Japan were purified and analyzed by Illumina sequencing. Blast comparisons of reconstructed contigs showed that the P. mume sample was infected by a putative novel virus with homologies to Cherry virus A (CVA) and to the newly described Currant virus A (CuVA), both members of genus Capillovirus. Completion of the genome showed the new agent to have a genomic organization typical of capilloviruses, with two overlapping open reading frames encoding a large replication-associated protein fused to the coat protein (CP), and a putative movement protein (MP). This virus shares only, respectively, 63.2% and 62.7% CP amino acid identity with the most closely related viruses, CVA and CuVA. Considering the species demarcation criteria in the family and phylogenetic analyses, this virus should be considered as representing a new viral species in the genus Capillovirus, for which the name of Mume virus A is proposed.With the increased use of high-throughput sequencing methods, new viruses infecting  Prunus spp. have been described [Betaflexiviridae family have been discovered and characterized, leading to the introduction of two novel genera, Prunevirus [Robigovirus [Secoviridae has been described in peach, for which the name of Peach leaf pitting-associated virus was proposed [Tymoviridae family were also recently reported from nectarine and peach: the nectarine virus M and the peach virus D, respectively [Since the advent of high-throughput sequencing methods in the plant virology field, a number of new viruses infecting escribed ,2. In paunevirus  and Robiigovirus . More reproposed . Two marectively ,7.Prunus mume, also known as Japanese apricot, belongs to the genus Prunus in the family Rosaceae and originates from China. It has been cultivated throughout East Asia for fruit production, as well as for ornamental purposes. Besides its cultural significance, P. mume is also cultivated for culinary or medicinal uses. To this date, 15 viroids or viral species from families Pospiviroidae, Closteroviridae, Bromoviridae, Potyviridae, Luteoviridae, Geminiviridae, and Betaflexiviridae have been described from symptomatic or from symptomless P. mume [Prunus spp. in general, many of the viral species infecting P. mume belong to the Betaflexiviridae family. Four genera are thus represented in P. mume among the 11 comprised in the family, with eight viruses: Apricot latent virus (ApLV) and Asian prunus virus 1 and 2  (Genus Foveavirus), Peach mosaic virus (PcMV) and Apple chlorotic leaf spot virus (ACLSV) (Genus Trichovirus), Apricot vein clearing associated virus (AVCaV) (Genus Prunevirus), and Apple stem grooving virus (ASGV) and Cherry virus A (CVA) (Genus Capillovirus) [ P. mume ,9,10,11.lovirus) ,13.P. mume tree showing slight symptoms of diffuse chlorotic spots was determined by a high-throughput sequencing approach. Phylogenetic analyses allow one to conclude that this virus, for which the name of Mume virus A is proposed, belongs to the genus Capillovirus in the family Betaflexiviridae, extending our knowledge of Prunus-infecting viruses.In the present study, the complete genome sequence of a novel viral species from a Japanese P. mume, “PM14”) showing diffuse chlorotic spots on leaves were collected in Japan (Kyoto province) and submitted to high-throughput sequencing analysis. The PM14 isolate was propagated by chip budding on GF305 peach seedlings under level 3 containment greenhouse conditions. Various Prunus sources from China, Japan, Czech Republic, Azerbaijan, Kazakhstan, Italy, and France,  were screened by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for the presence of Mume virus A.Symptomatic leaves from a Japanese apricot tree (wt)/v) in 50 mM potassium phosphate buffer, pH 7.6. Before transmission, activated charcoal (90 mg/mL), nicotine hemisulfate (2% v/v), and polyvinylpyrrolidone 40  were added to the homogenate. Carborundum (400 mesh) was used as the abrasive. The following plants were evaluated as potential hosts: Chenopodium amaranticolor, C. quinoa, Nicotiana benthamiana, Nicotiana tabacum cv. Xanthi, Nicotiana glutinosa, and Phaseolus vulgaris cv. Vedette. The appearance of symptoms was monitored over a three-week period.Young leaves of GF305 infected with the PM14 source were used as the inoculum. Leaves were ground  or a pipeline integrating the Newbler assembler. Contigs were then annotated by BlastN and BlastX comparison with the Genbank database . Mapping on reference genomes allowed the ordering of the contigs and the reconstruction of a unique scaffold spanning the viral genome.Double-stranded RNAs (dsRNAs) were extracted from symptomatic leaves of PM14 according to the protocol of Gentit et al. . The purTo complete the viral genome sequence, primers were designed from the contig sequences and used to amplify PCR products spanning the gaps between contigs . The twoThe sequence reported in the present manuscript has been deposited in the GenBank database under accession number MG783575.Extraction of total nucleic acids (TNAs) was performed according