Aureococcus anophagefferens , and diatoms  show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both ‘boom-and-bust’ and ‘steady-state infection’-like ecologies that include known as well as novel virus–host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus–host relationships in marine systems are abundant.Establishing virus–host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus–host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by  et al. use metatranscriptomics to resolve interactions between giant viruses and single-celled eukaryotic hosts.Viruses are partners in ecosystem ecology, yet their study has been primarily limited to laboratory models virus-host or derived from metagenomics. Here, Moniruzzaman  Mimiviridae, isolated using Acanthamoeba in the laboratory, are yet to be confirmedViruses that infect marine microbes are an integral component of aquatic ecosystems, with a diversity spectrum spanning the entire Baltimore classification scheme26in situ approaches that link the marine virosphere to their hosts within the microbial eukaryotes.Along with the NCLDVs, RNA viruses also comprise a major fraction of the marine viroplankton, infecting organisms ranging from diatoms and dinoflagellates to fishThe marine ecosystem consists of complex interactions among diverse organisms and their viruses. While studying individual host-virus systems remains critical to understanding the molecular basis of interactions, studying the overall contribution of viruses in a dynamic network of organisms is hindered by methodological limitations. Culture-independent approaches to study viruses, and especially viral communities, are challenging: ‘viromes’—large metagenomic data sets enriched with viral sequences, are usually generated by size exclusion ≤0.22 μm) of bacteria and small eukaryotes2 μm of bAdvances in sequencing and informatics are ongoing: while concern about chimeric assemblies and technical errors still exist, the generation of consensus genomes for populations within communities is now a common effort. Indeed, a recent workshop provided a comprehensive proposal to classify assembled virus genomes within the International Committee on Taxonomy of Viruses frameworkA. anophagefferens7Aureococcus by AaV) as well as identify novel virus–host interactions. These observations demonstrate that the depth of virus–host interaction in the global oceans is likely much deeper than previously anticipated, with viruses containing all forms of genetic material potentially infecting single-celled eukaryotes.Here, we examined metatranscriptomes from two highly productive sites on the east coast of USA—Quantuck Bay, New York, and Narragansett Bay, Rhode Island. Quantuck Bay experiences recurring ecosystem disruptive brown tide blooms caused by the pelagophyte PoxviridaeTo identify NCLDVs, we screened contig libraries generated at each study site for 10 conserved NCLDV core genesMimiviridae and Phycodnaviridae were consistently present in both Quantuck Bay and Narragansett Bay. At both sites the highest number of contigs fell within the Mimiviridae family, followed by Phycodnaviridae (Mimiviridae clade. Their presence and relative abundance in these field surveys demonstrate that the Mimiviridae are an important component of the marine virosphere and are as active as the better-studied Phycodnaviridae group.We placed the MCP contigs on a reference phylogenetic tree and studied their relative expression levels in terms of a metric that we defined as ‘rarefied counts per kilobase’ (RCK) (details in Methods section). Phylogenetic placement of the contigs demonstrated that NCLDV members from aviridae . A largeAureococcus count of ∼2.28 × 106 cells per ml and ∼2.23 × 106 cells per ml, respectively (Aureococcus count of ∼1.91 × 106 cells per ml. We detected a persistent infection of A. anophagefferens by AaV across this sampling period. High stringency (similarity ≥97%) mapping of reads to the genome identified 1,368 and 604 reads that could be assigned to peak bloom samples QB-S1 and S2, respectively, after library size normalization, while 236 reads were mapped to the QB-S3 sample taken during bloom decline  and 16 June (QB-S2) represented the bloom peak with an ectively . The thi decline . Across  decline . SimilarV genome  and specV genome  progressV genome . Indeed,in situ samples over a period of ∼4 weeks  that infect marine single-celled eukaryotes have been described9Chaetoceros sp., Asterionellopsis glacialis and Rhizosolenia setigera)AurantiotrychiumMarnaviridae, which currently have only one member—HaRNAV, that infects the marine raphidophyte Heterosigma akashiwoDicistroviridae family, with 90 and 36 contigs from Quantuck Bay and Narragansett Bay, respectively  ssRNA viruses, with the main contributors coming from a yet unclassified group of viruses in the Picornavirales orderectively . Intereshe ocean . No (–) Dicistroviridae, Secoviridae and Picornaviridae families across all the time points  of heterotrophic protists alongside Aureococcus19Aureococcus and the potential shift in competition that might occur during Aureococcus bloom decline. These observations suggest a broad ecological role for viral infection during phytoplankton bloom decline, which would not have been resolved with targeted studies of AaV or metagenomic approaches. Taken together, the apparent dynamics and abundance of this unclassified Picornavirales suggest this group is a major component of the marine virioplankton, and strengthens previous observations that the Picornavirales phylogenetically distinct from the established families can be dominant members in different marine environments12To assess how the activity of virus groups changed over time, we measured the proportion of reads that mapped to different virus groups for each library. In Narragansett Bay, the majority of the virus reads originated from the unclassified marine Picornavirales and the e points . The unce points . In Quane points . Howevere points . Overalle points . AureocoEndornaviridae family dsRNA virusesEighteen of the assembled contigs (nine from each site) were >7,000 bp and had best hits to different Picornavirales members. Phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) gene and feature analysis of existing (+) ssRNA virus genomes suggested that these contigs are complete or near-complete Picornavirales genomes . While tde novo assembled genomes by mapping the data collected over spatiotemporal gradients. All the (+) ssRNA virus genomes from Quantuck Bay samples showed higher relative expression during bloom decline (QB-S3) compared to the time points corresponding to the bloom peak , RdRP (RNA viruses) and viral replicase (ssDNA viruses) were compared to the functional eukaryotic marker gene RNA polymerase II large subunit RPB1, , a candi35Aureococcus and AaV, also contained another Mimiviridiae member, several ssDNA and (+) ssRNA viral contigs along with eukaryotes belonging to prasinophyceae and pelagophyceae (Aureococcus being infected by more than one virus type cannot be discounted (and indeed is perhaps likely). Moreover, the potential for AaV to infect closely related pelagophytes remains a possibility Phycodna- and a Mimiviridae member, also included a RPB1 contig phylogenetically placed in the Cercozoa group , while containing both ophyceae . The posoa group . AlthougMimiviridae and several ssRNA viral contigs connected to choanomonada, stramenopile, diatom and dinoflagellate members  and several diatom contigs  contained a  members . The maj members . While l contigs . To dateagellate . Only ones group . A numbeAureococcus cluster A(ii) harboured a fungal contig and a Barnaviridae member—a virus family with fungi as the only known hosts  that originated from a single coding sequence. Increased sequencing, along with the continued development of assembly tools, will provide better resolution to these relationships. These limitations notwithstanding, the analysis provides a ‘proof-of-principle’ for inferring the complex relationships among diverse unicellular eukaryotes and their viruses using metatranscriptome data.Increased sample resolution in the future will resolve more statistically robust relationships, which can further narrow potential interacting partners. One limitation of reference-independent assembly of high-throughput data is fragmented contigs originating from same transcript—which is illustrated by two In this study, we demonstrate how metatranscriptomics can provide a unique view of the marine virosphere by simultaneously detecting multiple viral infections across the landscape of the eukaryotic plankton within an ecosystem setting. This effort can largely overcome previous technical limitations involved in the study of different viral groups, owing to their size range and genome type, within the same sample. In the last two decades, we have learned much regarding the diversity and dynamics of the phages in the ocean, but the eukaryotic virosphere has remained elusive, with little known about who is infecting whom in the environmentAureococcus cells were counted from glutaraldehyde  fixed whole water samples using an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody as described previouslyIn situ samples from June 22nd (third sampling point) was also used to carry out nutrient amendment experiments—results from these samples were included to both expand the environmental conditions sampled (for a similar population) as well as to increase the total number of individual samples  for analyses. Briefly, bottles were filled with natural sea water from the bloom and were amended with 25 μM ammonium only (+N), 4 μM phosphate only (+P) and 25 μM ammonium and 4 μM phosphate (+N&P) in triplicate. Three additional bottles with no nutrient addition were used as control. The samples were then incubated for 24 h in a floating chamber at 0.5 m in eastern Shinnecock Bay at the Stony Brook—Southampton Marine Science Center under one layer of neutral density cover to mimic the light and temperature levels of Quantuck Bay. Samples for Aureococcus cell density measurement and total RNA extraction were collected at T=0 and T=24 h. Approximately 25 ml of natural seawater from each of the in situ and nutrient amendment samples were prefiltered through 5 μm polycarbonate (PC) filters and collected on 0.2 μm PC filters. The samples were flash frozen immediately after filtration and transferred to −80 °C. Prior RNA extraction, CTAB buffer  amended by polyvinylpyrrolidone (1% mass/vol) was added to each of the samples.Samples were collected from a brown tide bloom in Quantuck Bay , NY that occurred from late May to early July, 2011, covering the initiation, peak and demise of the bloom. Samples collected on June 14 (BT-S1) and June 16 (BT-S2) represented the peak of the bloom, while sample collected on June 22 (BT-S3) represented the initial phase of bloom decline. et al.in situ samples.The detailed sampling procedure for Narraganset Bay has been previously described in Alexander RNA was extracted from Quantuck Bay samples using the UltraClean Plant RNA Isolation Kit  according to manufacturer’s protocol. RNA samples were quantified spectrophotometrically and were sequenced in the Columbia Sequencing Center  using Illumina HiSeq platform with poly-A enrichment at a depth of ∼50 million 100 bp single end reads. Two more replicate samples were sequenced from June 22 (QB-S3) at a depth of 100 million reads. For the present study, these biological replicates from QB-S3 were pooled together before further analysis.in situ samples were pooled, representing 6 l of water for each sample. RNA was extracted using RNeasy Mini Kit  according to a modified yeast RNA extraction protocol. Briefly, lysis buffer and RNA-clean zircon beads were added to the filter. Samples were then vortexed for 1 min, placed on ice for 30 s, and then vortexed again for 1 min. The resulting RNA was eluted in water and possible DNA contamination was removed using a TURBO DNA-free Kit . RNA from each triplicate was pooled by sample or treatment. >1,000 ng RNA from each sample then went through a poly-A selection using oligo-dT beads followed by library preparation with TruSeq RNA Prep Kit . The samples were sequenced with an Illumina HiSeq2000 at the Columbia University Genome Center to produce ∼60 million; 100 bp paired-end reads per sample.For Narragansett Bay, replicate filters from each treatment and For bioinformatics analyses, sequence reads from both locations were quality trimmed , No ‘N’s allowed, 70 bp size cutoff) in CLC genomics workbench 8.0 . The data from all the Quantuck Bay samples were combined and assembled together, and a