Uganda has experienced seven Ebola Virus Disease (EVD) outbreaks and four Marburg Virus Disease (MVD) outbreaks between 2000 and 2019. We investigated the seroprevalence and risk factors for Marburg virus and ebolaviruses in gold mining communities around Kitaka gold mine in Western Uganda and compared them to non-mining communities in Central Uganda.A questionnaire was administered and human blood samples were collected from three exposure groups in Western Uganda . The unexposed controls group sampled was community members in Central Uganda far away from any gold mining activity which we considered as low-risk for filovirus infection. ELISA serology was used to analyse samples, detecting IgG antibodies against Marburg virus and ebolaviruses (filoviruses). Data were analysed in STATA software using risk ratios and odds ratios.Miners in western Uganda were 5.4 times more likely to be filovirus seropositive compared to the control group in central Uganda  whereas people living in high-risk areas in Ibanda and Kamwenge districts were 3.6 more likely to be seropositive compared to control group in Luweeero district . Among all participants, filovirus seropositivity was 2.6% (19/724) of which 2.3% (17/724) were reactive to Sudan virus only and 0.1% (1/724) to Marburg virus. One individual seropositive for Sudan virus also had IgG antibodies reactive to Bundibugyo virus. The risk factors for filovirus seropositivity identified included mining , male sex , going inside mines , cleaning corpses  and contact with suspect filovirus cases .These findings indicate that filovirus outbreaks may go undetected in Uganda and people involved in artisan gold mining are more likely to be exposed to infection with either Marburg virus or ebolaviruses, likely due to increased risk of exposure to bats. This calls for active surveillance in known high-risk areas for early detection and response to prevent filovirus epidemics. Ebolavirus and Marburgvirus belong to the family Filoviridae and cause classical viral haemorrhagic fevers (VHFs) in humans, which are associated with high morbidity and mortality and pose a serious threat to human and animal populations in endemic countries. Uganda reported 11 filovirus outbreaks from 2000 to 2019. These include seven EVD outbreaks caused by Sudan virus (6 outbreaks) and Bundibugyo virus (one outbreak) and four Marburg virus disease (MVD) outbreaks caused by Marburg virus and Ravn virus [Rousettus aegyptiacus bats to be the known reservoir for Marburg virus [Viruses in the genera vn virus  includinvn virus . Previourg virus –6. In 20Rousettus aegyptiacus bats that live in the mines, a known reservoir for Marburg virus. A comparison group in Luweero district was chosen as a control, unexposed group because it is in the Central region of the country far from Kitaka and any other mines, and we hypothesized that Rousettus aegyptiacus bats may not inhabit this region due to lack of suitable habitat, no previously reported MVD cases, and therefore inhabitants would mostly likely be at low risk for exposure to filoviruses. Despite EVD being reported in Luweero district in 2011 and 2012, the ecology of this place is different from that of Ibanda and Kamwenge districts as it is not forested and not have suitable habitat for the putative filovirus reservoirs , 200 persons between groups ‘2’ and ‘3’ and 200 persons in group ‘4’. This calculation was done estimating a 15% prevalence of Marburg virus exposure in group ‘1,’ 5% prevalence in groups ‘2’ and ‘3,’ and 0.5% prevalence of exposure in group ‘4,’ as well as a 95% confidence interval, 80% power, and a ratio of 2 controls to each exposed person. However, because of the high response rate (144.8%) during participant recruitment, a total of 724 individuals were sample . For groups 1 and 2, a purposive sampling procedure was used with a snowball approach. Participants were questioned to determine those currently working or those who used to work in Kitaka mines. The discovered miners were further questioned to identify additional miners or ex-miners. All discovered miners and their family and household members who were willing to participate were included in the project. For groups 3 and 4, a two-stage cluster sampling design was used, with a random selection of five sub-counties in each sampling area , followed by the random selection of three villages within those sub-districts. In selected villages, the investigators travelled to the location of the main trading post at the village’s centre, and participants were chosen following the EPI method [We sampled three groups of exposed individuals in Ibanda and Kamwenge districts that included: 1) Artisanal miners and persons that worked in the Kitaka mines from 2007 to 2015, 2) Members of the household or family housing compound of a miner during the period that the miner was actively working in Kitaka mines, 3) Members of households that resided within a 50 km radius from any open mining site associated with Kitaka mines, and that were not included in above groups 1 or 2. This third group also acted as a control for groups 1 and 2 above to assess the risk for filovirus infection between people with direct or indirect exposure to the mine versus living near it. We then sampled the 4Data were entered in Epi Info 7 and analysed using STATA . Risk ratios (RR) were calculated to evaluate the risk of filovirus seropositivity between different exposure groups and the unexposed control group, and to compare the risk for seropositivity between hypothesized higher-risk exposure groups (miners and their household members) to the lower risk exposure group (non-miners living within 50 km of the mines). To investigate significant risk factors for seropositivity among all participants (exposed and unexposed groups), we computed Odds Ratios (OR), which provide a reasonable estimation of the Risk Ratio (RR) since the exposure-outcome is less than 10% . We contAll the samples collected were tested for the presence of IgG antibodies by Enzyme-Linked Immunosorbent Assay (ELISA), which was validated by US Centres for Disease Control and Prevention (CDC) on known positive and negative human samples with a sensitivity of more than 90% and specificity of more than 90% [Briefly, a gamma-irradiated lysate of Vero cells (made in-house by US CDC Atlanta) infected with either Sudan virus, Bundibugyo virus, Ebola virus or Marburg virus was used as positive antigen whereas the negative or control antigen had uninfected Vero cells. 100 μl of positive antigen diluted in Phosphate Buffered saline  (Marburg Ag 1:3000 and Ebola Ag 1:2000 Dilutions) was applied on the upper half of the solid phase of a polyvinyl chloride microtiter plate  and the lower half coated with 100 μl of negative/control antigen in PBS then incubated at 4 °C overnight. Unbound antigen was removed from the well by washing three times with PBS-Tween . Samples were diluted 1:100 and 4-fold through 1:6400 in 5% skimmed milk in PBS-Tween and allowed to bind to the antigen. After washing, an anti-human IgG conjugated to horseradish peroxidase (HRPO) was added and allowed to bind. The plates were washed and the substrate ABTS  was added which in the presence of HRPO  and hydrogen peroxide, is converted from a colourless liquid to an intense green colour with a maximum light absorption at 410 nm. The amount of colour developed is proportional to the number of IgG antibodies which has bound to the antigen on the solid phase. OD values at 410 nm were recorded on a microplate spectrophotometer. The OD value of the control antigen-coated well was subtracted from its corresponding viral antigen-coated well to yield adjusted OD value. A sample was considered positive when the adjusted OD value of either the 1:400, 1:1600 or 1:6400 dilution was greater than 0.2 and the sum OD value was greater than 0.95. A panel of 1 or 2 negative control sera and 2 or 3 positive control sera were run each time the assay was used. Positive samples were retested against three circulating viruses  to assess cross-reactivity.Overall, we sampled 724 individuals, 291 (40.2%) from the low-risk exposure region in Central Uganda (Luweero district) and 433 from the high-risk exposure region in Western Uganda (Ibanda and Kamwenge districts), including 161 miners (22.2%), 138 miner household members (19.1%), and 134 non-miners living within 50 km of the mines (18.5%)  individuals tested had IgG antibodies against filoviruses. One person in the miner’s exposure group had IgG antibodies against Marburg virus . Seventeen individuals (17) were reactive against Sudan virus IgG antigens only , showing no cross-reactivity of the assay between Sudan Virus and Ebola virus. However, one person who was a family member of a miner had IgG antibodies to Sudan virus and Bundibugyo virus showing low cross-reactivity between the Sudan virus and Bundibugyo virus. No individuals had IgG antibodies against Ebola virus (EBOV).The three combined high-risk exposure groups in Ibanda and Kamwenge district in Western