to the protocol 2 described by Foissac et al. . Five miMultiple alignments of nucleotide (nt) or amino acid (aa) sequences were performed using the ClustalW program  implemenCapillovirus, CVA or Currant virus A (CuVA). These contigs, tentatively identified as belonging to a capillovirus, were manually assembled by mapping to CVA genome, in order to create a scaffold of 6542 nt missing the 5′ and 3′ genome ends and containing five internal gaps. The genome sequence of the PM14 isolate was completed by targeted PCR as described in the Materials and Methods section. The assembled complete genome sequence was deposited in the GenBank database under accession number MG783575.Double-stranded RNAs purified from the PM14 source were sequenced on an Illumina Miseq platform using paired-end sequencing (2 × 250 nt). After demultiplexing, quality trimming, and de novo assembly of the 33,113 reads obtained for that sample, Blast analysis allowed one to identify ten contigs sharing significant identity with known plant viruses. Four contigs integrating 1.6% of total reads were tentatively identified as belonging to a luteovirus (under characterization). The six remaining viral contigs integrated 1.59% of the reads and showed between 71% and 78% nt identity (54% to 83% aa identity for the encoded proteins) with the corresponding regions of two members of the genus capilloviruses , with an overall genome organization similar to that of oviruses . A phyloe 99% in  and, morBetaflexiviridae replicases are found within the ORF1 protein: a viral methyltransferase domain , a viral helicase 1 domain , and a RNA-dependent RNA polymerase 2 domain . No AlkB domain (2OG-FeII-Oxy-2-domain) has been identified within the ORF1 protein, as for most of Capillovirus members, with the exception of CuVA ORF1 in which AlkB domain was found between aa 623 and 736 [The ORF1 (2333 aa) encodes a large replication-associated protein aa 1–2105) fused to the coat protein . Three conserved domains typical for  and 736 ,21. The 05 fused Capillovirus members confirm a clear clustering of the PM14 isolate with CVA and CuVA, with highly significant bootstrap values (100%)  in the replicase and CP genes [Capillovirus. We therefore propose the name of Mume virus A (MuVA) for this species.Phylogenetic analyses based on alignments of CP or MP sequences from s (100%) A,B. The d the MP . The levCP genes , the PM1Citrus tatter leaf virus (CTLV) and Pear black necrotic leaf spot virus (PBNLSV) are recognized as citrus and pear isolates of ASGV, respectively [Prunus species originated from seven different countries were screened by a specific RT-PCR test , suggesting a limited prevalence of MuVA. Further investigations are necessary to evaluate the distribution, prevalence, and potential pathogenicity of this new member of the Capillovirus genus.Despite significant efforts to transmit MuVA to various herbaceous plants, none of the plant species tested could be successfully inoculated. No local or systemic symptoms appeared on inoculated plants, and RT-PCR assays performed to detect MuVA were all negative. MuVA was, however, successfully graft-transmitted to peach seedlings (GF305 indicator). However, no symptoms could be identified on leaves or in growth habit of the infected peach plants (data not shown). The initial tree in which MuVA was detected showed diffuse chlorotic spots on leaves. However, it is co-infected by a potentially novel luteovirus (currently under characterization), so that it is difficult to assess the role of MuVA in the symptomatology observed. Moreover, due to the dsRNA sequencing strategy used in the present study, the potential contribution in the observed symptoms of another agent such as a DNA virus or a viroid cannot be excluded. Among the capilloviruses described so far, four are known to infect fruit trees, which represent in fact only two viral distinct species, CVA and ASGV, since ectively ,23. CVA ectively ,25. No nectively ,24. To a


Astroviruses are recognized as a leading cause of gastroenteritis in humans and animals. They are also associated with extra-intestinal diseases, such as hepatitis in ducklings, nephritis in chickens, and encephalitis in cattle. In February 2017, a fatal infection of goslings characterized by visceral urate deposition was reported in the Shandong province, China. Our systematic investigation led to the isolation of an astrovirus, designated AAstV/Goose/CHN/2017/SD01, and similar disease was reproduced by experimental infection of healthy goslings, fulfilling Koch’s postulates. The isolated astrovirus replicated well and resulted in 100% mortality of goose embryos. Complete genome sequence analysis revealed that the isolate was genetically distinct from known astroviruses and closely related to members of the avastrovirus genogroup II. Experimental infection showed that the isolate was highly pathogenic in goslings, causing clinical signs, growth repression and in many cases mortality. Histopathological examination indicated that lesions occurred mainly in