Apis mellifera) health is threatened globally by the complex interaction of multiple stressors, including the parasitic mite Varroa destructor and a number of pathogenic viruses. Australia provides a unique opportunity to study this pathogenic viral landscape in the absence of V. destructor. We analysed 1,240A. mellifera colonies across Australia by reverse transcription-polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS). Five viruses were prevalent: black queen cell virus (BQCV), sacbrood virus (SBV), Israeli acute paralysis virus (IAPV) and the Lake Sinai viruses (LSV1 and LSV2), of which the latter three were detected for the first time in Australia. We also showed several viruses were absent in our sampling, including deformed wing virus (DWV) and slow bee paralysis virus (SBPV). Our findings highlight that viruses can be highly prevalent in A. mellifera populations independently of V. destructor. Placing these results in an international context, our results support the hypothesis that the co-pathogenic interaction of V. destructor and DWV is a key driver of increased colony losses, but additional stressors such as pesticides, poor nutrition, etc. may enable more severe and frequent colony losses to occur.Honeybee ( Unravelling the complexity of increased honeybee (Apis mellifera) colony losses has been a particular focus of current research because of the economic value of honey production and crop pollination. Recent research implicates multiple stressors, including parasites, pathogens, chemicals, poor nutrition, climate and beekeeper management6. Arguably the most significant of these factors is the parasitic mite, Varroa destructor, which feeds on developing honeybee larvae and adult bees and transmits lethal viruses11. Since shifting host from the Asian honeybee (A. cerana) to A. mellifera in the mid-20th century, this mite has spread worldwide causing significant honeybee losses12. The emergence of V. destructor has also significantly altered the viral landscape in honeybee populations globally by increasing virus transmission and causing selection of more virulent virus strains16. The combination of V. destructor and viruses is now considered the major cause of global colony losses18, but uncovering the importance of viruses alone remains a significant challenge due to the ubiquitous presence of V. destructor.Increased awareness of the importance of pollination for functional ecosystems and global food security has seen a surge in research into pollinator health and population declines19. The expansion of V. destructor has significantly increased colony stress and elevated the importance of viruses in colony losses18. This has led to increased understanding of the role of pathogenic viruses within colonies and identified deformed wing virus (DWV) as a major co-pathogen involved in colony losses in association with V. destructor22. Although DWV was only discovered following the spread of V. destructor, it is thought to exist naturally at low prevalence in all honeybee populations23. This hypothesis was recently tested following the arrival of V. destructor in Hawaii14 and New Zealand24, showing that DWV prevalence increased while strain diversity decreased. However, data for historical virus prevalence before V. destructor introduction is rare and typically predates the discovery of DWV26.Viruses typically persist as covert infections in honeybee populations, but outbreaks occur when colonies become stressed or encounter certain environmental conditionsA. mellifera populations in the world that are not infested by V. destructor. Most of these are small island populations with small-scale beekeepers. Australia is the exception by having a large commercial-scale beekeeping industry that is exposed to similar agrochemical and pathogen stressors as North America and Europe, but remains free of V. destructor. Australia has also not experienced the increased colony losses reported overseas. Australia’s mite-free status therefore provides an excellent stage for comparing viral landscapes with and without the confounding effects of V. destructor, hence providing insights into the causes of global colony losses. The last extensive studies of honeybee viruses in Australia relied on serological methods to examine apiaries during 1980 and 198327. This identified the presence of sacbrood virus (SBV), black queen cell virus (BQCV), Kashmir bee virus (KBV), chronic bee paralysis virus (CBPV) and cloudy wing virus (CWV). Since then honeybee virology in Australia has continued sporadically29, but remains under-researched and molecular screening for viruses has been non-existent.There are now only a handful of 30. There are no reported symptoms , but there is speculation that Australian honeybees have covert infections of DWV and recent overseas studies claim to have detected DWV-A in imported bee samples32. There are also other viruses linked with V. destructor including acute bee paralysis viruses (ABPV), Israeli acute paralysis virus (IAPV) and slow bee paralysis virus (SBPV) that have not been tested for in Australia using molecular methods. Furthermore, several new viruses such as the Lake Sinai viruses (LSV1 and LSV2) have been recently identified overseas using next-generation sequencing (NGS)33, of which there is no information for in Australia. NGS technology has been applied in multiple insect systems for virus discovery38 and presents a valuable tool for characterising the viral landscape of Australian honeybees.With the help of molecular methods it is possible to expand our knowledge of the diversity of viruses in Australia. Of particular note is the apparent absence of the DWV complex V. destructor. Our results showed that in the absence of V. destructor there was still a significant prevalence and diversity of honeybee viruses, but this did not include the DWV complex or several other viruses that have been linked to V. destructor pathogenicity. The implications of these results for unravelling the complex epidemiology of global honeybee colony losses are discussed.In this paper, we characterised the Australian honeybee viral landscape by both direct detection using reverse-transcription PCR (RT-PCR) and a NGS approach to deliver wider coverage and sensitivity. We hypothesised that there would be lower virus prevalence in Australian honeybees and DWV would be absent, compared to honeybee populations in the presence of Honeybees were analysed from 1,240 hives representing 155 independent apiaries across five chosen regions , with four viruses co-infecting between 3% and 17% of samples in Regions 1 and 2 and all five viruses detected in one Region 1 sample (SA). However, five other honeybee viruses were not detected in any region; DWV, SBPV, ABPV, KBV and CBPV, which included an additional 124 brood samples tested for DWV and SBPV.NGS was used to gain a more complete picture of the honeybee virus landscape in Australia by identifying virus genomes in nine pooled adult bee samples from Regions 1–4. This approach increased our sensitivity for detecting low prevalence viruses and avoided potential problems with primer specificity. All five viruses detected by RT-PCR were identified in each pooled sample, with a high number of reads covering the full genome in most cases. Normalised read counts also provided an indication of relative infection levels for each virus Table . Consist39. A number of sequence reads also mapped to the KBV reference genome, but still had higher identity with IAPV.A small number of sequence reads mapped to the DWV and SBPV and CBPV reference genomes when using a lower similarity threshold of 0.7 Table . Manual A. mellifera population has been identified that has confirmed the absence of V. destructor and DWV, and also has a large commercial-scale beekeeping industry. We distilled these studies into five stress profiles representing the spectrum of global A. mellifera populations to show that V. destructor and DWV are key factors in increased colony losses  with similar levels of divergence have also been identified from Europe and the USA42 and from two previous Australian studies44. Only partial gene sequences are available for these variants, but high similarity was found between the QLD, VIC/NSW-1 and VIC/NSW-3 variants with Cairns isolates (94–97%) and the NT variant was 99% and 97% similar to LSV5 and LSV3 sequences. Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) gene showed several of the variant LSV isolates formed a separate clade related to LSV1 isolates. We propose that this clade be consolidated with related LSV strains  and designated LSV3. The WA-2 and VIC/NSW-2 variant LSVs were distinct from all currently known LSV strains and 86% similar to each other. We propose these variants be designated to a new LSV group, LSV8.LSV1 and LSV2 are recently described viruses from the USA that have since been detected in Europe and have been also correlated with colony losses. The SA and TAS isolates were most similar to the original USA strains, while the remaining isolates separated into several distinct groups with some geographic clustering Fig. . De novoV. destructor. We found five honeybee viruses  that were common and, in contrast to our hypothesis, virus prevalence was still high despite the absence of V. destructor. Virus prevalence in this study was comparable with, or higher, than similar studies in Europe51, North America53 and Asia56. Differences in study design make direct comparisons difficult, but our data support that viruses can achieve high prevalence without V. destructor acting as a mechanical and biological vector. It is well established that honeybee viruses can persist as covert infections in colonies and high apiary level prevalence does not necessarily equate to high infection levels within hives57, but using NGS data as an indication of abundance suggests all five viruses were present at levels greater than expected for covert infections. It is uncertain how indicative these levels are of pre-Varroa levels or if other pathogens have induced high virus prevalence in Australia. The microsporidian parasites Nosema apis and N. ceranae have reported synergistic relationships with BQCV and potentially other viruses and could be driving the high virus prevalence in Australia59. Interestingly BQCV in this study was highest in the winter samples (August), which coincides with peak Nosema spp. infections60, and the winter BQCV isolates formed a distinct clade. Seasonality will certainly influence virus prevalence, such that longitudinal monitoring of viruses in Australia would further add to our understanding of viral dynamics in the absence of Varroa and DWV.This study provides the first comprehensive molecular-based analysis of Australia’s honeybee viral landscape and highlighted key contrasts with overseas landscapes affected by V. destructor14. While it is fundamentally difficult to prove complete absence of DWV, we have taken a weight of evidence approach that is consistent with the World Organisation for Animal Health (OIE) guidelines to support this conclusion. This approach not only considers the negative detection of DWV in our structured RT-PCR survey, but also includes the results of our sensitive NGS analysis, the lack of clinical signs of DWV-associated disease, limited pathways for virus introduction and the previous lack of detection in imported Australian bees by the USA using a NGS analysis52. Collectively, this evidence gives strong support for the absence of DWV in Australia. This finding contradicts two recent overseas studies that have reportedly detected DWV-A in acquired Australian bee samples32. In both studies, short sequences were recovered by quantitative PCR from a small sample set and these showed