Uganda had higher filovirus positivity at 3.7% 16/433) compared to low-risk exposure group in Central Uganda at 1.1% (3/291). Specifically, artisanal gold miners who enter bat-inhabited caves had a higher seroprevalence of 5.6% (9/161) compared to non-miners in central Uganda at 1% (3/291) and had 5.4 times the risk of being seropositive for filoviruses compared to the unexposed group in central Uganda . Miner family/household members and non-miners living within 50 km of the mines were not significantly more likely to be filovirus seropositive than the non-exposed participants in Luweero district , include male sex , going inside mines , cleaning corpses  and contact with EVD/MVD suspects   and another in savanna rangeland in central Uganda (unexposed), we see that the risk of being filovirus seropositive is higher specifically among miners by at least five times compared to the unexposed group, but not higher among household members of miners or people living near the mines. Therefore, filovirus exposure risk is specifically associated with exposures occurring in the mines themselves, likely exposure to bats or their excrement or body fluids. As has been reported before, the decommissioned bat-occupied Kitaka mines where the exposed population is centred is inhabited by bats of species rg virus , 5, 6.We expected a higher seroprevalence against Marburg virus than ebolaviruses, but the opposite was observed with ebolaviruses seroprevalence being higher than Marburg virus. Whereas it has been confirmed during previous investigations that bats occupying the mines are actively infected with Marburg virus and had been associated with two MVD outbreaks , 10, no Another filovirus serological study by Nkoghe et al. 2011) in rural Cameroon and Gabonese populations where the prevalence of Ebola virus was higher in populations near forests . A secon11 in rurThe four seropositive individuals we found in the “unexposed” Luweero district group could be due to travel and migration from high-risk areas but may also be due to the movement of reservoirs such as bats that are known to travel long distances hence spreading the infection. Ebola virus seropositivity has before been reported in a grassland savanna-like ecosystem in Nigeria similar to the grassland savannah ecosystem of Luweero where the three Sudan virus (3) seropositive individuals were identified . HoweverBeing male was associated with a high risk of being seropositive  compared to being female, likely partly due to men being more likely to be miners and go inside the mines and the forests for manual work and become exposed and hence acquire infection. Cleaning a dead body was significantly associated with being seropositive for filoviruses. This has been widely reported in outbreaks of filoviruses as burials and funeral rites amplify these outbreaks. Contact with EVD/MVD suspect was a predictor of filovirus seropositivity and has been reported in a partial meta-analysis done on the risk of ebolaviruses transmissions .Looking at the overall seroprevalence reported in this study, the findings suggest that there may be filovirus infections and outbreaks that occur in Sub- Saharan African countries and go undetected by the health care systems. This could possibly lead to large epidemics as was seen in West Africa . AdditioA similar unpublished study was carried out by the CDC and the Ministry of Health following the 2007 MVD outbreak in Kamwenge and Ibanda district in the same area. In that study, they found a seroprevalence of Marburg virus at 1.2% (7/564) and Sudan virus at 1.2% (7/564). The seroprevalence of Marburg virus was slightly lower in our study whereas that of ebolaviruses was higher. We do not have a clear explanation for these differences in seropositivity between the two studies conducted in the same area 8 years apart. Also, the seroprevalence in our study is lower than 8% and 3% for pooled seroprevalences reported in meta-analyses of seroprevalence of ebolaviruses performed in other parts of the world . FollowiOur investigation also reported a lower seroprevalence of ebolaviruses (2.5%) than pooled seroprevalences reported in other studies in neighbouring Democratic Republic of Congo (DRC) at 10% , 27–30, These variations in seroprevalence could be due to differences in filovirus ELISA testing protocols and potential cross-reactivity caused by a non-filoviral infection. The test, developed by CDC that was used in this study is more specific than other filovirus serological tests used in previous filovirus seroprevalence studies . This seWe conclude that filovirus infections may go undetected by the health care system in Uganda. Also, miners in Ibanda and Kamwenge, near Kitaka mine, are at higher risk of filovirus infection compared to populations living further away from these cave environments. Increased surveillance is still critical in detecting and quickly averting future widespread and devastating filovirus epidemics.


Lycopersicum esculentum L.) and pepper (Capsicum annuum L.) plants belonging to the family Solanaceae are cultivated worldwide. The rapid development of next-generation sequencing (NGS) technology facilitates the identification of viruses and viroids infecting plants. In this study, we carried out metatranscriptomics using RNA sequencing followed by bioinformatics analyses to identify viruses and viroids infecting tomato and pepper plants in Vietnam. We prepared a total of 16 libraries, including eight tomato and eight pepper libraries derived from different geographical regions in Vietnam. We identified a total of 602 virus-associated contigs, which were assigned to 18 different virus species belonging to nine different viral genera. We identified 13 different viruses and two viroids infecting tomato plants and 12 viruses and two viroids infecting pepper plants with viruses as dominantly observed pathogens. Our results showed that multiple infection of different viral pathogens was common in both plants. Moreover, geographical region and host plant were two major factors to determine viral populations. Taken together, our results provide the comprehensive overview of viral pathogens infecting two important plants in the family Solanaceae grown in Vietnam.Tomato ( Lycopersicum esculentum L.) and pepper (Capsicum annuum L.) plants belonging to the family Solanaceae are cultivated worldwide. Tomato fruit, classified botanically as a berry, is consumed in various ways, such as fresh in salad or as materials for diverse dishes, sauces, and drinks [Tomato  technology facilitates the identification of viruses and viroids infecting plants ,13,14. Nanalysis . NGS-basSeveral viruses infecting tomato and pepper plants in Vietnam have been reported previously ,20,21,22To identify viruses infecting tomato and pepper plants, we collected leaf samples showing viral disease symptoms, including yellowing, mosaic, mottling, and dwarfing, in different geographical regions in Vietnam . We collSamples collected from the same host plants in the same geographical regions were pooled and subjected to total RNA extraction followed by preparation of libraries for RNA-Seq. We named each library according to geographical regions and host plants. For example, the tomato sample collected from Dong Ahn was named DAT (Dong Ahn Tomato) and the chili pepper sample collected from Gia Lam was named GLCP (Gia Lam Chili Pepper). In total, we prepared 16 libraries, including eight tomato and eight pepper libraries. The prepared libraries were paired-end (2 × 100 bp) sequenced by HiSeq 2000 system.The numbers of raw read bases ranged from 8,473,848,288 (DDBP library) to 4,899,434,654 (NBT library). The sequenced raw data of each library were assembled using the Trinity program for de novo assembly. The number of assembled contigs ranged from 77,563 (library NBT) to 146,324 (DDRP library) A.To identify virus-associated contigs, we performed a BLASTN search using assembled contigs against virus reference genome database obtained from NCBI database. We identified a total of 602 virus-associated contigs. The number of virus-associated contigs for tomato (450 contigs) was higher than that for pepper (152 contigs) B. The nuPotyvirus. Three viruses in the genus Tobamovirus—tomato mosaic virus (ToMV), pepper mild mottle virus (PepMMV), and tobacco mild green mosaic virus (TMGMV)—were identified in both plants. In addition, CMV and pepper vein yellow virus (PepVYV) from the genus Polerovirus were identified. Interestingly, we identified three viruses with tripartite RNA fragments such as capsicum chlorosis virus (CaCV), pepper chlorsotic spot virus (PCSV), and tomato necrotic ring virus (TNRV) from the genus Tospovirus. We identified four different single-stranded DNA viruses such as ageratum yellow vein China virus (AYVCNV), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), lindernia anagallis yellow vein virus (LaYVV), and tomato yellow leaf curl Vietnam virus (TYLCVNV) from the genus Begomovirus. Moreover, two double-stranded (ds) RNA viruses—pepper