the kidneys of infected birds. However, virus-specific genomic RNA was detected in all representative tissues, and virus shedding was detected up to 12 days after inoculation, suggesting that the isolate was able to spread systemically and replicate efficiently in vivo. Collectively, our study demonstrates, for the first time, the etiological role of a genetically distinct astrovirus in the fatal infection of goslings. Astroviridae family. Currently, two genera: namely Mamastrovirus and Avastrovirus are distinguished within this family. The genus Mamastrovirus includes astrovirus species isolated from humans and a number of mammals. Isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus Avastrovirus2. AstVs have been detected in humans and a variety of animal species, including non-human primates, other mammals and avian species5. Their genomes are 6.8–7.9 kb in length, consisting of a 5′-untranslated region (UTR), three open reading frames (ORFs), a 3′-UTR and a poly (A) tail6. The high degree of genetic diversity among AstVs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species8. Human classical AstVs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis8.Astroviruses (AstVs) are non-enveloped, positive-sense, single-stranded RNA viruses belonging to the 16. Increasing evidence indicates that there is a high degree of cross species transmission of AstVs between domestic birds, and even the potential to infect humans17. By comparison, fewer AstV infection cases have been described in domestic goose flocks. Bidin et al.18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and pre-hatching mortality of goose embryos. Studies to detect AstV genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks20. In February 2017, an outbreak of disease was reported in a goose farm in Weifang, Shandong Province, China. Affected flocks (containing 2000–3000 goslings) experienced continuous mortality rates ranging from 20 to 30% during the first 2 weeks of the outbreak despite antibiotic and supportive treatment. We conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian AstV. The pathogenicity of this virus was evaluated by experimental infection of goslings.In poultry, AstV infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic lossesIn the field, affected goslings displayed signs of depression and were observed sitting alone Fig. . The palRdRp) gene21. Sequence and phylogenetic analysis of the amplified RdRp gene with other known AstV sequences retrieved in the GenBank database showed that the detected virus could be assigned to the subgroup 1.2 within the avastrovius group 1, with the closest relationship to the astrovirus detected from dropping samples of northern shovelers (Anas clypeata) in Hong Kong . The subsequent passage of the isolate caused 60–100% mortality of the embryos by 5 dpi. The dead embryos exhibited severe subcutaneous edema and hemorrhages with necrotic foci in the liver  as proposed by Martella et al.23.Virulent bacteria were not isolated and tissue samples were negative by PCR for goose parvovirus, goose hemorrhagic polyomavirus, reovirus, or Tembusu virus. However, a DNA fragment was amplified from the RNA sample extracted from the pooled spleen, liver and kidney tissues using pan-AstV RT-PCR targeting the AstV RNA-dependent RNA polymerase  in length with similar gene organization to other known avastroviruses, consisting of a 5′-UTR of 10 nt, three sequential ORFs , a 3′-UTR of 236 nt and a poly (A) tail stretching 30 nt Fig. . ORF1a o225 to Q225. The potential effect of the mutation on the virus adaptation need to be further evaluated.To determine the potential genetic mutation(s) that might occur during the goose embryo passage, the initial virus genome was sequenced using the total RNA extracted from the clinical case tissue homogenate. Nucleotide differences between the initial virus genome and that of the fourth embryo-passaged isolate was shown in Table The complete genome sequence of AstV SD01 had the highest similarity to those of turkey AstV 2 (TAstV-2) strains, at the level of 61.6–62.4%  during the experimental period. At necropsy, slight to moderately swollen kidneys were noted for the deceased birds Fig. . HistoloInfected goslings exhibited signs of depression from 3 dpi and this symptom persisted for 3–4 days. One bird died at 5 dpi and severe urate deposition, similar to that seen in the field cases, was evident on the surface of the heart, liver, and kidney Fig. . For theWhen the samples were tested by RT-PCR for virus shedding evaluation, the AAstV specific RNA was sequentially detected from the cloacal swabs of infected goslings from 2 to 12 dpi Fig. . Viral ROrally inoculation of goslings with the isolate resulted in depression of 4 birds from 5 to 8 dpi. One gosling died at 7 dpi with evident urate deposition on the surfaces of heart and liver at necropsy. Another severely ill bird was killed humanely at 8 dpi for animal welfare reasons. Viral RNA was detected by RT-PCR in the cloacal swabs from 4 dpi Fig.  and grow19, but