high similarity (>98%) to local DWV-A strains. Based on our results we believe these detections were likely false positives or contamination. It seems improbable for DWV to be detected in such limited samples and not detected in this wider study using equally sensitive methods. However, our NGS analysis did map several short sequence fragments (<200 nucleotides) to the DWV reference strains under low stringency conditions. While some of these fragments were within the accepted variation of the DWV complex (up to 20%), these sequences overall were significantly different (69–74%) to each DWV strain  and absence of ABPV in this population also supports a theory that these viruses occupy the same ecological niche, which leads to one virus dominating under different conditions69. For instance ABPV dominates in Europe, while IAPV dominates in Asia, Australia and the USA and KBV dominates in New Zealand. IAPV is still considered a serious pathogen of honeybees because of its high virulence, its occasional cause of natural honey bee mortality without an apparent synergist, and evidence that V. destructor is an effective biological vector70. However, the comparable prevalence of IAPV in Australia with overseas studies supports the hypothesis that, as a fast replicating virus, IAPV is not closely associated with V. destructor pathogenicity. Any A. mellifera brood killed by IAPV vectored by V. destructor, will die before emergence, thus preventing V. destructor offspring from leaving the capped brood cell18. Therefore, while V. destructor is capable of vectoring IAPV, the prevalence and abundance of this virus is most likely influenced more by other factors.Our study has also given the first direct confirmation of IAPV in Australia after being detected in the USA in imported Australian bees and being linked with Colony Collapse Disorder (CCD)41. With the detection of diverse Australian LSV strains it is apparent these viruses have had a long association with A. mellifera globally. In fact, a LSV variant was identified in a closed A. mellifera population on Norfolk Island, which has only had introductions of Australian honeybee stock44. In this study, LSV1 and LSV2 had consistently lower read abundance than the other detected honeybee viruses, suggesting a more stable coexistence with A. mellifera. In contrast, LSV2 has been recorded as the most abundant virus in US studies and associated with weak and collapsed colonies42. There is much to learn about this diverse group and further characterisation of LSV variants will help focus efforts on strains relevant to honeybee health. Furthermore, if LSV1 and LSV2 have long existed in the global honeybee population, it is surprising they have only recently been detected. Perhaps both viruses were identified previously using serological methods, but were renamed after being molecularly identified, as appears to be the case with IAPV and KBV. It has been noted that bee virus X (BVX) and bee virus Y (BVY), share multiple similarities with LSV1 and LSV2 but have not yet been genetically characterised71. Further comparison of the physical characteristics of LSV1 and LSV2 with BVX and BVY is warranted.LSV1 and LSV2 are also new detections for Australia and extend the distribution of this virus complex. These viruses were identified as common viruses in the United States in 2009 and since then a diversity of LSV strains have been identified in Europe and North AmericaV. destructor and delivered the first molecular analysis of honeybee viruses in Australia. It is clear from these findings that viruses can occur at high prevalence and abundance without the assistance of V. destructor mites and there is considerable viral diversity in this population. The absence of several viruses, DWV and SBPV in particular, is valuable biosecurity knowledge for Australia but additionally provides a unique opportunity for future comparative studies. Furthermore, it leads on to a testable hypothesis that V. destructor acting as a co-pathogen with DWV and interacting with environmental stressors is most likely to be the underlying driver of increased colony losses. Further examination and monitoring of the unique Australian A. mellifera population should aid our understanding of viral dynamics and pathogen interactions without the confounding effects of V. destructor or DWV and will contribute to global efforts to improve honeybee health.This study has provided valuable insight into the honeybee viral landscape in the absence of One-off samples from 155 apiaries totaling 1,240 hives were collected between August 2013 and April 2015 from five distinct regions in Australia  and cDNA generated using the Tetro cDNA synthesis kit (Bioline) following the manufacturer’s protocol using both random hexamer (50 ng/μl) and oligo (dT)18 (270 ng/μl) primers and RT incubation at 40 °C for 40 min. Each sample was tested for 10 honeybee viruses; sacbrood virus (SBV), black queen cell virus (BQCV), Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV-A and DWV-B), slow bee paralysis virus (SBPV), Lake Sinai virus 1 (LSV1) and Lake Sinai virus 2 (LSV2). PCR assays were carried out in 10 μl reactions containing 1 x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 1 U Taq DNA polymerase (New England Biolabs) and 1 μl cDNA template. PCR cycling conditions were 30 cycles of 95 °C (15 s), 56 °C or 58 °C (30 sec), 72 °C (40 s) and primers are given in Supplementary material Adult bee samples was split into four sub-samples of 50 bees and homogenized in 5 mL of 0.05 M potassium phosphate buffer. 500 μl of homogenate was cleared by adding 50 μl of diethyl ether and 100 μl of chloroform, vigorously shaking for 30 seconds and then centrifuging for 2 minutes at 7,000 de novo assembled in CLC Genomics workbench using default parameters and a minimum contig length of 1,000 nucleotides. Contigs were compared against the NCBI non-redundant protein database using BLASTx and contigs of interest were inspected manually to detect potential variant genomes. Phylogenetic analysis of viral genomes were done in MEGA772. Mulitple amino acid sequence alignments with relevant reference sequences were performed with MUSCLE and consensus maximum-likelihood phylogenetic trees were constructed using appropriate models for each virus and 1,000 bootstraps.Nine pooled adult bee samples from the nine sampling periods  were chosen for NGS. Pooled samples were created from 1 ml of 50 homogenized bees per apiary sample into a screw-cap centrifuge tube and adjusting with 0.05 M potassium phosphate buffer for a total volume of 20 ml. 3 ml diethyl ether and 3 ml chloroform were added, shaken vigorously and centrifuged at 6,000 rpm for 30 minutes (J-E Avanti centrifuge). Supernatants were transferred to Ultraclear SW28 tubes (Beckman Coultier) and centrifuged at 21,500 rpm for 3.5 hours at 4 °C (Beckman L-80 ultracentrifuge). Pelleted samples were dissolved in 1 ml of 0.05 M potassium phosphate buffer then passed through a 0.22 μm filter to remove bacterial contamination. We then mixed 340 μl of each filtered sample with 10 μl of RNase, 10 μl DNase and 40 μl of DNase I buffer and incubated at 37 °C for 2 hours. RNA was extracted from the treated samples using the Purelink viral RNA extraction kit. Illumina libraries were prepared using the TruSeq stranded mRNA library prep kit  and 100 bp paired-end sequences were generated on an Illumina HiSeq. 2500 run in rapid mode at the Biomolecular Research Facility . An average of 93 million paired-end reads were generated per library. Bioinformatics analysis of RNA-seq data was carried out with the CLC Genomic workbench . Raw data were quality trimmed and adapter sequences removed before the trimmed reads were mapped to reference virus genomes from the NCBI GenBank database of the honeybee viruses tested for by RT-PCR. Mapping parameters were first set with a length fraction of 0.9 and similarity fraction of 0.9, and a second mapping was run with the similarity fraction set to 0.7. Consensus sequences were manually inspected for genome coverage and similarity to mapped reference genomes using BLASTn. Trimmed data were also To place our results within the international context and the current debates on the causes of increased honeybee colony losses, we undertook a semi-systematic review of the literature to find comparable virus studies. Studies that presented prevalence data for multiple viruses across large areas or in relation to colony losses were included. The results from these studies were extracted into a summary table (Supplementary material Supplementary material


P < 0.06) as compared with all other ingredients. High delta values (P < 0.001) were observed in soybean meal for PDCoV (42.04 days) and TGEV (42.00 days). There was a moderate correlation between moisture content and the delta value for PDCoV  and TGEV . There was also a moderate negative correlation between TGEV survival and ether extract content . In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal. In addition to this, moisture and ether content appear to be an important determinant of virus survival in feed ingredients.Porcine Epidemic Diarrhea Virus (PEDV), Porcine Delta Corona Virus (PDCoV), and Transmissible Gastroenteritis Virus (TGEV) are major threats to swine health and contaminated feed plays a role in virus transmission. The objective of our study was to characterize inactivation of PEDV, PDCoV, and TGEV in various feed ingredient matrices. Samples of complete feed, spray dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and corn dried distillers grains with solubles were weighed (5 g/sample) into scintillation vials and inoculated with 1 mL of PEDV, PDCoV, or TGEV. Samples were incubated at room temperature for up to 56 days. Aliquots were removed at various time points followed by preparing serial 10-fold dilutions and inoculating in cell cultures to determine the amount of surviving virus. Inactivation kinetics were determined using the Weibull model, which estimates a delta value indicating the time necessary to reduce virus concentration by 1 log. Delta values of various ingredients were compared and analyzed as to their nutrient composition. Soybean meal had the greatest delta value (7.50 days) for PEDV ( Nidovirales, family Coronaviridae, and subfamily Coronavirinae [Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and the more recently discovered Deltacoronavirus [Coronaviruses belong to the order avirinae , and areavirinae . The subonavirus . Coronavonavirus . In humaonavirus . In swinonavirus ,5.Transmissible Gastroenteritis Virus has been identified as a cause of severe diarrhea, dehydration, vomiting, and high mortality in neonatal pigs since the 1960’s . ShortlySalmonella. Several research studies have evaluated the survivability of Salmonella in feed, along with the effect of processing on reducing its concentration in feed [Until recent years, the main concern involving feed safety has been the risk of contamination with  in feed –13. Duri in feed ,15. Subs in feed . With th in feed . Differe in feed –21. The  in feed .With the potential risk of post-processing contamination, feed mills are developing biosecurity plans to minimize the risk of corona virus transmission from feed, feed ingredients, and feed transport. To do this, identifying ingredients that are of greater risk of prolonging the survival of these corona viruses is essential for developing these biosecurity procedures. Previous research studies have shown that PEDV survival in feed varies depending on the specific ingredient, with soybean meal appearing to promote extended survival time . However™) cells. The NVSL strain of PDCoV and Purdue strain of TGEV  strains were propagated and titrated in ST (swine