similar assembly procedure was also followed for the Narragansett Bay specific samples. This resulted in 2,455,926 contigs for Quantuck Bay and 9,525,233 contigs for Narragansett Bay at a 100 bp size cutoff.Paramacium bursaria Chlorella Virus—PBCV, NCBI accession: BAA22198) and ‘D13L’ protein of Mimivirus (NCBI ID: AAV50707) have been experimentally and bioinformatically characterized to be the MCP4749Marseilleviridae, Iridioviridae and Ascoviridae members have only a single capsid homologue annotated as MCP. Finally, representative Asfarviridae member African swine fever virus harbours a single capsid gene, also known to encode the major structural componentMost giant virus genomes contain multiple genes having domains characteristic of viral capsids. Among the capsid orthologues, protein ‘VP54’ (in –3) to MCP and RPB1 were kept for further analysis.For selecting eukaryotic RPB1 contigs, HMM profile specific to domain ‘RPB1-C-term (NCBI CDD ID: cd02584)’ and ‘RPB1-N-term (NCBI CDD ID: cd02733)’ was used to query the contig libraries. All the MCP and RPB1 candidate contigs detected in this manner were queried against NCBI Refseq database and only contigs with first BLASTx hits  and viral replicase (pfam: PF03090) motif were selected using HMM profile specific to these motifs.To detect contigs originating from viruses other than NCLDVs, we combined the proteins derived from all the viruses having algal, fungal and protozan hosts available on NCBI database. This protein database was queried against the contig libraries using tBLASTn with an e-value cutoff of ≤10et al.To reconstruct phylogenies, reference sequences for MCP (giant viruses), RdRP (RNA viruses), viral replicase (ssDNA virus) and RPB1 (eukaryotes) were downloaded from NCBI Refseq database. A number of RPB1 sequences representing several eukaryotic groups were also collected from Marine Microbial Eukaryotes Transcriptome Sequencing Project (MMETSP)www.clcbio.com). The genome annotation with predicted features was assisted by pfamFor the complete or near-complete Picornavirales genomes ORFs were predicted on using CLC genomic workbench 8.0  in CLC genomics workbench 8.0. The read mapping values were normalized by library size and length and expressed as RCK. RCK values of viral and eukaryotic contigs >225 base pairs were converted into matrices separately for Quantuck Bay and Narragansett Bay data sets, which included mapping statistics from both www.ncbi.nlm.nih.gov/sra . The genomes are submitted in the NCBI database under the accession numbers: KY286099—KY286107 and KY130489—KY130497.The field sequence data reported in this paper have been deposited in the National Center for Biotechnology Information Sequence Read Archive, How to cite this article: Moniruzzaman, M. et al. Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics. Nat. Commun.8, 16054 doi: 10.1038/ncomms16054 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


BMI) and healthcare costs in relation to obesity‐related comorbidity and depression. A population‐based cohort study was undertaken in the UK Clinical Practice Research Datalink (CPRD). A stratified random sample was taken of participants registered with general practices in England in 2008 and 2013. Person time was classified by BMI category and morbidity status using first diagnosis of diabetes (T2DM), coronary heart disease (CHD), stroke or malignant neoplasms. Participants were classified annually as depressed or not depressed. Costs of healthcare utilization were calculated from primary care records with linked hospital episode statistics. A two‐part model estimated predicted mean annual costs by age, gender and morbidity status. Linear regression was used to estimate the effects of BMI category, comorbidity and depression on healthcare costs. The analysis included 873 809 person‐years  from 250 046 participants. Annual healthcare costs increased with BMI, to a mean of £456 (95% CI 344–568) higher for BMI ≥40 kg m−2 than for normal weight based on a general linear model. After adjusting for BMI, the additional cost of comorbidity was £1366 (£1269–£1463) and depression £1044 (£973–£1115). There was evidence of interaction so that as the BMI category increased, additional costs of comorbidity  or depression  were greater. High healthcare costs in obesity may be driven by the presence of comorbidity and depression. Prioritizing primary prevention of cardiovascular disease and diabetes in the obese population may contribute to reducing obesity‐related healthcare costs.The objective of this study was to evaluate the association between body mass index ( Obesity is associated with higher healthcare costs.It is less clear whether obesity, or obesity‐associated comorbidities and depression, are the main driver of healthcare costs.Few studies have been based on nationally representative data sources.This study reports data for a large English cohort, with 873 809 person‐years of follow‐up.Healthcare costs are greater as the BMI category increases, but comorbidity and depression are the greatest drivers of healthcare costs in obesity.Prioritizing primary prevention of cardiovascular disease and diabetes, as well as improving depression management in obesity, may contribute to reducing obesity‐related healthcare costs.Obesity is a growing global health concern, accounting for substantial national healthcare expenditures, with healthcare costs predicted to be higher by around a third in obese people compared to those of normal weight The aim of this paper is to investigate the association between body mass index (BMI) category and healthcare costs, focusing on the issue of whether BMI category, obesity‐related comorbidity and/or depression most strongly determines costs related to obesity. Answering this question may enable better informed efforts to increase the effectiveness and efficiency of the management of obese individuals. This analysis advances the literature on obesity‐related healthcare costs in three important ways. First, it utilizes data from a large and nationally representative database. Previous studies have relied on smaller https://www.cprd.com). The CPRD collects primary care electronic health records for 7% of the UK population and is considered representative of the UK population in terms of patient demographic characteristics and the size and composition of the general practices of reporting data We undertook a population‐based cohort study using the UK Clinical Practice Research Datalink (CPRD) . This left 250 046 participants for the analysis.A cohort of participants was drawn from the CPRD. Participants were eligible if they were registered with a CPRD general practice between 1 January 2008 and 31 December 2013. We evaluated eligible participants’ BMI records using the categories 18.5–24.9; 25.0–29.9; 30.0–34.9; 35.0–39.9; 40.0–44.9 and 45.0 kg m−2 into a single group to represent morbid obesity. Comorbidity status was evaluated using medical diagnoses coded into electronic health records during general practice consultations. Person time was classified according to morbidity status using the first diagnosis of type 2 diabetes (T2DM), coronary heart disease (CHD), stroke or cancer. Depression was re‐evaluated in each year of follow‐up, and patients were considered to have depression if they were diagnosed with depression during the year or if they were prescribed antidepressants in year and were ever diagnosed with depression. Morbidities, including T2DM Electronic health records were analysed from the latest of the date the practice joined CPRD or the patient's first registration to the earliest of the end of patient registration or date of death. Person time for men and women was allocated to a BMI category according to the most recent BMI value, combining BMI records >40 Kg mHealth care utilization was estimated from participants’ electronic health records with linked hospital episode statistics (HES) data. Primary and secondary care utilization was evaluated, including primary care consultations at the practice, by telephone, at home, emergency and out‐of‐hours. Secondary care utilization included admissions to hospital, out‐patient, day case and emergency visits. All drug prescriptions issued by the practice were evaluated. Utilization rates were calculated using person time at risk. Age‐standardized rates were estimated using direct standardization and the European Standard Population (ESP) for reference. The costs of healthcare utilization were evaluated for participants by morbidity and depression status within BMI categories for the period 2008–2013. Initially, the mean cost per person per year was calculated for each combination of BMI category and comorbidity status. The analysis was undertaken from a health service provider perspective  and did not consider indirect costs such as loss of productivity or patient and carer time.Unit costs were applied to the categories of healthcare utilization to estimate healthcare costs. The unit costs were all taken from reference sources based on UK£ 2013 price estimates. The Personal Social Service Research Unit (PSSRU) publication ‘Unit Costs of Health and Social Care (2013) was used as the reference for all healthcare costs A two‐part model The research represents part of a study approved by the Independent Scientific Advisory Committee (ISAC) of the Medicines and Healthcare products Regulatory Agency (MHRA) (ISAC Protocol No. 13–089).The cohort person‐years are presented in Table The two‐part model used to estimate healthcare costs is presented in Table The predicted annual healthcare costs in UK£ by BMI category, gender, comorbidity and depression status are presented in Table Table This study investigated healthcare costs in relation to BMI and may be the first study to isolate depression as a driver of costs separate from other common obesity‐related morbidities. Previous studies investigating healthcare costs relating to obesity using patient cohorts have attempted to estimate the marginal effect of obesity on costs There is a wide variation in cost estimates for obesity based on population sample studies, ranging from an additional €160 per year for severe obesity, compared to normal weight We found that having a comorbidity was the greatest predictor of increased healthcare costs at £1366 per year followed by clinical depression at £1044 per year. Of the four obesity‐related morbidities we assessed, stroke was found to be associated with the highest absolute costs, which is likely to result from acute secondary care required after a stroke. This finding is supported by a previous paper that identified cardiovascular diseases and cardiovascular agents as the biggest drivers of healthcare claims costs per unit of BMI We found that depression was the second greatest driver of costs, and its presence increased costs in all conditions, consistent with a previous investigation conducted into depression and healthcare utilization In T2DM, body weight did not appear to be associated with healthcare costs. This phenomenon has been reported elsewhere, and the possibility of greater morbidity in diabetics who are of normal weight was suggested as an explanatory factor This study has a number of strengths that make it a valuable addition to the literature on health care‐related costs of obesity. Firstly, it is based on a large, longitudinal, nationally representative dataset and uses costs associated directly with provision of care rather than based on insurance claims, which are an indirect measure of morbidity and may be less accurate. It is also one of few papers to use clinically measured height and weight values in the calculation of BMI rather than self‐reported values that are prone to reporting bias The economics literature has moved towards using an instrumental variables approach to examine the causal effect of obesity on medical costs by using the weight of a biological relative as an instrument The findings of this analysis emphasize that healthcare costs for obesity are largely driven by a few key obesity‐related morbidities and not by high BMI alone. Further research is now needed to elucidate the costly elements of these patients’ care and to develop clinical pathways to manage them more efficiently. Persistently high obesity rates and evidence from the literature www.icmje.org/coi_disclosure.pdf and declare no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work.All authors have completed the ICMJE uniform disclosure form at MG and ACR designed the study. JC and MG conducted the analysis. ACR and MG interpreted the results. MG, ACR and HB drafted the paper. All authors commented on and approved the manuscript.