the prevalence and pathogenicity of AstV among domestic geese remains poorly understood due to the lack of efficient in vitro culture techniques and diagnostic assays. In this study, AAstV SD01 was successfully isolated by inoculating tissue samples into goose embryos, as confirmed by RNA detection methods similar to those for pan-AstV. We successfully cultured the virus in vitro provided a convenient method for virus propagation and laboratory diagnosis. Isolation of the AAstV from field cases and reproduction of the disease in a gosling infection model with the inoculating virus prepared in embryos fulfils Koch′s postulates.Several studies have reported the existence of AstV in goose flocks10. In this study, the mortality of young goslings caused by subcutaneous or oral inoculation indicated that AAstV SD01 was highly pathogenic. The experiment was fairly a represent of the situation in the field, where susceptible goslings are exposed to AstV soon after they are placed in contaminated houses. Apart from mortality, avian AstV infection can decrease feed intake and alter feed conversion efficiency, leading to growth repression. The decrease in body weight of infected goslings is a major concern as a 29% lighter body weight at 14 dpi has a considerable economic impact.AstV infection occurred within the first days or week of life usually resulted in a worse outcome, as the age-dependent pathogenicity of AAstV has been reported25. Extra-intestinal infection with nephritis has been reported in birds infected with chicken astrovirus and avian nephritis virus26. Viral RNA was found in all of the tissues sampled from the infected goslings killed on 5 dpi, indicating that the goose AstV has a wide tissue tropism and spread systemically after inoculation. Virus shedding was detected by RT-PCR and persisted in the infected goslings for about 12 days, further indicating that the virus replicated efficiently in vivo.Histologic examination revealed the presence of a proteinaceous substance in the renal tubules, indicating that AAstV SD01 infection caused increased permeability of the kidney epithelia barrier. Degeneration and necrosis of tubular epithelial cells found in the deceased goslings provided further evidence of kidney function damage. These results could explain the development of visceral urate deposition in infected goslings. Increased epithelium permeability due to AstV infection has been reported in both human and avian species27. Nonetheless, based on the limited number of goslings infected in present study, it is not likely to get accurate evaluation for the virulence of the isolate.It is interesting that encephalitis lesions were observed in the deceased goslings (data not shown), along with the detection of AAstV SD01 RNA in the brain tissue Fig. . HoweverThe present work describes the isolation of the astrovirus AAstV/Goose/CHN/2017/SD01 from tissue samples of goslings dying from a disease characterized by visceral urate deposition. The successful reproduction of the disease by experimental infection demonstrates the etiological role of this AAstV. Based on the genetic analysis of the complete capsid region at amino acid level, the isolate should be assigned as a member within the genotype consisting of TAstV-2 and DAstV-1 strains. The high variability of the genomic sequence to other known astroviruses suggest more detailed antigenic investigations should be performed.2 for 48 h. Then the spleen, liver, and kidney tissue were pooled and tested for the presence of goose parvovirus28, goose hemorrhagic polyomavirus29, AstV21, reovirus30, and Tembusu virus31, respectively.For bacteriological diagnosis, liver, and kidney samples from dead goslings were first inoculated onto tryptic soy agar plates  containing 2% fetal calf serum, and incubated at 37 °C under an atmosphere with 5% COg, at 4 °C for 10 min. The supernatant was filtered using a syringe-driven filter unit with a pore size of 0.2 μm and the filtrate was inoculated into five 9-day-old goose embryos (0.2 ml/egg) via the chorioallantoic membrane route. Embryos were incubated at 37 °C and candled daily. Embryos that died beyond 24 h and those that survived until 5 day after inoculation were chilled to 4 °C overnight. The allantoic fluids were collected for a hemagglutination (HA) activity test performed by a standard method using 1% chicken red blood cells as an indicator, and then were subjected to additional passage in goose embryos.To isolate AstV, the kidney, spleen, and liver samples were homogenized with sterile phosphate buffered saline  to a 20% suspension (w/v) and centrifuged at 8000×th and 9th passage, the virus suspension was 10-fold serially diluted with PBS and inoculated into 9-day-old goose embryos via the chorioallantoic membrane route. The embryos were incubated for 7 days at 37 °C and the mean embryo lethal dose (ELD50) of infectious virus was calculated using the Reed–Muench method32.To determine the infectious titers of the 4pEASY-Blunt Simple Cloning Vector . The recombinant vector