This is the first household study of multiple respiratory viruses in a low-income setting that used intensive sampling regardless of symptoms. There was a remarkably high frequency of virus infection and illness, demonstrating the opportunity for complex interactions. Households are high-intensity close-contact environments favorable for transmission of respiratory viruses, yet little is known for low-income settings.Active surveillance was completed on 47 households in rural coastal Kenya over 6 months during a respiratory syncytial virus (RSV) season. Nasopharyngeal swabs (NPSs) were taken from 483 household members twice weekly irrespective of symptoms. Using molecular diagnostics, NPSs from 6 households were screened for 15 respiratory viruses and the remainder of households only for the most frequent viruses observed: rhinovirus (RV), human coronavirus , adenovirus (AdV), and RSV (A and B).Of 16928 NPSs tested for the common viruses, 4259 (25.2%) were positive for ≥1 target; 596 (13.8%) had coinfections. Detection frequencies were 10.5% RV (1780), 7.5% HCoV (1274), 7.3% AdV (1232), and 3.2% RSV (537). On average, each household and individual had 6 and 3 different viruses detected over the study period, respectively. Rhinovirus and HCoV were detected in all the 47 households while AdV and RSV were detected in 45 (95.7%) and 40 (85.1%) households, respectively. The individual risk of infection over the 6-month period was 93.4%, 80.1%, 71.6%, 61.5%, and 37.1% for any virus, RV, HCoV, AdV, and RSV, respectively. NPSs collected during symptomatic days and from younger age groups had higher prevalence of virus detection relative to respective counterparts. RSV was underrepresented in households relative to hospital admission data.In this household setting, respiratory virus infections and associated illness are ubiquitous. Future studies should address the health and economic implications of these observations. The current understanding of respiratory virus epidemiology arises mainly from analysis of specimens collected from individuals seeking care at a hospital or health facility, usually focusing on a single virus. This approach cannot provide a complete description of viruses in circulation in the community. A proportion of the infections will be asymptomatic or not severe enough to require medical attention, and, respiratory virus infections are typically of short duration. Hence, a full ecological/epidemiological description requires frequent sampling of individuals in a population regardless of symptoms, which is rarely undertaken. As a result, our understanding of seasonality, persistence patterns, and transmission dynamics of most respiratory viruses at the community level remains uncertain. Increased sensitivity and range of pathogens detectable by molecular diagnostics over traditional methods (culture isolation or antigen detection)  enable eThe present study involved respiratory virus screen of >16000 respiratory specimens that were collected from members of a rural coastal community in Kenya. The specimens were collected through household-based active surveillance for 6 months. Deep nasopharyngeal swabs (NPSs) were collected from all household members irrespective of symptoms. The intensive surveillance provided detailed infection data that allowed comprehensive investigation of the circulation of the respiratory viruses in the community. Previous reports have described the data on respiratory syncytial virus (RSV) in detail , and herThe current analysis is of data from a household cohort study undertaken in rural coastal Kenya within the Kilifi Health and Demographic Surveillance System . The stuBy multiplex real-time polymerase chain reaction assay, NPS collections from 6 households were screened for 15 respiratory virus targets as previously described . These ht test, χ2 test, and Fisher exact test. Week-delimited data on virus detections were plotted to show the temporal distributions and co-circulation at sampling, individual, and household level. Overall prevalence of the detected respiratory pathogens in households, individuals, and samples is also shown. The crude household and individual attack rates  were stratified by age, symptom status, household size, and gender.Data analyses were done with Stata version 13.1 software . Appropriate statistical tests were used that included the Student An informed written consent was obtained from all the study participants or their parents/guardian. Ethical approval for the study was provided by the Kenya Medical Research Institute Scientific and Ethical Review Committee in Kenya and the University of Warwick Biomedical Research Ethical Committee in the United Kingdom.2P value = .049; The median occupancy in the 47 households was 8 members . The average age of the members in each household at the start of sampling was 15.5  years. The baseline characteristics of the 6 households that were screened for all the 15 respiratory targets compared to the 41 households whose samples were tested for only the most prevalent respiratory viruses were similar, apart from the latter having a higher proportion of school-going children . Those NPS specimens with multiple virus detections had increased frequency of symptoms over single infections . The details of the number of samples that were positive for the respective targets are provided in One or more of the 15 respiratory viruses were detected in 864 of 2844 (30.4%) of the NPS collections, of which 714 (82.6%), 126 (14.6%), 19 (2.2%), 4 (0.5%), and 1 (0.1%) had 1, 2, 3, 4, and 5 viruses, respectively, (co-)detected. The proportion of samples that were virus positive was higher for specimens collected while the individual had symptoms compared with specimens collected during asymptomatic periods , 62 (77.5%), 57 (71.3%), 58 (72.5%), 52 (65.0%), 37 (46.3%), 11 (13.8%), and 8 (10.0%), respectively. The corresponding number of symptomatic infections of those ever infected, by pathogen, was 46 (61.3%), 33 (53.2%), 26 (45.6%), 29 (50.0%), 25 (45.5%), 11 (29.7%), 2 (18.1%), and 4 (50.0%), respectively. RV, AdV, HCoV, and RSV were the most prevalent respiratory viruses. They were each found in all the 6 households, infecting at least 1 member and were taken forward as the prevalent targets for screening of the NPS collection from the remaining 41 households. The temporal infection profile for the 6 households showing positive samples for each member is shown in 2P < .0001). The detected viruses, in order of frequency, were RV (1780 [10.5%]), HCoV (1274 [7.5%]), AdV (1232 [7.3%]), and RSV (537 [3.2%]). Of the HCoVs detected, 627 (49.2%), 399 (31.3%), and 212 (16.6%) were single infections of OC43, NL63, and 229E, respectively, and 36 (2.8%) had mixed HCoV strains. For the RSV-positive specimens, 231 (43.0%) and 287 (53.4%) had RSV group A and B only, respectively, while 19 (3.5%) specimens had both. Of all the virus-positive NPS collections, 657 (36.9%), 407 (33.0%), 410 (32.2%), and 229 (42.6%) had symptomatic infections with RV, HCoV, AdV, and RSV, respectively.All the 16928 NPS collections from the 47 households had infection data from the 7 prevalent respiratory targets . Of the 16928 NPS tested, 4259 (25.2%) were positive for 1 or more of the selected respiratory virus targets. Of the virus positives, 3687 (86.6%) were single virus detections, 526 (12.4) were dual, and 45 (1.1%) were triple, while only 1 (0.02%) had 4 targets codetected. Virus-positive specimens had a higher probability of being associated with respiratory symptoms compared with virus-negative specimens  detected over the 6-month study period . A higheRSV infections were first detected in the area from the hospital surveillance at the end of November 2009  but begaRV and HCoV were detected in all 47 households while AdV and RSV were detected in 45 (95.7%) and 40 (85.1%) households, respectively . All of P = .04). There were no statistically significant differences in the attack rates regardless of symptoms by sex and school-going status (P = .026). The attack rates by household sizes varied by pathogen and illness status. Households with fewer household members  had higher attack rates than larger households that were statistically significant for RV, HCoV, and RSV irrespective of symptoms. For symptomatic infections, only RSV showed a significant association by household size  in Michigan and Seattle, Washington, despite using less-sensitive diagnostic techniques (culture and serology) . In the Some of the pathogens were uncommon, and it is likely that a seasonal peak of some viruses fell outside the study period. In this location, peak occurrence of influenza (A or B) is in the second half of each year based on inpatient pediatric surveillance , 18. HMPDual or multiple infections were common . A prospective cohort study in a daycare center in the United States using comparable molecular techniques reported a coinfection rate of 27% among symptomatic young children , indicatAt least 1 virus was detected in 93.4% of the study participants over the 6-month study, and on average, each individual had evidence of 3 different viral infections. Given the close contacts of individuals in the households, the participants’ exposure to the investigated respiratory viruses was high: >95% of the households had 1 or more members detected with RV, HCoV, and AdV. The individual attack rates declined with increasing age for most of the target pathogens, most likely due to acquisition of immunity following previous infections. School-going children are usually respiratory virus introducers to households , but didThe frequency distribution of viruses in the community does not reflect that in the hospital, which provides a reminder that hospital data do not well describe infection transmission in the community, but rather the disease that arises, and this is clearly virus specific—that is, very much higher prevalence of RSV, HMPV, PIV3, and, interestingly, rhinoviruses among hospital cases than in the community.The study has some limitations. First, the study was designed with a focus on RSV, and here we are presenting an observational data set from essentially a “convenience” sample for a small number of viruses for a short period. A surveillance over a longer period and investigating a wider range of respiratory viruses would provide more comprehensive data on virus circulation. Multiple years of study would compensate for year-to-year variation. Second, our sample was households with infants, so it might be possible that households without infants would have a lower prevalence. Third, only a small number of households had their samples subjected to full respiratory screen. Given the clustering of respiratory infections by households, it is possible that a different set of households might have resulted in an additional choice of targets for screening. However, the similarities in circulation of the respiratory viruses in the community study and the hospital surveillance do not support this view. Last, virus infection was deduced from molecular diagnostics, which do not necessarily equate with the presence of potentially infectious virus, leading to overestimation of infectiousness. Each multiplex in the molecular screen may have reduced sensitivity for detecting coinfections as compared to single-target assays.In conclusion, respiratory virus infections and associated illness in this setting are ubiquitous in households. The molecular screen of these specimens revealed continuous and considerable respiratory virus circulation and infection frequency in this population that varied with virus species and subject age. The study here unveils previously unknown patterns of respiratory pathogen circulation in a rural low-income population. The remarkable frequency of virus infections of multiple species and strains lends itself to an ecological analysis of interactions that may be influential in virus ecology. The etiology of respiratory disease and immunological burden of respiratory viruses in children is worthy of further study. In addition, investigation on the human virome in the nasopharynx would provide insight on these viruses and how they affect human health and disease. Future studies should address the health and economic implications of these observations.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table S1Click here for additional data file.Supplementary Figure S1Click here for additional data file.Supplementary Figure S2Click here for additional data file.Supplementary Figure S3Click here for additional data file.Supplementary Figures LegendClick here for additional data file.


