Callithrix jacchus) housed at the Wisconsin National Primate Research Center (WNPRC) were diagnosed postmortem with lymphocytic enterocolitis. We used unbiased deep-sequencing to screen the blood of deceased enterocolitis-positive marmosets for viruses. In five out of eight common marmosets with lymphocytic enterocolitis, we discovered a novel pegivirus not present in ten matched, clinically normal controls. The novel virus, which we named Southwest bike trail virus (SOBV), is most closely related (68% nucleotide identity) to a strain of simian pegivirus A isolated from a three-striped night monkey (Aotus trivirgatus). We screened 146 living WNPRC common marmosets for SOBV, finding an overall prevalence of 34% (50/146). Over four years, 85 of these 146 animals died or were euthanized. Histological examination revealed 27 SOBV-positive marmosets from this cohort had lymphocytic enterocolitis, compared to 42 SOBV-negative marmosets, indicating no association between SOBV and disease in this cohort (p = 0.0798). We also detected SOBV in two of 33 (6%) clinically normal marmosets screened during transfer from the New England Primate Research Center, suggesting SOBV could be exerting confounding influences on comparisons of common marmoset studies from multiple colonies.From 2010 to 2015, 73 common marmosets ( Callithrix jacchus) are a valuable model species due to their small body size, communal monogamous familial behavior, birth of hematopoietic chimeric litters, short parturition intervals, and status as members of a non-endangered primate species ), AF023425 (simian pegivirus A isolated from three-striped night monkeys [Aotus trivirgatus]), AF023424 (simian pegivirus A isolated from mustached tamarins [S. mystax]), NC_001837 (simian pegivirus A isolated from white-lipped tamarins [S. labiatus]), KC796075 (bat pegivirus PDB737B), KC796075 (bat pegivirus PDB106), KC796082 (bat pegivirus PDB24), U63715 (human pegivirus genotype 1), D87713 (human pegivirus genotype 3), AB021287 (human pegivirus genotype 4), AY949771 (human pegivirus genotype 5), AB003292 (human pegivirus genotype 6), and AF070476 (simian pegivirus A isolated from common chimpanzees [Pan troglodytes]).Sequence data were analyzed using CLC Genomics Workbench 5.5 . Low-quality reads (Phred < Q30) and short reads (<100 bp) were removed with CLC Genomics Workbench 7.1 , and the remaining reads were assembled de novo using the MEGAHIT assembler. Assembled contiguous sequences (contigs) and singleton reads were queried against the GenBank nucleotide database using the basic local alignment search tools blastn. Nucleotide sequences were codon aligned individually for all known pegiviruses with complete genomes using ClustalW2 in the alignment editor program in MEGA6.06 and edited manually. The best-fitting distance model of nucleotide substitution for each alignment was inferred using the maximum likelihood (ML) method with goodness of fit measured by the Bayesian information criterion in MEGA6.06. The best-fitting nucleotide substitution model for the phylogenetic alignments was inferred to be the GTR model with discrete gamma and invariant among-site rate variation. The GenBank accession numbers of the sequences used are MT513216 (Southwest bike trail virus variant 1 [SOBV-1]), MT513217 (Southwest bike trail virus variant 2 [SOBV-2]), MK059751 (dolphin pegivirus), KU351669 (porcine pegivirus), KC815311 (rodent pegivirus), KC796088 (bat pegivirus I), KT439329 (human hepegivirus), KC796076 (bat pegivirus G), KC796080 (bat pegivirus F), KC410872 (equine pegivirus), KC145265 (Theiler’s disease-associated virus), U44402 (human pegivirus genotype 2), GU566734 (GB virus-D), U22303 (simian pegivirus A isolated from black-mantled tamarins [http://pfam.xfam.org/). The amino acid similarity of the novel pegivirus with related pegivirus lineages was determined across the polyprotein using SimPlot v3.5.1 [Eidolon helvum]).Protein family analysis and putative protein predictions were performed using Pfam  using representative members of each pegivirus species ,81.Plasma samples from 136 healthy WNPRC common marmosets were screened specifically for SOBV by RT-PCR. Twenty plasma samples collected from NEPRC animals were likewise screened by RT-PCR.Screening of these animals was performed with samples from animals positive for SOBV by deep sequencing as positive controls. RNA was isolated from 100–500 μL of plasma using the QIAamp Viral RNA Mini Kit (Qiagen). A primer set  targeting a region of the viral helicase which was conserved among the animals initially positive by deep-sequencing was designed, resulting in a 615-bp amplicon. Viral RNA was reverse-transcribed and amplified using the SuperScript III High Fidelity One-Step RT-PCR kit . The reverse transcription-PCR conditions were as follows: 50 °C for 30 min; 94 °C for 2 min; 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 68 °C for 1 min; and 68 °C for 5 min. Following PCR, amplicons were purified from excised gel slices (1% agarose) using the Qiagen MinElute Gel Extraction kit (Qiagen). Each amplicon was quantified using Quant-IT HS reagents (Invitrogen), and approximately 1 ng of each was used in a tagmentation reaction with the Nextera XT DNA Library Prep Kit. Final libraries representing each amplicon were characterized for average length using a DNA high sensitivity chip on a 2100 bioanalyzer  and quantitated with Quant-IT HS reagents. Libraries were sequenced on a MiSeq.All animals humanely euthanized or that die spontaneously at the WNPRC undergo complete postmortem examinations (necropsy) with the collection of a standardized set of tissues for histologic evaluation and ancillary diagnostics (if necessary). Lymphocytic enterocolitis is well-characterized in common marmosets  and a map-values are two-sided and p < 0.05 was used to define statistical significance. Statistical analyses were conducted using R version 3.6.3 in RStudio version 1.1.383. We used univariate logistic regression to evaluate the associations of SOBV viremia with enterocolitis risk. Analyses were repeated to determine association with lymphocytic disease in small intestines only, large intestines only, both the small and large intestines, and either the small or large intestines. All reported https://github.com/aheffron/SPgVwnprc_in_marmosets).Metagenomic sequencing data have been deposited in the Sequence Read Archive (SRA) under Bioproject PRJNA613737. Derived data, analysis pipelines, and figures are available for easy replication of these results at a publicly accessible GitHub repository  be formally named the Southwest bike trail virus (SOBV). Pegivirus RNA was not detected in the plasma of the ten clinically normal common marmoset controls.Aotus trivirgatus) [SOBV consists of a 9.8-kb-long contig that is highly similar to the genome of simian pegivirus A (SPgV-A) trivirgatus, a simian pegivirus previously discovered in a three-striped night monkey (irgatus)  . Age at the time of screening was associated with the likelihood of SOBV , with the likelihood of positivity increasing with each additional year of age  clinically normal screened animals tested positive for SOBV RNA. Nineteen of 60 females (31.67%) and 31 of 86 males (36.05%) tested positive at the time of screening. Sex was not associated with the likelihood of SOBV using univariate logistic regression  to the WNPRC. Samples from 33 NEPRC common marmosets were collected while the animals were in quarantine. Two (6%) of these were found to be positive for SOBV RNA when screened by RT-PCR. Given that pegiviruses are known to persist in hosts for years or decades ,39,40,41p = 0.779), colitis in the large intestine (p = 0.196), either a colitis or enteritis (p = 0.820), or an enterocolitis (p = 0.0798), or with lack of any lymphocytic disease (p = 0.904) . SOBV status was likewise not associated with any other pathology  . Sex wasathology .We describe the discovery of a novel simian pegivirus, the Southwest bike trail virus (SOBV), first identified in common marmosets diagnosed with lymphocytic enterocolitis. We show this pegivirus was prevalent in our colony during a period of increased incidence of lymphocytic enterocolitis and that it was less prevalent in a similar, clinically normal colony. The novel virus was not significantly associated with the likelihood of developing lymphocytic enterocolitis, though prevalence of the virus increased with increasing age in the common marmoset. With an average prevalence of 34%, SOBV was common throughout the WNPRC common marmoset colony.Pegivirus , have single-stranded, positive-sense RNA genomes and produce enveloped virions [Pegiviruses, the members of genus  virions . The fir virions ,86, and  virions ,34,35,81 virions ,58,59,60 virions ,103,104  virions ,112,113, virions ,117,118. virions ,65,66,67 virions ,74,75,76 virions  and geno virions . These eAotus trivirgatus) [Eidolon helvum). Given that common marmosets and three-striped night monkeys are native to northern South America, this may indicate a South American bat species harbors a more closely related pegivirus and could have been the source of an interspecies spillover.It is not known whether common marmosets are the natural host for SOBV or whether they acquired this virus from another species in captivity . Other pirgatus) , a speciirgatus) ,131,132,The routes of transmission of SOBV and of other simian pegiviruses have not been examined. HPgV transmission has been extensively studied and is known to occur efficiently through blood products or dialysis ,135,136,The high prevalence of this virus at the WNPRC raises important considerations about potential effects on common marmoset experiments. Facilities working with common marmosets should prescreen the animals to establish the pegivirus status of animals in research to account for potential confounding. Pegiviruses can replicate at high titers in a host for more than a decade ,41,147; This study has several limitations. First, this study was observational in nature, as we did not want to risk infecting more marmosets in our research colony with an apparently transmissible and potentially harmful virus. This study design could not examine a causal link between viral positivity and the development of lymphocytic enterocolitis. Definitive establishment of causation would require demonstrating that animals infected experimentally develop the disease. Second, many animals in this study were concurrently enrolled in other WNPRC studies, and therefore some were euthanized earlier than would have occurred otherwise when those studies reached experimental endpoints. We chose to use this convenience sample as it allowed us to achieve a large study sample size in which to investigate a potential infectious contributor to an important and poorly understood cause of common marmoset mortality without disrupting other ongoing studies at the WNPRC. Third, not all of the animals initially screened were deceased at the time of this analysis, and future necropsies of these animals may contribute additional data concerning the likelihood of enterocolitis development. Finally, some animals in this study may have cleared the virus before the samples we tested were collected. Consequently, these animals could have been mistakenly classified as virus-naïve, and others may have acquired the virus after initial screening. Development of a SOBV-specific ELISA or other serodiagnosis tools would enable deeper appropriate analyses of SOBV infection rates both prospectively and retrospectively.In summary, this work describes the discovery of a novel simian pegivirus and investigates its relationship with a widespread and devastating cause of common marmoset mortality. Our study lays the groundwork for the future development of a nonhuman primate model system using this natural infection as a potential model for studying the mechanisms of these enigmatic viruses and providing a greater understanding of their genus as a whole.We discovered a novel pegivirus, SOBV, in common marmosets with lymphocytic enterocolitis. This novel virus had highly variable prevalence between two different colonies . While SOBV was not found to be associated with pathology in this retrospective analysis, the difference in rates of infection among different colonies may affect the outcomes of studies done at different primate centers, and thus infection rates and prevalence should be monitored. Further investigations should probe the routes of transmission and the biological effects of experimental infection.


Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. The frequent emergence of new viral diseases is mainly due to international trade, climate change, and the ability of viruses for rapid evolution. Disease control is based on two strategies: i) immunization , and ii) prophylaxis to restrain virus dispersion . Disease management relies strongly on a fast and accurate identification of the causal agent. For known viruses, diagnosis consists in assigning a virus infecting a plant sample to a group of viruses sharing common characteristics, which is usually referred to as species. However, the specificity of diagnosis can also reach higher taxonomic levels, as genus or family, or lower levels, as strain or variant. Diagnostic procedures must be optimized for accuracy by detecting the maximum number of members within the group (sensitivity as the true positive rate) and distinguishing them from outgroup viruses (specificity as the true negative rate). This requires information on the genetic relationships within-group and with members of other groups. The influence of the genetic diversity of virus populations in diagnosis and disease management is well documented, but information on how to integrate the genetic diversity in the detection methods is still scarce. Here we review the techniques used for plant virus diagnosis and disease control, including characteristics such as accuracy, detection level, multiplexing, quantification, portability, and designability. The effect of genetic diversity and evolution of plant viruses in the design and performance of some detection and disease control techniques are also discussed. High-throughput or next-generation sequencing provides broad-spectrum and accurate identification of viruses enabling multiplex detection, quantification, and the discovery of new viruses. Likely, this technique will be the future standard in diagnostics as its cost will be dropping and becoming more affordable. Viral diseases are a major threat to sustainable and productive agriculture worldwide, resulting in losses of several billion dollars every year . The higPresently, curing plants once they have been infected by a virus is not feasible, unlike bacteria or fungi that can be treated with antibacterial or antifungal agents, respectively. So, disease management relies on preventing viruses from entering plants, or getting plants resistant to viral infection, using multiple strategies that must be developed specifically for each virus, host, and environment (pathosystem). Specific tools for virus diagnostics and identification are pivotal to set up and evaluate disease management. Here, the current state and progress of procedures used for virus detection are reviewed, discussing important features such as their sensitivity, specificity, versatility, portability, capacity for multiplexing, and virus quantification and designability. This review also includes basic concepts of genetic diversity and evolution of plant viruses and how they must be considered to improve detection. Finally, the main strategies for disease control are described, showing both the more suitable detection methods and how genetic diversity and evolution of virus populations can affect the efficiency and durability of some control strategies. This review follows a pragmatic approach aimed to guide plant pathologists to design and apply more accurate detection procedures for a more efficient management of viral diseases.Viruses have a great potential for high genetic variability due to their rapid replication and generation of large populations. Viruses with RNA genomes, comprising most plant viruses, and viroids have the highest mutation rate of any group of replicons, since RNA polymerases lack a proofreading activity . The mutThe genetic variation produced by mutation and recombination is restricted and structured by the other three evolutionary forces: natural selection, genetic drift, and gene flow. Natural selection is a directional process by which the less fit virus variants will decrease their frequency in the population (negative or purifying selection) as a result of functional restrictions necessary for replication, movement between plant cells, transmission by vectors, and specific interactions between virus and host or virus and vector . PositivIn the last decades, rapid and specific serological  and molecular techniques (molecular hybridization and DNA amplification) for the detection of plant viruses have been developed. ELISA is based on specific binding of viral proteins with antibodies , and molDetection methods based on DNA amplification can be classified into two types: polymerase chain reaction (PCR) and isothermal amplification. PCR makes millions of DNA copies of a specific region of the viral genome that are usually visualized by electrophoresis or by hybridization with fluorescent probes. PCR can use as template genomic DNA, or complementary DNA obtained after reverse transcription (RT) of viral RNA. Amplification occurs in three steps: i) denaturation by heating at 90°C to 95°C to separate the double-stranded DNA (dsDNA) template into single strands; ii) annealing by cooling at 40°C to 60°C to allow the primers (two short DNA sequences of 15–40 nt) to bind the start and end of the target DNA; iii) extension by heating at 70°C to 75°C, in which a thermostable DNA polymerase synthesizes new DNA strands starting from the primers. These steps are repeated for 20 to 40 cycles, so the newly synthesized DNA segments serve as template in next cycles . The PCRGeobacillus stearothermophilus, under isothermal conditions at 60°C to 65°C  Heli to 65°C . Recentl to 65°C .The advent of high-throughput sequencing (HTS) technologies, also known as next-generation sequencing, has led to a revolution in plant virus diagnosis . HTS doewww.geneious.com). Several platforms for second-generation sequencing have been developed by different companies such as Roche 454, Illumina, Solid and Ion Torrent  that are assembled by connecting overlapping sequence reads according to nucleotide identity by informatic analysis, e.g., Geneious package . LAMP exhibits a sensitivity in the order of qPCR and is less affected than PCR by inhibitors , which are often a cause of false negatives. Paradoxically, the high sensitivity of the amplification techniques can be a problem, as contamination of reagents and instruments with amplicons from previous samples and cross-contamination between samples can produce false positives reducing specificity.An important factor affecting the accuracy of the serological and molecular detection methods is the genetic variability within each virus species and the genetic relationships with other virus species. Since these methods are based on specific binding (protein with antibody or nucleic acids with probes or primers), some dissimilar virus variants can fail to react giving false negatives. For example, universal detection of PPV by ELISA with monoclonal antibodies failed for some PPV isolates . On the https://www.ncbi.nlm.nih.gov/). The genetic diversity and structure can be estimated easily with the MEGA X software . For example, detection of PepMV by RT-PCR  requiring a specific binding that can fail to detect some genetic variants. HTS generates hundreds of megabases to gigabases of nucleotide sequence reads in a single run providing a good sensitivity . HoweverDetection and identification of viruses are based on assigning a virus from a plant sample to a group of viruses sharing common characteristics. In most cases, the level of detection is the virus species, but it can also be set for higher taxonomic units such as genus or family, or lower units like strain .Fabavirus  using monoclonal antibodies . Moleculabavirus . This genditions .Comovirinae  were designed based on amino acid motifs of the RNA-dependent RNA polymerase: (T/V)YGDDN(V/L) and TSEG(Y/F)P P .Fabavirus, which can be used for a hypothetical universal detection of viruses within this genus. The PCR products obtained with conserved primers for a genus or family can be further purified and sequenced to identify the viral species or discover new ones  and specificity (some primers can bind to nontarget sequences). Several approaches can be used, such as i) limiting the degenerate sites to the last 9 to 12 nucleotides from the 3′ terminus, which are critical for PCR amplification, whereas some mispairings at the 5′ terminus are allowed; ii) choosing low degeneracy (one- or two-fold) codons, particularly at the 3′ terminus; and iii) using modified nucleotides such as inosine (I) for four-fold degenerate sites that can base-pair with the four normal nucleotides: A, C, G and T. As an illustration, new ones . In plannew ones .Tools to detect small genetic variations are also necessary given the great potential of viruses to generate high genetic and biological variation . Several techniques based on PCR or using PCR products as templates have been used for genotyping: i) Randomly amplified polymorphic DNA (RAPD)-PCR uses a single short primer with an arbitrary nucleotide sequence (8–12 nucleotides) to produce different random segments depending on the target amplified, which are visualized by electrophoresis. This technique does not require knowledge of the target DNA sequence, but its reproducibility is low and has been applied only for few plant viruses ; ii) Reshttps://www.ncbi.nlm.nih.gov/BLAST/), which compares the query sequence with all sequences from databases and find those that are more similar.HTS techniques are the most