was transformed into competent Escherichia coli Trans-T1 and transformants containing the PCR amplified fragment were selected by PCR following the manufacturer’s instruction. At least two representative transformants were subjected to bidirectional DNA sequencing using Applied Biosystems ABI3730 . The 5′ and 3′ ends of the viral genome were amplified using the 5′/3′ RACE kit  following the guidelines of the manufacturer. The initial complete genome was assembled and manually edited using the Software ContigExpress. Based on the initial genome sequence, additional primer pairs were designed  and the cDNA was synthesized using a Reverse Transcription System  with random primers following the manufacturer’s instructions. Viral genomic fragments were amplified by PCR with primer sets designed against conserved regions of the AstV sequences retrieved from the GenBank database Table . PCR proed Table , and PCRTo evaluate the potential adaptive mutation (s) of the virus that might occur during the process of goose embryo passage, we sequenced the complete genome of initial virus using the total RNA extracted from the clinical case tissue homogenate of kidney, spleen, and liver using the method described above. The genome sequence was compared with that of the isolate of fourth passage.Nucleotide sequences of the virus genome and the deduced amino acids of the ORFs were compared with known AstV ORF sequences retrieved from the GenBank database. Neighbor-joining trees of the complete genome nucleotide sequences, ORF1b and ORF2 amino acid sequences were constructed using MEGA 7.0 software, with bootstrap values calculated from 1000 replicates. The mean amino acid genetic distance (p-dist) of the viral RdRp and capsid were calculated using MEGA 7.0 with a bootstrap test obtained from 100 replicates.Anser anser domesticus) were obtained from a local hatchery. Birds were raised in negative pressured isolators with ad libitum access to feed and water. Three experiments were conducted to investigate the pathogenicity of the isolate. Animal infection experiments were approved by the China Agricultural University Animal Ethics Committee.One-day-old goslings (4 ELD50). Goslings inoculated with sterile PBS (uninfected control) were kept in a separate isolator. Clinical signs and mortality were recorded for 10 days. Dead birds were necropsied immediately, and tissue samples of the heart, liver, spleen, kidney, lung, thymus, bursa, and brain were collected. A portion of these tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4–5μm sections were cut and stained with hematoxylin and eosin (H&E). Pieces of liver and kidney tissue were frozen and subjected to virus isolation.The aim of this experiment was to evaluate whether the isolate was pathogenic in goslings. Thirteen 2-day-old goslings were infected by subcutaneous inoculation with 0.5 ml of the virus suspension prepared from the infected goose embryos at the fourth passage , <0.01 (**).This experiment was designed to investigate the in vivo replication of the virus and its impact on the growth of infected birds. Fourteen 5-day-old goslings were infected as in experiment 1. Birds inoculated with sterile PBS were used as the uninfected control. The tissue distribution of the virus was analyzed in three infected goslings 5 dpi. Representative tissues samples were collected and subjected to histopathological examination and virus RNA detection. For virus shedding detection, cloacal swabs were collected from both infected and control birds on 2, 4, 6, 8, 10, 12, and 14 dpi. Swabs were immersed in 1 ml of 1 × Dulbecco’s modified Eagle’s medium  and frozen at −75 °C. Simultaneously, individual birds were weighed. The difference between the mean body weights of infected and uninfected control birds was tested by Student’s n = 6) or intranasally (n = 6) at 5-day old with the astrovirus isolate at the dose as experiment 1. Five goslings were kept as uninfected control. Mortality were checked daily. Cloacal swabs were collected for virus shedding detection as described in experiment 2.To investigate the possible infection route, three groups of 1-day-old goslings were kept at separate isolator. Birds were inoculated orally  in PBS and were homogenized using beads in a high-throughput Tissuelyser . Then, the homogenate was centrifuged at 8000×Table S1Table S2Table S3Figure S1Figure S2Figure S3Figure S4Figure S5