testicular) cells. The cells were grown in Minimum Essential Medium (MEM) with Earle’s salts supplemented with L-glutamine , 8% fetal bovine serum , 50 μg/mL gentamicin (Mediatech), 150 μg/mL neomycin sulfate , 1.5 μg/mL fungizone (Sigma), and 455 μg/mL streptomycin (Sigma). The maintenance medium for PEDV included Dulbecco's Modified Eagle Medium  with antibiotics, and 10 μg/mL of trypsin . For PDCoV, the maintenance medium consisted of MEM with antibiotics and 5 μg/mL trypsin (Gibco). Maintenance medium for TGEV consisted of MEM with antibiotics and 4% donor horse serum . The cells were washed thrice with phosphate buffered saline  before virus inoculation. After virus inoculation, the cells were incubated at 37°C for 1 h for virus adsorption using appropriate maintenance medium. Inoculated cells were incubated at 37°C under 5% CO2 for up to 8 days. The inoculated cells were observed for the appearance of virus-induced cytopathic effects (CPE). The CPE appeared at about 8 to 10 days post-infection for PEDV, and 5 to 6 days post-infection for PDCoV and TGEV. The cells were subjected to three freeze-thaw cycles (-80°C/25°C) followed by centrifugation at 2500 × g for 15 min at 4°C. The supernatant was collected and aliquoted into 50 mL centrifuge tubes  followed by storage at -80°C until used.The NVSL  strain of PEDV was propagated in Vero-81 . This diet did not contain any animal by-products (batch no. 831458). The feed ingredients including spray dried porcine plasma (SDPP), meat meal, meat and bone meal, blood meal, corn, soybean meal, as well as low, medium and high oil corn dried distillers grains with solubles, were obtained from the University of Minnesota feed mill at the Southern Research and Outreach Center . Samples of feed and feed ingredient were submitted to the Minnesota Valley Testing Laboratory  for chemical composition analysis . Standar4, 1.5 × 106, 6.8 × 106, respectively, was added to the appropriate vials and mixed thoroughly. Only one virus was added to each ingredient sample to prevent interactions between viruses in a single ingredient sample. All samples were stored at room temperature (approximately 25°C) for 0, 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days. The samples were stored uncovered and the humidity in the room was not controlled. This experiment was performed in triplicate with each of the viruses to produce three replicates for PEDV, PDCoV, and TGEV.All feed samples tested negative for PEDV, PDCoV and TGEV using real time RT-PCR. Five gram aliquots of complete feed and feed ingredients  were prepared in scintillation vials. One mL of either PEDV, PDCoV, or TGEV with initial titers of 3.2 × 102 until CPE appeared. Virus titers were calculated as TCID50/mL using the Karber method [In all experiments, the surviving virus was recovered in an eluent consisting of a 3% solution of beef extract in 0.05M glycine (pH of 7.5). The elution buffer was the same for each of the three viruses evaluated. The elution procedure has been used in previous experiments with calicivirus and was modified to be used in feed samples . The samr method . The hig50/mL) was modeled by GInaFiT [N is the surviving virus after the treatment expressed as (log TCID50/mL), N0 is the initial virus titer (log TCID50/mL), Delta (δ) is the time of the first logarithm decline for the virus titer population (days), and n is the shape parameter.Virus concentration data (log TCID GInaFiT . Weibull GInaFiT :Log(N)=2 (Adj. R2) as follows:m is the number of observations, j is the number of model parameters, and SSQ is the sum of squares.Three valid replicates were used to evaluate how well the model fit with the experimental data by calculating the adjusted RP < 0.05 was considered to be significantly different. The fixed effect was the feed ingredient being analyzed. Results for each virus were analyzed independently. A correlation analysis using the corr procedure of SAS  was done to determine potential associations between feed and feed ingredient chemical composition and the delta values for complete feed or each ingredient.An ANOVA test using the mixed procedure of SAS  was used to compare differences among delta values. Least squared means with Tukey adjustment were used to determine differences among treatment means; 2 values compared with those obtained with the log linear model. Delta values from the Weibull model (time necessary to reduce virus concentration in 1 log) were compared among feed and feed ingredient samples for PEDV, PDCoV and TGEV to characterize virus inactivation kinetics  and PDCoV  survival with moisture content. This suggests that increased moisture content may lead to increased virus survival, but this correlation was not observed for PEDV  and TGEV . This means that as predicted, as moisture content increases, so does virus survival. However, the same relationship was not observed in PEDV survival. Contradicting our hypothesis, there was no correlation between pH and virus survival. This is probably because our experiment was done in dry feed ingredients. Other experiments evaluating the impact of pH on virus survival that have shown an effect were performed in liquid samples [P < 0.05). This correlation was unexpected and only observed in TGEV survival.When comparing the inactivation kinetics of each virus, there is a similar trend for soybean meal to have the greatest virus survival. With such high survival of all three viruses in soybean meal, a correlation was done to determine what feed characteristic might cause this high survival. As stated in our hypothesis, moisture was one of the variables believed to have an impact on virus survival. This was somewhat confirmed in the correlation which determined a moderate positive relationship between PDCoV (r = 0.4823,  samples . In addiOne of the major limitations to this experiment was the addition of liquid media necessary to inoculate the feed samples with virus. The addition of 1mL of liquid media to 5 gram aliquots of feed or ingredient greatly increased the moisture content of the sample. As is evident from our results, moisture plays a significant role in virus survival and hence a change in moisture content will alter virus survival kinetics as opposed to that observed in completely dry feed. Though the moisture content was altered, the same amount of moisture was added to each feed ingredient making it possible to still compare virus survival among various ingredients. A second limitation to our experiment was the low virus titers used in the PEDV experiment. The concentration of PEDV used in the experiments was at the maximum titer that could be obtained for this virus by using the presented methods. Though the concentration of PEDV in the sample could be increase by adding more inoculum to each vial, this would also increase the moisture in the sample and contribute to the limitations previously stated. These low titers limited the amount of reduction that could occur in virus concentration, which then limited the variation we could observe among feed ingredients. We hypothesize that if we were working with higher concentrations of virus, we would have observed a statistically higher delta value for PEDV survival in soybean meal, as opposed to just numerically higher value. Having higher delta values for PEDV could then minimize the difference observed between PEDV and the other two coronaviruses. The second problem with the low virus titers is that they may not represent the virus titers observed in the field. A study measuring the residual amount of PEDV in a contaminated feed bin estimated these CT values between 19.5 and 22.2 . This amOverall, we conclude that the survival of PEDV, PDCoV, and TGEV varies based on feed ingredient. Out of the feed ingredients analyzed, soybean meal had greatest delta value in all three viruses, along with the highest concentration of remaining virus after 56 days of incubation. These results validated previous research demonstrating extended coronavirus survival in soybean meal. These results also suggest that soybean meal may be a risk factor for virus transmission if contamination occurs, but the likelihood of contamination has yet to be evaluated.


Caenorhabditis elegans. The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for Orsay virus infection, we designed a forward genetic screen that utilizes a virally induced green fluorescent protein (GFP) reporter. Following chemical mutagenesis, two Viro (virus induced reporter off) mutants that failed to express GFP were mapped to sid-3, a nonreceptor tyrosine kinase, and B0280.13 (renamed viro-2), an ortholog of human Wiskott-Aldrich syndrome protein (WASP). Both mutants yielded Orsay virus RNA levels comparable to that of the residual input virus, suggesting that they are not permissive for Orsay virus replication. In addition, we demonstrated that both genes affect an early prereplication stage of Orsay virus infection. Furthermore, it is known that the human ortholog of SID-3, activated CDC42-associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that VIRO-2 may be a substrate for SID-3 in C. elegans. A targeted RNA interference (RNAi) knockdown screen further identified the C. elegans gene nck-1, which has a human ortholog that interacts with TNK2 and WASP, as required for Orsay virus infection. Thus, genetic screening in C. elegans identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.Many fundamental biological discoveries have been made in  Caenorhabditis elegans, which shares many evolutionarily conserved genes with humans. We exploited the robust genetic tractability of C. elegans to identify three host genes, sid-3, viro-2, and nck-1, which are essential for Orsay virus infection. Mutant animals that lack these three genes are highly defective in viral replication. Strikingly, the human orthologs of these three genes, activated CDC42-associated kinase (TNK2), Wiskott-Aldrich syndrome protein (WASP), and noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) are part of a known signaling pathway in mammals. These results suggest that TNK2, WASP, and NCK1 may play important roles in mammalian virus infection.Orsay virus is the only known virus capable of naturally infecting the model organism  Caenorhabditis elegans has played a critical role in the discoveries of evolutionarily conserved processes such as RNA interference (RNAi)  , its usenfection , 3.Nodaviridae , a C. elegans ortholog of human Wiskott-Aldrich syndrome proteins (WASPs), which are essential in C. elegans for Orsay virus infection. A further targeted RNAi knockdown determined that nck-1, a C. elegans ortholog of the human noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1), was also essential for Orsay virus infection in C. elegans. The human orthologs of these three genes are part of a known kinase signaling pathway  reporter strain that is induced by Orsay virus infection  chemical mutagenesis, the goal was to identify mutants that fail to activate GFP expression after Orsay virus infection (Viro [virus induced reporter off] mutants). Mutants with this phenotype could be defective in either their ability to support Orsay virus infection or in the pathway that leads to transcriptional activation of GFP expression.To identify host factors important for Orsay virus infection in nfection . This stnfection , 10, fusde novo Orsay virus infection, yielding three independent Viro mutants  in Viro-1, and a mutation was identified in the B0280.13 gene  in Viro-2. The B0280.13 gene was subsequently renamed viro-2. Hereafter, these mutants are referred to as sid-3(vir9) and viro-2(vir10) mutants or strains.Approximately 8,000 EMS-mutagenized haploid genomes were screened. Initial Viro hits were rescreened to remove false-positive