cryptic virus 2 (PCV2) in the genus Deltapartitivirus and southern tomato virus (STV), from the genus Amalgavirus—were identified. Furthermore, two viroids, CLVd and PCFVd from the genus Pospiviroid were identified.We identified 18 different virus species belonging to nine different viral genera . We idenWe identified 13 different viruses and two viroids from eight different tomato libraries. Of these, PCFVd (225 contigs) was the most frequently identified viral pathogen based on the number of identified contigs, followed by TNRV (56 contigs), TYLCKaV (26 contigs), PVY (23 contigs), and CMV (23 contigs) A.In the case of pepper, we identified 12 viruses and two viroids. PeVYV (23 contigs) was the most frequently identified virus, followed by CMV (22 contigs), PCSV (22 contigs), PCV-2 (16 contigs), and TMGMV (16 contigs) B. We comNext, we calculated the proportion of virus-associated reads in each library A. ExceptWe examined the number of libraries for each virus and viroid B. PCV-2 We examined the number of identified viral pathogens in each library C. ExceptBased on virus-associated reads, we examined the proportion of viral pathogens in each library D. TNRV wAccording to plant hosts, we examined the proportion of identified viral pathogens by combining all virus-associated reads. In tomato plants, TNRV (43.4%) was the most dominant viral pathogen, followed by PCSV (21.5%) and CLVd (20.8%) A. In pepWe assembled viral genomes for four STV isolates, one PMMoV isolate, three ToMV isolates, one TMGMV isolate, one AYVCV isolate, and two PCFVd isolates by RNA-Seq and conducted bioinformatics analyses. The assembled viral genomes, which were assembled to complete genomes, were subjected to BLASTN search to retrieve homologous viral genome sequences. After nucleotide sequence alignment, we generated six different phylogenetic trees . The phyWe carried out RT-PCR using virus-specific primers to confirm results of RNA-Seq. Virus-specific primers were designed based on the identified sequence for individual virus and viroid . The resRecently, a large number of viruses and viroids infecting tomato and pepper plants have been identified in a single study based on diverse NGS techniques ,24,25. AA previous study identified a total of 22 viruses infecting tomato derived from 170 field-grown samples in China by small RNA sequencing . When weSolanaceae has been reported [Pepper viromes in two different pepper cultivars grown in India have previously been reported, revealing diverse DNA and RNA viruses infecting pepper plants . Interesreported . By contreported . This streported . RegardlThe advance of NGS techniques facilitates the easy identification of known and unreported viruses in a target plant . Based oIt is noteworthy that tomato and pepper plants are hosts for a wide range of viruses and viroids, as shown previously ,25. In oSolanaceae [Furthermore, many viruses and viroids were commonly identified in both tomato and pepper plants, which are members in the family lanaceae ,37,38. Blanaceae . Some viSolanaceae. Many plant viruses reported in this study could infect diverse host plants and thus present the possibility of the continuous virus disease epidemics in the fields of Vietnam and potential threat to the agricultural industry.Multiple infection by diverse viruses in a single host is very common. Similarly, at least seven different viral pathogens were found to infect both tomato (BLT) and pepper plants (DDBP). Furthermore, the analysis of viral population using virus-associated reads revealed that there was, preferentially, a dominant viral pathogen in tomato and pepper plants grown in the same field. However, we could not confirm whether the dominant viral pathogen played a major role in causing typical virus symptoms. Taken together, we identified diverse viruses and viroids infecting tomato and pepper plants grown in the fields in Vietnam by RNA-Seq. Our results showed that infection by different viral pathogens was common in the two plants. However, a specific viral pathogen was dominantly present, depending on the host plants as well as on the isolated regions, suggesting that the geographical region and host plant were two major factors for determining viral populations. Since we could not confirm whether the dominant virus identified by RNA-Seq is the major pathogen in each plant to cause viral symptoms observed, further experiments and screenings for identified viruses are required to provide more information for preventing virus diseases epidemic in pepper and tomato fields. Although we did not provide direct evidence for economic losses caused by virus infections in pepper and tomato plants in Vietnam, our results provide the comprehensive overview of viral pathogens infecting two important plants in the family Leaf samples were collected from plants exhibiting viral symptoms in open fields of pepper and tomato in Vietnam. We pooled leaf samples collected from the same geographical regions and host plants. Description of samples used in this study was were summarized in http://bio-bwa.sourceforge.net/). The number of mapped reads for identified virus was calculated using bbmap.sh implemented in BBMap program (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbmap-guide/). The raw data are available at the NCBI database with the BioProject number PRJNA636575.Raw sequence files from each library were de novo assembled by the Trinity program with default parameters . The assTo generate phylogenetic trees, the assembled genome sequence of each virus was subjected to a BLASTN search against NCBI’s GenBank. We retrieved the top ten homologous viral genome sequences for individual virus or viroid. Nucleotide sequences were aligned by ClustalW program . PhylogeTM Onestep RT-PCR kit  following conditions based on the manufacturer’s instructions. The cycling conditions were 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles at 95 °C for 20 sec, 50 °C for 40 sec, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. The PCR products were confirmed by gel electrophoresis with 1Kb DNA marker  in 1% agarose gel with TAE buffer.RT-PCR was carried out using virus-specific primers . The RT-


Subsequently, she was introduced the ketogenic diet; continuous ketosis has been maintained for over the last 4 years. After the introduction of ketogenic diet, a normalization of the cardiomyopathy and improvement of hepatopathy was achieved, with enhanced overall quality of life.Now 15-year-old girl with glycogen storage disease (GSD) type IIIa (OMIM  The estimated GSD type III incidence is 1/100,000 live births. In addition to hepatomegaly, which is present in GSD type IIIb, also variable skeletal myopathy and cardiomyopathy are characteristic for GSD type IIIa, possibly leading to a cardiac failure [Glycogen storage disease (GSD) type III OMIM 32400 is We aimed to assess the influence of KD on the severe obstructive cardiomyopathy, hepatopathy and other clinical characteristics in a patient with GSD type IIIa. We made a decision to introduce KD in our patient due to her rapidly worsening condition, basing on a few promising reports that were then available .2AGL gene homozygous mutation c.3980G>A (p.W1327X) resulting in an almost undetectable amylo-1,6-glucosidase activity), who was diagnosed at 1 year of age. At that time she was introduced a high carbohydrates diet ; carbohydrates (9 g/kg per day (g/kg/d)) contributed 53% daily calories, proteins (4 g/kg/d) contributed 23% and fats (1.8 g/kg/d) contributed another 23%. Progressively she developed left ventricular obstructive hypertrophy, hepatomegaly and skeletal myopathy with highly elevated liver and muscle enzymes. She also presented recurrent hypoglycemic events despite treatment with frequent diurnal and nocturnal meals with cornstarch supplements. Due to progressive obstructive cardiomyopathy, she was introduced KD at the age of 11. The diet consisted of ketogenic ratios of 2.5:1; fats (5.2 g/kg/d) contributed 87% daily calories, proteins (1.6 g/kg/d) contributed 11% and carbohydrates (0.3 g/kg/d) contributed 2%. Continuous ketosis was maintained for over 4 years. A very good clinical support by attending physicians and experienced clinical dietitians was provided , also to avoid discontinuation of ketosis. The blood sampling at regular outpatient visits was performed in a fasting state, as recommended [This is a case report of a now 15-year old girl with GSD type IIIa  and sustained in normal range after 4 years.2 to 37 g/m2) and thickness of left ventricular walls , without residual outflow obstruction. Finally, no hypoglycemic events were recorded while on KD.Cardiac MRI was repeated after 16 and after 40 months, showing a normalization of left ventricular parameters, with a decreased of total left ventricular mass index (LVMI)  [While on KD in our patient, we observed normalization of severe progressive obstructive cardiomyopathy, similarly as reported in a few previous shorter observations. Dagli et al. report on a male child with