Glycogen storage disease type Ia is a genetic disorder that is associated with persistent fasting hypoglycemia and the inability to produce endogenous glucose. The development of diabetes with glycogen storage disease is exceedingly rare. The underlying pathogenesis for developing diabetes in these patients is unclear, and there are no guidelines for treatment.We describe a case of a 34-year-old woman of South Asian descent with glycogen storage disease type Ia, who developed uncontrolled diabetes mellitus as a young adult. Hyperglycemia was noted after childbirth, and worsened years later. Treatment for diabetes was difficult due to risks of hypoglycemia from her underlying glycogen storage disease. With minimal hypoglycemic events, the patient’s blood glucose improved with exercise in combination with a sodium-glucose co-transporter 2 inhibitor and an alpha glucosidase inhibitor.We report a rare case of diabetes in the setting of glycogen storage disease-Ia. Based on the literature, there appears to be a relationship between glycogen storage disease and metabolic syndrome, which likely plays a role in the pathogenesis. The management of glycemic control remains a clinical challenge, requiring management of both fasting hypoglycemia from glycogen storage disease, as well as post-prandial hyperglycemia from diabetes mellitus. Glycogen storage disease type 1 (GSD-I) is a rare genetic condition that develops due to an inborn error of metabolism causing deficient activity of the enzyme, glucose 6-phosphatase .GSD-I is inherited in an autosomal recessive manner, with an incidence of 1 out of 100,000 births . There aPatients with GSD-Ia are recognized early for their episodes of hypoglycemia in childhood which can be severe, causing seizures, neurocognitive decline, as well as stunted growth . TreatmeGSD-Ia is a disorder that highlights the importance of glucose as a basic energy source throughout the body. Understanding the physiology of uptake, utilization, and storage of glucose is essential for medical treatment of various pathologies involving altered glucose metabolism. Patients with GSD-1a suffer from fasting hypoglycemia; thus it would seem paradoxical for an individual who has primarily a disease of decreased endogenous glucose production to develop diabetes. We describe a case of a patient with known GSD type Ia who developed uncontrolled diabetes. In reviewing our patient’s case presentation, we explore the pathophysiology of glucose homeostasis, and the challenge to find an appropriate medical therapy.A 34-year-old woman of South Asian descent with a past medical history of obesity [body mass index (BMI) 41.6], polycystic ovarian syndrome, psoriasis, horseshoe kidney with uric acid kidney stones, and hypothyroidism, was diagnosed at age 3 with GSD-Ia. As an infant she had hypoglycemic episodes resulting in seizures. A liver biopsy showed decreased G6PC enzymatic activity of 0.45 μmol/min/g tissue (N 3.50 ± 0.8 μmol/min/g tissue) with increased glycogen content. Gene analysis revealed a mutation of the G6PC gene homozygous for 50delGT, thus confirming the diagnosis of GSD-Ia.Initially, our patient was managed with frequent feedings and by the age of 9, a night-time cornstarch regimen was initiated to prevent hypoglycemia. She had expected complications of the disease, including uric acid kidney stones, lactic acidosis, hepatic adenomas with hepatomegaly, and diffuse steatosis.At the age 31 years, our patient became pregnant and did not undergo routine gestational diabetes screening due to her underlying GSD. Three days prior to delivery, she was incidentally noted to be hyperglycemic for the first time, with a blood glucose more than 300 mg/dL (N 70–140 mg/dL). The elevated glucose was attributed to gestational diabetes for which she was started on insulin. Treatment was complicated by severe hypoglycemia with a blood glucose of 32 mg/dL, and she subsequently refused all forms of insulin.Thereafter, our patient was lost to follow-up for 2 years. At the age of 33 years, she sought care when she had continued hyperglycemia and a hemoglobin A1c (HbA1c) of 13% (N 4.3–6.0%). On social history, our patient was working at an office job; she denied any toxic habits including smoking or alcohol use. There was no family history of consanguinity nor birth defects; her young child was healthy. She had a maternal grandmother with diabetes, and her mother had prediabetes. On examination, our patient had a BMI of 41.6, short stature, and generalized obesity. Her heart and lung examinations were unremarkable; she had an obese abdomen with hepatomegaly. A skin examination was notable for acanthosis nigricans, with scattered psoriatic patches. Neurologically, she was alert and appropriate, and her examination was non-focal. Laboratory tests were significant for elevated triglycerides of 960 mg/dL, with microalbuminuria, and otherwise normal liver function tests. Magnetic resonance imaging (MRI) of her abdomen showed multiple liver adenomas with diffuse steatosis. Further investigation of her diabetes showed an insulin level of 32 μIU/mL (n 1.9–23 μIU/mL), and a high c-peptide of 5.2 ng/mL (N 0.8–3.1 Ng/mL), with a fasting blood glucose of 311 mg/dL (N 70–140 mg/dL). A homeostatic model assessment of insulin resistance (HOMA IR) calculation to determine insulin resistance was calculated as 24.6 (N <2.5), thus indicating severe insulin resistance [With initial lifestyle modifications, including a lower carbohydrate diet, exercise, and attempting weight loss, our patient’s HgA1c improved from 13% to 11.9%. It was clear medication initiation was needed, however this was a challenge due to the patient’s underlying