During 2014–2016, we conducted mosquito-based Zika virus surveillance in Rio de Janeiro, Brazil. Results suggest that Zika virus was probably introduced into the area during May–November 2013 via multiple in-country sources. Furthermore, our results strengthen the hypothesis that Zika virus in the Americas originated in Brazil during October 2012–May 2013. Aedes aegypti mosquito is considered the main vector of Zika virus in urban and suburban areas throughout the world, including Brazil, where the mosquito has been confirmed, together with Ae. albopictus mosquitoes, as a vector for the virus , which we pooled (n = 178) and subjected to Zika virus detection using real-time RT-PCR  clustered within the same strongly supported lineage, which included strains detected in Rio de Janeiro and other parts of Brazil in late 2015 and in 2016  Figure. Ae. aegypti mosquitoes that were collected during a mosquitoborne virus surveillance program in Rio de Janeiro. Information regarding Zika virus infection rates is lacking for female and male mosquitoes trapped in the field. However, experiments performed in the laboratory demonstrated transovarial transmission of Zika virus among Ae. aegypti mosquitoes and revealed a minimal filial infection rate of 1:290 (In this study, we detected Zika virus RNA in 2 pools of engorged Ae. aegypti mosquitoes trapped in Rio de Janeiro before the first case of autochthonous Zika virus disease was diagnosed in the city (In conclusion, we showed the presence of Zika virus in engorged Bayesian maximum clade credibility tree representing the time-scale phylogeny of the Zika virus outbreaks in the Americas.


Marburg virus (MARV) has caused outbreaks of filoviral hemorrhagic fever since its discovery in 1967. The largest and deadliest outbreak occurred in Angola in 2005, with 252 cases and 227 deaths. In 2014, we developed a mouse-adapted MARV, Angola variant through serial passaging in mice. The mouse-adapted MARV exhibits many of the hallmarks of MARV disease in humans. By applying deep-sequencing to every passage of the virus, we are able to study virus evolution in this host with surprising precision. We show that two regions go through substantial changes: the intergenic region between NP and VP35, as well as the first 100 amino acids of the VP40 protein. Our results also reveal that there were profound changes during the production of the final virus stock in cell culture. Overall, our results show that a handful of regions carry most of the mutations acquired during the adaptation of the virus to a new host and that many mutations become fixed very early during the adaptation process. Filoviridae as a member of the species Marburg marburgvirus1. It has been causing sporadic outbreaks in Central Africa since at least 19752. MARV causes a hemorrhagic fever characteristic of the filoviruses with a case-fatality rate in humans ranging from ~20% to 90%4. The virus is found in fruit bats and transmitted to humans as a zoonotic disease5. The genus Marburgvirus includes a single species (Marburg marburgvirus) which includes two viruses: Marburg virus and Ravn virus (RAVV). Live replication-competent viruses have been isolated from fruit bats caught in Africa5. Recent studies have shown that both viruses and their variants can be found circulating within the same bat colonies5.Marburg virus (MARV) belongs to the family 6. This variant was chosen because it caused the largest and deadliest recorded MARV outbreak.While MARV can cause outbreaks as lethal as Ebola virus, countermeasures against MARV, such as vaccines and treatments, are much less developed. This was due, in large part, to the absence of good small animal-adapted viruses. The lack of such viruses meant that testing of countermeasures had to be performed in animals that were at least partially immunosuppressed or in non-human primates. In 2014, we developed a mouse-adapted MARV based on the Angola variant (MARV-Angola)The adaptation of the virus required 24 passages in SCID mice. At that point, the virus was shown to be equally lethal to C57BL/6 and BALB/c mice. The disease caused by the adapted virus mimicked the liver damage and coagulation abnormalities observed in human cases. The virus was adapted by infecting new mice with liver homogenates collected on day 7 post-infection. Samples of the liver homogenates were retained. These samples were used to recover viral RNA from 23 of the 24 passages.The samples retained from the adaptation allow us to use deep-sequencing to look at the adaptation process at a level of detail that was previously not possible with past sequencing technologies. We believe that understanding the sequence and frequencies of mutations during adaptation will allow better monitoring of MARV and the related ebolaviruses for changes that may indicate adaptation to a new host and potentially identify locations in the viral genome that are associated with virulence.The most active adaptation was found within the VP40 gene as well as the NP-VP35 intergenic region. We also sequenced a virus stock after 2 passages in Vero E6 cells. This stock lost a large number of mutations seen in the last mouse passage; however, the MARV-Angola stock retained high levels of lethality to mice. This result points to a handful of mutations as being crucial for causing MARV disease in mice.We performed deep-sequencing on liver homogenate samples from each passage during the adaptation of MARV to mice. All passages except for passage 12 were amplified fully or partially by RT-PCR Fig. . The medth passage. The NP, on the other hand, started to adapt only after 13 passages. We detected 6 mutations in the VP35, 5 of them in the first 50 N-terminal amino acids. The VP35 started mutating at passage 5. We also detected 6 mutations in VP30, 4 of them in the first 100 N-terminal amino acids and all 6 mutations were only detected during passages 16 to 21. The VP24 only went through 2 mutations during the adaptation process. Interestingly, one mutation (V157M) appeared after the 4th passage and became dominant starting at passage 7. Unfortunately this fragment could not be amplified for passages 5 and 6.All MARV proteins went through some degree of mutations Fig. . A totalNo mutations were detected in the intergenic regions. However, the non-coding regions did undergo mutations during the adaptation Fig. . Most no6 were also found, except for mutations at positions 19104 and 19105. The coverage of the final stock dropped below the 200 minimum by position 19094, therefore no variants were called beyond that position.In order to understand the effect of cell culture passaging, we also sequenced the virus stock used to characterize the model. The virus stock was produced by passaging the passage 24 isolate twice in Vero E6 cells to produce a large virus stock that can be used for subsequent studies. A large number of mutations from the passage 24 isolate were lost during the stock production, but a few new mutations appeared Fig. . These n10 TCID50/gram tissue in the last passages6), which has an impact on the rate of virus mutation, as larger viral population sizes result in stronger clonal interference and hence more mutations competing for fixation7. It is possible that different mutations may arise and become fixed under different conditions . Nevertheless, we were still able to use this rare opportunity to study the changes in the virus population after each passage.We previously published a new MARV-Angola model, in which the virus was adapted to produce lethal infection in adult immunocompetent mice. Samples from each of the 24 passages in mice were preserved, unfortunately not in sufficient quantities to determine the viral load accurately. Therefore the titer of each sample may be different  which is required and sufficient for the blockade of interferon signalling9. One interesting mutation in this region is G79S, which appears at passage 6 and becomes essentially fixed by passage 10. This mutation was found to confer interferon antagonism in mouse cells in vitro10. Another mutation in VP40, D184N, is interesting because it was found to provide MARV Musoke VP40 with a replicative advantage in guinea pig cells12. Also, its frequency very closely matches that of the V157M mutation in VP24.The same temporal pattern is also followed by 6 mutations in the non-coding regions of NP  and by 1 mutation in the non-coding region of VP40. Finally, the VP40 mutation E260K also presents interesting patterns. It appears earlier than the D184N mutation and almost reaches fixation, until the appearance of D184N, at which point the virus population appears to be split between the two mutations . The frequency of viruses carrying E260K always moves in the opposite direction from D184N. Thus, these mutations appear to be mutually exclusive.The extent of mutations in VP40 was interesting but not unexpected. One of the main functions of VP40 in MARV immunopathology is to block the host cell responses to interferon signalling14. Thus, VP35 does not interact with host proteins as much as VP40. Surprisingly, no mutations were left in the glycoprotein by the final passage, suggesting there was no need to adapt to the host cell surface receptors. Supporting that hypothesis is the fact that the mutations were almost all found within the mucin domain, which is cleaved before receptor interaction in the endosome. The adaptation took place in SCID mice, which are unable to mount an adaptive immune response, T and B cells responding to a specific pathogen. This is another possible reason for the lack of major changes in the GP. Its mucin domain contains many targets for antibodies and T cell responses; this domain is known to be highly disordered and prone to mutations, which allow the virus to escape adaptive responses. In the absence of a normal immune response, there was no selection pressure on the GP; this absence of adaptation did not seem to affect virulence, as the virus was also fully lethal to wild-type mice. The L, NP, and VP30 also had a number of mutations which accumulated by the end of the passaging. While these proteins mainly function together as the transcription/replication unit, they also likely interact with certain host proteins, although most of those proteins remain ill-defined.While the VP35 is also involved in immune evasion, its main role is to interact with the viral dsRNA to prevent its detection by the host cells15; although we were able to detect mutations in more coding regions. The VP40 mutation D184N appeared in their 3rd passage (4th passage in this experiment) along with G79S shortly before the virus became fully lethal to SCID mice. We also detected the presence of VP40 mutation Y19H but not I187T (we did find G188R) or I228M and we detected NP mutation Y536H but not GP mutation F445P. The two mutations found to restore the anti-interferon activity of RAVV VP40 in mouse cells (V57A and T165A) appeared during the adaptation but were absent from the final stock. Since this stock was found to be lethal to immunocompetent mice, other mutations may be compensating for the effect of V57A and T165A. Interestingly, even though Lofts et al. passaged