GSD type IIIa, treated with a high carbohydrate diet, developing severe cardiomyopathy with LVMI of 209 g/m78 g/m2) . Mayoran78 g/m2) . In addiRegarding hepatopathy, Rossi et al. also report a decrease of ALT and AST concentrations in adults in 100% and 83% patients, respectively. In children, ALT and AST decrease in only 11% and 22% patients, respectively . An imprDevelopment of the cardiomyopathy in GSD type IIIa may be caused by the high carbohydrate diet, according to reports of the literature about the association between carbohydrate overtreatment and an elevated risk for cardiac involvement and/or cardiomyopathy . Rossi eSome concerns were previously expressed regarding the low expected adherence to KD for these patients ,6. The pDuring the 4-year follow up, the patient and her family reported much better quality of life while on KD; reasons stated were: no need for burdensome overnight dietary regimen; no hypoglycemic events recorded; much improved physical fitness and self-image after losing weight; and most importantly, to worrying less about the worsening cardiomyopathy. This further reinforces our opinion about KD feasibility and efficacy.5Cardiomyopathy is a potentially life-threatening complication in treated GSD IIIa patients that could be reversed on the KD, which has also potential to significantly improve other clinical characteristics and overall quality of life, with a good safety profile. KD was shown to be feasible for the patient and her family, which resulted in a good compliance to the diet. It is worth mentioning that our reported study has the longest follow-up to date. Further prospective studies are needed; however, the data yet available might already suffice to be encompassed into the recommendations on GSD type IIIa management strategies.10.13039/501100004329Slovenian Research Agency project V3--1505 and program P3--0343.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Study was partly founded by the


Newly returned from the frontline of the battle against Ebola in West Africa, Dr George Fu Gao talked to NSR on this largest Ebola epidemic in history. As Deputy Director-General of the Chinese Center for Disease Control and Prevention (China CDC), an Academician of the Chinese Academy of Sciences and a fellow of the Third World Academy of Sciences (TWAS), Dr Gao has been personally involved in the fight against acute infectious diseases both on the laboratory bench and in the field for many years. Recalling the sight of an Ebola-infected young man stumbling to his death on the street and observing the continuing climb of patient statistics, he anxiously calls for larger global help to affected countries. The help provided by the world community to West Africa was ‘too late and insufficient’, and in this age of globalization ‘no country can care only for herself alone’. In this interview, Dr Gao also reviews the history, progression, and prognosis of the Ebola epidemic, the cause of failure in the early prevention and containment of the disease, the role of government policies and public health systems, and global prospects and strategies for similar acute infectious diseases in the future. NSR: Having returned to China, are you still closely monitoring Ebola? What is your assessment of the current situation?Gao: Certainly. The China CDC is providing a daily report on Ebola, and I am closely watching the situation in West Africa. We cannot be too optimistic about the current status. Among the three most affected countries, Liberia appears to have passed the peak, although the decline is rather slow and fluctuates; Sierra Leone is currently lingering near the peak, showing an apparent plateau; Guinea's situation fluctuates greatly, with signs of a major return of Ebola. Based on the overall trend, the best scenario is that the epidemic may be curtailed by the coming June, but some scientists estimate that this is more likely to occur by the end of year 2015.NSR: Ebola virus was discovered in 1976, but has never caused the large-scale infection we face today, why?Gao: The most important features in preventing acute infectious diseases are part of the ‘Four Early’ strategy: early discovery, early diagnosis, early quarantine, and early intervention. In this recent epidemic, there was failure in all four of these strategies. The main problem lies in the management: the three West African countries did not pay much attention to this matter in the beginning, and it became hard to contain the disease once it spread.Ebola began in December 2013, with the first patient being a two-year-old boy in Guinea. When the Pasteur Institute confirmed the cause to be Ebola virus in March 2014, Ebola had already spread to Liberia. Sierra Leone claimed to have attended to this matter but did not install an effective measure. By May 2014, the disease had already been spreading for half a year and entered into Sierra Leone. If sufficient attention were given at that time, Ebola would not have become so unmanageable later. After May 2014, as the situation became more serious, the inability of the three affected countries to deal with the epidemic was apparent, and other countries did not react quickly enough.NSR: As reported in the media, many countries and organizations deployed help to West Africa. Did this help to curtail the disease?The main problem lies in the management: the three West African countries did not pay much attention to this matter in the beginning, and it became hard to contain the disease once it spread.—George Fu GaoGao: There was some impact, but I think the help was too late and not strong enough. As I have just said, the three countries themselves were no longer capable of dealing with the disease by May 2014. The public health facilities are poor in these countries—there are no diagnostic capabilities, not enough healthcare workers, and insufficient medicine and basic nutrition or food for sustaining the patients. The fact is that the World Health Organization (WHO) did not openly announce the seriousness of the epidemic until August 2014. Even then, most countries only reacted at the level of discussion—for setting rules and operation procedures, without really reaching the frontline to curtail the disease. To fight Ebola in countries like Sierra Leone, ‘teach them how to fish’ is not sufficient; you must also ‘give them the fish’. There is an immediate need for trained healthcare workers on the ground, helping the diagnosis, quarantine, and intervention of the disease.Many people have said that death rate for Ebola is anywhere between 20% and 90%. This is neither a rigorous nor correct statement. If I said the rate for an event to occur is between 0 and 100%, you would say that I was talking nonsense. With regard to Ebola, if there is intervention the death rate is 20%, and the rate will reach 90% if there is no intervention. During the early phase of the epidemic, some Ebola patients did not even receive a saline infusion , and the death rate was high. Now, with vein infusions, the death rate is dropping, even in the absence of an antiviral treatment. In other words, Ebola is not so horrific; its lethality can be controlled with proper actions.NSR: Was the slow reaction to the Ebola epidemic related to the systems and policies of local governments?Gao: Indeed. As a former British colony, Sierra Leone's policies on biomedical ethics followed closely those in western countries. This aspect is acutely reflected in matters related to Ebola. For example, a Japanese-developed anti-influenza virus inhibitor, Avigan or Favipiravir, has been shown to be effective against Ebola in animal models, but the clinical trials for such a drug in Sierra Leone is hampered by many factors, including ethics. Even when the drug was available, there were many meetings to deal with the issues of distribution of the drug, who should get the drug etc., while the situation continued to worsen.Furthermore, in China, there is a multisector coordination system  related to acute infectious diseases, and compulsory quarantine can be quickly implemented when suspected cases are found. This cannot be done in Sierra Leone as there is no supporting regulation. There is also the lack of hospitals and facilities for accepting the patients. Furthermore, healthcare workers strike from time to time due to the lack of proper personal protection or delayed wage payments. These conditions are hard to imagine in China.NSR: In other words, if Ebola spreads to China, we will be able to deal with it.Gao: Yes. China now has a well-established system for responding to the spread of infectious diseases, unless the infected individuals go to hide in remote areas, an unlikely scenario. Thus, we do not need to worry, as long as we can react early. Of course, there are still many unanswered questions regarding Ebola virus and its infection. We must take every step seriously.NSR: Vaccine is important for the fight against pathogens. Why is a vaccine still unavailable so many years after the discovery of Ebola