Societal and environmental pressures demand high-quality and resilient cropping plants and plant-based foods grown with the use of low or no synthetic chemical inputs. Mild strain cross-protection (MSCP), the pre-immunization of a plant using a mild strain of a virus to protect against subsequent infection by a severe strain of the virus, fits with future-proofing of production systems. New examples of MSCP use have occurred recently. New technologies are converging to support the discovery and mechanism(s) of action of MSCP strains thereby accelerating the popularity of their use. Multiple and significant global trends are currently demanding for highly sustainable, reliable, and increasingly efficient production of plant crops. Our response to the specter of climate change drives the need for reduced greenhouse gas emissions e.g., in the production or use of synthetic chemicals for insect management, and the need to be able to grow crops under progressively extreme climatic conditions ,2. WorldWhile viruses are the major pathogen causing emerging infectious diseases in plants worldwide, we have a few methods to manage the crop viruses . FurtherCross-protection, also known as “pre-immunization” is a method of plant virus control, whereby a plant is deliberately infected with a mild strain of a virus in order to protect the plant against damage caused by a more severe  strain of the same virus . MSCP prWhen naturally occurring mild strains are used, MSCP is not burdened with the social acceptability issues of genetic modification or gene editing. However, the use of MSCP does need to retain social license for growers and consumers. Social license cannot be assumed . As suchCross-protection is usually only effective between closely related strains of the same virus, although there are cases where phylogenetically distinct viruses can protect against each other, for example citrus vein enation virus was reported to protect against citrus tristeza virus (CTV) . ExploitThe traditional approach for identification of isolates for cross-protection involves identifying individual plants that display only mild symptoms, or remain symptomless, in fields where other neighboring plants display severe symptoms correlated with viral infection. Next, virus(es) are isolated from these individual plants; it should be noted that one part, limb or side of a plant may display mild symptoms while the rest of the plant exhibits severe symptoms, potentially indicating different sub-populations distributed throughout the individual plant . These vWhen searching for potential cross-protective isolates, one should consider that an isolate may only “protect” one host cultivar, for example in citrus the CTV cross-protective isolate IAC protects Pear sweet orange but not Hamlin or Valencia . FurtherCucumis metuliferus as an alternative host to obtain mild strains of papaya ringspot virus-P from nitrous acid-induced mild strains HA5-1 and HA6-1.Another method for selecting attenuated strains is by passing a viral population that may be a composite of many strains through an alternative host. Sometimes the predominant strain within the population changes through this virus–host interaction. Reasons for this may be that the alternative host is resistant to some strains of the virus and prohibits the replication and/or movement of such strains while others are able to move through the plant more rapidly. This technique was used by Yeh and Cheng  who usedAttenuated (mild) strains may also be selected through thermal treatment of infected plants; in this way a heat-attenuated mild strain of CTV was discovered . AdditioHigh-throughput sequencing (HTS) is a promising tool for the discovery of potential cross-protective isolates. HTS can rapidly identify the genotypic composition of the virus or viruses present ,31,32; aFolimonova  laid outThe cross-protection phenomenon was observed almost a century ago ,36,37,38Potexvirus, was discovered in the 1970s by Jones, Koenig, and Lesemann [Solanum muricatum) plants in Peru, extensive host range studies showed that not only numerous Solanum species and other members of the Solanaceae family can be infected by PepMV but also artificial infection  of Tetragonia expansa, a member of the Aizoaceae family and Cucumis sativus, a member of the Cucurbitaceae were possible  [Pepino mosaic virus (PepMV), genus Lesemann . Originaditions) . As a poHowever, for two decades, PepMV lived in the shadows until an outbreak was reported in greenhouse tomatoes in the Netherlands ; the firAlthough several attempts were underway to find sources of genetic resistance to PepMV within accessions of tomato and wild relatives, to the best of our knowledge, no PepMV-resistant tomato varieties are yet available ,103. In In 2015, the European Food Safety Authority (EFSA) concluded that there was no consumer risk in using a natural mild strain, PepMV CH2 isolate 1906  for MSCP, but also identified knowledge gaps in terms of environmental risk assessment or ecotoxicology . NeverthIt is encouraging to see that MSCP works well in these crops and can hopefully also be applied to other viruses that pose threats to tomato production including the newly emerging tomato brown rugose fruit virus, a tobamovirus with a similar mode of transmission as PepMV that has the potential to severely affect tomato production worldwide .A common explanation of cross-protection is RNA silencing. During RNA silencing, double stranded RNA (dsRNA) produced during viral replication is recognized by Dicer-like (DCL) enzymes and cleaved into small fragments, 21–26 nucleotides long ,132. TheNicotiana tabacum and Nicotiana benthamiana. Interestingly, both strains appeared to be highly localized on a cellular level, with the two viruses occupying different cells, a phenomenon that has also been observed for other virus systems [Arabidopsis thaliana plants, casting more doubt that RNA silencing was involved with at least this virus system [RNA silencing by itself is not able to explain the mechanism of cross-protection fully. In a study done by Ziebell, Payne, Berry, Walsh, and Carr  a 2b sil systems . The auts system . Anothers system  supporteAn alternative model explaining cross-protection without RNA silencing involves the protective strain preventing the challenge strain from uncoating upon entering the plant cell. The coat protein from the protective mild strain may recoat the challenge strain and prevent its replication , a propoSuperinfection-exclusion was proposed as the mechanism by which CTV cross-protection occurs , where iHarper unpublished). It may be that a protective mixture is able to incorporate a pathogenic challenge isolate into the population, and reduce its fitness such that it cannot accumulate or express to a level necessary for pathogenesis.In citrus at least, extant protective isolates such as GSMS-12 and Pera/IAC are populations, mixtures of different genotypes ,144, sugThe cross-protection phenomenon may not have a simple or single explanation. While some studied MSCP strains protect against infection with challenge isolates by triggering RNA silencing, others do not . In someCross-protection as a disease management strategy can be very successful, as proven by the management of CTV or PepMV. Eradication of CTV by removing infected trees has proven unsuccessful, as the virus spreads quicker than infected trees can be identified and subsequently removed . Using cFor traditional breeding or cis-genics these are seldom the required genetic sources of resistance available within a cultivar, e.g., zucchini squash does not harbor sources of resistance to papaya ringspot virus type W (PRSV-W) within its genome . While oSocietal and environmental pressures demand high quality and resilient cropping plants and produce with the use of low or no synthetic chemical inputs. Pathogenic viruses result in significant losses in crop production and crop quality but these are increasingly managed by MSCP which is both environmentally safe and socially friendly. Since MSCP is dependent on host and virus strains, an effort in finding local mild strains has to be made; any one mild strain cannot be used for the protection of cultivars grown in different locations. New technologies are converging to support the discovery and mechanism(s) of MSCP strains thereby accelerating the popularity of their use. However, without bioinformatic predictions of host range or pathogenicity the major barrier to the use of MSCP strains is in demonstrating their efficacy. Once efficacy is secured, MSCP proffers effective and sustainable protection against pathogenic viruses of crop plants across a range of production environments. The analysis of the societal and environmental benefits from the application of MSCP technology may provide impetus to further accelerate its use and, perhaps, assist in clarifying its underlying mechanism(s).