powerful and versatile since the nucleotide sequences can be used not only to estimate the genetic variation and structure of virus populations but also to identify and ascribe a virus sample to different taxonomic levels or discover new virus species, genera or families , accordiRecombination can produce biological and genomic variants of a virus that can be very similar in one genome region and very divergent in other. Thus, the complete genome or different regions of it should be analyzed for detection and identification of recombinant variants. Recombination can be detected by comparing nucleotide identity and/or phylogenetic relationships along the genome, which can be performed with different procedures implemented in the package RDP 4 . For exaProcedures to detect and identify various viruses or virus strains in a single assay simultaneously reduce time and cost of the analysis see .Multiplex PCR or RT-PCR is the amplification of multiple targets simultaneously in a single PCR by using several primer pairs specific for each target. Development of a multiplex PCR or RT-PCR assay is often complex since primers must comply with several conditions: i) similar melting temperatures (similar length and G-C content) so that all primers can function under the same PCR conditions; ii) compatibility, avoiding cross-binding and competition; iii) similar sensitivity; and iv) flanking genomic regions of different sizes so that the amplicons of each target can be separated and visualized by gel electrophoresis. The last constriction can be avoided by using primers labeled with different color fluorescent dyes or coupling the PCR with hybridization with specific probes . MultiplMicroarray analyses can screen many samples simultaneously. They can be based on serology, but most are based on molecular hybridization . CaptureMicrosphere technology, like Luminex xMAP, can detect up to 500 targets in a single sample. It is based on microspheres (beads) coated with specific antibodies or oligonucleotides, which capture respectively viruses or PCR products obtained from their genetic material. The beads have been dyed into spectrally distinct sets, or “regions,” allowing them to be individually identified. After binding, the target is labeled by specific conjugated antibodies or probes which will give a fluorescent signal. The color code of the bead in combination with the fluorescent signal identifies a unique combination . LuminexHTS is the most powerful technique for multiplex detection as it can identify and discover an unlimited number of viruses and virus variants within a plant .Estimating the amount of a specific virus provides more precise information than just determining the presence or absence of that virus. ELISA and molecular hybridization can be used for rough quantification of viral particles or nucleic acids based on the signal intensity . Real-tiOther important features to be considered in the detection techniques are the costs, throughput screening  and easiness, not only during the application but also during the design or development Table 3.Sample processing is a critical step and affects the rapidity, easiness and throughput of the detection process. Molecular hybridization and PCR techniques require purification of total RNA or DNA from plants to remove substances inhibiting the detection process  or producing a background signal . Inhibition of PCR can be avoided or minimized by diluting the extracts or by immunocapture . HTS alsExtracts obtained by just grinding plant tissue in buffer can be used for ELISA and LAMP. For some viruses, tissue-prints made by cutting leaf petioles or rolled leaf blades transversely and gently pressing the fresh cut onto nitrocellulose membranes have been analyzed directly with ELISA or molecular hybridization . An alteOn-site detection of plant viruses is an interesting feature allowing a prompt response. Presently, several techniques are commercially available . Lateralhttp://bioinfo.ut.ee/primer3-0.4.0/) and Primer Express (Thermofisher) for PCR, or Primer Explorer (https://primerexplorer.jp/e/), LAMP Designer (Optigene) and LAVA  is the most widely-used immunization method. There are two types: i) active resistance driven by resistance proteins, encoded by dominant alleles, which recognize specifically a sequence or conformational pattern of a virus gene  and induces death of the infected cells (hypersensitive response), precluding virus movement to adjacent cells and systemic infection ; and ii)Plant breeders usually aim at complete resistance, in which the virus cannot establish a systemic infection. ELISA and molecular hybridization have been used to test germplasm and cultivars for resistance since a good number of plants can be analyzed simultaneously . When a Agrobacterium-mediated transient co-expression of each resistance gene mutant and the Avr from the challenge virus in Nicotiana benthamiana leaves so the resistance response is observed as a necrotic lesion. This approach has been used to broaden the resistance specificity of the potato gene Rx  interaction with other viruses in mixed infections , ii) posPlant breeders are keen on the host genetic variability, but often they neglect the virus genetic variability, which should be considered when new resistance genes are tested to minimize the possibility of resistance breakdown. Multiplex real-time qPCR is well-suited to evaluate the effect of mixed infections in overcoming resistance. Detection of virus variants with punctual mutations leading to resistance breakdown would be a valuable tool to monitor the dispersion of these variants. However, this has proved to be a difficult task as in most cases resistance breakdown occurs by only one nucleotide substitution (producing one amino acid change). The presence of other neutral substitutions around the critical mutation hinders the design of molecular markers for resistance breakdown. Real-time qPCR with Taqman probes has been developed to detect single nucleotide polymorphisms associated with resistance breakdown for beet necrotic yellow vein virus (BNYVV) and tomato spotted wilt virus (TSWV) ;. HoweveAnother strategy to obtain resistant plants is based on the RNA silencing mechanism. RNA silencing is a regulatory mechanism of gene expression in eukaryotes and a natural antiviral defense mechanism. The host RNA silencing machinery targets dsRNA that arise from replicative viral intermediates or secondary structures in the genomic RNA due to internal complementarity, which are detected by RNases (Dicer-like proteins) and cleaved into small RNA duplexes (siRNA or miRNAs) of 21–24 nucleotides (nt) in length. One of the two strands of the small RNAs is recruited to the RNA-induced silencing complex (RISC) that subsequently cleaves cognate viral RNAs in a sequence-specific manner. These cleaved RNAs are recognized by RNA dependent RNA polymerase (RDR), which amplifies the dsRNA molecules, thus contributing to the amplification of the host defense mechanism that results in effective inhibition of local and systemic viral infection . To counin vitro-produced dsRNAs from viral sequences onto plants  and sanitary certification of virus-free germplasm  are the first measures to prevent the introduction and emergence of new viruses in a geographical area. Virus detection should be based on sensitive and broad-spectrum methods, since discarding healthy material is preferable to the risk of spreading new diseases. On-site detection devices can be useful to make decisions quickly, thus preventing importation and exportation delays. HTS is the most powerful detection procedure since it can detect all the viruses (known and unknown) present in a plant in an unbiased way. Its use in quarantine and clean plant programs is increasing as it is becoming more economically affordable . PhytosaSince epidemiology and evolution are coupled in viruses, phylogeographic studies, comparing genetic variation in space and time, can provide useful information on the introduction sites of new viruses and the dispersal paths . As an iSanitation, that is, removing viruses from valuable cultivars, is necessary when no healthy plants are available. Virus-free plants are usually produced by thermotherapy, chemotherapy, electrotherapy, and tissue culture alone or combined . ThermotTobamovirus, can be reduced by hygienic measures such as using disposable gloves or washing hands with disinfectant, heat sterilization of tools and debris and limiting the access to crops  Reduto crops  or entomto crops ; and bioto crops . ii) Preto crops . iii) Into crops .The rate of insect transmission can be evaluated using different detection techniques, such as ELISA, molecular hybridization and PCR, to detect the virus in the receptor plants and real-time qPCR to estimate the virus titer in the source plants, which affects transmissibility .The main challenge of agriculture in this century is to produce nutritious food for the growing world population in a sustainable manner while protecting the environment and human health . DamagesThe correct identification of viruses is critical for disease management. However, the great ability of viruses to evolve and generate molecular and biological variation is a major difficulty for virus detection and disease management. Presently, when a new virus-like disease appears, the first approach is to test for known viruses with well-established techniques . PCR tecPresently, disease management relies on preventing introduction of new viruses by border control and certification of virus-free propagative material  and preventing virus dispersal in the field. Quarantine and certification require sensitive and broad-spectrum detection methods to minimize escapes such as the use of conserved primers specific for virus genera or families and multiplex procedures , faces hHTS excels for its broad-spectrum and multiplex detection, sensitivity, and precise quantification. No previous knowledge is required, enabling unbiased detection and discovery of new viruses. The sequences obtained allow a precise taxonomic assignation and an estimation of genetic relationships with other viruses and viral isolates. It is reasonable to expect that HTS, especially portable nanopore sequencing devices, will become the standard diagnostic procedure as costs will be dropping and analytical procedures improving.LR conceived the idea. LR, IF, and LG wrote the manuscript. LR and IF designed the figures. All authors contributed to the article and approved the submitted version.This work was partially funded by grants RTA2017-00061-C03-02 from Spanish Ministerio de Ciencia e Innovación co-financed by FEDER and 51912 from IVIA. IF acknowledges financial support from the Spanish Ministerio de Economia y Competitividad, through the “Severo Ochoa Programme for Centers of Excellence in R&D” 2016-2019 (SEV-2015-0533).The opinions expressed, and arguments employed in this paper are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.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.