virus?Gao: An Ebola vaccine is obviously a matter of concern for public health, but pharmaceutical companies are driven by profit and are responsible to their shareholders. They will not actively develop the vaccine until the government decides to invest on it. We all saw the consequence of their slow reaction.NSR: What is the current status of vaccine development?Gao: There are three vaccines currently in clinical trials. One is being developed by GlaxoSmithKline, based on chimpanzee adenovirus (AdV) 3 and Ebola glycoprotein; the second is being developed by NewLink Genetics and Merck Vaccine, based on attenuated vesicular stomatitis virus and the glycoprotein; and the third one is being developed by Dr Wei Chen of the Chinese Academy of Military Medical Sciences, based on human AdV5 and the glycoprotein. There is a problem using AdV5; 80% of people have antibodies against it, and whether this will affect clinical effectiveness remains unknown. Other vaccines are also under various stages of development.NSR: Given their clinical trial status, these vaccines cannot be used widely at present. Is that right?Gao: No. This is a scientific as well as a management issue. I have frequently said, in the course of history, biological safety and ethics were ignored, but it seems to be overemphasized at present. It has been a long delay for the above-mentioned vaccines to be set for clinical trials during the outbreak because of the existing ethical and management hurdles. In China, the Ebola vaccine, developed by Dr Wei Chen, could be used in human trials prior to animal tests, though it is controversial. During the SARS epidemic, we considered taking blood samples from SARS patients to develop ‘therapeutic plasma’. This simply could not be done in Sierra Leone for the Ebola convalescent antisera. The same is true for sequencing of the virus. This has to be done in the USA and requires approval from Sierra Leone.NSR: Through viral sequencing, a Harvard team found in June 2014 that Ebola virus has mutated with a rate doubling that found over the past 40 years, why?Gao: As the virus spreads in the human population, it continues to mutate and adapt to new human hosts. In the past, the infection normally spread for only 2–3 months, but this time the spread lasted for more than six months by June 2014, allowing more time for the mutated virus to survive. Now another half a year has passed, we would like to know the extent of further mutation beyond that reported earlier. This is why I suggested that our NHFPC should coordinate the shipment of a BGI's sequencer to West Africa for on-site sequencing.NSR: Does rapid mutation of Ebola virus make it more dangerous?Gao: Some people worried that Ebola virus may convert into a ‘influenza-like virus’ in terms of rapid mutations (antigenic drift) (requiring different vaccines for different subtypes). As you know, we need to be vaccinated every year to be protected against the seasonal flu. If Ebola virus mutates as quickly/frequently as influenza virus, the vaccine needs to be continuously renewed. We need to have a vaccination for influenza each year because each vaccine can only target one virus subtype or virus strain. A vaccine developed now for Ebola may become obsolete by the time it is used for vaccination. For example, the H1N1 and H3N2 vaccines are currently being used to prevent influenza, but the vaccine suitable for the mutated virus next season needs to be developed. Such vaccine development requires large investment and has a very high risk. Thus, research and development of Ebola vaccine belongs to the realm of public health. It must be a government-led effort, and little initiative can be expected from the pharmaceutical industry.NSR: To some extent, didn't the Ebola epidemic repeat the pathway of the AIDS epidemic?Gao: In the beginning, both Ebola and AIDS could be considered to be diseases of the poor. Both originated from Africa where medical care and living conditions are poor, and both were neglected by the world in the beginning. When AIDS reached Europe in the 1980s and began to spread by sexualencounter, it caught the world's attention. For 40 years, Ebola was active only in local regions in Africa. Without the present epidemic, it probably would not cause a ripple in the media.NSR: Can you comment on the AIDS situation in China?Gao: AIDS is still very serious in China. The situation has become more serious than that in the USA and Europe. The pathway for AIDS transmission in China is now clear. During the early period of post-cultural revolution, poverty was widespread in China and many poor people relied on selling blood plasma to make a living. After separating the plasma, the blood cells were infused back to the donor. Since the blood was mixed among many people before re-infusion, the virus carried by one donor quickly spread to others. Most AIDS cases in Henan and Shanxi villages were caused by this blood infusion practice.When people became more affluent in the 1990s, drug addiction became the main pathway of infection, and high incidence rates were concentrated in Yunnan, Guangxi, and Xinjiang. In this century, with increasing social diversification and tolerance, (both homo- and hetero-) sexual transmission became the main pathway of propagation. In particular, incidence rate due to male homosexual transmission has risen rapidly in recent years, and college students have become a high-risk incidence population. This indicates that we have much to do in social transparency and education, including distribution of free condoms in public places and education against dangerous sexual behaviors.NSR: How is the current status and effectiveness of AIDS therapy?If Ebola virus mutates as quickly/frequently as influenza virus, the vaccine needs to be continuously renewed. A vaccine developed now for Ebola may become obsolete by the time it is used for vaccination.—George Fu GaoGao: The current therapy is rather effective. The drug is pushing gradually towards the cure of AIDS. However, the present approach in developing an AIDS vaccine is not very hopeful. I think the AIDS vaccine research has reached a dead end. Just following the successful example of polio vaccine—using a vaccine to activate immune responses—may not be the best approach. When a virus has already weakened the immune system, is it reasonable to further force the immune system to fight the virus? We need a new conceptual scheme and new approaches.NSR: With regard to influenza, is it possible to develop a universal vaccine against all subtypes of a virus?Gao: I have studied influenza virus for a long time. Personally, I think it is not possible to develop a universal vaccine. We can now find universal neutralizing antibodies (to influenza virus) in human blood, but when the virus really invades humans, these antibodies seem to be in hibernation, showing no protective reaction. We do not understand the underlying mechanism yet. We need a new theoretical framework to guide the search for a universal vaccine.NSR: What do you think is the most urgent measure needed for controlling the Ebola epidemic in West Africa?Gao: I think China has accumulated much useful experience in dealing with acute infectious diseases. With minor modifications, the Chinese practices could be adapted to West Africa. In particular, supportive therapy for patients is more urgent at present than preventive approaches such as vaccine development.NSR: What do we know about the origin of Ebola?Gao: Scientists now believe that Ebola virus originates from bats, some of which are distributed along the belt above the equator, from Sierra Leone, Republic of Congo, to the Philippines and our regions of Yunnan and Hong Kong. It is not clear whether these bats migrate along the equator-like migratory birds. If so, then it can spread the virus to different parts of the world without human carriers.NSR: Doesn't SARS virus also originate from bats?Gao: Indeed. With regard to the origin of SARS virus, Zhengli Shi's team at Wuhan Institute of Virology of CAS were the first to report, in the journal Nature, the isolation of a strain of virus from horseshoe bats (named ‘WIV1’) that is very similar to SARS coronavirus (SARS-CoV). They also showed that WIV1 binds to the SARS-CoV receptor, thus providing direct evidence that bat-to-human transmission is plausible. K. Y. Yuen of Hong Kong University has also discovered many viruses in bats  and we, in collaboration with K. Y. Yuen showed that HKU4 can use the receptor for the newly emerged Middle East Respiratory Syndrome coronavirus to enter human cells, although the efficiency is low. This represents another example of bat-originated respiratory syndrome viruses. In a paper published in 2013 in Nature, my team at the Institute of Microbiology of CAS reported the molecular structural mechanism by which MERS virus interacts with the human cell surface receptor (CD26). Such structural information is very useful for designing both antiviral drugs and prophylactic vaccines.NSR: How