Plantago lanceolata populations, and to understand the variation in their prevalence and distribution across the Åland Islands, South-West Finland. By subsequent design of PCR primers, we screened the five most common viruses from two sets of P. lanceolata plants: 164 plants collected from 12 populations irrespective of symptoms, and 90 plants collected from five populations showing conspicuous viral symptoms. In addition to the previously reported species Plantago lanceolata latent virus (PlLV), we found four potentially novel virus species belonging to Caulimovirus, Betapartitivirus, Enamovirus, and Closterovirus genera. Our results show that virus prevalence and diversity varied among the sampled host populations. In six of the virus infected populations only a single virus species was detected, while five of the populations supported between two to five of the studied virus species. In 20% of the infected plants, viruses occurred as coinfections. When the relationship between conspicuous viral symptoms and virus infection was investigated, we found that plants showing symptoms were usually infected (84%), but virus infections were also detected from asymptomatic plants (44%). Jointly, these results reveal a diverse virus community with newly developed tools and protocols that offer exciting opportunities for future studies on the eco-evolutionary dynamics of viruses infecting plants in the wild.Wild plant populations may harbour a myriad of unknown viruses. As the majority of research efforts have targeted economically important plant species, the diversity and prevalence of viruses in the wild has remained largely unknown. However, the recent shift towards metagenomics-based sequencing methodologies, especially those targeting small RNAs, is finally enabling virus discovery from wild hosts. Understanding this diversity of potentially pathogenic microbes in the wild can offer insights into the components of natural biodiversity that promotes long-term coexistence between hosts and parasites in nature, and help predict when and where risks of disease emergence are highest. Here, we used small RNA deep sequencing to identify viruses in  Arabidopsis thaliana , from P. lanceolata sampled in Åland  vary in their prevalence across the P. lanceolata populations in the Åland Islands. We also found that the plants showing symptoms were more likely to be infected by one or more virus species than asymptomatic plants . Finally, we successfully developed inoculation protocols using sap and aphids for PlLV and using sap for Plantago latent caulimovirus and Plantago enamovirus.Here, we characterize virus communities in natural populations of nce 1993 . Since 2pathogen . Extendireported , and we in Åland . Here, oall RNAs  and subsPlantago lanceolata is a perennial rosette-forming herb with world-wide distribution  using the method described in . The obtEndornaviridae and Ophioviridae. Hence, we designed primers for four virus taxa, Caulimovirus, Betapartitivirus, Enamovirus and Closterovirus. In addition, we used the PlLV detection primer pair described in ®  polymerase and the following thermal cycling conditions: 95 °C for 2 min, 25 cycles of 95 °C (40 s), 50–57 °C (40 s), 72 °C (1 min), and a final extension of 72 °C for 5 min , 1720 (25 plants), 2619 (9 plants), 4309 (10 plants) and 9031 (37 plants) in June 2015 . First, we analysed the association between infection status and visible symptoms for each virus species separately. In each analysis infection status by the target virus  was defined as a binary response variable and visual symptoms  were defined as categorical explanatory variables. We then analysed whether infection status (0/1) by any of the five tested viruses was associated with the visual symptoms defining the model variables as in the previous analyses. In these analyses, the whole data set of 90 symptomatic plants was used. In these analyses, the sample size was not sufficient to use population as explanatory or random factor.In order to understand how infection of individual host plants depends on the virus species and population, we fitted a Generalized Linear Model in SAS 9.1 PROC MIXED . Virus infection status of the plant  was defined as a binomial response variable with a logit link function, and virus species and host population were used as explanatory class variables in repeated measures model. Plant individual was used as the subject of repeated measures.To understand how virus species richness differs among the populations, we calculated the Shannon  and SimpDysaphis plantaginea) transmission. Dysaphis plantaginea is a specialist herbivore on P. lanceolata and apple  that occurs in the study are in the Åland Islands. In addition to using P. lanceolata as virus maintenance plant, we tested inoculation to three plant species commonly used in virus maintenance, Nicotiana benthamiana, Chenopodium quinoa, and Chenopodium amaranticolor, using the two sap inoculation methods. All virus infected plant and aphid material was collected from the Åland Islands in July 2017 and the plant material was tested with the detection primers as described above. First, we tested whether PlLV can be transmitted by aphids in an inoculation study where a colony of five aphids was first starved for 24 h, then allowed to feed on PlLV infected P. lanceolata leaf for 24 h acquisition access period (AAP) followed by 2 h starvation period after which they were placed on uninfected P. lanceolata plants for a 2-day inoculation access period (IAP). All P. lanceolata individuals used were cloned from mother plants that tested negative for the viruses used in the experiment. In addition, we used (RT-)PCR to verify that the plants tested negative for infection prior to inoculation. DNA samples were taken 14 and 20 days post inoculation (DPI) and the infection was detected using PlLV specific primers as described above. We then performed a set of inoculations with the five viruses using sap from infected plants in 0.02 M phosphate buffer (pH 7.4). Finally, we used the successfully infected plants from the sap and carborundum experiment to test sap inoculation with syringe. In this experiment, the same buffer was used and the leaves of the receiving plants were first wounded with a sterile scalpel and then injected with 100 µL sap and buffer. In this experiment, P. lanceolata, C. quinoa, C. amaranticolor and N. benthamiana were used as receiving plants  and all four putative novel viruses belonging to the Caulimoviridae, Partitiviridae, Luteoviridae and Closteroviridae families (see below). Ophioviridae- related contigs were found in populations 1045 and 9623, and Endornaviridae- related contigs in populations 3375 and 9205.The small RNA sequencing resulted in 10–21 million high quality reads per sample. The reads were assembled into 638 762 contigs ranging from 45 to 5,410 nucleotides (mean 52 nt) using re found , represere found . The meaCaulimoviridae-related PCR product and used the translated 331 amino acid sequence in a Blast search. We found the closest hit with polyprotein of Carnation etched ring virus, genus Caulimovirus, . The amino acid sequence was further used to determine phylogenetic relationships with other unique caulimovirus sequences  showed a high degree of similarity to the RdRp of Dill cryptic virus 2, genus Betapartitivirus, . We analysed its phylogenetic relationship with 22 unique sequences of the known species of the Partitiviridae family. The analysis showed that the putative novel betapartitivirus amplified from P. lanceolata, hereafter referred to as Plantago betapartitivirus, is nested within the clade composed by eight plant-infecting betapartitiviruses  to the RdRp of Grapevine enamovirus 1 (genus Enamovirus) in a Blast search. Pairwise amino acid sequence identity calculation revealed that the 295 amino acids sequence had 49–69%, 49–60% and 49–50% identities with members of the Enamovirus, Polerovirus and Luteovirus genera, respectively. Finally, the phylogenetic analysis revealed that the 295 amino acids sequence branched within the Enamovirus genus  of Beet yellow stunt virus, genus Closterovirus . To understand the phylogenetic relationships of the virus, we analysed the partial HSP70 amino acid sequence together with the other known unique closterovirus sequences. The partial HSP70 sequence we obtained from P. lanceolata clustered together with members of the Closterovirus genus . The population with highest infection prevalence (1719) was also the only population where all five species were detected. In six of the virus infected populations, only one virus species was detected, while five of the populations supported between two to five of the studied virus species. Across populations, the frequencies of the five virus species varied PCs varied . Plantags varied . Overalls varied .In order to understand whether plants showing typical conspicuous viral symptoms were infected by the five viruses studied, (RT-)PCR detections were performed on samples collected from symptomatic plants. We classified the symptoms into four categories: yellowing, redness, necrotic lesions, and curliness. Out of all symptomatic plants collected from the wild, 84% were infected by one or more viruses while in non-symptomatic plants infection prevalence was 44% . The plaD. plantaginea with an inoculation success rate of 20%. When we tested different transmission methods on P. lanceolata and three commonly used virus maintenance plants C. quinoa, C. amaranticolor and N. benthamiana, we found that PlLV was transmitted successfully with carborundum to C. quinoa as 100% of inoculated plants became infected, but the success in other inoculation methods and viruses was lower  and C. amaranticolor (33% plants infected) and using syringe to N. benthamiana (33% plants infected) as well as for Plantago enamovirus to C. quinoa using carborundum  as a vector. This is in line with a recent study on Alfalfa leaf curl virus that reported aphid transmission for the first time for geminiviruses (We tested different inoculation methods for future experimental studies. We found that PlLV can be transmitted using rosy apple aphid (iviruses . We alsoiviruses .P. lanceolata populations are assigned to genera typically showing wide host range as well as transmission by generalist vectors, e.g., caulimoviruses, closteroviruses and enamoviruses. Previously it has also been shown that luteoviruses and geminiviruses can move between wild and cultivated hosts (Plantago lanceolata populations often occur adjacent to agricultural fields, and it is a host plant for several generalist herbivores (Plant pathology research has largely focused on viruses infecting crop species and thus very little is known on the viruses present in wild plant populations . This ised hosts . Plantagrbivores . UtiliziOur results increase understanding of the pathogen diversity present in wild populations—local virus communities are diverse and spatially variable. The observed low similarity with previously characterized viruses underlines the need for more studies on natural ecosystems to uncover true levels of pathogen diversity. Coinfections are proving to be common in both natural and agricultural environments , and our10.7717/peerj.6140/supp-1Table S1Click here for additional data file.10.7717/peerj.6140/supp-2Supplemental Information 1Click here for additional data file.