their virus repeatedly in immunocompetent animals only one mutation appeared in the GP gene, suggesting that the MARV GP does not need adaptation between primate- and mouse-adapted viruses.The mutations found in the coding regions are consistent with those reported during the adaptation of MARV-Ci6716. Based on minigenome experiments, it was shown that these mutations could alter the expression of genes both upstream and downstream.The mutations in the non-coding regions of NP were extensive and somewhat unexpected. While we were expecting that the non-coding and/or intergenic regions would mutate to properly modulate protein expression for the new host, the number of mutations was much higher than that of any other non-coding region. The two genes located downstream of this region, VP35 and VP40, are both very important for immune evasion, their expression very likely needs to be re-modulated to fit the pressures on the virus in a new host. A similar change has been noted in sequences obtained during the Ebola virus disease outbreak in West Africa, in which a recent study has uncovered that mutations in two sites in the equivalent non-coding region of Ebola virus lead to increased virulenceWe also looked into the effect of cell culture passaging. We observed two main overarching effects. First, many mutations still present in most of the virus population after passage 24 disappeared after production of the virus stock. Second, the few mutations which remained after passaging the virus in cell culture were mostly fixed in the virus population. These two results suggest that even without plaque purification, cell culture passaging of MARV leads to selection and mutation of the virus. While our results cannot confirm that the changes are adaptive, it is interesting to note that the cells being infected are from NHPs, requiring a degree of balance between mouse-adapted and “cell-adapted” viruses. Many of the new mutations appeared in the NP-VP35 intergenic region, suggesting that adaptation to cell culture affects gene regulation rather than protein sequences.Overall, our results offer a detailed glimpse into the changes which occur during virus adaptation to a new host and the first study of this type for a filovirus. In turn, these results show “hot-spots” for adaptation and monitoring of adaptation, these include: the 5′ NP non-coding region, the first 100 amino acids of the VP40 protein, and the VP35 protein.All experiments involving live, replication-competent Marburg virus were carried out inside the Bio-Safety Level 4 laboratory at the National Microbiology Laboratory of the Public Health Agency of Canada in the Canadian Science Center for Human and Animal Health.The RNA was extracted from 140 µl of liver homogenate supernatant using a Qiagen Viral RNA Mini kit, according to the manufacturer’s instructions. Elution was performed in 60 µl.The reverse transcription was carried out using the SuperScript III RT enzyme (ThermoFisher) following the manufacturer’s instructions. Four microliters of RNA were used in the reaction along with 250 ng of random hexamers (ThermoFisher). The subsequent PCR reactions were carried out using Phusion Green Hi-Fidelity PCR mix (ThermoFisher) in 50 µl, using 2 µl of the RT product as template, primers at 200 nM, and 1 U of Phusion enzyme. The PCR conditions were 35 cycles of: 98 °C 30 s, 55 °C 30 s, 72 °C 3 min; and a final extension at 72 °C for 10 min. The genome was amplified in 6 overlapping fragments of 3–6 kb (named A through F). In some cases, some fragments could not be recovered; for a few passages where fragment F could not be amplified, two subfragments F1 and F2 were successfully amplified. In all other cases where fragments were not amplified by the primers in Supplementary Table The amplicons were quantitated using the PicoGreen dye and fragments from the same passage were pooled at equimolar concentration using a BioMek FX (BioTek). Library construction was done using the Nextera DNA Sample preparation kit (24-Sample) (Illumina) as per manufacturer’s instructions. Sequencing was carried out using a MiSeq sequencer (Illumina) using the MiSeq Reagent Kit v3 (600 cycles) (Illumina).Paired reads from each passage were aligned to a reference sequence (GenBank accession DQ447660.1) using Bowtie 2 version 2.0.5. Conversion to bam files, sorting, and indexing were performed using Samtools version 0.1.18. Variant calling was performed using freebayes version 0.9.8 using a ploidy of 4, a max complex gap of 0, a minimum quality score of 30, a minimum frequency of 5%, a minimum coverage of 200 fold, and to ignore insertions and deletions. The frequencies of variants were determined as the number of reads with a specific variant over the depth at that location. Finally, coverage was determined using bedtools version 2.25.0.http://osf.io/8sprb (DOI: 10.17605/OSF.IO/8SPRB).All assembled files (*.bam) are available on the Sequence Read Archive (SRA) under the BioProject accession number PRJNA320428. The coverage results files and variant calls, as well as the scripts used to produce the figures are available on the Open Science Framework, at Supplementary Material


We evaluated the risk for the Spanish Olympic Team acquiring Zika virus in Rio de Janeiro, Brazil, during 2016. We recruited 117 team members, and all tested negative for Zika virus. Lack of cases in this cohort supports the minimum risk estimates made before the Games. The current Zika virus epidemic became a major concern for national Olympic delegations before they traveled to Rio de Janeiro, Brazil, during summer 2016. Fear about individual consequences of the infection, such as congenital or neurologic disorders, were common among athletes and other participants of the Olympic Games and led some persons not to attend the Games for these reasons. The possibility of the Olympics contributing to a global spread of the Zika virus epidemic also was a concern, initially raised by ≈100 academic researchers, expressed in an open letter addressed to the World Health Organization (WHO) in May 2016 (−6 to 3 × 10−5) .Athletes and other participants were invited to participate in the study through the Spanish Olympic Committee. A total of 117 Olympics participants accepted and were included in the study during September and October 2016. After providing oral and written information, study participants signed an informed consent form, and demographic and health data were recorded in a medical questionnaire. A total of 10 mL of blood was drawn from each participant, and serologic tests for Zika virus , dengue virus , and chikungunya virus  were conducted at the Instituto de Salud Carlos III . For all samples initially testing positive for Zika virus, we conducted microneutralization testing.>1 signs or symptoms while in Brazil: 18% rash, 23% fever (temperature >38°C), 14% itching, 9% of conjunctival hyperemia, 9% arthralgia, 14% myalgia, 40% malaise, 9% lymphadenopathy, 32% headache, and 19% gastrointestinal symptoms. Ninety-nine percent of participants received Zika virus counseling before they traveled to Rio de Janeiro, including the advice of having protected sex during and after the Games (Twenty-one participants had he Games .For 4 persons, test results for Zika virus IgG was positive; IgM and neutralization testing yielded negative results. The 4 Zika virus IgG–positive participants had received previous yellow fever vaccination and were asymptomatic. One sample showed Zika virus IgM in the absence of specific IgG; the results were confirmed in a follow-up sample. Thus, the sample was classified as false positive.Study participants were advised to wait to conceive in accordance with WHO specifications: 6 months for men, 2 months for women. Participants with pregnant partners were advised to use condoms during the entire pregnancy.A lack of Zika cases in this cohort supports the risk calculations made before the Games and the WHO statement that there were no Zika cases associated with the Olympic Games (


Culex quinquefasciatus in Shenzhen, China, for the first time, in 2011. In this study, we characterized the ultrastructure of NDiV, determined its complete genome sequence and made comparisons with other known nidoviruses. Electron microscopic observation revealed that the NDiV strain isolated in China produced viral nucleocapsid-like particles and vesicles in host cells. The extracellular virions were enveloped and were spherical with short spikes. The complete genome sequence of the newly isolated NDiV was submitted to the GenBank database (GenBank accession number KF522691). Sequencing of the viral genome showed that the homologies of NDiV isolated in China and Vietnam were greater than 94.0% and 89.0% at the nucleotide and amino acid sequence levels, respectively. Moreover, gene substitution was detected, whereas insertions and deletions were not. A phylogenetic tree analysis showed that these viruses belong to the genus Alphamesonivirus1 of the family Mesoniviridae. The similarity between the two viruses regarding morphological and molecular biological characteristics indicates that the molecular genetics of NDiV are conservative and that the regional differences are unlikely to have a significant effect. This is the first report of the isolation and complete sequencing of a mesonivirus in mainland China.The Nam Dinh virus (NDiV) was isolated from  The Nam Dinh virus (NDiV) was identified in mosquitoes, and no evidence of vertebrate infection by this virus has been detected. NDiV was found to be a positive-sense, single-stranded RNA ([+]ssRNA) virus. The 20,192 nucleotide (nt)-long genome contains two major open reading frames (ORFs), ORF1a and ORF1b, which encode two proteins  using a ribosomal frame shift mechanism. The intact NDiV virion exhibited a spherical form with a diameter of 60–80 nm2. The structure, replication and transcription patterns of the viral genome were similar to those of coronaviruses in the order Nidovirales. A phylogenetic analysis showed that NDiV is distantly related to coronaviruses, indicating that this virus represents a missing link in the transition from small to large nidoviruses.A virus thought to be the first known insect-borne nidovirus was discovered during surveillance for Japanese encephalitis virus (JEV) in mosquitoes from VietnamMesoniviridae, which is based on two closely related viruses, the Cavally virus (CavV) and NDiV, has been classified as a new member of Nidovirales by the Executive Committee of the International Committee on Taxonomy of Viruses (ICTV)3. At present, ten mesoniviruses have been successfully isolated, including CavV, the Hana virus (HanaV), the Meno virus (MenoV), the Nse virus (NseV) and the Moumou virus (MoumouV) in Cote d’Ivoire, NDiV in Vietnam and America, the Dak Nong virus (DKNV) in Vietnam, the Bontang Baru virus (BBaV) and the Karang Sari virus (KSaV) in Indonesia, the Kamphaeng Phet virus (KPhV) in Thailand and the Casuarina virus (CASV) in Australia8. Mesoniviruses are widely distributed in different geographic regions and have a large host range of mosquito species. Thus, these viruses are commonly detected in mosquitoes around the world, with