Poor data quality and use have been identified as key challenges that negatively impact immunization programs in low- and middle-income countries (LMICs). In addition, many LMICs have a shortage of health personnel, and staff available have demanding workloads across several health programs. In order to address these challenges, the Better Immunization Data (BID) Initiative introduced a comprehensive suite of interventions, including an electronic immunization registry aimed at improving the quality, reliability, and use of immunization data in Arusha Region, Tanzania, and Southern Province of Zambia. The objective of this study was to assess the incremental costs of implementing the BID interventions in immunization programs in these two countries.We conducted a micro-costing study to estimate the economic costs of service delivery and logistics for the immunization programs with and without the BID interventions in a sample of health facilities and district program offices in each country. Structured questionnaires were used to interview immunization program staff at baseline and post-intervention to assess annual resource utilization and costs. Cost outcomes were reported as annual cost per facility, cost per district and changes in resource costs due to the BID interventions . Sub-group analyses were conducted by health facility to assess variation in costs by volume served and location . One-way sensitivity analyses were conducted to identify influential parameters. Costs were reported in 2017 US dollars.In Tanzania, the average annual reduction in resource costs was estimated at US$10,236  per health facility, while the average annual reduction in resource costs per district was estimated at $6,542. In Zambia, reductions in resource costs were modest at an estimated annual average of $628  per health facility and $236 per district. Resource cost reductions were mainly attributable to reductions in time required for immunization service delivery and reporting. One-way sensitivity analyses identified key cost drivers, all related to reductions in health worker time.The introduction of electronic immunization registries and stock management systems through the BID Initiative was estimated to result in potential time savings in both countries. Health worker time was the area most impacted by the interventions, suggesting that time savings gained could be utilized for patient care. Information generated through this work provides evidence to inform stakeholder decision-making for scale-up of the BID interventions in Tanzania and Zambia and to inform other Low-to-Middle-Income Countries (LMICs) interested in similar interventions. Immunization has proved to be the most cost-effective public health intervention through reducing childhood mortality and morbidity attributable to vaccine-preventable diseases. Despite immunization being such an effective public health tool, not all children are being reached with the lifesaving vaccines they need . One keySeveral factors have been identified as inhibiting immunization program performance improvement, including the poor quality of data and the poor use of existing data , 4–7. DaGiven these challenges with immunization program data, there is a global effort to strengthen country immunization systems by supporting the collection of better-quality data and better use of these data to inform program decision-making. One such effort is through the Better Immunization Data (BID) Initiative , led by Very few studies have evaluated the costs of interventions aimed at improving data quality and use in other countries that have implemented similar interventions. Hence, we sought to provide some evidence on these costs using data from Tanzania and Zambia. Our objective was to estimate the economic costs of immunization program logistics and service delivery before and after the implementation of the BID interventions, and use these data to estimate the incremental costs or savings attributable to the interventions. The findings from this study are intended to inform the scale-up of such interventions within the two countries and across other countries in the region.We conducted a micro-costing study  to estimWe developed primary data collection tools to identify resources used for transporting and storing vaccines, staff time for logistics and service delivery, office equipment and communications, and printing and office supplies. Similar questionnaires were used to collect data on the resources used at district level, focusing on activities related to the logistics and management of health facilities. The tools used in the two countries were similar, but adaptations were made to reflect country-specific characteristics of each immunization system.We collected data from a sample of health facilities in each district and a sample of districts in each region/province. We included 4 of the 7 districts in Arusha Region and 6 of the 13 districts in Southern Province. Health facility sample sizes are shown in At the time of post-intervention data collection, the facilities in Tanzania had been using the electronic immunization registry for an average of 8 months (range 4 to 11 months); in Zambia, the average was 3.5 months (range 1 to 8 months). In addition, at the time of post-intervention data collection, health facilities in both countries were still using the paper-based system as back-up because policy decisions had not yet been made to eliminate the paper system and solely rely on the electronic system. Therefore, we asked health workers to assess the change in resource use under a scenario in which only the electronic system was in use.We collected immunization service delivery costs across five main categories: (1) human resources; (2) cold chain equipment; (3) communications, printing and office supplies; (4) facility office equipment and (5) transport. Human resources costs included salaries and per diems for staff working in the immunization program. Staff were asked to self-report the time spent on providing fixed and outreach immunization services, logistics and stock management for the immunization program and data reporting. The costs of cold chain equipment captured the capital costs of refrigerators, freezers, cold boxes, and vaccine carriers used in the immunization program, and the annual costs of electricity or gas to run the cold chain equipment, as relevant. Costs of office equipment and communications included capital costs for computers, tablets, printers, scanners, and other equipment used by the immunization program and communication and printing costs. Finally, transport costs reflected the costs to collect vaccines and immunization supplies from the district and transport them to facilities or to conduct outreach services .For resources shared with other programs, costs were allocated to the immunization program based on the reported percentage spent or use of the immunization program. Capital costs were annualized using different lifespans: 3 years for office equipment; 5 years for vehicles; and 10 years for cold chain equipment. All local cost data were collected in local currencies and converted into 2017 US dollars using average exchange rates for the year . As neceResource use data were combined with unit costs to calculate economic costs at the district and facility levels. All cost estimates  were reported as annual economic costs per facility or district. Our incremental cost estimates relied on the cross-sectional data from the two surveys conducted, one at baseline and one at post-intervention. These surveys were conducted at different time points. However, given that time use was self-reported and subject to recall bias, at post-intervention, along with asking survey respondents to estimate time spent on immunization activities with the BID interventions, we asked them to recall and report how much time they had been spending on these same activities before the implementation of the BID interventions. We used these data to provide an alternative set of estimates for the analysis. In addition, we conducted univariate sensitivity analyses to identify the cost drivers and time use for the interventions.In Zambia, we estimated similar trends as for Tanzania but Zambia’s baseline costs were lower at $5,324 . Post-intervention costs were estimated at $4,695 for Zambia ; therefore, savings attributable to the BID interventions were smaller (we estimated a savings of approximately $628 per facility each year). This represents a 12% reduction in costs per facility with BID compared to baseline. Similar to Tanzania, the largest savings would be achieved through reduction in staff time on immunization activities. We also estimated a reduction in annual transport costs of $14 per health facility, resulting from a reduction in emergency trips to the district vaccine store to collect vaccines and transportation for immunization-related outreach activities.We conducted univariate sensitivity analyses to assess uncertainty and identify influential parameters on changes in resource utilization due to the interventions . InfluenWhen results were stratified by health facility characteristics -rural versus urban and low volume versus high volume- we found that in Tanzania, there was a smaller variation across health facility categories based on location (results not shown in tables). For example, rural facilities were estimated to save an average of $8,144  with the introduction of the interventions, compared to $9,423  savings in urban facilities. The variation was slightly higher based on volume served; we found that low-volume settings were estimated to save approximately $7,683  per facility compared to $9,367  in high-volume facilities. In Zambia, the immunization volume categorized as low or high volume resulted in