Anopheles sinensis mosquitoes from Hubei, China. An in vitro comparison study of the HB14-71-01 isolate and the group A BAV revealed differences based on observed cytopathic effect, plaque size, and viral growth rates. Additionally, the phylogenetic analysis indicated that the Hubei isolate belongs to a novel genotype of BAV and emerged nearly 105 years ago (95% highest posterior density (HPD): 35–434), unlike the two previously reported genotypes A and B. Our findings extend the knowledge about the genomic diversity and potential vectors/hosts of BAVs and will improve understanding of the relationships between genetic variation and pathogenicity.Banna virus (BAV) is considered to be an emerging human pathogen that is transmitted by blood-sucking insects. BAV was isolated from various species of mosquitoes, midges, and livestock. It is widely distributed geographically, since it was identified in China, Vietnam, and Indonesia. Previously reported evolution studies of BAV indicated that BAV can be divided into two groups, including isolates from China and Vietnam clustered in group A, and Indonesian isolates in group B. In this study, we report the isolation of a new strain of BAV named HB14-71-01 from  Seadornavirus within the Reoviridae family [Banna virus (BAV) is a virus grouped under the genus e family . BAV is e family . Subseque family ,4,5.BAV is a double-stranded RNA (dsRNA) virus composed of 12 segments and is approximately 19.5 kb in genome length. Segments 1–4 and 8–10 encode for the structural proteins of BAV, while segments 7 and 12 encode for the protein kinase and the dsRNA-binding protein, respectively. The remaining segments encode for nonstructural proteins with unknown function ,7.Previous research studies on BAV found that BAV strains cluster significantly according to their geographical distribution. Phylogenetic analysis based on the complete coding sequence of segments 9 or 12 indicate that BAV is divided into two groups: A and B ,8,9,10. Anopheles sinensis mosquitoes collected in Yichang, Hubei, China in 2014. This new isolate has phenotypic and genomic characteristics diverging from the BAV group A viruses and is differently clustered, forming a new genotype—group C.In China, Banna virus has a wide geographic distribution since it was isolated from various regions including Gansu, Shanxi, Inner Mongolia, Liaoning, Beijing, Hubei, and Yunnan. All these previous isolates from China belong to group A ,13,14,15Anopheles sinensis, Armigeres subalbatus, Culex quinquefasciatus, and Culex tritaeniorhynchus. The samples were grouped into pools of 50 and kept frozen at −80 °C until further analysis.Adult mosquitoes were caught in Yichang, Hubei province, China  in 2014. Trapped mosquitoes were killed by flash-freezing in liquid nitrogen, and they were then morphologically classified into four species: The pooled mosquitoes were homogenized, and the extracts were clarified using centrifugation as described previously . The hom2O, and 2.0 µL of RNA sample; thermal cycling was performed at 50 °C for 30 min and 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s. PCR products were visualized in a 1.5% agarose gel with ethidium bromide.RNA was extracted using a QIAamp viral RNA extraction kit  following the manufacturer’s instructions. To screen for BAVs, one-step reverse-transcription polymerase chain reaction (one-step RT-PCR) was conducted using a Prime ScriptTM one-step RT-PCR kit Ver.2  and two primer sets, 12-B2-S: 5’–CAGAGTATAAATCAATCGCCCAAG–3’ and 12-B2-R: 5’–GTTCTAAATTGGATACGGC GTGC–3’ . The reaAedes albopictus C6/36 cells. Cytopathic effects (CPEs) were observed daily, followed by RT-PCR detection of BAV.Supernatants of positive pools were inoculated onto monolayers of g for 30 min at 4 °C; it was then centrifuged with 20% sucrose cushion at the bottom of the tube  using an SW32 Ti rotor . The supernatant was discarded, and the virus pellet was resuspended in 200 μL of phosphate-buffered saline (PBS). The resuspended pellet was ultracentrifuged in a continuous sucrose gradient (20–70% w/v) in an SW41 Ti rotor at 39,000 rpm for 2 h; sucrose was removed from the purified fractions by centrifugation with 200 μL of PBS at 40,000 rpm for 1 h at 4 °C. The supernatant was discarded, and the pellet was resuspended in PBS and stored at −80 °C. Electron microscopy was performed using the negative contrast method. The pure virus particles were added to a Formvar carbon-coated copper grid for 10 min and negatively stained for 2–3 min with 2% phosphotungstic acid (PTA) at pH adjusted to 6.8 with 1 M KOH; the samples were examined using a Hitachi U8010 electron microscope .The culture supernatant was harvested after at least two blind passages, and clarified to remove cellular debris by centrifugation at 5000× Culex tritaeniorhynchus mosquito pool from Yunnan, 2015; this species has a highly similar (>95%) genome sequence with the other reported BAVs isolated from Yunnan which belong to group A. To compare the basic viral characteristics between HB14-71-01 and YN15-126-01, a plaque assay and a growth kinetics assay were performed simultaneously. Prepared aliquots of 10-fold serial dilutions of the virus in Rosewell Park Memorial Institute (RPMI) medium were inoculated onto the C6/36 cell monolayers in 24-well plates for 2 h. The cells were then covered with an overlay medium including 1.5% methylcellulose and incubated at 28 °C for 4–6 days to allow for plaque development. Afterward, the infected cells were stained with 2% crystal violet in 30% methanol for 5 min at room temperature. The plaques were manually counted and measured.BAV strain YN15-126-01 was isolated from the For the growth kinetics assay, the C6/36 cells in a T75 flask were infected with the BAV viruses YN15-126-01 or HB14-71-01 (multiplicity of infection (MOI) = 0.001), and 2 mL of supernatant was collected from the flask, before 2 mL of fresh medium was re-added every day until all cells were either floating or dead. The virus titers of the collected samples were detected using the plaque assay, and the experiments were repeated three times.The viral RNA was extracted from the passaged virus and sent to Shanghai Biotechnology Corporation (Illumina Miseq System) for sequencing. The viral sequences were edited and assembled using AbySS v2.0.2  and SOAPThe nucleotide sequence identity of the complete coding sequences (CDSs) of a newly isolated Hubei BAV and five other strains  were analyzed using BioEdit v7.0.5 [Seadornavirus genus  were used for the analysis, and the background information is listed in Seadornavirus genus was conducted using the ClustalW module, and phylogenetic analyses were conducted with the maximum-likelihood method with 1000 bootstrap replicates using MEGA v7.0.21 [The 12th segment genes of 30 BAV strains and representative members of the  v7.0.21 . Bayesia v7.0.21 . The GTR v7.0.21 . The relAnopheles sinensis. The isolate was named HB14-71-01.We screened 44 mosquito pools to determine the prevalence of BAV in the field-collected mosquitoes. Six mosquito pools were PCR-positive for BAV, indicating that the prevalence of BAV infection in mosquitoes in Yichang is approximately 13.6%. Only one BAV isolate was successfully obtained from a pool of De novo assembly of the next-generation sequenced data acquired 99.5% coverage of the complete BAV genome. The lengths for segments 1–12 were 3752, 3059, 2449, 2037, 1637, 1656, 1118, 1105, 1070, 957, 830, and 852 bp, respectively. The complete CDS of VP1 to VP12 encoded 1219, 954, 720, 628, 481, 427, 306, 302, 282, 245, and 208 amino acids, respectively.8 plaque-forming units (PFU)/mL for YN15-126-01; however, the highest titer for HB14-71-01 was achieved in six days post infection at 105 PFU/mL , Liao ning virus (LNV), Mangshi virus, and Kadipiro virus (KDV) of the her BAVs . Additio−4 substitutions per site per year (s/s/y) (95% HPD: 1.093 × 10−4 to 5.628 × 10−2).Based on the segment-12 genome sequences, a maximum clade credibility tree was generated for BAVs . The MRCPreviously reported BAV genotypes (group A and group B) are significantly clustered according to the geographical point of virus isolation. BAV group A is further divided into subgroup A1, comprising strains from north China, and subgroup A2, comprising strains from south China and Vietnam ,8,9,10. Cx. tritaeniorhynchus and An. sinensis in Hubei, China, in 2009 and phylogenetic analysis of their 12th segment , indicating that these isolates belong to subgroup A2 alongside other BAV isolates from Yunnan, Inner Mongolia, Beijing, and Vietnam. However, in this study, a new Hubei BAV isolate from An. sinensis obtained in 2014 does not cluster in subgroup A2, but rather, clusters in group C, thus expanding the diversity of natural BAVs.Additionally, Gao et al. previously reported the isolation of four BAV strains from HB14-71-01 and QTM104536 were clustered together to form group C and were reported in a mosquito collected in 2014 and in Odonata collected in 2013, respectively. However, the sequencing information for QTM104536 was obtained from the metagenomic analysis, and no virus isolate and phenotypic information was reported for this strain . Quite aIt should be noted that only a few BAV strains have their full genome information in the National Center for Biotechnology Information (NCBI) GenBank; hence, whole-sequence analysis is currently limited only to the analysis of segments 9 or 12. When additional full BAV genomes are revealed in the future, evolutionary analysis based on other segments will substantially improve understanding of these viruses.


The dataset being studied consisted of 1450 Chinese Holstein bulls with full pedigree information, born between 1996 and 2012, representing 11 AI centers. Genetic parameters were estimated in a multivariate analysis using the average information restricted maximum likelihood estimation of variance (AI-REML).Semen traits are important for the widespread use of superior bulls. Thus, the objective of this study was to estimate the heritability of five semen traits, ejaculate volume (VE), progressive sperm motility (SM), sperm concentration (SC), number of sperm (NSP), and number of progressive motile sperm (NMSP), and their genetic correlations  to 0.99 (NSP and NMSP).Our findings provide useful information on the heritability of semen traits in Holstein bulls and the relationships among them, and should assist in selection for improvement of semen traits in Chinese Holstein bulls. Female fertility has been widely studied for breeding purposes in many countries –3, whereConception rate in the Holstein cattle has declined dramatically recently in some countries , 17. BecData from a total of 1450 Chinese Holstein bulls were used in this study. The bulls were from 11 artificial insemination (AI) centers in China, and the number of bulls in each center ranged from 69 to 202. All the bulls were born between 1996 and 2012, and five semen traits were recorded across years  by tracing back as many generations as possible for the bulls. The number of animals in the pedigree file was 12,985.The pedigree information was extracted from the Dairy Data Centre of China  were defined as 10–24 months, 25–36 months, 37–60 months, 61–96 months, and older than 96 months; Seai is the fixed effect of the season of collection, where the four seasons (i = 1 to 4) were defined as December to February, March to May, June to August, and September to November; ColIntj is the fixed effect of the interval of between consecutive collections for each bull (j = 1 to 5), which was defined as 0 d, 1–4 d, 5–7 d, 7–11 d, and longer than 11 d; Numk is the fixed effect of the number of collections on the respective collection day (k = 1 to 2); BulStal is the fixed effect of AI station (l = 1 to 11); po is the random permanent environment effect with var.(p) ~ N, where σ2pe is the permanent environmental variance and I is the identity matrix; an is the random additive genetic effect with var.(a) ~ N, where σ2a is the additive genetic variance and A is the relationship matrix; BulHanm  is the effect of BulHanm (m = 1 to 54), as a random effect of bull handlers, nested within AI station; eghijklmno is the random residual effect with var.(e) ~ N, where σ2e is the residual variance. (Co) variance components were estimated using the DMU software package [For each of the five semen traits, the model for the genetic parameters was: package .P < 0.01).The total number of ejaculates was 332,632 and the average number of recorded ejaculates per bull was 229. The bulls had an average semen volume of 7.22 mL, progressive sperm motility of 67%, sperm concentration of 11.37 × 10 9/mL, total sperm of 83.52 × 10 9, and total motility sperm of 59.21 × 10 9  was similar to the value of 0.31 reported by Gredler et al. 2007), whereas the negative correlations of − 0.20 and − 0.38 were reported by Druet et al. (2009) and Kealey et al. (2006), respectively. The genetic correlation between SC and NSP  reported by Druet et al. [, whereasThe low heritability of semen traits (0.12 to 0.22) indicates a substantial environmental influence; this suggests that potential environmental factors that may affect semen quality, such as bull handler, season, and the interval between semen collections need to be controlled carefully so that genetic differences are more accurately assessed. Therefore, to improve semen quality and increase the quantity of superior bulls, environmental influences such as bull handlers, season, and the interval of two continual semen collections for each bull, should be optimized. In addition, other environmental influences that are not routinely recorded in AI stations, such as temperature, humidity, and feed, should be recorded, and studies of candidate genes for semen traits and GWAS can be performed with the aim of selecting bulls with superior semen quality and quantity.In this study, we estimated the genetic parameters of five semen traits in Chinese Holstein bulls for the first time. The heritability of semen traits was varied from 0.12 to 0.22. The correlations among the semen traits were all positive. NSP had a strong genetic correlation with all other semen traits and its heritability was 0.16, which suggests the focus of genetic improvement ought to be around NSP. Our results provide valuable insights into the genetic basis of semen traits in the production period and should assist in designing selection programs to improve semen traits in Chinese Holstein bulls.