does a virus invade human cells? Does it always need a surface receptor?Gao: The great majority of viruses need to bind to specific cell surface receptors before entering the cell. The SARS receptor is called ACE2 and MERS receptor is CD26. There are indeed viruses that may not require receptors for invading the cell. For example, the receptor for Dengue fever and other rather ancient viruses (e.g. yellow fever virus and West Nile virus) has never been found. As Ebola virus can infect multiple somatic cell types, it also may not require a specific cell surface receptor, but this remains to be determined. An unusual feature of Ebola virus is the prolonged incubation period—as long as 21 days. This is rather rare among acute infectious diseases. Male Ebola patients can carry virus in their semen for three months during the recovery period. It is not clear why.NSR: Does this mean the potential for the sexual transmission of Ebola?Gao: There is no evidence for sexual transmission yet. Human eyes and testicles are immune privileged organs that could tolerate an invading virus without eliciting inflammatory immune responses and thus should not be infected. We are currently studying why Ebola virus can reach the semen—it means that the testicles are infected. The paradox of the immune privilege and virus infection-resistance is not fully understood for these organs.NSR: What are the remaining mysteries in Ebola virus?Gao: I think the mechanism of cross-species transmission needs to be understood. Many newly emerged infectious diseases involve infection in both humans and animals, but some viruses affect humans but not animals, why? Understanding whether and how a virus affecting one species become infectious in another species is critical in developing effective preventive measures. Our understanding of molecular mechanisms via research on influenza virus over the years greatly helped the analysis of the viral infection mechanism when avian flu epidemic appeared.We have recently established a CAS Center for Influenza Research and Early-warning. One of our goals is to solve the problem of cross-species transmission through multidisciplinary approaches. Regarding virus propagation by migratory birds, for example, zoologists may examine the migratory behaviors of birds, virologists and molecular biologists can study the mechanism of infection of avian viruses on human cells, and China CDC, which has established a national network of infectious diseases monitoring networks throughout China since the SARS pandemic, can provide immediate information on disease incidence in various local regions. Integrating multiple resources of information will allow us to understand the nature of the virus, and whether and how it spread cross species.NSR: Some scientists propose to use ‘gain of function’ (GOF) to predict potential threats of viral mutation on human populations. What is your view on this approach?Gao: Frankly, I am not in favor of the GOF approach, although there are pro and con arguments. My point is that the process of gene mutation and natural selection involves many more combinatorial scenarios than the human mind can imagine, and how could one select among all possibilities and provide comprehensive tests? I think a more direct and effective approach is to strengthen the capability of monitoring disease-causing viruses and finding solutions to curtail their spread. For example, we have monitored H5- and H9-type influenza viruses over a long period; when similar infectious diseases appear, we can rely upon existing methods and platforms to analyze the virus and propose a solution. During the H7N9 avian flu epidemic in 2013, we were able to identify the problem andMany newly emerged infectious diseases involve infection in both humans and animals, but some viruses affect humans but not animals. Understanding whether and how a virus affecting one species become infectious in another species is critical in developing effective preventive measures.—George Fu Gaosuggest the closing of markets for live poultry trading, a measure that prevented further spread of the flu.NSR: Ultimately, the capability in the prevention and control of acute infectious diseases testifies the social and economic strength of the country, doesn't it?Gao: Indeed, acute infectious diseases could be regarded not only as a medical problem but also a social problem. It could evoke social unrest and become a political problem. The SARS epidemic resulted in the dismissal of the minister of health in China; Sierra Leone has replaced their minister twice since the Ebola outbreak. It is also a diplomatic problem as many countries are involved in dealing with Ebola. As a poor country to begin with, Sierra Leone received another devastating blow by Ebola. The only consolation may be some improvement of its public health system after this epidemic.Finally, this epidemic testifies to the fact that infectious diseases are a global issue that requires collaborative efforts from many countries. Given the absence of adequate public health systems in many developing countries, affecting their ability to deal with acute infectious diseases, more developed countries must provide rapid and effective responses when such an epidemic occurs again in the future.


Allium sativum L) in Ethiopia. Attempts were made to identify the viruses using molecular techniques from 95 composite leaf samples collected from 44 farmers’ fields and 51 germplasm accessions. Reverse transcription (RT-) PCR using genus and/or virus specific primers was used to amplify partial genome sequences of potyviruses, allexiviruses, carlaviruses and a tospovirus followed by sequencing of PCR products. Results indicated that ~73.7% of the samples are infected with at least one virus. Onion yellow dwarf virus  is the most common virus detected followed by Garlic virus C (genus Allexivirus) and Shallot latent virus . Other viruses detected at lower frequency include Garlic virus X and Garlic virus D (genus Allexivirus), Leek yellow stripe virus (genus Potyvirus) and Iris yellow spot virus . Mixed infection of two or more viruses was detected in 65.7% of the samples. Phylogenetic analysis suggested that the different viruses may have been introduced to Ethiopia from Europe or Asia. This is the first report of Garlic virus X, Garlic virus D, IYSV and SLV in garlic in Ethiopia. The high incidence of OYDV and IYSV which cause severe yield loss alone or in mixed infection with allexiviruses and carlaviruses is a cause of concern to growers.Little information exists on the type and incidence of viruses infecting garlic ( Allium sativum L.) is a spicy vegetable crop widely cultivated throughout the world. In Ethiopia, the crop is grown as a major vegetable in an estimated area coverage of 19,000 ha in 2016 (CSA 2018). Diseases caused by viruses are among the major production constraints of the crop globally causing yield loss of up to 50% (Fujisawa Potyvirus namely Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), and Shallot yellow stripe virus (SYSV) and of the genus Carlavirus namely Garlic common latent virus (GarCLV) and Shallot latent virus , all of which are aphid-transmitted , -B (GarV-B), -C (GarV-C), and –D (GarV-D), −E (GarV-E) and –X (GarV-X) and Shallot virus X (ShVX) , a member of the genus Tospovirus transmitted by thrips, is also reported to infect garlic in many areas of the world  were collected in May 2013 from 44 farmers’ fields (area ranging from 0.5 to 2.0 ha for each field) representing eight administrative zones (Table S1) from various major growing locations in Ethiopia and 51 germplasm accessions maintained at Debre Zeit Agricultural Research Center (DZARC), Oromia, Ethiopia (Table S1). Garlic crops consisting of both local garlic land races and three improved varieties  commonly cultivated in Ethiopia were surveyed just before flowering stage. Fields were selected in the main garlic growing areas of the different zones by travelling on the main roads and stopping in every 5–10 km wherever the crop is found. From each field or germplasm accession plot, leaf samples comprising both symptomatic and asymptomatic ones were collected by randomly harvesting leaves from 8 to 10 plants, making a composite sample, by moving in zigzag pattern along the fields and accession plots. Each sample was separately desiccated over silica-gel kept in air tight falcon tube and transported to BecA-ILRI Hub, Nairobi, Kenya for in vitro laboratory analysis. RNA was extracted using ZR Plant RNA MiniPrep kit . cDNA was synthesized with Maxima First strand cDNA synthesis kit,  as per the manufacturer’s recommendation with the modification that reverse primers employed for respective PCR amplification  was used in a total reaction volume of 20 μl with cDNA as a template. Degenerate genus specific PCR primer pairs previously reported to amplify different potyviruses, allexiviruses and carlaviruses were used . In addihttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Virus sequences