mesoniviruses likely to be present in more area than other viruses4.A newly established family, NDiV has been successfully isolated from various mosquito species in Vietnam and the United States. Further investigations of the various features of the ecology, morphology and molecular biology of this virus are critical for an improved understanding of the virus. Furthermore, studies of NDiV as part of mosquito monitoring programs in a greater number of areas are required to enrich the resources available for such research.Culex pipiens quinquefasciatus in Shenzhen China9. In this study, we report on the successful isolation of the ultrastructure of NDiV for the first time in China and its complete genome sequence. We also present comparisons of the nt and amino acid (aa) sequences of NDiV with those of other viruses and a phylogenetic analysis.During the period from 2009 to 2012, NDiV was isolated from samples of Aedes albopictus, Aedes togoi, C. pipiens quinquefasciatus, Culex tritaeniorhynchus, Anopheles sinensis, Anopheles minimus and Anopheles anthropophagus) and representing three genera  were collected. Of these, 95.2% were C. pipiens quinquefasciatus  or Culex tritaeniorhynchus .During arbovirus surveillance of mosquitoes in the Longgang District of Shenzhen, China, a total of 18,560 mosquitoes belonging to seven species  and residential dwellings  caused cytopathic effects (CPEs) in C6/36 cells and were positive for the putative RNA-dependent RNA polymerase (RdRp) gene of NDiV; however, they did not cause CPEs in baby hamster kidney (BHK-21) cells. The nt and aa distances of the RdRp gene of the isolate SZ11706Z and three other isolates were 0.11 ± 0.06% and 0.20 ± 0.08%, respectively. This suggested that the four isolates should be classified as a single virus. Further analysis of one of these isolates (SZ11706Z) is described below.A total of 220 pools containing all the collected mosquitoes and 4 pools of To observe the morphological characteristics of the SZ11706Z strain, we used ultrathin sections and negative staining for the electron microscopic examination. Viral nucleocapsid-like particles and numerous vesicle-enveloped particles (40–60 nm in diameter without surface projections) were detected in the cytoplasm of infected insect cells at 24 h post-infection Fig. . ObservaThe complete genome sequence of the SZ11706Z strain comprised 20,130 nt, including a 5′-untranslated region (UTR) (1–360 nt) followed by at least six ORFs: ORF1a , ORF1b , ORF2a , ORF2b , ORF3  and ORF4  and a 3′-UTR , excluding the 3′ poly (A) tail. The “slippery sequence” GGAUUUU that is present in NDiV and controls ORF1a/ORF1b −1 ribosomal frame shifting (RFS) was also conserved in the SZ11706Z strain.Analyses of the nt and deduced aa homologies of the six conserved protein domains and ORFs of SZ11706Z and other representative mesoniviruses were performed using BLAST searches. Sequence alignment showed that the SZ11706Z strain shared the greatest degrees of nt and aa similarity with NDiV/02VN178/Vietnam . The superfamily 1 helicase (Hel1) domain that is conserved in all nidoviruses had a higher aa identity than other protein domains, ranging from 79.0 to 99.0%  method, a phylogenetic tree of nidoviruses was constructed based on the conserved protein domains of RdRp. As shown in Fig. avV Fig. .Figure 2Culex vishnui from Vietnam1, C. triaeniorhynchus from Vietnam, C. pipiens quinquefasciatus from Shenzhen, China and Houston, Texas, USA, and A. albopictus from Houston, Texas, USA4. Thus, it is most frequently detected in Culex mosquitoes, particularly C. pipiens quinquefasciatus, indicating that Culex mosquitoes are probably the major host of NDiV in nature. An analysis of 39 CavV isolates from different habitat types along an anthropogenic disturbance gradient showed an increase in virus prevalence from natural to modified habitat types10. The data for CavV isolates support the hypothesis that disturbed habitats, particularly human settlements, support the proliferation of endemic viruses more efficiently than undisturbed primary forest. As in our study, NDiV-positive adult mosquitoes were also collected in or near areas of human habitation in the USA, Indonesia and Vietnam. The increasing trend of NDiV movement from natural to modified habitat types, similar to CavV of the same species, Alphamesonivirus 1, requires further confirmation based on statistical evidence obtained by expanding the types of sites sampled and increasing the number of mosquito samples.NDiV has been successfully isolated from various mosquito species including 5 reported that the replication and growth of DKNV found in Vietnam was supported by the cell lines C6/36, NIAS-AeAI-2 and NIID-CTR, whereas replication was not supported by the vertebrate cell lines Vero, BHK-21, HmLu-1 and CCL-141. Replication of CavV, HanaV, MenoV and NseV found in Cote d’Ivoire was detected in infected C6/36 cells but not in the other vertebrate cell lines tested6. Growth of the NDiV-02VN178 and SZ11706Z strains was detected only in C6/36 cells1, indicating a greater susceptibility of insects than of vertebrates to infection by mesoniviruses. During the period from 2005 to 2001, Nga isolated NDiV from the cerebrospinal fluid of acute encephalitis syndrome patients without JEV infection in the C6/36 cell line11, indicating that NDiV infects vertebrates. Thus, it is necessary to conduct further investigations of the molecular epidemiology of NDiV in patients with acute encephalitis syndrome (AES) of unknown etiology.Kuwata2. Our results also showed that the replication of NDiV isolated in China occurred in the cytoplasm of host cells rather than in cytoplasts. The vacuoles of infected cells in our study appeared similar to those described by Nga2, with the accumulation of viral nucleocapsid-like particles and individual spherical nucleocapsid-like particles with electron-dense interiors containing the viral genetic material observed in the cytoplasm. This indicates that the morphology of nucleocapsid-like particles of NDiV in C6/36 cell cytoplasm from the two areas is identical. Alphamesonivirus 1, as the type species of Alphamesonvirus (the new genus of Mesoniviridae), was proposed to recognize NDiV and CavV. The morphology of NDiV virions from China and Vietnam appeared similar in electron microscopy after negative staining. These virions were of homogeneous spherical shape and appeared to contain very short, spike-like projections. The virions of CavV were typical CoV-like virions with rod-shaped surface projections. However, there were significant differences in the appearance of the spike proteins of the two viruses, and further investigations are required to understand the relationship between the efficiency of viral envelope assembly and spike proteins.Nga identified NDiV as a single-stranded RNA virus based on an analysis of the ultrastructural characteristics of the virus as viewed in ultrathin sections. Furthermore, the replication of NDiV was found to occur mainly in the cytoplasm of host cellsComparison of the aa sequences of the six major ORFs of NDiV-SZ1106Z and NDiV-02VN178 revealed differences that were due predominantly to substitution mutation; neither insertion nor deletion mutations were present. The lowest homology was identified in ORF3b.et al. speculated that ORF3b is involved in the synthesis of mRNA3 and in encoding membrane (M) and envelope (E) proteins, its specific role has not yet been determined7. Further investigations are necessary to elucidate the effects on protein synthesis and structure of the aa sequence differences encoded by the ORF3b of the viruses isolated in the two areas.Although Zirkel pro), RdRp and Hel1 domains, which are conserved in all nidoviruses12. In this study, we found that the homology of NDiV-SZ11706Z and NDiV-02VN178 with CavV was greater than 90.0% based on the aa sequences of 3CLpro, RdRp and Hel1. The phylogenetic positions of the viruses were compared based on the 3CLpro, RdRp and Hel1 domains that are conserved in mesoniviruses and on the sequences of putative proteins 1ab of other representative species of mesoniviruses. Our analysis showed that NDiV-SZ11706Z and NDiV-02VN178 share a common ancestor node and form a separate rooted branch. These results support the classification of NDiV-SZ11706Z as a species of Alphamesonivirus 1.The typing of coronaviridae species is usually based on aa sequence homology with the 3C-like chymotrypsin-like protease  was found in Cote d’Ivoire, new species of Alphamesonivirus including HanaV, MenoV and NseV  were found in succession6. After NDiV was found in the province of Nam Dinh in northern Vietnam, DKNV (Alphamesonivirus-8) was identified in the province of Dak Nong in southern Vietnam5. In this study, we successfully isolated NDiV from a location in Shenzhen, China. This suggests that the new species of Alphamesonivirus may exist in southern China.At present, the genus This study demonstrates that NDiV can be isolated from mosquitoes in China and provides further evidence that the virus is prevalent in mosquito populations around the world. NDiV isolated in China shows a major difference in the ORF3b region compared to NDiV isolated in Vietnam, although no morphological differences between the two isolates were apparent based on observation by electron microscopy. The similarity between the two viruses in morphological and molecular biological characteristics indicates that the molecular genetics of NDiV are conserved and that regional differences are unlikely to have a significant impact. However, further studies involving animal experiments are required to investigate potential differences in the pathogenicity of NDiV.Arbovirus surveillance was conducted in the Longgang District of Shenzhen, China, during the period from August 2009 to September 2011. UV light traps  were used to capture mosquitoes ten days per month. Adult mosquitoes were collected from 38 monitoring sites that represented six habitat types, including residential dwellings, hospitals, schools, construction sites, parks and stadiums. The traps were set up at 07:00 pm, and the mosquitoes were collected at 09:00 am the following morning. The mosquitoes were separated according to species, site and collection date and were then divided into pools of approximately 100 mosquitoes each. The mosquito pools were stored in liquid nitrogen prior to virus isolation.A. albopictus C6/36 cells and BHK-21 cells were used to isolate viruses from 220 pools of 18,560 mosquitoes. Specifically, suspensions of mosquito homogenates were filtered through 0.22-µm Millipore Millex-GS filters  and used to infect C6/36 and BHK-21 cells at 28 °C and 37 °C, respectively, under 5% CO2. CPEs were evaluated every 8 h in three blind passages after incubation for 24 h and observation over the next 7 days. A positive virus isolation from a specimen was defined as one in which 70% CPE was observed at 2 days post-infection.Suspensions of cells showing CPE were processed for RNA extraction with the High Pure Viral RNA Kit  according to the manufacturer’s instructions. A reverse transcription-polymerase chain reaction (RT-PCR) method was used for NDiV-specific