Algal viruses are considered to be key players in structuring microbial communities and biogeochemical cycles due to their abundance and diversity within aquatic systems. Their high reproduction rates and short generation times make them extremely successful, often with immediate and strong effects for their hosts and thus in biological and abiotic environments. There are, however, conditions that decrease their reproduction rates and make them unsuccessful with no or little immediate effects. Here, we review the factors that lower viral success and divide them into intrinsic—when they are related to the life cycle traits of the virus—and extrinsic factors—when they are external to the virus and related to their environment. Identifying whether and how algal viruses adapt to disadvantageous conditions will allow us to better understand their role in aquatic systems. We propose important research directions such as experimental evolution or the resurrection of extinct viruses to disentangle the conditions that make them unsuccessful and the effects these have on their surroundings. Photobacterium phosphorium about 10 years later [6 to 109 of virus particles per mL of marine waters [The invention of the electron microscope around 1930 not only confirmed the existence of viruses but also how diverse they are. The first aquatic virus to be reported was the marine bacteriophage rs later  and eukae waters ,5 or evee waters . Viral pe waters ,9. The re waters ,16,17,18Since the discovery of high viral numbers and diversity, their role in aquatic ecosystems has been acknowledged as indispensable in structuring the microbial community and biogeochemical cycles  there are low numbers of viruses in the environment; (ii) they have low or no viral infectivity and/or (iii) they have low or no viral replication ability and subsequent low or no release of viral particles into the environment. Thus, for the purposes of this review, viruses with one or more of these characteristics will be referred to as “unsuccessful”. The factors that make viruses unsuccessful can be either intrinsic to them, when they are associated with their own heritable life cycle traits such as burst size or latent period, or be extrinsic to them and be caused by the environment such as by temperature or host resistance mechanisms . ExampleThe first step that determines the success of viral infections is the host–virus encounter. This primarily depends on both host and viral abundances but it is also influenced by other properties such as cell size, swimming abilities or the presence of non-host organisms . MathemaHeterosigma akashiwo and its virus HaV [Heterocapsa circularisquama [Rhizosolenia setigera [Chaetocerus debilis [Chaetocerus lorenzianus [Phaeocystis pouchetii [Ectocarpus siliculosus, from the phaeovirus genus, as also being capable of infecting other algae such as Kukuckia kylinii [Feldemannia simplex or when EfasV virus infects Ectocarpus siliculosus [Myriotricha clavaeformis [In terms of algal viruses, intraspecific infectivity has been observed in several cases such as that of the raphidophyte irus HaV , the ssRrisquama  or thosesetigera , the ssD debilis  and Chaeenzianus , or the ouchetii  among ot kylinii ,45. Thesiculosus  and Myriaeformis . Even thaeformis ,48. Secoaeformis . It is tChlorella variabilis has a latent period of 6–8 h and a burst size of around 1000 virus particles with only 30% infectious ones [Heterocapsa circularisquama has a latent period of 24 h and produces 3.4 × 103 infectious particles [Latent period and burst size are the two main life cycle traits that determine whether a virus is successful or not and are commonly specific to every virus. For example, the dsDNA virus PBCV-1 infecting articles . It is iThe latent period is referred to as the duration of the viral infection within the host and can be divided into two types: lytic or lysogenic . While lEmiliana huxleyi [Chlorella-like algae [Ectocarpus siliculosus, Feldmannia species and Feldmannia irregularis. Their infection strategies are somewhat remarkable as they only infect and integrate their genetic material into the genomes of the algae’s free-living gametes. This ensures that their DNA is mitotically transmitted to all cells during gamete development, although it is only active in the cells of the reproductive organs. Viral replication and release occur when conditions are favourable for gamete release, allowing new viral particles to easily infect newly released gametes [Following our definition, lysogenic viruses are less successful than lytic viruses . Their i huxleyi  or Chlorke algae . On the  gametes ,55,56.Despite having grouped this life strategy as one of the ways in which viruses are not very successful, it is also important to note that it represents a survival strategy for them in areas with low host abundance or low productivity and also allows them to be protected in harsh environmental conditions, and it can prevent secondary viral infections in the hosts .Burst size refers to the amount of viral progeny that is released into the environment after infection . Althoug2 to 103 viruses per cell [Burst sizes are very variable across all known viruses, ranging from hundreds to tens of thousands per cell. Viruses that belong to the phycodnaviridae family, for example, have burst sizes of 10per cell . The numper cell . Regardiper cell . These cFactors that are not related to the heritable life cycle traits of viruses are grouped here as extrinsic factors. They determine whether a virus is successful or not and are common to the environment. Extrinsic factors can affect viruses during their particle state by increasing viral decay, e.g., low host numbers in the environment decrease the number of successful infections through lower host–virus encounter rates, but they can also modify some of the viruses’ intrinsic life cycle traits, e.g., low temperatures or nutrient-depleted conditions can elongate viral latent periods.Host resistance to viral infections is a common factor by which viruses become unsuccessful. To date, different forms of resistance have been reported in microbial hosts such as bacteria or phytoplankton, and they are all related to the different stages of viral infection: attachment, when the virus physically attaches to the algal cell; viral entry, when the virus or its nucleic acids enters the cell; or the last internal steps, which involve viral gene expression, replication, virion assembly and cell lysis .Porphyridium [Phaeocystis pouchetii could not be infected by the virus PpV-AJ96. This stage consists of Phaeocystis cells enclosed by a thin but strong semi-permeable skin with plastic and elastic properties [Chlorella variabilis algae, PBCV-1 virus and Brachionus calyciflorus rotifer) Frickel et al. [Chaetoceros tenuissimus was also able to resist viral infection of CtenRNAV during its stationary phase of growth but only in the presence of bacteria, as they observed a bacteria–algae association that inhibited viral infection. Some organisms are also capable of evading viral infection by escaping. The algae Emiliana huxleyi, for example, is able to undergo meiosis in the presence of a virus and therefore transform itself from a virus-susceptible diploid cell to a resistant haploid cell. This study also offered Frada et al. [Attachment to the host cell represents the first stage of viral infection and often involves receptors at both ends. Viral receptors include a wide range of proteins, carbohydrates and lipids, allowing them to infect a wide range of hosts. However, hosts have proven to evade this stage by modifying their cell wall receptors, either by decreasing the number of receptors or simply by losing them . Other mhyridium . Evans ehyridium  also fouhyridium . Algal shyridium . Jacobsehyridium  found thoperties . This skoperties . Using al et al.  found a l et al.  showed ta et al.  the oppoa et al. ,69,70.Heterocapsa. By artificially introducing the viral RNA into the cells, they reported that the transfected HcRNAV34 viral genome was replicated only in susceptible cells and not in resistant ones.Despite the lack of knowledge regarding mechanisms to block viral entry or replication in marine organisms, some general ones such as the destruction of incoming virus DNA by restriction modification systems or the CRISPR/Cas system have been shown in bacteria . AlthougBathycoccus sp., Micromonas sp. and Ostreococcus tauri) were able to evade lysis after inoculation. They showed that resistance mechanisms in Ostreococcus tauri were achieved either through lysogeny or attachment inhibition.Lysogeny, pseudolysogeny or chronic infections can be associated as a host resistance mechanism to prevent or delay host burst and viral release . In an eApart from the ways in which hosts themselves can evade infection by viruses, there are other stressors in the environment that can lower viral success. Here, we review different biotic pressures that directly or indirectly affect viruses.Phaeocystis globosa. A reduction in the abundance of the virus and its progeny occurs due to a secretion of organic compounds by the algae prior to viral lysis [Predation represents a direct stressor on viral populations. In the marine realm, nanoflagellates and sponges are known for removing high quantities of viruses through phagotrophy and filter feeding, respectively ,73. Becaal lysis . The effal lysis . HoweverAlthough virophages represent a great threat to other viruses, none that infect phytoplankton cells have been isolated to date. Reported virophages are small, double stranded DNA viruses that require the co-infection of another virus to replicate. They are considered as parasites of larger viruses, specifically some members of the Mimiviridae family, as they take advantage of their replication machinery and often deactivate them or decrease their burst size .Chlorella variabilis, Frickel et al. [Paramecium bursaria, a symbiotic ciliate that hosts Chlorella algal species, can influence the abundance of chloroviruses. Finally, host resistance mechanisms also have the potential to deconstruct the whole microbial community by making virus-resistant cells resistant to grazing activity as well [The presence of species with which viruses do not directly interact can also affect them. These effects are referred to as indirect and they have been largely studied in ecology . Some exl et al.  demonstrl et al.  assessedl et al.  showed t as well .2 and salinity.There are many abiotic stressors in the environment that can make viruses unsuccessful. Some stressors can have strong direct effects on viruses when they are outside the host in their particle state through inactivation or destruction, but they can also modify viral life