were further aligned and analyzed by the ClustalW using BioEdit sequence alignment editor program version 7 ( h t tp ://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were constructed from ClustalW-aligned sequences on MEGA-X (http://www.megasoftware.net/mega.php), using the Maximum-Likelihood method with 1000 bootstrap replications. Pairwise sequence comparisons (PASC) were carried out on aligned sequences using Geneious v11.0.2  computer software. To assess possible recombination events, sequences from the different virus groups from this study together with representative isolates from the NCBI database were analyzed using the RDP4 program   computer software and aligned with all known garlic viruses on the NCBI database using BLASTn available on the NCBI website  were infected with at least one virus with mixed infection of two or more viruses detected in 46 (65.7%) of the infected samples. Of the 44 composite samples each collected from the 44 farmer’s fields and tested by RTPCR for the different viruses, 33 (75%) were found to be infected with at least one virus whereas the remaining 11 (25%) did not test positive to any of the viruses screened in this study. Of those fields with virus infection, 11 (33.3%) had single infection, 15 (44.4%) had mixed infection of two viruses, whereas infection from three viruses is recorded in seven (~22.2%). None was infected with four or more viruses. In all the eight zones where samples were collected, different viruses were detected and there was no clear association of one virus or the other with specific administrative zone of the country. On the other hand, of 51 garlic germplasm accessions screened, 37 (~72.5%) were infected by at least one virus. Double infection was found in 11 (~29.7%) of the accessions and infection with three or more viruses was detected in the remaining 11 (~29.7%) of accessions screened. A total of 14 (~27.5%) of the accessions was found to be free from all the viruses tested in this study.A total of 25 samples, each representing a farmer’s field, and 21 of germplasm accessions were tested positive by RT-PCR using the generic potyvirus primers pairs targeting the NIb-coding region (Table S1) giving the product of the expected size (350 bp). Out of these 46 amplicons, sequence information was obtained for 40 samples representing the different zones. These sequences were subjected to BLASTn analysis of which 33 isolates showed highest nucleotide identity (84–89%) to a range of OYDV isolates, while the remaining seven isolates showed the highest nucleotide identity (82–87%) to different LYSV isolates. Subsequently, representative samples positive for potyvirus degenerate RT-PCR were subjected to RT-PCR using OYDV, LYSV and SYSV specific primers targeting coat protein (CP)-coding region. Virus-specific RT-PCR amplicon of the expected size were only obtained for OYDV and LYSV but not for SYSV. It was thus confirmed that of the 40 potyvirus positive samples, 33 samples contained OYDV, seven contained LYSV and none was positive for SYSV.BLASTn analysis of the CP-coding nucleotide sequences of OYDV isolates from Ethiopia revealed 85–87% identity to OYDV isolate-72 from Poland (KF862684). Similarly, BLASTn analysis of the CP coding nucleotide sequence of LYSV showed 80–87% similarity to a range of LYSV from Latin America including isolates from Brazil (KP258216) and Mexico (KF597283), as well as isolates from Europe , Israel (AF071525) and Japan (AB194632).The allexivirus degenerate primer pair targeting the CP-coding region successfully amplified products of the expected band size of ~750 bp from a total of 41 samples tested. PCR products from all these samples were purified and sequenced. BLASTn analysis revealed that 31 samples are infected with GarV-C with 81–97% nucleotide identity to arange of GarVC isolates from Czech Republic and Sudan, making this virus the most common allexivirus in Ethiopian garlic crops. Eight samples showed the highest nucleotide identity of 85–96% to an Australian GarV-X isolate-SW3 (JQ807994), while the remaining two isolates (Et10 and Et20 GenBank accession number MH794550 and MH794555) showed the highest nucleotide identity of 83% to GarV-D isolate1058.3 from Czech Republic (JX682868). None of the amplicons generated by degenerate allexivirus primers gave sequences of ShVX, GarV–A and –B suggesting that these viruses are absent in our samples.Allium crops namely Garlic common latent virus (GarCLV) and Shallot latent virus (SLV) (The reported generic carlavirus primer pair pCAR-1/ PC-R4  targetinus (SLV)  targetinBy RT-PCR using IYSV specific primers , a fragmPhylogenetic analysis and PASC were carried out to determine the evolutionary relationship of the different garlic viruses identified in this study. Since most isolates of the same virus species sequenced in this study showed high nucleotide sequence identity (data not shown), only representative ones were used in the phylogenetic analysis.Phylogenetic analysis showed that allexiviruses sequences formed six different clades each containing the different allexivirus species as expected . Five ofIn our analysis, sequences of SLV isolates from different parts of the world were grouped into two clusters. All Ethiopian SLV sequences were included in one of the groups, together with GLV isolate from Poland (KF862710) which formed a terminal branch supported with moderate to high bootstrap value . The secPhylogenetic analysis of garlic potyviruses from Ethiopia revealed that both OYDV and LYSV isolates from Ethiopia form distinct terminal branches . All OYDPASC analysis carried out for the different garlic virus isolates sequenced from Ethiopia and other isolates from NCBI (data not shown) indicated that our isolates showed close similarity to species in the database and fulfill the species demarcation criteria suggested by ICTV and hence confirmed identification based on BLAST and phylogenetic analysis. The analysis also revealed that garlic virus isolates of the same species sequenced from Ethiopia showed close sequence similarity to each other which is also consistent with results of phylogenetic analysis. Recombination analysis on RDP4 revealed that no potential recombination event/s happened between isolates of the different virus groups of garlic from Ethiopia with their respective isolates from NCBI.Garlic samples collected from most (75%) of farmers’ fields in Ethiopia were found to be infected with at least one virus belonging to either potyvirus, allexivirus, carlavirus or tospovirus groups. A total of seven viruses known to be prevalent on garlic elsewhere to a different degree were identified in Ethiopia. Of the seven distinct viruses reported, OYDV, LYSV and Garlic virus C have been previously reported from Ethiopia  suggesting its economic importance in garlic production. Hence, future disease management efforts should target this virus. It is known that in single infection, OYDV and IYSV can cause severe symptoms in garlic compared to carlaviruses and allexiviruses which individually cause little or no disease symptoms. On the other hand, mixed infection of any of these viruses is reported to cause more severe diseases resulting in drastic reductions in yield (Cafrune et al. Thrips tabaci is first reported as a serious pathogen of onion in the Pacific Northwest (Gent et al. Thrips tabaci (Kritzman et al. Thrips tabaci is a common pest infesting Allium crops in Ethiopia. Further information on the distribution IYSV and its strains in Ethiopia, its other hosts including onion, the accurate identification of its thrips vector and studying its biology would be pursued to provide valuable information to help growers to control this devastating virus disease.The high incidence of IYSV in garlic production in Ethiopia poses special concern. This virus, transmitted by Our analysis did not indicate recombination event in all of the different Ethiopian garlic virus isolates we tested suggesting that none of the viruses are recombinant at least in the part of the genome analyzed. However, it should be noted that sequences for the different viruses infecting garlic generated in this study represent only small part from the viral genome providing us with a smaller chance for detecting potential recombination event compared to the whole virus genome. It would thus be more informative to conduct recombination analysis after generating full length sequences of Ethiopian garlic viruses in the future.In conclusion, our work on garlic viruses in farmers’ fields and national germplasm collection provided unprecedented information on the relative incidence, distribution and diversity of viruses in the country and an insight into their evolutionary relationship, as well as their potential to cause damage singly or in mixed infection. The molecular assays used in this study for the identification of garlic viruses are proved to be highly sensitive and reliable. The degenerate carlavirus primer pair (Tsuneyoshi et al. Click here for additional data file.