much larger variation between strata, with savings of $275 [95% CI: $205-$298] in low-volume facilities compared to $2,177  in high-volume facilities. Stratified cost savings were $380 [95% CI: $612 cost savings to $247 increased costs] and $776  in rural and urban facilities, representing 9% and 13% decreases in costs, respectively.The estimated savings in Zambia from the implementation of the interventions were more modest. The new equipment increased district annual costs by $491 and communication costs by $62. Most of the savings were derived from reduced labor time and fewer printing costs for immunization registers, stock ledgers and tally sheets. We estimated that time valued at $789 would be saved per year by staff at each district office. Overall, the rollout of the interventions was estimated to result in annual net savings of $236 for the average district office in Southern Province.This study aimed at estimating the cost implications of introducing electronic immunization registries and stock management systems in Arusha Region, Tanzania, and Southern Province, Zambia. In both countries, we found that electronic systems may result in savings compared to paper immunization and stock registers. Savings were mostly attributable to reduction in health workers’ time spent on immunization activities, such as administrative tasks and reporting. Efficiencies gained due to electronic registration and reporting were, as expected, higher in absolute terms in high-volume facilities compared to low-volume facilities.To our knowledge, this study is the first to estimate the savings to be realized using electronic immunization registries and stock management systems in resource-limited settings. Few studies published in the literature evaluated interventions similar to those introduced through the BID Initiative –24, mostWhile not having a direct financial implication for health ministry budgets, the reduction in health worker time represents an important finding and suggests that human resources could be freed up at health facilities so that staff could spend more time on patient care rather than administrative tasks. Also, given the competing time demands of health workers who work across different programs, the benefits of the time savings could be spread to other programs. At the district level, the time saved from the automatic generation of monthly reports is expected to allow district immunization officers to divert energy to other activities, such as supervision.We found that the estimated savings were much larger for districts and health facilities in Arusha Region, Tanzania, than those in Southern Province, Zambia. While there could be other reasons for these differences, we suspect that the shorter evaluation time in Zambia (between the deployment of the BID initiative interventions and the collection of the post-intervention costing data) may partially explain these findings. In fact, we hypothesize that staff were still adjusting to the new system in Zambia and hence had not gleaned the full benefits of the new system.Our study has several limitations. First, the cost estimates are not representative, as a purposive sampling approach was used and samples were relatively small. To address this limitation, we conducted sensitivity analyses and varied input values over low and high ranges to assess how the cost implications would differ under varying assumptions. Second, the time between rollout of the interventions and post-intervention data collection was short, especially in Zambia. This choice was driven by the project timelines and the broader delays experienced during rollout of the interventions. Therefore, estimates reported here are likely to capture learning costs and thus underestimate savings from the BID interventions. A longer-term evaluation of the BID initiative may be warranted to provide more accurate estimates of its cost implications. Third, all time use data included in this analysis were self-reported and thus may have been under- or overestimated. Also, at the time of post-intervention data collection, countries were continuing to use both their paper-based system and the electronic system, making it difficult to assess system changes. This is because the relevant ministries of health had not yet made the decision to solely rely on the electronic system. As a consequence, we relied on staff’s assessment of what their time use would be if they were using only the electronic system. Fourth, due to the lack of data or complexity in assessing them, the study did not include all benefits that could result from the BID interventions, further underestimating its benefits. For example, potential benefits in terms of a decrease in the number of stockouts and wastage, better forecasting, more timely immunizations and higher coverage, and improved decision-making could not be taken into account because of the shortness of time between deployment and evaluation. In addition, our ability to capture the costs of the immunization program relied on the availability and quality of the data. Finally, in this study we did not include the upfront costs of implementing the BID interventions, such as the system development costs or the costs of rolling out the BID interventions to facilities and districts and the costs of maintaining the system. These costs will be reported in a separate analysis.The introduction of electronic immunization registries and stock management systems through the BID initiative was estimated to be cost saving in Tanzania and Zambia. These savings were primarily due to time efficiencies and associated staff cost savings. Information generated through this work provides evidence for key stakeholders in Tanzania and Zambia to inform decision-making for the scale-up of the BID interventions in these countries and to inform decisions in other countries that may be interested in similar interventions.Poor data quality and low data use are key challenges that negatively impact immunization programs in low- and middle-income countries;Electronic immunization registries (EIRs) together with related data use interventions can be a solution for these challenges.Evidence on the incremental costs of implementing EIRs and data use interventions in immunization programs in Tanzania and Zambia;The study found that the interventions introduced resulted in savings in health worker time.The authors declare no competing interests.


Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by  contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector. Highly pathogenic avian influenza (HPAI) viruses form a continuous threat to the poultry industry and to public health. The Eurasian H5N1 lineage (A/Goose/Guangdong/1/1996) first emerged in poultry in China in 1996 . The EurAnas penelope) exclusively, whereas other species of wild birds were also sampled intensively [Experimental infection experiments demonstrated that various wild duck species can become infected with HPAI H5 viruses and excrete virus, without showing clinical symptoms . Furtherensively . In the ensively , 9, 23. ensively , and virGallus gallus), Pekin ducks (Anas platyrhynchos domesticus), and Eurasian wigeons (Anas penelope) with a H5N8-2014 group A virus, and group B viruses H5N8-2016 and H5N6-2017. We compared pathogenicity, clinical signs and virus shedding of the different host species after infection with these three recent HPAI H5 viruses. In addition, environmental transmission of the H5N8-2016 virus was studied by the exposure of chickens to contaminated water or bedding material. The results of this study will improve risk assessment and enhance our ability to develop control measures to prevent the introduction of HPAI viruses in the poultry sector.The recurrence of outbreaks caused by HPAI H5 viruses in wild birds and poultry underscores the need for more insight into the pathogenicity of these viruses for waterfowl and gallinaceous birds. In this study, we experimentally infected chickens  . The animal procedures were performed conform the guidelines from the European Union directive 2010/63/EU.https://www.gisaid.org): H5N8-2014 , H5N8-2016 , and H5N6-2017 . Virus stocks were generated by two passages in 9- to 11-day-old specific pathogen free (SPF) embryonated chicken eggs. Full genome sequencing identified a few ambiguous nucleotide positions present in both swabs and egg passages, but no mutations due to passaging. The virus stocks were titrated using standard methods to determine the median egg infectious dose (EID50) titers [6 EID50/mL inoculum.The viruses used in this study were derived from the index cases in the poultry sector. The whole genome sequences were generated previously and were deposited to the GISAID Database  titers . The virSix-week-old Specific-Pathogen-Free (SPF) White Leghorn chickens were obtained from MSD Animal Health . Six-week-old Pekin ducks were obtained from a commercial breeding farm, and approximately 6-week-old Eurasian wigeons were obtained from several hobby holdings. Eurasian wigeons are seasonal breeders, and therefore the age of the birds likely varied between 4 and 8 weeks old. All animals used in this study were from both sexes. Pekin ducks and Eurasian wigeons tested negative for antibodies against AI virus in serum using ELISA at the day of arrival, and tested negative for AI virus by RT–PCR in swabs on the day of arrival and after an acclimatization period of 7 days. The experiment was performed in biosafety level 3 (BSL 3) facilities at Wageningen Bioveterinary Research . Peking ducks and Eurasian wigeons infected with the same virus were housed in the same room, separated in two floor pens with solid livestock dividing panels. The chickens participating in the infection experiment were housed in one room separated in three floor