cycle traits by reducing burst sizes or lengthening latent periods. Abiotic stressors include biological, chemical and physical factors. Here, we discuss temperature, sunlight and UV radiation, nutrient concentrations, non-host particles, COStudies on the effects of temperature on viruses have yielded a variety of results depending on the viral species examined as well as on the time and/or spatial scale of the study. However, the general trend suggests that very high temperatures are detrimental to viral abundance .Micromonas pusilla was shown to be inactivated at temperatures between 40 °C and 95 °C [Chaetoceros tenuissimus were negatively affected by a range of different temperatures. Also, strains of the alga Phaeocystis globosa showed different sensitivity to temperature changes. While one strain became sensitive at temperatures higher than 35 °C and completely inactive at 45 °C, another strain became sensitive and inactive at 10 °C lower, respectively. Interestingly, they also characterized both strains and found that while the former one was larger in size, with both its genome and proteins being more complex, the latter one had a shorter structure but was also associated with a longer latent period and more specific infections [Chlorella variabilis and its virus PBCV-1 experience contrasting growth rates depending on the temperature they are exposed to. On the one hand, Chlorella variabilis population grew slower  than when they infected hosts during the light period (day time) or at the beginning of the dark period. On the other hand, studies on DNA and RNA viruses that infect the raphidophyte Heterosigma akashiwo showed that their latent periods were of similar duration in darkness as in the light [Solar and UV radiations are possibly the most important abiotic stressors for algal viruses as their hosts are photosynthetic organisms that rely on light for energy and survival. Algal viruses are therefore in constant exposure to sunlight and UV radiations . The negdarkness , the Chlthe dark , or the ut light . Moreoveut light  found thhe light ,92.Heterosigma akashiwo, HcV and HcRNAV, both of which infect the dinoflagellate Heterocapsa circularisquama [Jacquet and Bratbak  looked arisquama .Chlorella-like algae, ATCV-1 and CVM-1, and a virus that infects Chrysochromulina parva, CpV-BQ1, showed that decay rates are the highest during summer and the lowest during winter. This might also be explained by seasonal temperature changes [The negative effects caused by sunlight and UV radiation are, however, seasonal as light intensity is stronger and lasts longer in summer than in winter. A long-term study of two viruses that infect  changes .Chlorella variabilis hosts had low C:P ratios (carbon to phosphorus). Other studies that have used the algae Emiliana huxleyi and its viruses as model systems have shown that viral production increases in high nutrient concentrations and N-depleted conditions but that it is 70% lower in P-depleted environments [Micromonas pusilla, P limitation led to a 150% prolongation of the latent period and an 80% reduction in viral burst sizes [Phaeocystis pouchetii [Concentrations of inorganic nutrients such as phosphorus (P), nitrogen (N), silicates (Si) or iron (Fe) determine and limit algae growth in all aqueous systems . Becauseronments . In virust sizes . Anotherst sizes . The comouchetii . Furtherouchetii  showed touchetii  reportedThe presence of non-host particles in the environment, such as clay sediments or host debris, can have direct or indirect effects on the success of a virus. Due to the peptide structure that allows viruses to attach, their binding affinity is fairly high and they can sometimes adsorb to particles that are not their hosts . These iPhaeocystis globosa are disrupted could be a cause of death in viruses. TEP are very sticky particles that exhibit the characteristics of gels and consist predominantly of acidic polysaccharides. They are present in freshwater as well as marine systems and they can be formed abiotically from dissolved matter or through dead phytoplankton. Large quantities of viruses, from 10–80%, were adsorbed to TEP in Phaeocystis globosa mesocosms [There were reports in the 1990s suggesting that phytoplankton bloom formations cause an increase in DOM (dissolved organic matter) which in turn leads to an increased number of bacteria and a high non-host absorption of viruses . Baudouxesocosms . Mari etesocosms  also con2 concentrations and the associated low pH have been linked to higher viral decay in aquatic systems. Larsen et al. [2 concentrations. Burst sizes have also been shown to be negatively affected in EhVs and PgVs, being reduced from 2890 to 2032 viruses per host cell [2 concentrations are due to less available energy for viral replication within the host cell [High COn et al.  as well n et al.  found thost cell  and by aost cell , respectost cell .Few studies have focused on the effects of salinity on algal viruses. While Guixa-Boixareu et al.  found anViruses might be characterized by a rapid evolutionary potential due to their high standing genetic variation, large population sizes, short generation times, high mutation rates and small genome sizes. They are likely able to quickly evolve and adapt to conditions that make them unsuccessful. Host specificity, latent period and burst size are virus traits that might therefore be strongly selected. As viruses depend on their hosts to reproduce, the evolution of these viral traits depends highly on host state and density. For example, viruses have evolved broader specificities and more extended latent periods in areas with low host abundance or shorter latent periods and larger burst sizes when their hosts are fast growing .Emiliana huxleyi and its virus. Regarding extrinsic factors that affect viruses and make them unsuccessful, the most notable adaptations are linked to resistance mechanisms against solar and UV radiations. Many studies have shown that viral replication inside the host cell occurs during the day but that cell lysis and the subsequent release of viruses occurs at night time. Thus, viruses encounter and infect new algal hosts when their major abiotic stressor is not present. Replicating during the day also gives viruses the opportunity to use the alga’s photosynthetic products as energy for replication and assembly [Emiliana huxleyi are characterized by a diel pattern with more progeny being produced during dusk; or Derelle et al. [Host resistance is an important host trait that determines virus reproduction. Host defences make viruses unsuccessful as they lower or inhibit their infectivity abilities. However, experimental evolution studies of host–virus population coevolution have shown that viruses are also able to quickly evolve counter adaptations and overcome host resistance. This in turn has been shown to lead to an arms race by which adaptations in each species triggers new adaptations in the other, and so forth . On muchassembly ,74. Aligassembly   across species. The reconstruction of evolutionary changes over time using approaches from resurrection ecology in combination with (meta)genomic analysis and data on the biotic and abiotic changes could help us identify disadvantageous conditions and how viruses have adapted to them . The iso


Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests. In the field, plant virus infections are often unmanaged or managed incorrectly owing to difficulties in diagnosis. Rapid diagnosis of a plant viral infection and therefore effective containment of the infection is required to minimize the spread of disease to other plants and limit crop losses. Unfortunately, for many plant viruses this is not practical as the methods of detection are either serology which depends on a specific antibodies (many of which are not available) or nucleic acid extraction, amplification and genome sequencing. Both of these techniques require transporting samples to a distant laboratory, adding time and expense to diagnosis. Consequently, there is a requirement for more efficient, quick and cheap tests that are capable of reliably identifying plant virus infections.Food security is a global challenge; over 6% of global food production is lost due to pathogens including viruses2. However, there are a number of limitations associated with their use as in-field diagnostics. The generation of an antibody generally requires the availability of significant quantities of the purified target virus, and this can be difficult or impossible to obtain. Antibodies are also expensive to generate and manufacture, and they can experience significant stability issues in the relatively harsh environments in which an in-field diagnostic test would be used. For this reason, we chose to explore the use of Affimer reagents as an alternative, non-antibody binding reagent4. Affimer proteins are novel affinity reagents which mimic the molecular recognition of antibodies, but can be isolated quickly and produced recombinantly. They are synthetic and are based on either a human cystatin A (type I) or a plant-derived phytocystatin consensus scaffold (type II). Affimer proteins are small , stable (70 °C < Tm < over 100 °C), monomeric proteins that lack disulphide bonds3. Their structure consists of a single α-helix and four β-strands, with the molecular recognition sites being located within two variable loops, each up to nine amino acids in length3. Their advantages include stability and their relatively inexpensive, simple production. Large phage display libraries (~1.3 × 1010 variants) have been generated4 and screened against hundreds of target proteins. A wide range of Affimer binders have now been identified from these phage libraries4 to a wide range of targets and have been successfully used for applications as diverse as molecular research tools9, biosensors6 and immunodiagnostics7. Importantly, Affimer proteins have been shown to be highly specific, and thus able to distinguish between closely-related targets8.Antibodies (where available) have been used for detection of plant viral infections9). Thus, many economically important and highly pathogenic plant viruses accumulate to only low titres in infected plants before killing them. There are also very significant biocontainment issues associated with producing the large amounts of an infectious entity that are typically required for antibody production. Virus-like particles (VLPs) can potentially be produced more easily and to a much higher concentration than the parent virus, and because VLPs are produced in the absence of the viral genome, and thus cannot be infectious, they are a fundamentally safer alternative to the use of WT virus. As a result, in recent years VLPs have been