The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays.Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion  system has an array of highly sensitive  in vitro diagnostic (IVD) real-time PCR assays for respiratory viruses, including an assay for influenza A (FluA) virus, influenza B (FluB) virus, and respiratory syncytial virus (RSV) (FFABR assay). The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays. We developed two LDTs for FluA virus strain typing on the Panther Fusion instrument, enabling side-by-side testing with the FFABR assay. The LDT-FAST assay uses proprietary primers and probes designed by Hologic for the Prodesse ProFAST+ (PFAST) assay. The exWHO-FAST assay is an expanded redesign of the WHO-recommended reverse transcriptase PCRs (RT-PCRs). To evaluate the performance of these two LDTs, 110 FluA virus-positive samples were tested. Of these, 104 had been subtyped previously; 54 were H3, 46 were 09H1, and 4 were fsH1. All were appropriately subtyped by both LDTs. Of the untyped FluA virus samples, three were subtyped as H3 by both LDTs and two were subtyped as H3 by the LDT-FAST assay only. The sample not subtyped by either LDT was retested with the FFABR assay and was now negative. Limit-of-detection (LOD) analyses were performed with five FluA virus strains. The LDT-FAST LODs were similar to the FFABR assay LODs, while the exWHO-FAST LODs were higher for two H3N2 strains, findings that were explained by analysis of primer/probe homology. In conclusion, either FluA virus typing assay would be a valuable complement to the Panther Fusion respiratory menu given the performance of these LDTs, the system’s full automation, and the ability to split eluates for both IVD and LDT testing.Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion  system has an array of highly sensitive  Influenza viruses are classified into three distinct types, A, B, and C, with subdivisions in type A based on antigenic characterization of the surface glycoproteins. Currently circulating strains of influenza A (FluA) virus include H3N2 and H1N1pdm09. Since the 2009 influenza pandemic, H1N1pdm09 quickly became the dominant H1 strain, displacing the former seasonal H1N1 (fsH1) strain; however, rare occurrences of fsH1 have been observed. This is fortunate, because the fsH1 strain had become universally resistant to oseltamivir (Tamiflu). The clinical significance of the H3N2 strain is its association with higher attack rates, hospitalization rates . Becausein vitro diagnostic (IVD) respiratory virus assays on the Fusion instrument, which include the FluA–FluB–respiratory syncytial virus (FluA/B/RSV [(FFABR]), Paraflu, and adenovirus–human metapneumovirus–rhinovirus (AdV/hMPV/RV) assays, have been shown to have exquisite sensitivity (Panther Fusion (referred to below as Fusion) is a fully automated high-throughput system with on-demand testing capabilities. This system integrates nucleic acid extraction with either transcription-mediated amplification (TMA) or real-time PCR. The sitivity . Furthersitivity , 6. The Clinical specimens comprised 110 nasopharyngeal swab (NP) specimens, in 3 ml of viral transport medium (VTM), previously determined to be positive for FluA virus by the FFABR assay . FluA virus subtypes had been determined by the PFAST assay (Hologic) for 104 specimens. These samples were used in previous studies comparing influenza virus PCR assays , 6 and w10 copies/ml, respectively, for use as a positive control and for additional precision and reagent stability studies.Three FluA virus isolates obtained from the ATCC  included an fsH1 strain, A/Florida/3/2006 (VR-1893); an H1N1pdm09 (09H1) strain, A/California/7/2009(H1N1)pdm09 (VR-1894); and an H3N2 clade 3C.3a isolate, A/Switzerland/9715293/2013 . An H3N2 clade 3C.1 isolate, A/Texas/50/2012(H3N2)-like (Texas), was obtained through the New York State Department of Health (NYS DOH) Proficiency Testing program. A variant H3N2 strain, A/Indiana/09/2012(H3N2v)-like, was a gift from Judith Lovchik, Indiana State Department of Health. Viral stocks were serially diluted 1:10 in VTM and the dilutions tested six to eight times for limit-of-detection (LOD) and precision analyses. The 1:10 dilutions for the fsH1, 09H1, and Swiss strains were also diluted 1:4 to meet assay validation requirements in New York State. These dilutions were tested simultaneously by the LDT-FAST, exWHO-FAST, and FFABR assays, using the same nucleic acid extract. Determinations of viral nucleic acid concentrations were based on quantified control viral RNA  using either the Prodesse ProFlu+ (Hologic) or the PFAST assay, depending on strain/clade-based amplification efficiency  RNA at the initiation of extraction. Following the standard protocol for respiratory virus detection, 500 μl of specimen was added to a Panther Fusion specimen lysis tube containing 750 μl buffer and 360 μl of the mixture used for extraction. The nucleic acid was subsequently eluted into 50 μl, and 5 μl was amplified for an effective sample volume of 14.4 μl.2 (Hologic), 50 mM KCl (Hologic), and RNA-IC primers and probes (Hologic), and the mixture was overlaid with Panther Fusion oil reagent (Hologic). exWHO-FAST   was performed using the following amplification profile: 1 cycle of 8 min at 46°C, 1 cycle of 2 min at 95°C, and 45 cycles of 95°C for 5 s, with 60°C for 22 s.High-performance liquid chromatography (HPLC)-purified primers and probes for the LDT-FAST test  were combined with 8 mM Tris , 4 mM MgCllle, IA)  primers mT) more compatible with the Fusion system’s optimal amplification profile. Based on assay optimization studies, it was determined that the the modified H3 primers and probe from WHO Protocol 2 and the modified H1N1pdm09 primers and probe from WHO Protocol 1 demonstrated superior performance in combination (data not shown). This multiplex was expanded into a triplex to include a target for the former seasonal H1 (fsH1) strain for a complete FluA virus strain-typing assay.The primers and probes from both the WHO’s one-step duplex real-time RT-PCR  and its one-step real-time RT-PCR for the H1 gene of influenza A(H1N1)pdm09 virus   were modin silico analysis of primer and probe homology, hemagglutinin (HA) gene sequences were obtained from the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiFlu database. This database comprises influenza sequences for the semiannual vaccine strain selection uniquely submitted from contributors such as the World Organisation for Animal Health , national reference laboratories, and all WHO Collaborating Centers for Surveillance, Epidemiology, and Control of Influenza  were determined using Microsoft Excel 2016  . Probit TC) values with the FFABR assay were 37.1 and 39.7 for the two samples subtyped by the LDT-FAST test only. The original FFABR assay TC value for the sample not subtyped by either LDT was 37.9. This sample was retested with the FFABR assay and was found negative upon repeat testing. Although these samples were not previously subtyped, they were all collected during a 2-week period in 2015 when the incidence of FluA virus infection was 25% and the subtype was exclusively H3  are a modular approach to syndromic testing on a fully automated platform. This system and associated IVD assays have numerous benefits, such as exquisite sensitivity and onboard reagent stability. However, this system does not include a FluA virus subtyping assay, which is often part of a respiratory virus panel. With the Open Access system, LDTs can be used alongside IVD assays, with the same full automation. Our laboratory developed and compared two LDTs on the Fusion instrument for the subtyping of FluA virus: one assay using Hologic’s proprietary primers and probes from their Prodesse ProFAST+ assay and one using modified versions of primers and probes recommended by the WHO .Both subtyping LDTs demonstrated clinical sensitivities comparable to that of the FFABR assay and were superior to many other commercial assays for FluA virus detection. The sensitivities of the cIAB, PFAST, RP, and Xpt assays in this sample set were 91%, 94%, 90%, and 85%, respectively, also by comparison to the FFABR assay. The sensitivities of 100% and 98.2% for the LDT-FAST and exWHO-FAST assays, respectively, were an impressive finding, because these samples had been subjected to repetitive freeze-thaw cycles for previous studies with the other assays. Another important point is that 97% of the H3 samples were taken from a previous prospective study involving consecutive specimens received in the lab during a 2-week period in the winter of 2015 . AnalyseTC values with the diagnostic FluA test are low, since assay modification may be needed. Fortunately, the risk associated with a false-negative typing result  is minimal and is not an uncommon result with other IVD assays.We developed two assays due to an initial lack of certainty that our lab would continue to have access to the proprietary sequences from Hologic. Although both subtyping assays demonstrated sensitivities superior to those of many other commercial assays for FluA virus detection, the LDT-FAST assay was a little more sensitive than the exWHO-FAST assay. Interestingly, the FluA virus samples missed by the exWHO-FAST assay were from the 2-week period in 2015 in which the FluA virus population consisted primarily of clade 3C.2a viruses , 11. ThiThe value of including primers and probes for subtype fsH1, at an added cost of approximately $1, is certainly questionable. Our lab chose to include them because we did have two cases in 2018. Similarly, one may assert that viral clade determinations are needed, particularly since vaccine efficacy can differ with circulating viral clades, as seen with influenza virus A/H3 and FluB clades. However, the value in a clinical lab is currently minimal. Also, with regard to value, the benefit of running a subtyping assay concurrently versus sequentially with a FluA diagnostic assay would have to be addressed at each institution. Many of the experiments in this study were run concurrently, providing proof of concept that an LDT can be performed side by side with an IVD assay on the same sample and extract. It should also be noted that the current instrument software allows for only three assays from the same extract; however, Hologic is currently in the process of updating the system to allow for five assays per extract.Besides the exquisite sensitivity seen with these FAST assays, the automation associated with the Fusion system has numerous other benefits. First, this is a fully automated process from the placement of the sample on the instrument to the reporting of results, which is certainly an improvement and reduces labor needs significantly. The instrument is designed for high-throughput testing with on-demand testing capabilities, and it complements Hologic’s modular approach to syndromic respiratory disease testing. Last, the system has reagent and consumable tracking with advance warning when more is needed.In conclusion, both FluA virus typing assays were successfully adapted to run on the Panther Fusion instrument, with sensitivities comparable to that of the FFABR assay, providing a valuable complement to the Panther Fusion respiratory menu. This study also demonstrated proof of concept that LDTs and IVD assays can be processed side by side with full automation from sample to answer, either from the same sample or from the same eluate.