gene (RdRp) detection with the Prime Script RT-PCR Kit . The primer sequences were: forward primer PRd1 5′-TCACACAACCGCATGCTACGC-3′ and reverse primer PRd2 5′-GTGGTCACGGCCAGTGGTGT-3′ (targeting a 2394 bp fragment). The polymerase chain reaction (PCR) products were visualized on 2% agarose gels and extracted from the gels using the PCR Purification Combo Kit (Invitrogen). The PCR fragments were sequenced and compared to known RdRp gene sequences of NDiV in the National Center for Biotechnology Information (NCBI) GenBank database by BLASTN.2.The NDiV-infected cells were collected and prepared for electron microscopy. Ultrathin sections and negatively stained material were evaluated by electron microscopy on a Tecnai 12 transmission electron microscope as described previouslyTotal viral RNA was used as a template for cDNA synthesis by RT-PCR using the SuperScript III First-Strand Synthesis System (Invitrogen). PCR was performed with twenty-two pairs of overlapping primers that were designed based on the complete genome sequence of NDiV-02VN178 (GenBank accession number DQ458789) Table . The PCRpro, HEL1, RdRp, the putative spike glycoprotein (S) and putative proteins 1ab with those of other mesoniviruses. An NJ tree was established with 1.000 bootstrap replicates in MEGA 4.1. The flowchart for isolation and identification of NDiV is shown in Figure Pairwise analyses of the newly identified NDiV at the nt and aa levels were performed using DNASTAR software. Alignment and phylogenetic analyses were performed by comparison of the RdRp motif domains with those of other nidoviruses and by comparison of the conserved regions within 3CLThe full genome sequence of strain SZ11706Z was assigned the GenBank accession number KF522691. The RdRp gene sequences of strains SZ10618Z, SZ11706Z, SZ11714Z and SZ11826Z were assigned the GenBank accession numbers JQ996712 to JQ996715, respectively.Supplementary Table S1 and Figure S1.


We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics.An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients.The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration  and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours.Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring. Arenaviridae, Filoviridae, and Flaviviridae families and Bunyavirales order may cause symptoms of febrile disease with hemorrhagic symptoms. We have developed and evaluated a novel method that can potentially identify all viruses and their genomic variants known to cause hemorrhagic fever in humans. The method relies on selected amplification of the target viral nucleic acids and subsequent high throughput sequencing technology for strain identification. Computer-based evaluations have revealed very high sensitivity and specificity, provided that the primer design is kept updated. Laboratory tests using several standard hemorrhagic virus strains and patient specimens have demonstrated excellent suitability of the assay in various sequencing platforms, which can achieve a definitive diagnosis in less than 3.5 hours.Viral hemorrhagic fever is a severe and potentially lethal disease, characterized by fever, malaise, vomiting, mucosal and gastrointestinal bleeding, and hypotension, in which multiple organ systems are affected. Due to modern transportation and global trade, outbreaks of viral hemorrhagic fevers have the potential to spread rapidly and affect a significant number of susceptible individuals. Thus, urgent and robust diagnostics with an identification of the causative virus is crucial. However, this is challenged by the number and diversity of the viruses associated with hemorrhagic fever. Several viruses classified in  Arenaviridae, Filoviridae, and Flaviviridae families and Bunyavirales order [Outbreaks of viral hemorrhagic fever (VHF) occur in many parts of the world ,2. VHFs es order . Human ies order ,3,4. Howes order . Even PCes order ,7.Next Generation Sequencing (NGS) has provided novel options for the identification of viruses, including simultaneous and unbiased screening for different pathogens and multiplexing of various samples in a single sequencing run . FurtherThe human specimens, used for the evaluation of the developed panel were obtained from adults after written informed consent and in full compliance of the local ethics board approval .https://www.ncbi.nlm.nih.gov/genbank/) were assembled into groups of >90% nucleotide sequence identity via the Geneious software (version 9.1.3) [https://ampliseq.com/browse.action) which provides a custom multiplex primer pool design for NGS . For initial evaluation of the approach and as internal controls, human-pathogenic viruses belonging in identical and/or distinct families/genera but not associated with hemorrhagic fever or febrile disease were included in the design  . The cone design .in silico for specific binding to the target virus strains, including all known genotypes and genetic variants. The primer sets were aligned to their specific target reference sequences and relative primer orientation, amplicon size and overlap, and total mismatches for each primer were evaluated using the Geneious software [https://blast.ncbi.nlm.nih.gov/Blast.cgi) [The designed primers were tested software . Pairs tast.cgi) . The senast.cgi) .0–103 for each virus were prepared and mixed with 10 ng of human genetic material recovered from HeLa cells.The performance of the novel panel for the detection of major VHF agents was evaluated via selected virus strains. For this purpose, nucleic acids from Yellow fever virus (YFV) strain 17D, Rift Valley fever virus (RVFV) strain MP-12, Crimean-Congo hemorrhagic fever virus (CCHFV) strain UCCR4401, Zaire Ebola virus (EBOV) strain Makona-G367, Chikungunya virus (CHIKV) strain LR2006-OPY1 and Junin mammarenavirus (JUNV) strain P3766 were extracted with the QIAamp Viral RNA Mini Kit  with subsequent cDNA synthesis according to the SuperScript IV Reverse Transcriptase protocol (Thermo Fisher Scientific). Genome concentration of all strains was determined by specific quantitative real-time PCRs using plasmid-derived virus standards, as described previously (protocols are available upon request). Genome equivalents (ge) of 10In order to compare the efficiency of amplification with the novel panel versus direct NGS, all virus cDNAs were further subjected to second strand cDNA synthesis using the NEBNext RNA Second Strand Synthesis Module  according to the manufacturer’s instructions. Reagent-only mixes and HeLa cell extracts were employed as negative controls in the experiments.The performance of the panel was further tested on clinical specimens from individuals with a clinical and laboratory diagnosis of VHF . For thi2, and 10 U Platinum Taq polymerase (Invitrogen) with added water to a final volume of 25 μl. Cycling conditions were 94°C for 7 minutes, 45 amplification cycles at 94°C for 20 seconds, 60°C for 1 minute, and 72°C for 20 seconds, and a final extension step for 6 minutes (at 72°C). Thermal cycling was performed in an Eppendorf Mastercycler Pro  with a total runtime of 90 minutes.The specimens were amplified using the custom primer panels designed for HFVs with the following PCR conditions for each pool: 2 μl of viral cDNA mixed with human genetic material, 5 μl of primer pool, 0.5 mM dNTP , 5 μl of 10 x Platinum Taq buffer, 4 mM MgClThe amplicons obtained from the virus strains were subjected to the Ion Torrent Personal Genome Machine (PGM) System for NGS analysis (Thermo Fisher Scientific Inc.). Initially, the specimens were purified with an equal volume of Agencourt AMPure XP Reagent . PGM libraries were prepared according to the Ion Xpress Plus gDNA Fragment Library Kit, using the “Amplicon Libraries without Fragmentation” protocol (Thermo Fisher Scientific). For direct NGS, specimens were fragmented with the Ion Shear Plus Reagents Kit (Thermo Fisher Scientific) with a reaction time of 8 minutes. Subsequently, libraries were prepared using the Ion Xpress Plus gDNA Fragment Library Preparation kit and associated protocol (Thermo Fisher Scientific). All libraries were quality checked using the Agilent Bioanalyzer , quantitated with the Ion Library Quantitation Kit (Thermo Fisher Scientific), and pooled equimolarly. Enriched, template-positive Ion PGM Hi-Q Ion Sphere Particles were prepared using the Ion PGM Hi-Q Template protocol with the Ion PGM Hi-Q OT2 400 Kit (Thermo Fisher Scientific). Sequencing was performed with the Ion PGM Hi-Q Sequencing protocol, using a 318 chip.Amplicons obtained from CCHFV-infected individuals and controls were processed for nanopore sequencing via MinION . The libraries were prepared using the ligation sequencing kit 1D, SQK-LSK108, R9.4 (Oxford Nanopore Technologies). Subsequently, the libraries were loaded on Oxford Nanopore MinION SpotON Flow Cells Mk I, R9.4 (Oxford Nanopore Technologies) using the library loading beads and run until initial viral reads were detected.The sequences generated by PGM sequencing were trimmed to remove adaptors from each end using Trimmomatic , and reaThe AmpliSeq design for the custom multiplex primer panel resulted in two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. All amplicons were designed to be within a range of 125–375 base pairs. Melting temperature values of the primers ranged from 55.3°C to 65.0°C. No amplicons <1,000 base pairs with primer pairs in relative orientation and distance to each other could be identified, leading to an overall specificity of 100% for all virus species. The primer sequences in the panels are provided in The overall sensitivity of the panel reached 97.9%, with the primer pairs targeting 6,007 out of 6,130 genetic variants  . Impaire3 ge of the target strain, the ratio of viral reads to unspecific background increased from 1×10−3 to 0.25 (CCHFV), 3×10−5 to 0.34 (RVFV), 1×10−4 to 0.27 (EBOV), and 2×10−5 to 0.64 (CHIKV) with fold-changes of 247, 10,297, 1,633, and 25,398, respectively. In direct NGS, no viral reads could be detected for CCHFV and CHIKV genomic concentrations lower than 103, and this approach failed to identify YFV and JUNV regardless of the initial virus count. In targeted NGS, the limit of detection was noted as 100 ge for YFV, CCHFV, RVFV, EBOV, and CHIKV and 101 ge for JUNV. For the viruses detectable via direct NGS, amplification provided significant increases in specific viral reads over total reads ratios, from 10−4 to 0.19 , 2×10−5 to 0.19 , and 3×10−4 to 0.56 . The average duration of the workflow of direct and targeted NGS via PGM was 19 and 20.5 hours, respectively.Amplification of viral targets with the multiplex PCR panel prior to NGS resulted in a significant increase of viral read numbers compared to direct NGS Figs . In specIn all patient sera evaluated via nanopore sequencing following amplification, the causative agent could be detected after 1 to 9 minutes of the NGS run . The chain silico. The sensitivity of the primer panel was impaired by virus sequences not included in the original design, as noted for Hantaan virus in this study. As 36 out of a total of 59 isolates have been published after panel design was completed, these genetic variants of Hantaan virus could