exploited in biotechnology for antibody production and many other applications11.In addition to the challenges of using an antibody as a diagnostic reagent, there are also significant problems with using wild-type (WT) infectious viruses to produce those antibodies (or any other binding reagent). Perhaps unsurprisingly, there is a lack of correlation between the pathogenicity of a virus and the level to which it accumulates in the tissues of infected plants  of CPMV are readily produced and purified12 using a well-established expression system. In addition, there is a large amount of structural information for both WT CPMV and CPMV eVLPs16. Using CPMV eVLPs we have identified an anti-eVLP Affimer and characterized its affinity for WT, infectious CPMV. We also determined the structure of CPMV bound to the selected Affimer using cryo-electron microscopy (cryo-EM). Finally, we demonstrate that the selected Affimer can detect the presence of CPMV directly within crude extracts of infected leaves, showing that the eVLP/Affimer combination is a potential route to the development of new in-field diagnostics.Here we sought to investigate whether VLPs could be used to generate an Affimer reagent that would be cross-reactive to a WT virus. For this analysis we used Cowpea Mosaic Virus (CPMV), a plant virus from the order 16. CPMV has a bipartite ssRNA genome . Each of the two genomic segments is encapsidated separately producing three fractions named for their sedimentation behavior in density gradients: CPMV-B(ottom) (containing the larger RNA-1), CPMV-M(iddle) (containing RNA-2) and a relatively small amount of naturally occurring empty particles (CPMV-T(op)). The protein components of CPMV-B, CPMV-M, CPMV-T and a recombinantly-expressed empty VLP (eVLP-CPMV) are almost identical structurally .CPMV is an icosahedral virus composed of 60 copies of both the Large (L) and the Small (S) coat protein subunitlly Fig. 13–16 witPMV Fig. . An AffiISA Fig. . To confISA Fig. . Seven cISA Fig. , were seISA Fig. . These wTo analyse the binding mechanism of CPMV to an Affimer reagent we chose to determine the structure of an Affimer:CPMV complex using cryoEM. Initially all four Affimer proteins were analysed using negative stain EM in complex with CPMV to find the Affimer reagent most suitable for cryoEM. Anti-CPMV Affimer 3 was selected as it did not cause appreciable aggregation or clumping of CPMV particles in negative stain screens. A negative stain dataset was collected and a preliminary 3D reconstruction was produced in RELION. However, the resolution was not sufficient to visualise the bound anti-CPMV Affimer 3, probably due to the size of the Affimer (11 KDa).18. To overcome this issue CPMV was applied to lacey carbon grids overlaid with a thin (<3 nm) layer of carbon. This causes the CPMV particles to be immobilized on the carbon support, anti-CPMV Affimer 3 was subsequently applied, excess solution was removed and the grid was finally washed prior to blotting and plunge freezing (see Methods and18 for more details). This method resulted in high quality cryoEM grids with particles that were evenly distributed and thus ideal for data collection  of samples of the following VLPs; Murine Norovirus (MNV), Satellite Tobacco Necrosis Virus (STNV), Hepatitis B (Hep B) and Potato Leaf Roll Virus (PLRV) using a biotin-streptavidin linkage, and analyzed the ability of the anti-CPMV Affimer 3 to recognize them using an ELISA Fig. . Anti-CPTo further test the suitability of anti-CPMV Affimer 3 for future in-field diagnostics, we tested the ability of anti-CPMV Affimer 3 to detect CPMV in crude extracts from infected leaves using a sandwich ELISA Fig. . In the Blind testing was performed in which twelve leaves were randomly numbered 1–12 . The leaves were extracted and analysed using the optimised sandwich ELISA conditions described above . This extraction and sandwich ELISA was repeated three times, using a fresh sample of the numbered leaf each time Fig. . The ELI20. Current methods for detection are almost exclusively antibody-based systems such as ELISAs or similar technologies2. Such methods are often used in lateral flow devices which allow the detection of antigens in crude samples via a colour reaction; such devices are suitable for in-field deployment. Due to difficulties in production of monoclonal antibodies the majority of available plant virus antibodies used for diagnostic tests are polyclonal22, increasing the likelihood of false positive results. The use of antibodies has a number of disadvantages due to the expense of production but, more importantly, antibodies are not available for a number of important plant viruses. This is primarily due to the difficulty in production of WT viruses to high concentrations and sufficient purity but also due to the biocontainment issues involved with propagating WT viruses. Other techniques for plant virus detection include nucleic acid extraction and analysis23. This is usually based on polymerase chain reaction (PCR) followed by detection to analyze the genetic content of the virus26. The use of PCR for detection requires DNA sequencing therefore specialized personnel are required and samples must be sent to a laboratory. Due to the complex instrumentation and time-consuming nature of these techniques there is a strong interest in developing new systems for detection of plant pathogens2. There are a large number of research groups tackling this need and a number of commercial kits are available.There is a requirement for more robust and sensitive technologies for the detection of plant virus infections, as accurate detection is critical for farmers to implement control strategies and prevent disease outbreaksTo overcome the disadvantages outlined above, we have demonstrated eVLPs can be successfully used to isolate Affimer reagents. Using a synthetic binder, such as an Affimer reagent, has a number of benefits compared with classic antibodies. For example, the most efficient and selective binders can be selected. This reduces the possibility of false positive results which is a common occurrence when using polyclonal antiserum. Additionally, Affimer reagents are cheap and relatively simple to produce. The use of VLPs as opposed to WT virus is also advantageous owing to the ease with which large quantities of VLPs can potentially be produced. In addition, isolation of eVLPs does not require live virus production therefore negating any biocontainment issues.The results presented here demonstrate that we have identified an Affimer reagent which binds to WT and recombinant CPMV Fig. . We have14.CPMV and CPMV eVLPs were produced as described in17. The secondary antibody, anti-rabbit HRP , was used at 1/10 000. Blots were developed using enhanced chemiluminescence (ECL) solution (Amersham).CPMV samples were boiled in SDS-PAGE sample buffer . Samples were separated by 8% SDS-PAGE, transferred to nitrocellulose, blocked with 1× PBS, 10% dried milk, 0.1% Tween 20, and probed with anti-CPMV at 1/20003.Phage display screening was performed as previously described3.Phage ELISA screening was performed, as previously described, on 24 randomly selected clones from the final pan round~45 e−/Å2. The final object sampling was 1.4 Å per pixel. A total of 2,980 exposures were recorded using the EPU automated acquisition software on a 17 Hz FEI Falcon II direct electron detector. Each exposure movie had a total exposure of 2 seconds and contained 35 images.Ultrathin carbon film on lacey carbon support film 400 mesh grids (Agar Scientific) were glow discharged for 30 seconds (Pelco easiGlow). The grids were prepared using a Leica GP freezing device at 8 °C and 95% relative humidity by placing 3 μl of 0.1 mg/ml CPMV on to the grid and incubating for 30 seconds. Excess liquid was manually blotted prior to placing 3 μl of 0.01 mg/ml anti-CPMV Affimer 3 onto the grid and incubating for 30 seconds. Excess liquid was manually blotted and 3 μl of 100 mM sodium phosphate buffer, pH 7.0 was placed on the grid to wash away any unbound anti-CPMV Affimer 3. The grid was blotted automatically using the Leica GP freezing device prior to plunge freezing in liquid ethane cooled by liquid nitrogen. Data was collected on a FEI Titan Krios  transmission electron microscope at 300 kV, using an electron dose of 27 and the contrast transfer function of each determined using gCTF28; any images showing signs of astigmatism were discarded. Approximately 1,000 particles were manually picked and classified using reference-free 2D classification in Relion (v1.4)29. The resulting 2D class average views were used as templates for automated particle picking using gAutomatch30  criterion33 as 3.44 Å  filtered to 60 Å. To correct for mechanical drift, beam-induced movement and radiation damage, statistical movie processing and particle polishing procedures were implemented4.Anti-CPMV Affimer 3 was produced and labelled with biotin as previously describedMaxisorb plates (Nunc) were coated with virus samples (10 µg/ml) overnight at 4 °C and then blocked with 2x casein blocking buffer (Sigma) overnight at 37 °C. The plates were washed once and incubated with 5 µg/ml biotinylated anti-CPMV Affimer 3 for 1 h at room temperature. Subsequently the plate was washed once and bound anti-CPMV Affimer 3 were detected by a 1:1000 dilution of HRP-conjugated streptavidin (Pierce) for 1 h at room temperature. Following 12 times washing Affimer 3 binding was visualised with 3,3′,5,5′ -tetramethylbenzidine (TMB) (Seramun) and measured at 610 nm.N. benthamiana leaves were produced by agroinfiltration of 3-week-old plants with pNinP-S1NT35 and pEAQ-RNA-213. Infiltrated leaves were harvested 7 days post infection. The uninfected negative control was grown alongside. Leaves were frozen at −20 °C prior to analysis.CPMV-infected Maxisorb plates (Nunc) were coated with anti-CPMV Affimer 3  at 4 °C, washed three times and then blocked with 1x casein blocking buffer (Sigma) for 1 hour at room temperature. The plates were washed three times and incubated with 25 µl purified CPMV or leaf extract  for which 80 mg of leaf is extracted into 800 μL solution.CPMV was extracted from leaf samples using two methods. The first method of extraction was to grind 8 mg of leaf in 80 μL of PBS containing complete EDTA-free protease inhibitor cocktail (at the manufactures recommended concentration) for five minutes, prior to spinning down at 17000 The concentration of CPMV in infected leaves was estimated using a calibration curve generated using purified CPMV . The CPMV concentrations in each extracted leaf sample were calculated , as there was 100 mg of leaf per ml, 50 μg/ml CPMV in the extracted solution is equivalent to 0.5 μg extracted CPMV per mg of leaf.