pens with solid dividing panels. All pens housed 10 birds participating in this study and 12 birds participating in another study until day 4 after infection. The chickens participating in the environmental transmission experiment were individually housed in cages placed in the experimental room. All rooms were temperature-controlled (18.8–22.6°C) and birds were housed under optimal light conditions and humidity, and feed and water were provided ad libitum. Sawdust and some straw were provided as bedding material in the pens. Individual birds were numbered randomly and allocated to an experimental group. Animal caretakers and laboratory personnel were aware of the group allocation during the experiment (not blinded). Eurasian wigeons and Pekin ducks were treated preventively for coccidiosis by the addition of Baycox (Bayer) in the drinking water for 48 h starting at the day of arrival. After the Baycox treatment, swimming ponds were provided to both Pekin ducks and Eurasian wigeons containing approximately 100 litres of tab water. Chickens were provided with perches and pieces of jute as cage enrichment. In the environmental transmission experiment, feed was spread over the bedding material to stimulate contact with the contaminated bedding. The ARRIVE guidelines for reporting animal research were followed .6 EID50/ml inoculum via both intranasal and intratracheal administration (inoculation dose of 105.3 EID50 per bird). All birds were observed for 10 days for signs of disease and mortality, and a pathogenicity score was calculated using the scoring protocol of the intravenous pathogenicity index (IVPI) [For the infection experiment, 10 birds of each species were inoculated with either H5N8-2014, H5N8-2016 or H5N6-2017 virus (9 groups). Birds were allowed a 7-day acclimatization period before infection with 0.1 mL of 10x (IVPI) . When a For the environmental transmission study, 24 independent cages were placed in an experimental room. Twenty chickens were individually housed in cages. Ten chickens received contaminated bedding material derived from the pen with the H5N8-2016 infected Eurasian wigeons, and 10 chickens received contaminated swimming water as drinking water. These chickens did not receive other sources of water. Bedding and water were not replaced during the experiment. Two cages contained two sentinel chickens as controls, and two cages contained contaminated bedding material and water without chickens to measure virus survival over time. The chickens were monitored for clinical symptoms and mortality for 7 days after exposure. Everyday OP and CL swabs were taken to determine virus shedding, and blood was collected at the last day of the experiment (day 7) to determine antibody production.Serum was collected from the blood samples and stored at −20°C until testing. For influenza virus-specific antibody detection, serum was tested by anti-NP ELISA as described previously . Hemaggl50 titer. A standard curve was used to convert the Ct values into equivalent EID50 titers and determine PCR efficiency. Amounts of virus detected in swabs are reported as mean equivalent log10 EID50/mL titers, with a lower detection limit of 101.7 EID50/ml.Swabs were placed in 2 ml of Tryptose Phosphate Broth (TPB) 2.95% and stored at −70°C until testing. For virus detection, total RNA was extracted from the swabs using the MagNA Pure 96 system . Influenza A virus was detected by a quantitative real-time RT–PCR targeting the matrix gene (M-PCR), as described previously [50 titers, as the amount of viable virus is expected to decrease in time. During the experiment, also 15 ml water samples were taken, which were stored at −70°C. The pH of the water was measured using pH indicator strips . The bedding and water samples were analysed for viable virus by inoculation of 200 μl into four SPF embryonated eggs.The bedding material and the swimming water in the pen housing the H5N8-infected Eurasian wigeons were sampled before collection at day 4 of the infection experiment. Thereafter, the bedding and the water in the two control cages were sampled every day during the environmental transmission experiment (7 days). The bedding was sampled using boot swabs, which were gently placed in containers after 1 min of walking, and were stored at −70°C until analysis. For analysis, 100 ml TPB containing 1% gentamycin was added to the boot swabs, the containers were shaken vigorously after which the suspension was collected. The suspension was filtered, after the final filtering step  a sample of 5 ml was incubated with 1% of penicillin and gentamycin and incubated at room temperature for 1 h. RNA was isolated using MagNA Pure as described above. For sampling of the water, a 1- l sample was collected and filtered directly as described previously . Brieflyp < 0.05.One indicator of pathogenicity of the viruses for each of the bird species studied is the level of and time to mortality of an infected host following infection. Comparisons of this induced mortality in time post infection  were done between the different virus infected groups within the same bird species (e.g. among chicken groups) using the log-rank test. Virus shedding was characterized by calculating the area under the curve (AUC) as a measure of total virus shed via OP or CL by each bird during the course of the infection and by calculating the length (duration in days) of shedding via each of these routes (OP or CL). Comparisons of shedding characteristics were done among the different virus inoculated groups within each bird species (e.g. among chicken groups). Mean levels of shedding (AUC) were compared using an ANOVA test, after assessing for normality (Shapiro test) and similarity of variances (Fligner test). If deviations of normality or similarity of variances were observed, comparisons were done using the Kruskal–Wallis test. Length of shedding was compared by fitting parametric survival regression models with a Weibull distribution (distribution that fitted the data best). Analysis was done using the software package R (version 3.6.1) . The libRenv (the average number of secondary infections caused by a contaminated environment) was calculated using the final size method, where each trial consisted of an environment-susceptible bird and was treated as a one-to-one transmission trial. Maximum likelihood estimation was used to estimate Renv following the model described by [For the analysis of the environmental transmission experiment, the environment reproduction number ribed by  and usinribed by .The pathogenicity of recent HPAI H5 viruses for different avian host species was studied by experimental infections of chickens, Pekin ducks and Eurasian wigeons. The viruses were isolated from three index cases in poultry in the Netherlands, H5N8-2014 (H5 clade 2.3.4.4 group A virus), H5N8-2016 and H5N6-2017 (H5 clade 2.3.4.4 group B viruses). Ten birds of each species were infected by intranasal/tracheal inoculation of the viruses and were monitored for 10 days for symptoms of disease and mortality. All surviving birds tested positive for antibodies against AI virus at the end of the experiment (day 10), demonstrating that these birds became infected and seroconverted, except for one Eurasian wigeon inoculated with H5N8-2016 virus. This bird was found positive by PCR analysis, as discussed below. For Pekin ducks, infection with the H5N8-2014 virus resulted in two deaths, and the estimated pathogenicity score was 0.13. Both ducks were listless at day 8, and one duck developed neurological symptoms at day 9 after which it was euthanized. Infection with the H5N8-2016 virus did not result in clinical signs or mortality in Pekin ducks, hence the pathogenicity score was 0.00. Higher mortality was observed in ducks infected with the H5N6-2017 virus, 6 out of the 10 challenged Pekin ducks died between 4 and 6 days after infection, the estimated pathogenicity score was 1.27. The survival time of Pekin ducks infected with the H5N6-2017 virus was significantly shorter than that of ducks infected with the H5N8 viruses . ClinicaFor the Eurasian wigeons, no mortality was observed after infection with the H5N8-2014 virus, and pathogenicity was limited resulting in a score of 0.01. Diarrhea was observed for one bird at day 9. Infection with the H5N8-2016 virus resulted in two deaths on days 7 and 8 after infection, with no obvious clinical symptoms preceding death. The pathogenicity score was 0.23. The highest pathogenicity score 1.68 was estimated for the H5N6-2017 virus. With this virus nine deaths were observed between days 3 and 6 after infection. The survival of Eurasian wigeons infected with the H5N6-2016 virus was significantly shorter compared to the two H5N8 viruses . Clinica10 EID50/ml) was calculated  and cloacal (CL) swabs were collected every day, and the amount of virus was quantified by real-time RT–PCR. The mean equivalent virus titers measured in the swabs for 10 days after infection are shown in Renv for environmental transmission of HPAI via water was 3.0 : 1.8–4.2) and via bedding was 0.0 (95% CI: 0.0–0.77). These results show that environmental transmission of HPAI virus to chickens via contaminated water is efficient (Renv > 1), whereas transmission via contaminated bedding material is less likely to happen (Renv < 1).Environmental transmission of the HPAI H5N8-2016 virus was studied to obtain more information on the natural route of virus transmission to poultry by indirect contact with infected wild birds. We therefore collected bedding material and swimming water from the pen housing the infected Eurasian wigeons. The materials were collected at day 4 after infection, at the peak of virus shedding from the cloaca . Ten chi5.5 vRNA copies/ml) and water samples (105.7 vRNA copies/ml) collected from the pen housing the