chronic liver disease, lactic acidosis, and refusal of insulin. Due to liver dysfunction, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a renally cleared medication, was trialed first. Our patient was treated with canagliflozin, with uptitration of the dosage to 300 mg daily. She concomitantly decreased her bedtime cooked cornstarch from 5 tbsp to 3 tbsp. With these interventions, after a few months, her HgA1c improved from 11.9 to 9% without episodes of hypoglycemia but notable elevation in lactate levels from 3.2 to 5.6 mmol/L (N 0.6–2.5 mmol/L).For more effective glycemic control, an alpha glucosidase inhibitor was added. A dose of acarbose 50 mg was started three times a day with meals. A continuous glucometer monitoring system (CGMS) 1 week after starting acarbose showed downtrending night-time blood glucose with an episode of hypoglycemia in the morning see Fig. . OmissioTypically, patients with GSD-Ia struggle with persistent episodes of fasting hypoglycemia; our case of a patient with GSD-Ia and type 2 DM is rare. The paradoxical nature of the two conditions presents itself as a challenge to glucose homeostasis.There are few case reports in the literature of patients with different subtypes of GSD who later developed DM. Most of these reports describe patients who develop DM in the setting of pancreatic insufficiency and insulinopenia, which does not appear to be the sole etiology in our patient with insulin resistance , 6. A liIn determining the etiology of diabetes in our patient there are several proposals. Typically, patients with GSD-Ia have <10% of normal functioning G6PC enzymatic activity. There is, however, a spectrum of enzymatic dysfunction; while some patients have no functional G6PC, others might have partial . In our Additionally, one study demonstrated that patients with GSD-I have an uncharacteristic insulin response to food when challenged with a glucose tolerance test. Subjects were adults with GSD-I subtypes who ingested a glucose load, and post-prandial insulin levels were measured. The results showed that these patients mounted a delayed insulin response to food, resulting in post-prandial elevated blood glucose as compared to controls . A dysreet al. showed that in a cohort of patients with GSD-Ia, there was a statistically significant increase in the features of metabolic syndrome when compared to those with GSD-Ib and controls [Recent literature describes a possible correlation between GSD-Ia and the development of metabolic syndrome, which could explain the propensity for our patient to develop diabetes. Melis controls . There wcontrols . In thisIn addition, the very nature of GSD-Ia itself with hallmarks of hypertriglyceridemia and liver steatosis likely contributed to our patient’s insulin resistance. In GSD-Ia there is excess build up of the substrate G6P which shunts into other metabolites including free fatty acids and triglycerides see Fig. . This prOur patient presented as a dilemma for treatment, requiring modulation of both fasting hypoglycemia from her GSD, as well as post-prandial hyperglycemia secondary to her diabetes. No guidelines exist on how to treat hyperglycemia in patients with GSD-Ia, and while insulin is an obvious option, it can exacerbate hypoglycemia. The use of a diagnostic CGMS on our patient was an effective tool that provided important information about general trends of blood glucose. The patient’s CGMS highlights her pattern of blood glucose trending down in the early morning and post-prandial hyperglycemia.Exercise in our patient modestly improved glycemic control, due to its beneficial effects on insulin sensitivity and hyperglycemia, however this was not enough . MedicalGSD-Ia remains a rare disorder; however, the life span of patients with GSD will expectantly become longer with better recognition and medical treatment. This unusual presentation of diabetes in a patient with GSD may become more frequently encountered as these patients enter adulthood, and understanding the pathophysiology to provide effective treatment will be necessary.GSD-Ia is a rare disorder which manifests with hypoglycemia and the inability to produce endogenous glucose. Despite a correlation between metabolic syndrome and GSD-Ia, there are few cases of patients with diabetes and GSD-Ia in the literature. This case reviews the pathophysiology of the metabolic disorder and highlights the rare presentation of a woman with GSD-Ia, who presented with diabetes. The challenges of treatment include worsening hypoglycemia from insulin, as well as potential kidney and liver dysfunction from GSD-Ia, which limits medication use. Exercise and diabetic medications including an alpha glucosidase inhibitor and an SGLT2 inhibitor, were the therapeutic choices used for our patient. With the increasing lifespan of GSD patients, this therapeutic challenge will likely present itself more often. Our case emphasizes the need for a discussion of the pathophysiology of the disease, and the risks versus benefits of different treatment regimens.