Regional variation in medical care costs can indicate heterogeneity in clinical practice, inequities in access, or inefficiencies in service delivery. We aimed to estimate regional variation in medical costs for people living with HIV (PLHIV), adjusting for demographics and case-mix.We conducted a retrospective cohort study using linked health administrative databases of PLHIV, from 2010 to 2014, in British Columbia (BC), Canada. Quarterly health care costs (2018 CAD) were derived from inpatient, outpatient, prescription drugs, antiretroviral therapy (ART), and HIV diagnostics. We used a two-part model with a logit link for the probability of incurring costs, and a log link and gamma distribution for observations with positive costs. We also estimated quarterly utilization rates for hospitalization-, physician billing- and prescription drug-days. Primary variables were indicators of individuals’ Health Service Delivery Area (HSDA). We adjusted cost and utilization estimates for demographic characteristics, HIV-disease progression, and comorbidities.Our cohort included 9577 PLHIV . Adjusted total quarterly costs for all 16 HSDAs were within 20% of the provincial mean, 8/16 for hospitalization costs, 16/16 for physician billing costs and 10/16 for prescription drug costs. Northern Interior and Northeast HSDAs had 38 and 44% lower quarterly non-ART prescription drug costs, and 2 and 5% higher quarterly inpatient costs, respectively.We observed limited variation in medical care costs and utilization among PLHIV in BC. However, lower levels of outpatient care and higher levels of inpatient care indicate possible barriers to accessing care among PLHIV in the most rural regions of the province.The online version of this article (10.1186/s12913-019-4391-8) contains supplementary material, which is available to authorized users. Regional variation in health care costs after adjustment for demographic and clinical factors can be indicative of inequities or a lack of well-defined clinical practice , 2. ThisLike other settings with concentrated HIV epidemics, there is substantial regional heterogeneity in the HIV epidemic within British Columbia (BC), demonstrated by differences in rates of new diagnoses, mortality rates, and availability of HIV-testing and harm reduction services , 11. HeaTargeting geographic regions for structural improvements is more straightforward for health policy makers, given the organization of health care delivery, and potential barriers to access driven by geographic factors , 18, 19.Our aim was to quantify regional variation in medical care costs for PLHIV. We generated disaggregated estimates of costs and utilization rates for hospitalizations, physician billings and drug dispensations (both with and without the costs of ART medications) to identify potential associations between components, particularly in regions where lower levels of outpatient care could be leading to higher levels of inpatient utilization.We conducted a retrospective cohort study using linked, provincial health administrative databases and disease registries, including the BC Centre for Excellence in HIV/AIDS drug treatment program and virology registries  and CD4 tests) , the BC Our primary outcome variable was the quarterly cost of medical care; calculated as the sum of inpatient, outpatient (physician fee-for-service billing claims), prescription drug costs (ART and non-ART), and diagnostic testing costs for each individual in our cohort. We estimated medical costs for PLHIV including the costs of ART medication, excluding ART medication, as well as costs of ART medication only for those receiving ART. We presented each of these analyses separately, as differential rates of ART uptake across HSDAs, combined with the high relative costs of antiretroviral medication, could potentially obscure differences in health care costs that indicated divergence from clinical practice standards or higher intensity of service provision among PLHIV. The methods for deriving these costs have been described previously , 28. We To support our primary analysis, and to provide further context as to which health care components showed greater regional differences among PLHIV, we estimated costs and utilization rates for hospitalizations, physician billing records and non-ART prescription drugs, respectively. We defined utilization outcomes as hospitalization days (days with an inpatient record), physician billing days  - chosen to reflect the frequency of contacts rather than the number of individual MSP billing line items - and drug days prescribed .Our primary independent variables were a set of quarterly indicator variables for an individual’s primary HSDA. We assigned one representative HSDA to each person in each quarter, depending on where an individual accessed health care services. We assigned time-varying indicators of HSDA due to high rates of intra-provincial migration for treatment among PLHIV , 30. Whe10 plasma viral load curve (AUC pVL) for 12-months prior to the start of the quarter  to capture individuals’ cumulative viral load over the previous 12-months. CD4 counts were taken from the most recent test prior to the start of the quarter, with previous test results carried forward for any missing observations, otherwise CD4 count was classified as unmeasured. AUC pVL measurements were also carried forward until the next non-missing observation, or the end of follow-up, otherwise AUC pVL was classified as unmeasured.We adjusted for a range of clinical factors, including measures of HIV disease progression  and ART status ). Additionally, we included the area under the logDemographic control variables included: age, sex, calendar year, transmission risk group, including: men who have sex with men (MSM); people who inject drugs (PWID); MSM who inject drugs (MWID); heterosexual/other (HET), year of diagnosis, grouped as: pre-1996; 1997–99; 2000–03; 2004–07; 2008–14, and two separate measures of medical comorbidity, the drug prescription-based Chronic Disease Score (CDS) , and theTo estimate quarterly health care costs, we fit a two-part generalized linear model (GLM) with logit link and binomial distribution for the probability of an individual incurring any costs, and a log link with gamma distribution for observations with non-zero costs . We estiWe estimated utilization rates for each component, using a two-part generalized linear model (GLM) with logit link and binomial distribution for the probability of an individual having any utilization, and a log link and negative binomial distribution for observations with non-zero utilization.We repeated our primary analysis using representative HSDAs assigned by highest cost, rather than highest frequency of visits, in a given quarter. We conducted additional sensitivity analysis removing costs for services received through the Provincial Health Services Authority , which were otherwise assigned to an individual’s representative HSDA, to determine if estimates for some HSDAs were disproportionately affected.We created our analytical sample in SAS 9.4, and conducted statistical analysis in Stata 14.1.Our sample included 9577 individuals, with 140,137 person-quarters of observation between January 1, 2010 and March 31, 2014. Our cohort was 20% female, with a median age of 45.5 years at baseline, 22.9% were known to be PWID and 28.5% MSM (Table Quarterly medical costs, adjusting for clinical and demographic factors and including ART, ranged from $3775 $3407, $4143] in Thompson Cariboo, to $4912  in East Kootenay, with all 16 HSDAs falling within 20% of the overall provincial mean (Fig.43 in ThoAdjusted mean hospitalization days ranged from 0.22 0.09, 0.34] in Kootenay Boundary to 0.51  per person-quarter in East Kootenay, and 10/16 HSDAs were within 20% of the overall mean (Fig. 4 in KootAdjusted mean days with a physician billing record ranged from 3.65  for PLHIV in the Northern Interior, to 5.87  per person-quarter for PLHIV in Central Vancouver Island, and 15/16 HSDAs were within 20% of the adjusted overall mean (Fig. Adjusted prescription drug days ranged from 103  in the Northeast, to 197 128, 266] per person-quarter in East Kootenay, and 14/16 HSDAs were within 20% of the adjusted overall mean (Fig. 8, 266 peAssigning HSDA by cost rather than frequency of contacts produced minor changes in estimates, with the largest change occurring in the Northwest HSDA, where adjusted quarterly costs were reduced to $3798 $3181, $4415] from $3989 . Removing PHSA costs from our analysis reduced adjusted quarterly costs to $3909 , from $4311  in the Northeast HSDA, and all other changes were smaller in magnitude (Additional file 81, $4415Our results showed moderate regional variation in medical costs for PLHIV in British Columbia, adjusting for demographic variables and case-mix. For estimates of total costs, including the costs of ART medication, costs per person-quarter in all 16 HSDAs were within 20% of the overall provincial mean, and cost differences between the highest and lowest cost HSDAs were $948  (Fig.Variation in annual health care costs in the general population was previously estimated to be 21% between the highest- and lowest-spending HSDAs in British Columbia, after adjustment for age, sex, aggregated diagnosis group, and health service environment . In the That we observed moderate regional differences in non-ART medical costs among PLHIV, after adjusting for demographics, disease progression and other comorbidities, suggests that standards of care were not substantially different across regions for PLHIV. This may be due, in part, to the maturity of the HIV care infrastructure in the province. Previous studies of inter-provincial variation in HIV testing rates have suggested that coordinating all HIV care in the province through a single center may produce better outcomes, by allowing for closer monitoring of PLHIV and better control over standards of care , and regIn our analysis of the individual components of medical costs and utilization, it was noteworthy that PLHIV in Northern Interior and Northeast HSDAs had 38 and 44% lower prescription drug costs, 15.3 and 9.1% lower physician billing costs, and 2.3 and 4.9% higher hospitalization costs, respectively. PLHIV in both HSDAs were below the overall mean in physician billing days, as well as prescription drug days. Furthermore, PLHIV in these HSDAs had lower levels of ART uptake than the provincial average (Table There is a substantial literature examining the interaction between the use of inpatient, outpatient, and prescription drug utilization. Previous studies have examined changes to insurance coverage or increased deductibles resulting in lower prescription drug use –44, the This analysis had several limitations. First, HSDA assignment was subject to some degree of misclassification due to the possibility of individuals receiving care in multiple HSDAs in a given quarter or missing information on HSDA, however, our results were robust to alternative HSDA assignment methods in sensitivity analysis (Additional file We estimated the magnitude of geographic variation in medical costs and utilization rates among PLHIV in British Columbia from 2010 to 2014, using linked, individual-level data to characterize regional differences. Despite substantial regional differences in demographic composition, ART uptake, and HIV care engagement, we found that variation in medical costs among PLHIV, both including and excluding ART medication, was comparable to the overall population of British Columbia, adjusting for demographic variables and case-mix. While there was generally modest variation among HSDAs for hospitalizations, physician billings and non-ART prescriptions, our results indicated possible substitution between outpatient care and inpatient care among PLHIV in the most rural regions of the province.Additional file 1:This supplement contains the data plotted in Figs.