not be detected with a comparable sensitivity or not at all with the current panel. This indicates that the panel has to be adapted to newly-available sequences in public databases. We have evaluated how the panel could be updated to accommodate these recently-added sequences and observed that two additional primer pairs could sufficiently cover all divergent entries. Although the approach for the panel design as well as the actual design with the AmpliSeq pipeline was successful for all genetic variants included, the amplification of viral sequences significantly diverging from the panel could not be guaranteed, which may also apply for novel viruses. Unlike other pathogenic microorganisms, viruses can be highly variable in their genome. Only rarely do they share genes among all viruses or virus species that could be targeted as a virus-generic marker by amplification. Our strategy for primer design and the AmpliSeq pipeline do not permit the generation of degenerated primers or the targeting of very specific consensus sequences. However, the design of the primer panel is relatively flexible, and additional primer pairs can be appended in response to recently published virus genomes. Moreover, an updated panel will also encompass non-viral pathogens relevant for differential diagnosis, and syndrome-specific panels targeting only VHF agents or virally induced febrile diseases such as West Nile fever and Chikungunya can be developed.In this study, we report the development and evaluation of an ultrahigh-multiplex PCR for the enrichment of viral targets before NGS, which aims to provide a robust molecular diagnosis in VHFs. The panel was observed to be highly specific and sensitive and to have the capacity to detect over 97% of all known genetic variants of the targeted 46 viral species We have further tested the panel using quantitated nucleic acids of six well-characterized viruses responsible for VHF or severe febrile disease, with a background of human genetic material to simulate specimens likely to be submitted for diagnosis, using the semiconductor PGM sequencing platform. The impact of amplification was evaluated with a comparison of direct and amplicon-based NGS runs. Overall, targeted amplification prior to NGS ensured viral read detection in specimens with the lowest virus concentration 1 ge) in five of the six viruses evaluated and 10 ge in the remaining strain, which is within the range of the established real-time PCR assays. Furthermore, this approach enabled significant increases in specific viral reads over background in all of the viruses, with varying fold changes in different strains and concentrations Figs  and 3. T ge in fiFinally, we evaluated the VHF panel by using serum specimens obtained during the acute phase of CCHFV-induced disease and employed an alternate NGS platform based on nanopore sequencing. This approach enabled virus detection and characterization within 10 minutes of the NGS run and can be completed in less than 3.5 hours in total . The impAlthough we have demonstrated in this study that targeted amplification and NGS-based characterization of VHF and febrile disease agents is an applicable strategy for diagnosis and surveillance, there are also limitations of this approach. In addition to the requirement of primer sequence updates, the majority of the workflow requires non-standard equipment and well-trained personnel, usually out of reach for the majority of laboratories in underprivileged geographical regions mainly affected by these diseases. However, NGS technologies are becoming widely available with reduced total costs and can be swiftly transported and set up in temporary facilities in field conditions ,27. DuriIn conclusion, virus enrichment via targeted amplification followed by NGS is an applicable method for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


The common assumption in potato virus epidemiology is that all daughter tubers produced by plants coming from infected mother tubers (secondary infection) will become infected via systemic translocation of the virus during growth. We hypothesize that depending on the prevalent environmental conditions, only a portion of the daughter tubers of a plant that is secondarily infected by viruses may become infected. To test this hypothesis experimental data from standardized field experiments were produced in three contrasting environments at 112, 3280, and 4000 m a.s.l. in Peru during two growing seasons. In these experiments, the percentage of infected daughter tubers produced by seed tubers that were infected with either potato potexvirus X (PVX), potato Andean mottle comovirus (APMoV), potato potyvirus Y (PVY) (jointly infected with PVX) or potato leafroll luteovirus (PLRV) was determined. Incomplete autoinfection was found in all cases, as the percentage of virus infected daughter tubers harvested from secondarily infected plants was invariably less than 100%, with the lowest percentage of infection being 30%. Changing the growing site to higher altitudes decreased autoinfection for all viruses. Therefore, the assumption of complete autoinfection of secondarily infected plants were rejected, while the hypothesis of environmentally dependent incomplete autoinfection was accepted. The findings help explain the occurrence of traditional seed management practices in the Andes and may help to develop locally adapted seed systems in environments of the world that have no steady access to healthy seed tubers coming from a formally certified seed system. The results obtained almost three decades ago are discussed in light of most recent knowledge on epigenetic regulation of host plant – virus interactions which allow for speculating about the underlying biological principles of the incomplete autoinfection. A research roadmap is proposed for achieving explicit experimental proof for the epigenetic regulation of incomplete autoinfection in the pathosystem under study. Solanum tuberosum L.) seed tubers used for planting can harbor latent pathogens that subsequently reduce emergence, plant vigor, crop quality and/or yield. Since infected tuber seed is an important source from which these pathogens spread, the proportion of such tubers in a tuber seed lot can accumulate with each consecutive generation, resulting in degeneration of seed quality , potato Andean mottle comovirus (APMoV)) or vectored by aphids [potato potyvirus Y (PVY) and potato leafroll luteovirus (PLRV)] . In thesA potato virus disseminates by infecting during the current growing season a healthy plant subsequently producing infected daughter tubers which disseminate the virus further if they are used as mother tuber, i.e., seed tuber for the plantings of the next season. The terms of primary and secondary infection are commonly used in potato virus epidemiology for the two infection types since many years  or tobacco rattle tobravirus (TRV) was reported some 40 years ago . One plot per treatment was planted at each location in 1987, and the experiment was repeated in 1988 with a minor modification in the treatments described below.Virus-infected tubers were planted in 1987 in three locations representing different agroecological potato growing zones in Peru: Imperial , Santa Ana  and Chicche  (Solanum tuberosum ssp. tuberosum ×S. tuberosum ssp. andigena) was chosen because it was widely grown in all zones and susceptible to each of the four viruses used in our study  6–10 weeks after planting, by checking whether secondarily infected plants were visible or not and whether the within field position of such plants was confirmed. Daily minimum and maximum temperatures were measured 1–2 m above ground and missing data were treated as described by Seed tubers (mother tubers), confirmed to be infected according to the testing described below and consequently later giving rise to secondarily infected plants, were planted jointly with healthy seed tubers at the above mentioned research sites in plots with 10 rows, each of 10 m length with 1 m between-row spacing and 0.33 m between-plant spacing, giving a total of 300 plants per plot. Separate plots were planted for each virus species. The infected and the healthy seed tubers were uniformly distributed in each plot of 300 plants , i.e., hPhthorimaea operculella) and other storage insects with Sevin®(carbaryl dust) as appropriate. From the four tubers harvested per plant and stored as mentioned above, three tubers were tested with ELISA to determine virus presence as described below.At harvest of the experimental plots, four randomly selected daughter tubers from each secondarily infected plant were collected. These tubers were stored before testing for virus infection (procedure see below) in paper bags  placed in wooden trays for 1–2 months after harvest at ambient temperature. Bags were sheltered from wind and rainfall, and protected from tuber moth  for ELISA (1:5 w/v) , 1986. E3) or Bromoethane (200 ml/m3) treatment  and site. The percentage of infected daughter tubers was calculated by summing up for each of the four virus classes studied the frequencies of tubers that were determined to be virus infected or uninfected in one particular season and site. Frequencies were then compared using Fisher’s exact test for 2×2 tables. This test enables comparison of low and high frequencies . For eacFigure 1). Also PVX, APMoV, and PVY (together with PVX) were detected in over 80% of tuber infections at 112 m. The lowest percentage of infection (30%) was observed for APMoV at 4000 m in 1988 and 1989 .Incomplete autoinfection was found in all cases, as the incidence of virus infected tubers harvested from secondarily infected plants was invariably less than 100%. The phenomenon was highly variable, depending on virus and location, but with a tendency to have more autoinfection at the lowest altitude. The highest value of autoinfection was obtained with PLRV at 112 m (88%) in 1987 although this was not significantly different from the PLRV percentage observed in 1988 (83%) (Table 2). In contrast, PVX symptom expression increased at higher elevation. The highest percentage of secondarily infected plants with symptoms was observed for PLRV at 112 m (99%), while the lowest was observed for PVX also at 112 m (0%). This seems surprising at first instance, since a high systemic infection of tubers suggests high virus titres which – even if only assumingly – might relate to high symptom expression. One explanation for this unexpected result could be that genetic processes governing altered virus replication and translocation as components of ‘resistance’ sensuThe effect of the changing environment at the experimental sites was evident for all viruses. The extent of decreasing tuber infection while increasing elevation varied across sites and seasons depending on the virus. For PVX, there were significant differences between all growing sites, while the tuber infection level was in all seasons the same at one particular site. For APMoV, however, there were also significant differences in tuber infection between seasons at one particular site (at 112 and 3280 m) in addition to the differences across sites. Sharp decreases occurred for