results  Diagram of EMS forward genetic screen using a GFP reporter strain. (B) Work flow for Viro screen using a GFP reporter strain. Download Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the sid-3(vir9) and viro-2(vir10) mutants yielded very low levels of Orsay virus RNA  on the viral nucleic acid stability on the plate during the course of an authentic infection. Thus, the “decay control” is an upper limit estimate of the amount of residual input Orsay virus RNA. In contrast, Viro-3 Orsay virus RNA levels were similar to that of the unmutagenized parent, suggesting that the defect in Viro-3 affects the pathway necessary for the transcriptional activation of the GFP reporter. In this study, we focused on analysis of sid-3(vir9) and viro-2(vir10) mutants.Orsay virus RNA levels in these strains were determined by real-time quantitative reverse transcription-PCR (qRT-PCR). The irus RNA . These lNematocida parisii, a microsporidial pathogen of C. elegans (sid-3(vir9), viro-2(vir10), and Viro-3 mutants with N. parisii. All three mutants still induced GFP expression in the intestinal cells, demonstrating that the GFP transgene was still functional and the virus-specific nature of the Viro phenotype  mutant strain, which is highly susceptible to Orsay virus infection. Approximately 50 F2 progenies that had the Viro phenotype and that did not support Orsay virus replication (data not shown) were identified. The F3 progenies were pooled, sequenced, and analyzed using the CloudMap bioinformatic pipeline (sid-3 (systemic RNAi defective) gene. This mutation leads to a premature stop after amino acid 911 of the SID-3 protein , harboring a >1-kb deletion was obtained from the Caenorhabditis Genetics Center  mutant in terms of both GFP expression and Orsay RNA levels (sid-3(ok973) mutant yielded little or no virus replication . These data clearly implicated sid-3 as the gene responsible for the defective GFP expression and reduced Orsay virus RNA levels.Several additional lines of evidence demonstrated that ing RNAi . RNAi kne, dpy-3 . sid-3 Rirus RNA . In addis Center  and crosA levels  and B. Slication . Finally F26F2.1  expressi F26F2.1  as did a10.1128/mBio.00940-17.3FIG S3 sid-3 and viro-2 mutant allele. Orsay virus replication was quantified by real-time qRT-PCR. ****, P < 0.0005. Download FIG S3, TIF file, 0.1 MB.Infection of CGC strains that carry independent Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00940-17.4FIG S4 P < 0.01; NS, not significant (P > 0.05). Download FIG S4, TIF file, 0.1 MB.RNAi knockdown of candidate causal genes in N2 strain. Real-time qRT-PCR of Orsay virus RNA levels after feeding RNAi knockdown of individual genes. **, Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the sid-3(vir9) mutant. This mutant form of SID-3 had previously failed to rescue the systemic RNAi defect in the sid-3(ok973) mutant  mutant . Animalscritical  and D. Icritical . The levcritical .10.1128/mBio.00940-17.5FIG S5 sid-3(vir9) strain on Orsay virus replication and GFP expression. (A) Imaging of Orsay virus-infected strains. Bars, 200 μm. (B) Real-time qRT-PCR of Orsay virus-infected strains. *, P < 0.05; NS, not significant (P > 0.05). (C) Quantification of SID-3::mCherry and SID-3K139A::mCherry expression through image analysis of mCherry fluorescence intensity. Three individual animals from each representative image were quantified. AU, arbitrary unit; ***, P < 0.005. Download FIG S5, TIF file, 1.4 MB.Impact of ectopic expression of SID-3::mCherry and SID-3K139A::mCherry in Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the viro-2(vir10) mutants: a C-to-T mutation which results in a premature stop codon at amino acid position 574 in the B0280.13 gene (viro-2) and a similar mutation at amino acid position 164 in the adr-2 gene (adenosine deaminase acting on RNA gene 2). The viro-2 gene encodes an ortholog of the human N-WASP , which harbors a premature stop codon at amino acid position 246 in the C terminus of the viro-2 gene product, was obtained from the million mutation project  strain (viro-2(vir10) mutant from a microinjected plasmid rescued both the GFP and Orsay virus RNA levels (viro-2 gene locus (data not shown). Similar results were obtained with a plasmid expressing a C-terminal mCherry fusion of VIRO-2  strain . OverexpA levels  and D, af VIRO-2 . On the 10.1128/mBio.00940-17.6FIG S6 viro-2(gk627069) on Orsay virus replication and GFP expression. (A) Imaging of Orsay virus-infected strains. Bars, 200 μm. (B) Real-time qRT-PCR of Orsay virus-infected strains. **, P < 0.01. Download FIG S6, TIF file, 1.2 MB.Impact of ectopic expression of VIRO-2::mCherry in Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the sid-3 and viro-2 gene mutations was generated by crossing the sid-3(vir9) and viro-2(vir10) strains. Orsay virus infection of the double mutant showed RNA levels similar to those of sid-3(vir9) or viro-2(vir10) single mutant alone  background to generate extrachromosomal array carrying strains. Heat shock of strain sid-3(vir9);virEx23[PHIP::RNA1WT] [sid-3(vir9) strain harboring wild-type RNA1] activated GFP expression. Furthermore, increased Orsay RNA1 levels were observed compared to analogous sid-3(vir9);virEx26[PHIP::RNA1D601A] [sid-3(vir9) strain harboring the D601A replication-incompetent RNA1] demonstrating that Orsay virus RNA1 replication can occur in the absence of SID-3  harboring both Orsay virus RNA1 and RNA2. The virIs1;rde-1 strain was then crossed with the jyIs8;rde-1 strain to yield the virIs1;jyIs8;rde-1 strain. The virIs1;jyIs8;rde-1 strain was further crossed with either the sid-3(vir9) or viro-2(vir10) strain to generate the virIs1;jyIs8;rde-1;sid-3(vir9) or virIs1;jyIs8;rde-1;viro-2(vir10) strain, respectively. When challenged with exogenous Orsay virus, virIs1;jyIs8;rde-1;sid-3(vir9) and virIs1;jyIs8;rde-1;viro-2(vir10) strains failed to induce GFP as expected  Imaging of exogenous Orsay virus infection of sid-3(vir9) and viro-2(vir10) strains carrying integrated Orsay virus genome. Bars, 200 μm. (B) Imaging after heat induction of sid-3(vir9) and viro-2(vir10) strains carrying integrated Orsay genome. Bars, 200 μm. Download FIG S7, TIF file, 1 MB.Impact of Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro biochemical studies  and VIRO-2 C-terminally fused with mCherry, each driven by the universal sur-5 gene promoter. Both proteins were detected in somatic cells with stronger expression observed in the intestinal cells. Both tagged proteins were functional, as they rescued the Orsay virus RNA level phenotype when coinjected into the sid-3(vir9);viro-2(vir10) double mutant (data not shown). Colocalization of these two proteins, especially along the apical side of the intestine, was observed in transgenic strains that were infected by Orsay virus (The human orthologs of SID-3 and VIRO-2 are reported to be a kinase-substrate pair through  studies . Howeveray virus .10.1128/mBio.00940-17.8FIG S8 rde-1(ne219) strain  at 48 h after Orsay virus infection. Bars, 10 μm. The zoomed-in area is indicated by the magenta box in panel A. Graph showed fluorescence intensity profiling of the arrow marked position. The intestinal apical membrane is indicated by a black bar on top. BF, bright-field microscopy. (C) Additional confocal microscopy images of rde-1(ne219) strain  with Orsay virus infection. BF, bright-field microscopy. Bars, 10 μm. Download FIG S8, TIF file, 2.7 MB.SID-3 and VIRO-2 colocalize in transgenic strains. (A and B) Confocal microscopy of Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. elegans proteins that interact with SID-3 or VIRO-2. In humans, multiple interacting partners of TNK2 and WASP have been defined , or C. elegans genes that interact with WSP-1  affect Orsay virus infection, we performed RNAi knockdown and evaluated Orsay virus replication. Of these tested genes, nck-1, the C. elegans ortholog of human NCK1, yielded an approximately 3-log-unit reduction in Orsay virus RNA. These RNA levels are even lower than those observed by RNAi knockdown of sid-3 and viro-2 (nck-1 (ok694) was crossed with a jyIs8;rde-1 strain and yielded virus levels similar to those of sid-3(vir9) and viro-2(vir10) mutant strains when infected by Orsay virus  EMS screen. A third host gene was subsequently identified by additional targeted RNAi screening. As the current EMS screen is far from saturation, additional EMS screening is likely to identify more host factors that play critical roles in Orsay virus infection. From a technical perspective, the use of a GFP reporter renders the screen essentially binary, thereby facilitating large-scale screening.In this study, we identified two genes that are important for Orsay virus infection of sid-3 nor viro-2 has been extensively studied in C. elegans. To date, there is only one published study on sid-3 that defined a role in endosomal trafficking in the context of systemic RNAi  screens for influenza A virus (IAV), hepatitis C virus (HCV), and vesicular stomatitis virus (VSV)  –37. Thussid-3 alleles, vir9 and ok973, were 5 to 6 log units lower than those detected in the parental strain  ]  and 4B. e cycles , 38, 39.e cycles , one posviro-2 phenocopy mutations in sid-3 in terms of both GFP phenotype and Orsay virus RNA levels, the genes encode very different proteins. viro-2 is one of four C. elegans orthologs  of the two human WASP proteins (WASP and N-WASP). VIRO-2 shares an EVH1/WH1 domain with human WASP proteins  domain is involved in regulation of actin polymerization. Normally, the VCA domain of WASP binds to its central GBD (G protein binding domain) and renders this protein inactive. Phosphorylation by a cytosolic kinase combined with either GTP-activated CDC42 or SH2/SH3 domain-containing protein binding synergistically activates N-WASP. This leads to the recruitment of the Arp2/3 complex through its VCA domain initiating actin polymerization and branching , RB2519 drh-1(ok3495), VC787 sid-3(ok973), RB860 nck-1 (ok694), and VC40417 viro-2(gk627069) from the million mutation project (Escherichia coli OP50 on nematode growth medium (NGM) plates in a 20°C incubator and moved every 3 days to a new NGM plate seeded with E. coli OP50. project  were obtjyIs8  (rde-1(ne219) strain to generate strain WUM31 jyIs8 ;rde-1(ne219). Strains VC787 sid-3(ok973) and strain VC40417 viro-2(gk627069) were crossed with the WUM31 jyIs8;rde-1 strain, yielding strain WUM57 jyIs8;rde-1;sid-3(ok973) and strain WUM58 jyIs8;rde-1;viro-2(gk627069), respectively. The sid-3(vir9) and viro-2(vir10) strains were crossed with the WUM28 virIs1;rde-1 strain, yielding WUM59 virIs1;jyIs8;rde-1;sid-3(vir9) and WUM60 virIs1;jyIs8;rde-1;viro-2(vir10), respectively  was added to the tubes, and then the tubes were frozen in liquid nitrogen. For each experiment, three replicate wells were used for each infection condition. Total RNA from infected animals was extracted using Direct-zol RNA miniprep (Zymo Research) purification according to the manufacturer’s protocol and eluted into 30 μl of RNase/DNase-free water.Orsay virus was prepared by large-scale liquid culture as described previously , filtereE. coli strain HT115 carrying double-strand RNA expression cassettes for genes of interest was induced using established conditions and then seeded into six-well NGM plates. dpy-3, sid-3, adr-2, lst-4, dhs-7, mig-13, mig-2, wsp-1, and nck-1 RNAi clones were from the Ahringer RNAi library  and HJ246 (TACGGATCCAAGGGTTGGTAGTGTTCCG). The PCR product was digested with BamHI, cloned into pL4440, and transformed in E. coli HT115. Twenty arrested L1 drh-1 mutant or N2 animals were seeded into each well of a six-well plate. After 72 h of RNAi feeding, Orsay virus was added to the plates as described above. At 48 h postinfection (hpi) the infected C. elegans animals were collected, and 350 μl of TRIzol (Invitrogen) was added to each well for RNA extraction.RNAi feeding was used for gene knockdown as described previously . E. coli library . To consin vitro transcripts. Primers GW314 and GW315 and probe Orsay_RNA1 that target the Orsay virus RNA1 segment were used to quantify Orsay virus RNA1 abundance .RNA extracted from infected animals was subjected to one-step real-time quantitative reverse transcription-PCR (qRT-PCR) to quantify Orsay virus replication as previously described . Brieflyfe cycle . The meajyIs8;rde-1 reporter strain was maintained on 10-cm plates seeded with 1 ml of E. coli OP50. Animals were grown to a stage with a high proportion of L4 larvae and were then collected by washing the plates with water. Animals were then treated with 50 mM of ethane methylsulfonate (EMS) for 4 h at 20°C with constant rotating. Animals were washed with M9 buffer and recovered on plates for 2 h. L4 P0 animals were transferred to a new NGM plate and were allowed to lay F1 eggs. F1 animals were washed away from plates after laying about 20 eggs for each animal. Hatched F2 animals were challenged with 800 μl Orsay virus filtrate per plate and screened for Viro mutants.The jyIs8;rde-1 reporter strain. Their progenies were assessed for the Viro phenotype to confirm Mendelian genetics for these mutants. Similarly, Viro-1 and Viro-2 mutants were reciprocally crossed with each other to define the ability of their progeny to complement the Viro phenotype.Two hermaphrodites of each mutant strain were backcrossed with eight males generated from the unmutagenized rde-1(ne219) strain, and F1 progenies were chosen and transferred to six-well plates. After 3 days, the F2 progenies were challenged with Orsay virus. A total of 72 F2 Viro animals were picked off F1 plates and transferred to six-well plates. F2 plates were replicated after 1 day of laying eggs. The original F2 plates were challenged again with Orsay virus, and both virus-induced GFP fluorescence and Orsay virus RNA levels were assayed at 3 days postinfection. Wells that yielded exclusively Viro animals and that yielded low RNA levels from replicated F2 plates were pooled, and genomic DNA from pooled samples was extracted using a Qiagen genomic DNA preparation kit according to the manufacturer’s protocol. DNA libraries were prepared using NEB-Next library preparation kit (New England Biolabs) and then sequenced using Illumina Hiseq 2500. The raw sequence data were analyzed using the pipeline CloudMap for C. elegans gene mapping (Viro-1 and Viro-2 mutants were crossed with the WM27  mapping .sid-3 expression plasmid (Psur-5::sid-3), the entire sid-3 gene and 500 bp of its native 3′ untranslated region (3′ UTR) were amplified from fosmid WRM061cD03. The PCR product was digested by BamHI and SacII and was then ligated into the plasmid containing the sur-5 promoter. To generate the sid-3 catalytic mutant expression plasmid (Psur-5::sid-3K139A), site-directed mutagenesis was performed to introduce a lysine-to-alanine (K139A) mutation in the SID-3 kinase domain. C-terminal mCherry fusion constructs of both the wild-type SID-3 and K139A mutant were generated by overlapping PCR. Psur-5::viro-2 and Psur-5::viro-2::mCherry were generated in an analogous fashion.To construct the sid-3 gene was injected into the mutant strain at a concentration of 50 ng/μl along with 5 ng/μl of Pmyo-3::mCherry as a transgenic marker and 50 ng/μl of DNA ladder (NEB) to help establish the array  according to the manufacturer’s protocol.For rescue of Viro-1, a fosmid (WRM061cD03) or an expression plasmid containing the he array . For resC. elegans mutant infections were acquired using a Zeiss Axioskop inverted fluorescence microscope. Briefly, young adult to adult C. elegans animals were anesthetized with 1 mM levimisol and then put on a 2% dry agarose pad with a coverslip (5 by 5 cm) on top. Images were acquired from both fluorescence channels and bright-field channels. Confocal images for colocalization of SID-3 and VIRO-2 were acquired using a Zeiss LSM 880 confocal microscope with Airyscan  equipped with a 63×, 1.4-numerical-aperture Zeiss Plan Apochromat oil objective. Quantification of mCherry expression was performed by image analysis with ImageJ 1.51j8.The microscopic visual scanning analysis for Orsay virus infection was carried out using a Leica stereo fluorescence microscope. Images of 10.1128/mBio.00940-17.9FIG S9 sid-3(vir9) strain and with ectopic expression of SID-3. ***, P < 0.005; NS, not significant (P > 0.05). Download FIG S9, TIF file, 0.1 MB.Quantification of virus-induced gene F26F2.1 RNA levels. Real-time qPCR of Orsay virus-induced gene F26F2.1 expression levels after virus infection in Copyright © 2017 Jiang et al.2017Jiang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Understanding the whole-body patterns of joint flexibility and their related biological and physical factors contributes not only to clinical assessments but also to the fields of human factors and ergonomics. In this study, ranges of motion (ROMs) at limb and trunk joints of young adults were analysed to understand covariation patterns of different joint motions and to identify factors associated with the variation in ROM.Seventy-eight healthy volunteers  living on Okinawa Island, Japan, were recruited. Passive ROM was measured at multiple joints through the whole body (31 measurements) including the left and right side limbs and trunk.Comparisons between males and females, dominant and non-dominant sides, and antagonistic motions indicated that body structures influence ROMs. In principal component analysis (PCA) on the ROM data, the first principal component (PC1) represented the sex difference and a similar covariation pattern appeared in the analysis within each sex. Multiple regression analysis showed that this component was associated with sex, age, body fat %, iliospinale height, and leg extension strength.The present study identified that there is a spectrum of “masculine” and “feminine” types in the whole-body patterns of joint flexibility. This study also suggested that body proportion and composition, muscle mass and strength, and possibly skeletal structures partly explain such patterns. These results would be important to understand individual variation in susceptibility to joint injuries and diseases and in one’s suitable and effective postures and motions.The online version of this article (doi:10.1186/s40101-016-0112-8) contains supplementary material, which is available to authorized users. In the field of orthopaedics and rehabilitation medicine, measuring range of motion (ROM) is a clinical procedure to evaluate a mechanical joint problem caused by disorders of the locomotor apparatus. The purpose of ROM measurement is not only to observe the extent of inhibition but also to identify the factors that restrict joint movement and to evaluate the effectiveness of treatment and training. A measurement method of ROM was established by the American Academy of Orthopaedic Surgeons (AAOS) , in whicThere have been many studies of joint ROM to date. Most of the studies have focused only on upper or lower extremity joint motions, focusing on the effects of age, sex, and/or side dominance and on some special population such as sports athletes and disease patients –9. Even Understanding whole-body patterns of joint flexibility and identifying their related biological and physical factors contribute not only to clinical assessments but also to the fields of human factors and ergonomics. Biological and physical factors such as age, sex, physical constitution, and daily activities can affect patterns of joint flexibility. Then, whole-body patterns of joint flexibility can have influences on whole-body motions and eventually can be important to know one’s suitable and effective postures and motions.The aims of this study were to understand the whole-body patterns of joint flexibility and to identify factors associated with variations in ROM. For this purpose, we measured ROMs of the limb and trunk joints of young adults and analysed their association with biological and physical factors. We also compared dominant and non-dominant sides to obtain a cue for related factors. Principal component analysis (PCA) was performed using multiple joint ROM data to identify covariation patterns among different joints. It was found that a major pattern that explains the variation between sexes also appears within each sex, and this pattern appears to be associated with some somatometric and sthenometric measurements.We recruited volunteers living in Okinawa Island, Japan. Inclusive criteria were healthy males and females between the ages of 20 and 29 who did not have any joint diseases and any history of orthopaedic surgery on joints. As shown in Additional file ROM data were collected from the subjects using a goniometer . All ROM measurements were performed by four observers after confirming that the inter-observer errors in the measurements were small (0.97 < ICC < 0.99). The motions examined are shown in Table Height, weight, upper limb length, iliospinale height, forearm circumference, forearm minimum circumference, calf circumference, and ankle circumference were measured following the standard anthropometric method . On the Grip strength was measured using the Smedley Hand Dynamometer (OG Giken. Co. Ltd). In the measurements, the subject was in a standing position with arms at their side, not touching their body. We obtained the average of three trials. As for leg strength, the flexor and extensor muscle strengths at the knee were measured using the Biodex System 3 dynamometer . Isometric knee flexion and extension strengths were tested at 60° of knee flexion. Peak torque was recorded for each motion in three trials and the average peak torque was calculated.W, H, and T are weight (kg), height (cm), and the sum of subcutaneous fat thicknesses (mm), respectively. The results of the somatometric and sthenometric measurements are shown in Table The calliper method was used to calculate body fat percentage (BF%). Subcutaneous fat thickness was measured at the mid-point of the posterior surface of an upper arm, at the inferior angle point of the scapula, and at the side of the lateral point from the umbilicus. Then, BF% and lean body mass (LBM) was calculated using the following formula –25:\documentclass2pt{minimmentclass2pt{minimt test, in which we subtracted ROM for dominant side from ROM for non-dominant side (ROM(ND) − ROM(D)). PCA was performed to elucidate whole-body patterns of joint ROM. Correlation coefficient and partial correlation coefficient controlling for sex were calculated between each principal component (PC) and each factor. Finally, to disclose factors associated with the whole-body patterns of ROM, multiple regression analysis was conducted. As for bilateral measurements, the averages of the left and right sides were input into these statistical analyses except for the test of the side difference.Statistical analyses were performed using SPSS® Statistics version 19  and Excel Statistics . Basic summary statistics were calculated for each sex. To identify biological and physical factors associated with each ROM, multiple regression analysis was used. Differences between dominant and non-dominant sides were examined by paired The results of measurements are summarized in Table The multiple regression analyses also showed that older age is significantly associated with lower ROMs for shoulder external rotation and horizontal flexion, elbow extension, wrist flexion and extension, and higher ROMs for elbow flexion and trunk flexion and rotation.A higher LBM was significantly related with lower