Eurasian wigeons. The amount of viral RNA detected in the two control cages declined during the experiment, approximately 1000-fold for the bedding samples at day 5, and 100-fold for the water samples at day 6. To test whether the virus detected by PCR is still infectious, samples of the water and bedding material were inoculated into four embryonated eggs , and subsequently from the two control cages during the 7 days of the environmental transmission experiment. The pH of the water samples was consistently 7.5 during the experiment, and the average temperature in the experimental room was 21.1°C. Bedding and water samples were analysed by real-time RT–PCR for the presence of AI virus, as is shown in ted eggs , columnsIn this study, we characterize three representative HPAI H5 viruses isolated from the index cases in poultry in the Netherlands. The H5H8-2014 virus belongs to H5 clade 2.3.4.4 group A, whereas the H5N8-2016 and H5N6-2017 viruses are classified as group B viruses. The pathogenicity of the three virus strains was directly compared in three avian host species: chickens and Pekin ducks , and Eurasian wigeons (as a model species for wild birds). Consistent with the pathogenicity of other clade 2.3.4.4 H5 viruses, the three viruses caused 100% mortality in experimentally infected chickens, with a median survival time of 2 days. For Pekin ducks, however, differences were observed between the three viruses. Pekin ducks infected with the H5N8-2014 and 2016 viruses showed very limited to no clinical symptoms. Mortality was 20% for the H5N8-2014 virus, and 0% for H5N8-2016 virus, resulting in low pathogenicity scores (0.13 and 0.00 respectively). After infection of Pekin ducks with the H5N6-2017 virus, symptoms of disease were observed starting 3 days after infection, and 60% mortality was observed resulting in a pathogenicity score of 1.27. The survival of Pekin ducks after infection with H5N6-2017 virus was significantly shorter compared to the two earlier H5N8 viruses. Previously performed IVPI studies in 6-week-old Pekin ducks demonstrated lower pathogenicity for the H5N8-2014 virus (IVPI score 1.87) compared to H5N8-2016 and H5N6-2017 viruses (IVPI scores 2.99 and 3.00 respectively) , 32–36. For Eurasian wigeons, infection with the two H5N8 viruses resulted in very limited clinical symptoms. Mortality was 0% for the H5N8-2014 virus, and 20% for H5N8-2016 virus, resulting in low pathogenicity scores (0.01 and 0.23 respectively). The mortality caused by the H5N6-2017 virus was 90%, and the survival time was significantly shorter than that observed for the two H5N8 viruses. Symptoms of disease started 3 days after infection, resulting in a pathogenicity score of 1.68. This demonstrates that the most recent HPAI H5 virus, the H5N6-2017 virus, is most pathogenic for both the domestic Pekin duck and the wild Eurasian wigeon. High levels of virus shedding were measured for the two duck species after infection with the HPAI H5 viruses, even when no clinical symptoms were observed. Infection with H5N8-2014 virus mainly resulted in shedding from the oropharynx, whilst after infection with the two group B viruses virus shedding from both oropharynx and cloaca was observed. The total amount of virus shed from the cloaca of Pekin ducks and Eurasian wigeons was significantly increased for the two group B viruses compared to the H5N8-2014 virus . Virus tAnas platyrhynchos), a species of migrating dabbling ducks of the Anatidae family, that also includes Eurasian wigeons. Whether the pathogenicity of the H5N6-2017 virus is also increased for other members of the Anatidae family remains to be determined, as differences between specific duck species have been reported in a recent study for an HPAI H5N8 group A virus [This study showed that the pathogenicity of the H5N6-2017 virus is increased for both Pekin ducks and Eurasian wigeons compared to the earlier H5N8 viruses.. Pekin ducks are a domestic duck breed descended from the wild mallard ( A virus . The mec A virus . GeneticRenv was higher than 1 (lower 95%CI was 1.7). Virus transmission via contaminated bedding material was not observed indicating a low probability of transmission via this route (Renv < 1). The virus in the bedding material was rapidly inactivated, whereas the virus in water remained infectious for at least 6 days at an average temperature of 21.1°C. The viability of low pathogenic avian influenza (LPAI) viruses in poultry bedding was investigated previously, and dependent on the type of bedding this viability was between 1 and 3 days [Poultry can become infected with HPAI viruses by the fecal-oral route, when feeding in an environment contaminated with infected wild bird feces. Therefore, increased cloacal shedding by wild ducks infected with HPAI H5 clade 2.3.4.4 group B viruses likely enhances the risk of introduction of these viruses into the commercial poultry sector. In agreement with this, more outbreaks were reported on commercial poultry farms and hobby holdings during the H5N8 group B virus epidemic in 2016–2017 . In thisd 3 days , which id 3 days ,43. Few d 3 days ,45, and d 3 days ,47. The d 3 days , and thed 3 days . In the d 3 days . The infd 3 days , suggestd 3 days , 51.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Personal protective equipment (PPE) is a critical need during the coronavirus disease 2019 (COVID-19) pandemic. Alternative sources of surgical masks, including 3-dimensionally (3D) printed approaches that may be reused, are urgently needed to prevent PPE shortages. Few data exist identifying decontamination strategies to inactivate viral pathogens and retain 3D-printing material integrity.To test viral disinfection methods on 3D-printing materials.The viricidal activity of common disinfectants  were tested on four 3D-printed materials used in the healthcare setting, including a surgical mask design developed by the Veterans’ Health Administration. Inactivation was assessed for several clinically relevant RNA and DNA pathogenic viruses, including severe acute respiratory coronavirus virus 2 (SARS-CoV-2) and human immunodeficiency virus 1 (HIV-1).SARS-CoV-2 and all viruses tested were completely inactivated by a single application of bleach, ammonium quaternary compounds, or hydrogen peroxide. Similarly, exposure to dry heat (70°C) for 30 minutes completely inactivated all viruses tested. In contrast, 70% isopropanol reduced viral titers significantly less well following a single application. Inactivation did not interfere with material integrity of the 3D-printed materials.Several standard decontamination approaches effectively disinfected 3D-printed materials. These approaches were effective in the inactivation SARS-CoV-2, its surrogates, and other clinically relevant viral pathogens. The decontamination of 3D-printed surgical mask materials may be useful during crisis situations in which surgical mask supplies are limited. Of these, SARS CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and now SARS CoV-2 are considered highly pathogenic.4 Human-to-human transmission of SARS CoV-2 occurs at an elevated rate compared to SARS CoV, which shares considerable sequence homology (79%).6Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) recently emerged as a highly transmissible human pathogen that rapidly escalated into a global pandemic.7 The increased demand for and shortages of PPE in healthcare and other essential workplace settings has created a need to address decontamination strategies and reuse of PPE. The Veterans’ Health Administration (VHA) recently began developing a supplemental surgical mask.8 The use of 3-dimensional (3D) printing technology within the healthcare industry is not new; it is currently used in applications such as drug delivery systems,10 surgery,12 personalized medicine,15 and biomedical engineering.17 In response to current needs during the COVID-19 pandemic, 3D printed technology has expanded to include the production of ventilators and other respiratory support equipment,19 nasopharyngeal swabs,20 face shields,21 and face masks.23Until there is an effective vaccine and/or therapeutic approach to treat COVID-19, SARS-CoV-2 control strategies are focused on transmission prevention, including social distancing, hand washing, and use of personal protective equipment (PPE).24In 2006, the US Department of Health and Human Services (DHHS) asked the Institute of Medicine (IOM) to convene a committee to conduct an evaluation of measures that would permit the reuse of disposable N95 respirators and reusable face masks in healthcare settings. The IOM committee followed criteria to prevent transmission of the 2003 SARS-CoV. The IOM committee found no validated method of decontamination that met criteria for decontamination of N95 respirators or surgical masks.26 In this study, we assessed the ability of materials used in the 3D printing of surgical masks to be decontaminated for human viral pathogens including SARS-CoV-2 using common disinfection methods. Data showing that human coronaviruses may survive on various surfaces for up to 9 days makes identification of decontamination strategies against pathogenic viruses increasing important not only during the SARS-CoV-2 pandemic but also for future viral transmission prevention practices.27 Our goal was to identify practical decontamination procedures that are easily adapted across healthcare and other workplace settings. We assessed the VHA 3D-printed mask material and 3 additional 3D-printing materials for virus inactivation in these studies.Although reuse of PPE masks was considered before the SARS-CoV-2 pandemic, literature on potential decontamination strategies are limited and guidelines are often institution- or manufacturer specific.Healthy volunteers were invited to participate in the