PVY and PLRV at elevations between 112 and 3280 m, with 27 and 50% average reduction in tuber infections, respectively. Infection did not decrease any further at higher elevations (4000 m). The pattern of symptom expression was correlated to the percentage of tuber infection for PVY, PLRV, and APMoV and was inversely related to the elevation of the potato production plots  did not provide evidence for infected tubers that were not detected by the ELISA-testing. There was 99% compliance between the proportion of daughter tubers that were infected with PLRV at 112 m in 1987 (88%) with the proportion of plants with PLRV symptoms in the respective grow-out test. The results of the grow-out tests and the other precautions taken to ensure accurate results  assured reliability of ELISA-results, avoiding false negatives and assuring that daughter tuber considered to be healthy were indeed healthy and that the calculated autoinfection rates are trustworthy.Several aspects of current knowledge were reviewed to assess their relevance to reduced autoinfection of daughter tubers from secondarily infected mother plants. These are:–Host plant genetics;–Mature plant resistance;–Environmental factors;–Anti-viral gene silencing and epigenetic effects.Genetic factors of the host plant were shown to be involved in autoinfection with plant viruses for other pathosystems with autoinfection being an important component of host plant resistance . Potato This kind of resistance explains partial tuber infection in primarily infected plants. Table 1; Figure 1).However, both mechanisms of limiting tuber infection can be excluded for secondarily infected plants: First, the period during which viruses can multiply, translocate and invade daughter tubers is not limited in secondarily infected plants, but rather lasts the entire seasons from planting to harvest. Thus, time is not a limiting factor for virus particles to invade tuber tissue. Second, if there were a meristem exclusion mechanism against virus invasion, more developmental time should result in less exclusion while the contrary was observed: at high altitudes, daughter tuber infection was lower than at low altitudes, while there was in fact 3–7 weeks more growth time available at high altitudes for the virus to multiply and translocate systemically , providing a range of contrasting environments for studying plant–virus interactions. Temperature has a major and direct impact on virus behavior and the interactions of potato viruses with their host plant or insect vector. Primary infection with PVX or with PVYN or PVYO is altered by temperature (N and PVYO (Carlaviridae), PVX, PVY and PLRV infection (Potyviridae) . Consequently, it can be hypothesized that temperature is a major factor in affecting viral replication and translocation and this may explain why temperature also affected autoinfection in our study.The diurnal temperature variations between the different experimental sites of this study were extremely different  . All theviridae) . Howeverviridae)  has beenTable 2), no conclusive pattern arises. Virus-induced leaf lesions, such as a necrosis, are associated with a high respiratory rate of plant cells. Substances involved in respiratory processes accumulate particularly in cells around the necrotic lesions (Table 2). Therefore, the scientific data, available so far, on effects of irradiance or dioxygen concentration, are not consistent with the relationship found in this study between the experimental site  and virus symptom expression or autoinfection.Low light intensity (irradiance) was shown to significantly increase the severity of mosaic disease caused by potato virus X and potato virus Y . This wa lesions . Only 60At the time, when the materials presented in this paper were produced, biological theory excluded an active reaction of the plant against the invasion of a virus. However, nowadays, there is scientific evidence for RNA-silencing, a plant mechanism that limits the accumulation and spread of viruses after an infection, and for RNA silencing suppression by plant pathogens: defense, counter-defense, and counter-counter-defense . It has Figures 1 and 2).Experimental proof is still lacking that the incomplete autoinfection with potato viruses found in this study is due to temperature-driven epigenetic effects. However, the above-mentioned recent findings in understanding gene-silencing phenomena encourage studying whether these may contribute to observed partial autoinfection with potato viruses reported in our study. In this respect, we consider it promising that a model for temperature-modulated autoinfection  was accuIn traditional seed systems in the Andes, there is no steady supply of healthy seed delivered by a formal seed production system, as there is in industrialized countries. However, partly because of incomplete autoinfection, as shown below, there is in epidemiological terms under certain growth conditions no ultimate necessity of such a supply for preventing low yields because of potato virus epidemics. Assuming random selection of seed tubers from a harvested tuber lot, as well as ambient circumstances where autoinfection is lower than 100% and a low within-season infection rate occurs , e.g., due to low density or minimal prevalence of vectors, the incidence of virus-infected plants will approach zero in consecutive planting generations. This phenomenon may be offset by non-random selection of seed  and/or by high primary infection rates. The interplay of degenerative and regenerative factors could explain why virus incidence in farmers’ seed used in the Andes is often found to be quite low despite the lack of a seed system providing virus-free seed .The effect of the three growing sites with contrasting altitudes on autoinfection efficiency in our study also implicitly explains the benefits gained by moving seed from sites at lower to higher elevations, a practice commonly applied by farmers in traditional Andean potato systems . It appeCrop management practices in farmers’ fields differ across agro-ecological zones of Peru represented in this study. For instance, farmers in these different zones may use different planting densities, plant nutrition and other parameters that influence crop performance, affecting tuber size, tuber numbers per plant and other variables. These management differences may also alter plant metabolism, which in turn may impact virus-plant interactions. To control for these potential crop management effects on autoinfection rates, crop management practices were standardized  in all experimental fields of this study.As noted above, farmers in the Andes have traditionally used altitude to manage and even reverse seed degeneration, but there are additional management options. For example, seed tuber management and crop management (mainly related to managing the maturity resistance), and early harvesting can reduce the level of autoinfection . AdditioApplying positive selection might regenerate a degenerated seed stock  if consistently and effectively applied across several generations. This is possible if clean seed or almost clean seed is still present in the seed lot that is planted in reasonably high proportions and if plants from those healthy seeds are effectively selected by the positive selection method. However, if this practice is combined with an environment-induced reduced efficiency of autoinfection as described in this paper, the sanitation effect of such practices should be significant and most of all agronomically most relevant. Although little literature data is available on long-term effects of positive selection, some research projects are currently ongoing in Uganda and elsewhere in Africa to assess to what extent regeneration is feasible and to what extent techniques such as positive selection can stop or delay autoinfection under severe or mild aphid pressure, starting with high-quality or low-quality seed lots e.g., . In viewThe commonly held theory in potato virus epidemiology that all daughter tubers of a seed-tuber infected mother plant (secondary infection) are systemically infected, is rejected by this study. The hypothesis that potato tuber infection by viruses  may be limited or even avoided by a mechanism preventing daughter tubers of an infected mother tuber to become infected, is clearly supported by the presented field data.This seemingly simple theory has great practical consequences, since costly, formal seed potato systems in industrialized countries in temperate climates are based on this assumption, supported by evidence under these climates. However, these formal systems have also been applied  in low-income countries with different climatic conditions and traditional, informal seed systems.Based on an extensive literature review, it can be concluded that reduced autoinfection is seemingly environment-dependent and most likely temperature-driven. Recent findings regarding the epigenetic regulation of plant virus – host interactions suggest that temperature-driven anti-viral gene silencing may explain the described phenomenon observed in Peruvian potato fields. However, this remains yet to be experimentally proven for the specific potato virus pathosystems addressed in this paper.While the traditional epidemiological theory of potato viruses must be reconsidered in the light of these findings, the latter open up new perspectives for improving food security by contributing to environmentally adapted effective and sustainable seed potato systems and also for breeding purposes. These results should be of particular benefit in potato growing zones where a continuous supply of healthy seed produced by a formal certification has, to date, been unsuccessful. Also, the combination of the findings with those developed in other studies for improving seed systems, e.g., implementing on-farm practices such as positive selection, will contribute to the design of a farmer-friendly package of management practices that further improve the productivity of the crop.The discovered environmental dependency of incomplete autoinfection is also of great importance in view of the challenges experienced by climate change. A change in plant virus epidemics may be forecasted for global potato growing zones, which could inform policy designed to mitigate potentially negative effects of climate change.Explicit experimental proof of the hypothesized temperature-driven RNA-silencing in the addressed potato virus pathosystems would facilitate exploitation of the principle of incomplete autoinfection. The genetic base and inheritance of incomplete autoinfection with potato viruses should be characterized, as well as its epigenetic dimension. Quantifying temperature-driven regulation of the observed phenomenon would facilitate efforts to improve seed quality and food security under varying environmental conditions.Conceived and designed the experiments: LBe, CG, EK, and US. Performed the experiments and analyzed data: LBe. Discussed and validated the results in view of new research findings and scientific evidence and wrote the paper: LBe, LBü, BDuf, BDup, GF, US, and PS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.