ROMs for shoulder external rotation and horizontal extension, and with higher ROMs for wrist flexion and hip adduction Table . BF% negSignificant ROM differences between dominant and non-dominant sides were detected for several motions. The non-dominant side had higher mobility than the dominant side for shoulder internal rotation, hip abduction, and ankle plantar flexion, whereas the opposite was observed for shoulder external rotation, wrist flexion, and hip adduction Table . In the all and PC1females (r = 0.75) and between PC1all and PC1males (r = 0.96)  Fig. . Extremeall was significantly correlated with age, BF%, iliospinale height, and leg extension strength after controlling for sex. PC2all was also associated with sex, and significant partial correlations with age, forearm circumference, grip strength, leg extension strength, and leg flexion strength were observed. PC3all was not affected by sex and had significant correlations with weight and forearm, minimum forearm, calf, and ankle circumferences.Table all  − ROMD) = −2.4 ± 4.2, P = 0.0245). On the other hand, some joint motions showed a larger ROM on the non-dominant side than on the dominant side. Of these motions, shoulder internal rotation and hip abduction are antagonistic movements of shoulder external rotation and hip adduction, respectively, that showed larger motion on the dominant side. These side differences may be due to off-centred neutral posture because the total ranges of antagonistic motions had no significant difference between the dominant and non-dominant sides. It is well known that side dominance causes asymmetry of posture. In addition, previous studies have reported that asymmetric daily posture, such as side sitting, can be related to ROM asymmetry [The data on differences between dominant and non-dominant sides also provide information on factors affecting the variations in ROM. Joint motions that had a larger ROM on the dominant side than on the non-dominant side were shoulder external rotation, wrist flexion, and hip adduction. This result suggests an involvement of daily activity in the variation in ROM. Regarding the asymmetry of shoulder joints, it has been reported that the side of the dominant hand/arm has a significantly larger ROM than the other side, especially among individuals who have experience in sports with overhead-throwing motion , 33. In symmetry , 34. Altall was associated with sex differences, and even when females and males were separately analysed, similar covariation patterns appeared as PC1. These results indicate that not only sexual dimorphism but also other factors, such as body fat, lower limb length, and muscle mass, can be involved in the component. In addition, our study also showed that PC2all and PC3all were significantly associated with muscle strength and limb circumference, respectively. This also indicated that body composition affects the whole-body patterns of joint flexibility.As for the relationships in ROM among different joints, Allander et al.  reportedall as “feminine type”, and the negative direction as “masculine type” (Fig. all. BF% had a negative association with the PC1all score, which means that an increased BF% is related to masculine type. Golden et al. [all suggests that the longer the limbs are, the higher the tendency for feminine type the individual has. Leg extension strength, being an index of muscle mass, was associated with a tendency to have masculine type.Based on the results of PCA, we refer to the positive direction of PC1pe” Fig. . Femininn et al.  have alsall should be strongly affected by acetabulum and femoral neck anteversion because antagonistic motions of hip joints, and hip abduction and adduction, were associated with sex oppositely. In a previous study, it was also reported that an increase in femoral neck anteversion contributed to a decrease in muscle strength of the gluteus medius and vastus medialis [Furthermore, the covariation pattern of PC1medialis . TherefoAs shown above, multiple factors are likely to be associated with ROM and whole-body patterns of ROM. To understand how cultural differences affect ROM, further global comparisons will be indispensable , 10, 37.Our present study, clarifying the covariation patterns of joint flexibility, will contribute to the prevention of joint injuries and to the evaluation of dysfunction in patients with musculoskeletal diseases. For example, it has been known that anterior cruciate ligament injuries are more frequent in females than in males partly because of joint laxity , and theA covariation pattern of ROMs that shows sexual dimorphism was found by PCA. Such covariation pattern was also observed within each sex as a spectrum of “masculine” and “feminine” types and was shown to be partly associated with body proportion and composition and with muscle mass and strength. Comparisons between dominant and non-dominant sides and between antagonistic motions provide suggestions that ordinary posture, daily motions, and skeletal morphology such as acetabular and femoral neck anteversion contribute to individual differences in ROMs. Such knowledge will contribute to the prevention of joint injuries and to improve one’s performance in sports and daily activities.


WGA) in a clinical setting. We estimated the costs of applying WGA to guide treatments for patients with advanced cancers and characterized how costs evolve over time.Limited data exist on the real‐world costs of applying whole‐genome analysis (POG) program in British Columbia, Canada. Cost data were obtained for patients who enrolled in the program from 2012 to 2015. We estimated mean WGA costs using bootstrapping. We applied time series analysis and produced 10‐year forecasts to determine when costs are expected to reach critical thresholds.The setting is the British Columbia Cancer Agency Personalized OncoGenomics . Over time, WGA costs decreased, driven by a reduction in costs of sequencing. Yet, costs of other components of WGA increased. Forecasting showed WGA costs may not reach critical thresholds within the next 10 years.The mean cost of WGA costs decreased over the studied time horizon, but expenditures needed to realize WGA remain significant. Future research exploring costs and benefits of WGA‐guided cancer care are crucial to guide health policy. Whole‐genome sequencing (WGS) or targeted gene panels represent a potential future standard of care in oncology  will vary across clinical and research settings. WGA will involve different workflow depending on whether it is applied for diagnostic, prognostic, or predictive purposes. Generally, the process of WGA involves a combination of sample preparation, WGS, and data processing including bioinformatics analysis, interpretation, and validation of genomic data  Personalized OncoGenomics (POG) program was initiated in 2012 to apply a more comprehensive form of WGA. POG‐related WGA uses both WGS and transcriptome sequencing (RNA‐seq) to guide real‐time treatment planning for patients with treatment‐resistant cancers , and discontinuing sequence analysis of diagnostic tissue samples. In Version 2.0, costs of reagents and materials purchased from American suppliers increased owing to changes in the American exchange rate  biopsy and sample processing , (2) panel sequencing (including bioinformatics and report generation), (3) WGS and RNA‐seq, (4) bioinformatics analysis related to WGS and RNA‐seq , (5) validation (verification experiments and confirmatory testing), (6) PET scans (performed in approximately 20% of POG cases to assess changes in disease status), and (7) other fixed program costs . We aggregated costs for each patient to obtain an estimate of total WGA costs. We estimated average monthly sequencing and WGA costs based on patients’ month of biopsy. We linearly interpolated missing monthly data (7.1% of observations). Sensitivity analysis involving cubic interpolation resulted in little difference in interpolated values and had no material effect on model conclusions. All costs are reported in 2015 Canadian dollars.t‐tests and Mann–Whitney U nonparametric tests to determine whether WGA costs significantly differed across program versions . After the first breakpoint, in March 2013, mean costs decreased by $659 per month  until June 2014. After the second breakpoint, in June 2014, costs declined by $191 per month  until December 2015. WGS and RNA‐seq costs became less variable after the third breakpoint in December 2014.Figure In this study, we analyzed total cost and cost components of POG‐related WGA from July 2012 to December 2015. We found, on average, WGA cost CDN$34,886 per patient over the time period. WGA costs decreased over time, driven by a reduction in WGS and RNA‐seq costs, which fell from CDN$29,500 per patient in Version 1.0 to CDN$15,490 in Version 2.0 of POG. In a research setting, WGS costs fell from CDN$6988 per genome to CDN$4570 per genome over the same period (Wetterstrand Although WGS costs are declining, changes are partially offset by increasing costs of bioinformatics analysis, validation costs, and other fixed program costs. Increasing fixed costs reflect sequencing platform upgrades and the need to employ full‐time staff to process more patients’ data, which was not necessary in the first version of the POG program. Increasing bioinformatics costs present additional challenges. As costs of data generation decline, long‐term data storage costs may not decline. It could soon be cheaper to regenerate data from DNA samples than to store sequence data (Batley and Edwards Proponents of precision medicine claim that sequencing genomes at a cost of $1000 each will soon be feasible Church . ForecasOur study is not without limitations. Our data on the cost trajectory of WGA are limited to 3 years, the majority of which involved building a new program, and 10‐year forecasts involve considerable extrapolation. We assume WGA costs will behave similarly in the future as they did in our sample and long‐term forecasts are subject to significant uncertainty. By forecasting several possible scenarios, we gain insight into the potential effects of future shocks on WGA costs and the sensitivity of our models to such departures. We also determine the maximum cost to apply WGA in 10 years with no further breaks in the trajectory of costs.Another limitation of our study corresponds to our setting. Our data record costs of applying a comprehensive form of WGA, involving WGS, RNA‐seq, and panel sequencing, to identify treatment options for patients with incurable cancers, some with highly resistant disease. This application of WGA may be particularly complex when compared to other settings and our estimates of mean WGA costs may be relatively high (Tsiplova et al. Costs of WGA are decreasing, but mean expenditures needed to realize WGA and guide treatments for patients with advanced cancers remain high. We found some cost elements are increasing and there are a number of factors to consider going forward. Reaching critical thresholds, particularly within a clinical setting, will take time and incorporating WGA on a wider scale will require significant monetary investment. Despite these costs, WGA offers many potential benefits and future research exploring the trade‐off between costs and benefits of WGA‐guided cancer care is essential to inform health policy and planning.The authors have no conflicts of interest to declare.The views expressed in the submitted article are the authors’ own and not an official position of the funder.Figure S1. Process for POG‐related whole‐genome analysis.Appendix S1. Detailed description of time series analysis.Figure S2. Number of patients enrolled in POG from July 2012 to December 2015.Click here for additional data file.