study. Following written informed consent, anticoagulated blood was obtained using heparin collection tubes. This study was approved by the University of Iowa Institutional Review Board.50) or p24 enzyme-linked immunosorbent assay (ELISA) for HIV-1.29 All SARS-CoV-2 work was performed at the University of Iowa Biosafety Level (BSL) 3 core facility, and all other virus studies were conducted at the Iowa City Veterans’ Affairs Infectious Diseases Research Laboratory under BSL2 conditions.We used numerous viruses in these studies. Enterovirus 68 (EV68), vaccinia virus, influenza A H1N1 and human coronavirus (HCoV-229E) were all supplied by the American Type Culture Collection (ATCC). We also tested yellow fever virus , HIV-1 (provided by the NIH AIDS Reagent Program), mumps virus (provided by Merck), adenovirus , Zika virus , SARS CoV-2 (Seattle Washington strain MN985325), murine hepatitis virus , and Dengue viruses types 1–4 . Virus titers were determined in appropriate cell lines by median tissue culture infectious dose , and VeroE6 and 17Cl-1 were provided by Dr Stanley Perlman. Cells were maintained in media as previously described.8 being developed is 3D printed using Multi-Jet Fusion (MJF) technology and a powder-based polyamide-12 (PA12) material . The surgical face mask incorporates a removable filter (not tested in this study). The 3D-material is a biocompatible thermoplastic used in medical applications33 and is believed to be resistant to disinfectants. Thus, it may be reused many times following standard approaches to disinfection. The surgical face mask design has undergone review in a clinical setting and was found appropriate when fabricated with the printer type and materials specified.23The VHA supplemental surgical face mask50 in the cognate cell line as previously described35 or by p24 ELISA (R&D Systems) for HIV-1.29Virus titers were determined by determining the TCID10 or p24) was calculated. Control and postinactivation virus titers were performed in a minimum of 3 biological replicate experiments.Disks were printed in Seattle and shipped to Iowa for testing. Each virus (100 µL) was added to 2 cm diameter × 1.5-mm-thick 3-D printed circular disks . Disks were allowed to dry in a laminar flow safety cabinet at room temperature for 2 hours. Disks were inactivated either by thermal or chemical treatment (as described in the Results section). Viruses were recovered by placing the disks into a 12-well culture plate, adding 200 µL media to each well, pipetting 6 times, then removing the media and storing at −80°C until determining the viral titer . Virus infectivity following each inactivation method was compared to control disks treated only with phosphate-buffered saline (PBS), and the reduction in infectivity . We used 2-tailed Student 36 In light of the COVID-19 epidemic, we included 1 human low pathogenicity coronavirus (229E), 1 nonhuman coronavirus (MHV), and SARS-CoV-2 in most inactivation experiments. We also included a variety of healthcare-relevant human pathogens , different genome compositions (DNA vs RNA), and genome structures (single strand vs double strand).12, 32% C14] dimethyl ethylbenzyl ammonium chlorides – 0.14%; n-Alkyl  dimethyl benzyl ammonium chlorides – 0.14%), control wipe (PBS), or a no-wipe control as indicated. After a single application, disks were allowed to dry  and virus recovery was measured.Chemical inactivation studies were performed by treating the virus-coated disks with a single application (by wipe) of bleach , isopropyl alcohol (70% IPA), a commercial quaternary ammonium compound  Fig. A. All vi2O2 treatment of virus-coated disks utilized ionized H2O2 (~3% after application) for 15 minutes of vapor contact time and 15-minute air exchange using the SteraMist Binary Ionization Technology (BIT) at the University of Iowa Hospitals and Clinics Central Sterilizing Services (UIHC) in a 1.90 M L × 1.90 M H × 0.99 M W chamber. Alternatively, virus-coated disks were treated with ionized H2O2 (~3% after application) in a direct contact format at a 61-cm (24-inch) distance with a minimum contact time of 4 seconds. Ionized H2O2 delivery was generated by passing a low-concentration source liquid (7.8% H2O2) through a 17,000V cold plasma arc.37 Both vaporized and direct contact H2O2 completely inactivated all viruses  and other virus controls (YFV). As seen in Figure 38 The effects of blood on chemical inactivation were studied by adding virus to whole blood  and testing inactivation as described above. Although disinfectant sensitivity varied between viruses in the presence of blood, viruses remained highly sensitive to bleach and quaternary ammonium  reduction in inactivation with bleach application, highlighting the importance of maximizing inactivation.Previous studies found that the presence of human blood may interfere with the viricidal activity of disinfectants.ium Fig. A–B with ium Fig. C. NotablAn important concern for the reuse of PPE is the PA12 material’s stability and resistance to disinfectants. To address the effect of repeat exposure to disinfectants on efficacy of viral inactivation, the PA12 material was exposed to 100 applications of disinfectant before virus was applied and virus inactivation assessed using a single-wipe application as described above. Following repetitive inactivation, there was no grossly apparent loss of integrity, and virus inactivation was reproducible Fig. D–F.In addition to the PA12 material, we tested the inactivation of select viruses on 3 materials commonly used in 3D printing applications. These included a fused deposition modeling (FDM acrylonitrile butadiene styrene (ABS) material , an FDM polylactic acid (PLA) material , and a stereolithography acrylic (SLA) material . These additional materials were selected because the FDM and SLA 3D-printing technology platforms are very frequently utilized, and ABS and PLA are the 2 most common materials for FDM. Further, the surgical guide (SG) material is a very popular SLA material for additional medical applications. Although these 3 materials and PA12 represent the most commonly used materials, other 3D printing materials have been utilized in the healthcare setting. Application of select viruses to these materials found that SLA was more impermeable, requiring longer time intervals for virus drying (>4 hours), while PLA was permeable to the applied virus preparations. In contrast, ABS was similar to the PA12 material studied above. Virus inactivation by chemical disinfection was identical to that observed using the PA12 material Fig. A–C. Howe8 In this study, we evaluated practical chemical and thermal decontamination methods for the ability to inactivate SARS-CoV-2, the causative agent of the COVID-19 pandemic, 2 surrogate coronaviruses (MHV and 229E), and a diverse set of clinically relevant human viral pathogens with different viral properties that may influence inactivation.The shortage of PPE during the COVID-19 pandemic created a critical need for the development of alternative, reusable equipment. However, decontamination guidelines for reusable equipment have not reached consensus and are often limited by incomplete testing against surrogate pathogens. To address the shortage of respiratory PPE, the VHA developed a 3D-printed surgical mask that may be disinfected and reused.2O2 formulations. Also, 70°C dry heat completely inactivated viruses including the SARS-CoV-2 surrogate coronaviruses used (MHV and HCoV-229E); however, this decontamination method was not tested against SARS-CoV-2. Furthermore, 70% IPA and 50°C dry heat did not completely inactivate the viruses included in this study, although viral titers were reduced by >90% (>1 log) with these methods. Notably, in these studies, a single-wipe application of 70% IPA did not decontaminate surfaces completely, and a more stringent application of 70% IPA may be required for complete virus inactivation. To address the concern that blood may alter the efficacy of inactivation, we showed that virus present in 50% whole blood was reduced by >93% when treated with a single application of 10% bleach and ammonium quaternary. These studies illustrate the potential for slight operator differences in inactivation, which highlights this potential importance when suboptimal virus inactivation chemicals (IPA) are utilized.SARS-CoV-2 and all other viruses tested were completely inactivated by a single application of 10% bleach, ammonium quaternary, and 3% H2O2 dissipates into water vapor and oxygen after application leaving no toxic residue or odor, and the proprietary delivery system is portable, making the adoption of this technology useful.37 This approach to disinfection was also completely effective . This technology is EPA-registered as a hospital disinfectant under the name “Binary Ionization Technology (BIT) Solution,” and appears on EPA lists K, L, G and M. This approach was used because the antimicrobial effect is rapid (15 minutes), there is little damage to materials and surfaces, the H2O2, and 70°C dry heat) were effective on 3D-printed surgical mask materials. Some approaches were effective for inactivation of the SARS-CoV-2 virus, while others were also effective against its surrogates and other clinically relevant viral pathogens. These results are consistent with previous viral inactivation studies, although those studies included variations in decontamination procedures and did not incorporate virus application onto a 3D-printed material.40 The decontamination of 3D-printed surgical masks may be useful during crisis situations in which the supply of surgical masks is limited. Our results may be used to further study decontamination strategies for 3D-printed materials used in clinical settings.In summary, several decontamination approaches (10% bleach, ammonium quaternary, 3% H