During October–December 2015, an epizootic hemorrhagic disease outbreak occurred in cattle in Japan. Forty-six animals displayed fever, anorexia, cessation of rumination, salivation, and dysphagia. Virologic, serologic, and pathologic investigations revealed the causative agent was epizootic hemorrhagic disease virus serotype 6. Further virus characterization is needed to determine virus pathogenicity. Orbivirus, family Reoviridae) is an arthropodborne virus that is transmitted among ruminant hosts by the bite of Culicoides midges (–Odocoileus virginianus) and cattle  strain Ibaraki virus was first identified in affected cattle in Japan in 1959. Ibaraki virus has caused disease repetitively in cattle, with signs and symptoms including fever, anorexia, nasal and ocular discharge, congestion of conjunctival and nasal mucous membranes, swollen eyelids and tongue, and dysphagia  and 6 were dairy cattle (Holstein heifers). The average age of all affected animals was 114.7 (range 11–187) months. The clinical signs observed in beef cattle were fever, anorexia, cessation of rumination, swollen eyelids, salivation, paralysis of the tongue, difficulty swallowing, nasal and ocular discharge, and abortion . Seven b,Early in the outbreak, Ibaraki disease was suspected, so blood samples were collected. We washed the blood cells and used them for virus isolation and reverse transcription PCR (RT-PCR) for group-specific and serotype-specific EHDV detection (>32 for EHDV-6 strain AUS1981/07 CSIRO 753 (>4-fold) in EHDV-6 antibody titers for paired serum samples of 5 cattle , although such increases were not observed in the other 15 cattle (All 46 of the affected animals tested positive for EHDV by group-specific RT-PCR (SIRO 753 . The neu5 cattle .At necropsy, we observed edema of the esophagus, pharynx, and tongue in an affected cow with dysphagia. The lumen of the esophagus was also dilated , panel AWe further characterized the causative agent by performing sequence analysis of genome segment 2, which correlates with serotype, and segment 3, which correlates with geographic genetic type, of outbreak isolate HG-1/E/15. We designed RT-PCR primers to amplify segments 2 and 3 from the full-length cDNA on the basis of sequence data of EHDV strains from Japan and Australia available in GenBank. We performed RT-PCR using RNA obtained from an affected cow no. 9;  as templWe aligned the sequences using ClustalW , panel A,,,We determined this febrile illness affecting 46 cattle in Japan in 2015 was an epizootic hemorrhagic disease outbreak caused by EHDV-6 infection on the basis of clinical findings, RT-PCR results, neutralization tests, and sequence analyses. Clinical EHDV-6 cases in cattle have been reported in Turkey, Bahrain, Israel, Morocco, Tunisia, Algeria, and Réunion Island ,,,, panel AThe neutralization tests showed significant increases of antibody titers in 5 cattle, suggesting recent EHDV-6 infection. At the same time, no significant increase of antibody titers was observed with the other 15 cattle. These results suggest that the onset of the epizootic hemorrhagic disease outbreak might have occurred >3–5 weeks after the EHDV-6 infection in the 15 seronegative cattle, considering that neutralizing antibody titer increases have been observed to occur in cattle 11–14 to 21–37 days after experimental EHDV-6 infection (The clinical and pathologic findings of this outbreak are similar to those observed for Ibaraki disease in cattle. Thus, EHDV-6 HG-1/E/15 possibly has pathogenic characteristics similar to those of Ibaraki strain EHDV-2 JPN1959/01 in cattle. Further investigations are needed to clarify the genetic characteristics of EHDV-6 HG-1/E/15 to determine why this outbreak occurred.