Dementia is a major cause of dependency and disability among older persons, andimposes huge economic burdens. Only a few cost-of-illness studies for dementiahave been carried out in middle and low-income countries.The aim of this study was to analyze costs of dementia in demented patientsof a private clinic in Lima, Peru.We performed a retrospective, cohort, 3-month study by extracting informationfrom medical records of demented patients to assess the use of bothhealthcare and non-healthcare resources. The total costs of the disease werebroken down into direct  and indirect costs.In 136 outpatients, we observed that while half of non-demented patients hadtotal care costs of less than US$ 23 over three months, demented patientshad costs of US$ 1500 or over . In our study, the monthly cost of a demented patient (US$ 570)was 2.5 times higher than the minimum wage .Dementia constitutes a socioeconomic problem even in developing countries,since patients involve high healthcare and non-healthcare costs, with thecosts being especially high for the patient's family. Dementiais devastating not only for individuals who have it, but also for their families andcaregivers.1 The huge costof the disease will pose a challenge to health systems in dealing with the predictedfuture increase in prevalence. Costs are estimated at US$ 604 billion per year atpresent and are set to increase even more quickly than prevalence.5Dementia is one of the major causes of dependency and disability among olderpersons,1 However, to date, mostcost-of-illness studies for dementia have been carried out in high-incomecountries.12 Evidence is also emerging in middle and low-incomecountries (MLIC),17 but as yet data remains sparse,particularly in the LA region.There is a growing body of evidence on the global economic cost related to dementia.The consensus is that dementia is already imposing huge economic burdens.A clearer understanding of the costs of dementia, and how these impact families,governments and their health and social care systems, is fundamental for raisingawareness, achieving proper prioritization, and focusing efforts toward improvingthe lives of people with dementia and of their caregivers.The aim of this cost-of-illness study was to analyze total, direct and indirect costsof dementia in a population of demented patients from a private clinic in Lima,Peru.Study design. A retrospective, cohort, 3-month study was carried outduring the period spanning from January 2012 to April 2013 in demented outpatientsof the International Clinic in Lima, Peru.The construction of this cohort was possible owing to the use of a dementia protocolensuring a standardized approach for managing demented outpatients in our clinic.Based on this protocol, the evolution of the disease was assessed at definitivediagnosis and again three months later, with an additional visit whenever necessaryfor adjustments to patient's therapy. These evaluations included routine evaluationof both patients and their caregivers (i.e. people providing the patient with hoursof care and helping them with activities of daily living). Overall evaluations aredetailed in the Procedures section.Setting and population. The International Clinic is a private, secondaryhealth care center in the city of Lima, capital of Peru. The clinic is located inthe central region of the city, a strategic location for arrival of patients fromany area of Lima. Given the catchment area, the majority of patients came frommiddle income areas [me areas .Clinical measurement scales. The following functional measurementscales were applied:18[1] the Clinical Dementia Rating (CDR) scale;20[2] the Mini-Mental State Examination (MMSE);21 the NeuropsychiatricInventory (NPI);22[3] the Pfeffer Functional Activities Questionnaire (PFAQ);23 and[4] the Beck Depression Inventory (BDI);24[5] the Zarit Burden Scale (ZBS).Care services utilization. An assessment was carried out on the useof both healthcare and non-healthcare resources (i.e. caregiver services). This wasperformed by extracting information from medical records. The information aboutnon-health resources was obtained by interviewing the patient's caregiver, and thisinformation was appended to the patient's medical record.Costs of dementia. The costs of dementia are the value of all goodsand services that are given up to prevent, diagnose, treat and otherwise cope withdementia. The total costs of the disease were broken down into direct and indirectcosts.16 These costs arebased on the value of resources used and resources lost, respectively.516 differentiating medical from social care costs.While direct medical costs encompass the medical care system , direct social costs arise from formalservices provided outside of the medical care system .5Direct costs refer to money used in an explicit way (explicitly exchanged) on drugs,hospitals, and a variety of medical or social services,5 This conceptrefers to money used in an implicit way as loss of income by the patient and/or lossor reduction of income by family members or caregivers, namely hours withoutmonetary reimbursement that informal caregiver spend with patients.16On other hand, indirect costs generally refer to productivity losses associated withthe illness. Because these costs arise from impaired productivity  this type of indirect cost is generally lessrelevant in the context of dementia, where most patients are elderly (who areretired in most cases ). An additional component of these costs is the informal carecosts, which arise from the unpaid inputs of family caregivers, friends andothers.Selection of patients. Eligible patients were subjects over 60 yearsold, with a diagnosis of dementia according to DSM-IV-TR25 and based on the neurologic and neuropsychologicalassessment. Those patients with limited functional independence and without areliable and consistent caregiver were excluded. For the "control" group(non-demented subjects), relatives of dementia patients who had no dementia symptomsand completed the protocol with negative results for dementia diagnosis wererecruited.Dementia subtypes were defined according to internationally accepted criteria:26 for probable dementia of Alzheimer-type;[1] NINCDS-ADRDA criteria27 for frontotemporal dementia; and[2] Lund and Manchester criteria28[3] NINDS AIREN criteria for probable vascular dementia.Data collection. Data were collected through a report form designedfor this study, and extracted from medical records. The following data werecollected:[1] demographics ;[2] administrative ;[3] clinical; and[4] care services.Clinical data. This data group included medical history of dementia,treatments for dementia management, and clinical status of patient or caregiver asmeasured by the previously mentioned scales .Care services data. This data group included information regardingutilization of healthcare and non-healthcare resources, collected by extractinginformation from medical records.Cost estimation. The time horizon of the study was three months, and2013 prices were used for cost calculations. Overall costs were calculated bymultiplying unit cost by resources consumption during the 3-month follow-up. Thecosts per person were separated according to resource consumption: diagnosis ormanagement of dementia. Cost per patient due to dementia management, both overalland per component, was calculated using the total three-monthly costs asfollowing:Total Costs =  +  + The replacement cost approach was used to estimate indirect costs, based on the legalminimum monthly wage in Peru for 2011 (US$ 222.22). This proxy was used only forinformal caregivers aged 15 to 64 years old . For projection purposes, only costs of managing dementia were used forcalculating  Total VariableCosts.To allow comparisons between countries, costs were expressed as US dollars, convertedfrom local currencies based on exchange rates during study execution (averageexchange rate in 2013 was 1 US dollar = 2.7 Peruvian "nuevo sol").Data analysis. Descriptive statistics were applied to all variables,including measures of frequency distribution for qualitative variables, as well ascentral tendency (mean or median) and of statistical variability  for quantitative variables. The Kolmogorov-Smirnovtest was applied to test normality assumption of variables.2-test).Differences among dementia subtypes were estimated using ANOVA (for Gaussian data)and Kruskal-Wallis (for non-Gaussian data) tests for quantitative variables.Post-hoc Bonferroni tests and post-hoc Mann-Whitney tests were used as appropriate.Qualitative variables were evaluated using the Chi square test(χThe factors assessed (characteristics of patient and caregiver) were used formultivariate analysis through a linear regression model using "total variable costsover three months" as the dependent variable for the explanatory analysis. Becausethe costs variable shows a skewed right distribution, estimation was also done withits natural log . This analysis was alsoapplied to dementia subtypes. The stepwise selection method was employed  to identify the best model, using 0.20 as the significancelevel for removal from the model. This analysis was carried out with an exploratorypurpose, using adjusted R squared to estimate the predictive power of the statisticsmodels selected.In all analyses, p<0.05was considered significant. Results were treated using thestatistical software package STATA, version 12.0 .Ethics statement. Written informed consent was obtained fromparticipants (or their relative in the case of dependent patients) and theirclinical records were used for this study. The information on patients wasanonymized and de-identified prior to analysis. The study protocol was approved bythe local research ethics committee of the Universidad San Martín de Porresof Lima, Peru.In the present study, clinical records were collected from 136 outpatients withcomplete data. All selected patient with dementia had at least one caregiver. Theunit costs are shown in Demographic and clinical characteristics of patients. Regardingpatients' demographic and clinical variables, a significantly higher age was notedin patients with Alzheimer's dementia and also more years of education in patientswith vascular dementia relative to other patients. However, patients withAlzheimer's dementia showed a significantly higher CDR with respect to patients withvascular dementia , primarily due toan elevated consumption of healthcare resources including the use of antidementiaand antipsychotic drugs, an amount representing more than 2.5 times the legalminimum monthly wage in Peru for 2011. Thus, our results are in line with thosepreviously reported in the literature.5On the other hand, these costs are much lower than those seen in other countriesbecause costs tend to be lower in developing countries, both per person andsocietally. This occurs because, in these regions, there is much greater reliance onunpaid informal care provided by family and others.32 which used the Blessed DementiaRating Scale. Thus, the total cost of the disease appears to be better explained bythe greater use of both healthcare and non-healthcare resources, that may beassociated with patient severity, as directly measured by the CDR scale, andindirectly by the greater need for care (leading to hiring of a caregiver). It ispossible that caregiver age serves as an indicator of indirect costs owing to lossor reduction of income by family members of working age .In the present study, total costs of dementia were significantly associated withdementia subtype, CDR and caregiver age, independently of patient age, sex or yearsof education. Level of clinical deterioration, as evaluated with the CDR scale, wasassociated with a significantly higher cost for the disease, similar to thatobserved in other studies15 The cited studyfound CDR values to be associated with higher total cost only in patients withfrontotemporal dementia. Moreover, only BDI and instrumental activities of dailyliving (IADL) values proved statistically associated with total costs ofdementia.15 Since the IADLscale was not used in the present study, it was not possible to explore thisassociation. However, no differences in PFAQ values were found.The factors identified by this study differ to those of the study by Rojas etal.33Previous studies had not explored the utility of explanatory models by dementiasubtypes, and the potentially predictive power in patients with Alzheimer's dementia(most frequent dementia subtype) is considerable. Additionally, we noted thatnon-healthcare costs did not differ statistically between dementia subtypes. Thus,healthcare costs are critical in the total costs of the disease. A higher rate ofrequests for magnetic resonance inpatients with frontotemporal dementia could beassociated with greater requirements for establishing an accuratediagnosis.34Support for their use in patients with frontotemporal dementia35 or vascular dementia36 is limited. By contrast,antipsychotic drug consumption was highest in patients with frontotemporal dementia,corroborating previous studies. 38 The degenerative dementias  appear to be more expensive thanvascular dementia. Our results are similar to those previously reported by Rojas etal.15 The cost ofantidementia drugs in Peru is very high and the monthly cost of standard treatmentexceeds the legal minimum monthly wage for 2013 (222.22 US dollars).The costs of drugs appear to be the main resource in healthcare costs. Antidementiadrug consumption was highest in patients with Alzheimer's dementia, in accordancewith guideline recommendations.Our study did not consider the drugs or medical services for other medical conditions(such as chronic non-communicable diseases) that have a high prevalence in this agegroup, especially in patients with vascular risk factors or vascular dementia thatrequire medication on a chronic basis. In the present study, no significantinfluence of other characteristics of the patient  or of the primary caregiver on total costs of disease was found. Therefore,according to the data, the total cost of dementia does not depend on thesevariables. Increased disease evolution time can be expected to be accompanied by, asthe patient's situation worsens, a greater need for the participation ofprofessional and paid caregivers, further increasing the total cost of the disease.However, given that only the CDR scale showed a significant difference on univariateanalysis, it is noteworthy that in terms of evaluation scales, the CDR may serve asan overall indicator of other scales applied in demented patients.16 While inSpain the monthly overall mean cost of AD (US$ 1760.78) was almost three times theminimum wage - MW (average MW for 2003-2006: US$ 612.92), in Argentina (average MWfor 2002-2008: US$ 163.62) monthly cost (US$ 644.67) were almost four times the MW.In Peru, the monthly cost of a demented patient (US$ 570) was only 2.5 times the MW.Thus, social costs appear to be even greater in developed countries. Although ourstudy did not further evaluate indirect costs, it highlighted an interesting socialphenomenon: a substantial proportion of caregivers of working age, which constitutesa social problem.Some studies in LA have found similar cost patterns for dementia, showing thatindirect costs constitute the largest proportion of expenditure and almost allcost-of-illness is funded by the patient's own family.1 Costs of informal care and the direct costs of social carerepresent similar proportions (42%) of total costs worldwide, while direct medicalcare costs are much lower (16%).5However, our study found that almost 90% of total expenditure was dedicated todirect medical care costs. Thus, future prospective research should appropriatelymeasure the resources associated with informal costs in demented patients and theirfamilies.The estimated annual worldwide cost to society of dementia (costs of dementia) of US$604 billion in 2010, highlights the enormous impact that dementia has onsocioeconomic conditions worldwide.Some limitations of the present study should be highlighted:[1] the sample size is low and the sample design used is notrepresentative (precluding inference);[2] with the selected population (outpatients living at home), it was notpossible to obtain any information about institutionalized patients;[3] information bias is possible since retrospective data was used;[4] there may be other demographic , clinical (existence ofcomorbidities) and caregiver  variablesnot considered in this study, as well as variables for adequatelyassessing the non-healthcare resources  and costs;and[5] this study was a cohort that only covered 3 months of follow-up,precluding assessment of the dynamics of resource utilization overperiods of one year or longer. We sought to estimate resources minimallyexpected for a period of one year using the information from onetrimesterNevertheless, we conducted a study with analytical purposes , whichconstitutes the first publication reporting the factors associated with costs ofdementia in a Peruvian elderly population, specifically in outpatients living athome in Lima. This study begins a line of research to be continued by prospectivestudies assessing costs of dementia .In conclusion, this study found that type of health system coverage, CDR value, andhired caregiver in demented community-dwelling outpatients were associated withtotal costs of the disease, which proved far higher than in non-demented elderly.Thus, dementia constitutes a socioeconomic problem even in developing countriessince patients involve high healthcare and non-healthcare costs, with these costsbeing especially great for the patient's family.The seeking of medical help is relatively unusual in low and middle income countries,where dementia is often viewed as a normal part of ageing. Demand for medical careis likely to increase in the future, with improved awareness, posing a challenge forgovernments to respond to the substantial growing numbers of people with dementia. Abroad public health approach is needed to improve the care and quality of life ofpeople with dementia and their family caregivers.


Recently, avian influenza virus has caused repeated worldwide outbreaks in humans. Live Poultry Markets (LPMs) play an important role in the circulation and reassortment of novel Avian Influenza Virus (AIVs). Aerosol transmission is one of the most important pathways for influenza virus to spread among poultry, from poultry to mammals, and among mammals.In this study, air samples were collected from LPMs in Nanchang city between April 2014 and March 2015 to investigate possible aerosol transmission of AIVs. Air samples were detected for Flu A by Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR). If samples were positive for Flu A, they were inoculated into 9- to 10-day-old specific-pathogen-free embryonated eggs. If the result was positive, the whole genome of the virus was sequenced by MiSeq. Phylogenetic trees of all 8 segments were constructed using MEGA 6.05 software.To investigate the possible aerosol transmission of AIVs, 807 air samples were collected from LPMs in Nanchang city between April 2014 and March 2015. Based on RRT-PCR results, 275 samples (34.1%) were Flu A positive, and one virus was successfully isolated with embryonated eggs. The virus shared high nucleotide homology with H9N2 AIVs from South China.Our study provides further evidence that the air in LPMs can be contaminated by influenza viruses and their nucleic acids, and this should be considered when choosing and evaluating disinfection strategies in LPMs, such as regular air disinfection. Aerosolized viruses such as the H9N2 virus detected in this study can increase the risk of human infection when people are exposed in LPMs.The online version of this article (doi:10.1186/s12985-017-0800-y) contains supplementary material, which is available to authorized users. While aquatic birds are the natural hosts of avian influenza virus (AIVs), the transmission of AIVs from wild birds to poultry has been seen in many places in the world, and the virus has caused frequent sporadic human outbreaks owing to contacts between poultry and humans –6. DurinIn previous studies searching for the source of human infection in LPMs, close attention was paid to poultry throat and cloaca swabs, as well as environmental samples such as feces, feathers, and poultry drinking water, while aerosol samples in LPMs were poorly investigated. Aerosol transmission is one of the most important pathways for influenza virus to spread among poultry, from poultry to mammals, and among mammals –11. In rIn this study, 46 sample sites in ten different LPMs were selected in Nanchang city, Jiangxi province, China. These LPMs were distributed in four districts  Fig.  of the cRNA was extracted with Qiagen RNeasy Mini kit, and influenza A was detected by RRT-PCR with primer and probe sets recommended by WHO  for 1 h, then virus isolation was conducted by inoculating the treated samples into 9- or 10-day-old specific-pathogen-free embryonated eggs. After incubation at 37 °C for 48 h, the allantoic liquid of the eggs was harvested and tested by Flu A fast diagnostic kits . If the result was positive, the allantoic liquid was aliquoted and stored at −80 °C.FluA-probe: TGCAGTCCTCGCTCACTGGGCACG). If the aerosol samples were Flu A-positive, they were treated with 6 antibiotics  software package; the phylogenetic trees of all the 8 segments were conducted by using neighbor-joining method with Kimura two-parameter distances. The bootstrap value was 1000.2 = 39.792, p < 0.01), while no difference was detected between the positive rate of air samples collected at 5 and 7 m . In the wholesale LPM, the Flu A positive rates of air samples collected at 5 and 7 m were not significantly different . The control samples were all negative for Flu A. One virus was successfully isolated from these Flu A RRT-PCR positive samples by inoculating samples into Specific Pathogen-Free (SPF) embryonated chicken eggs, and the HA titer of this virus was 256. This virus isolation positive sample was collected from Jingdong Bird and Flower market in Qingshanhu district, Nanchang city on July 31, 2014. Full genome sequencing of the virus showed it was a subtype-H9N2 virus and was termed as A/environment/Jiangxi/14737/2014 (H9N2) (Abbreviated as JX14737 here after). The sequences of all eight segments of this virus were submitted to the Global Initiative on Sharing All Influenza Data (GISAID) with accession numbers from EPI858159 to EPI858166.In total, 807 air samples were collected from 46 stalls in 10 different LPMs, from 4 districts in Nanchang city  was detected in the HA protein, which indicated that the virus was prone to binding to α2,6-sialic acid receptor; this human-like receptor binding preference increased the risk of human infection of AIVs . We downSubtype-H9N2 influenza virus was first identified in chicken farms in China in 1994 , respectSporadic human infections with H9N2 virus have been reported since the 1990s. Epidemiology data showed that some of these patients did not have direct contact with live poultry or any history of live poultry exposure . These sThere were some limitations to this study. First, the air sample collection time (1.5 h) and the distance between stall center and instrument might affect the results. Second, the presence of polluted particles in the air might decrease the sensitivity rate of virus isolation.2 = 39.792, p < 0.01). The results suggested that the risk of human infection by aerosolized virus was increased when people were closer to stalls selling live poultry.In our study, one H9N2 AIV was successfully isolated from 807 aerosolized samples. The virus shared high nucleotide homology with H9N2 AIVs from South China. Our study provided further evidence that the air in LPMs can be contaminated by influenza virus and its nucleic acids, and this fact should be considered when choosing and evaluating possible disinfection strategies for LPMs, such as regular air disinfection. Further statistical analysis suggested that the Flu A positive rate of air samples collected 3 m from poultry stalls were significantly higher than that of air samples collected at 5 and 7 m in the retail LPM (χ