Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB.Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon ( Oncorhynchus tshawytscha), sockeye salmon (O. nerka), and steelhead/rainbow trout (both O. mykiss) [The aquatic rhabdoviral pathogen infectious hematopoietic necrosis virus (IHNV) infects many species of Pacific salmonid fish. In the multi-host ecosystems of western North American rivers, IHNV has diverged into three major virus genogroups that vary in host-specific virulence for three major host species: Chinook salmon ( mykiss) ,2. In th mykiss) ,4. Chino mykiss) ,6. Phylo mykiss) ,8,9. To Salmonid novirhabdovirus) belonging to the taxonomic family Rhabdoviridae, in the genus Novirhabdovirus [O. mykiss), sockeye salmon, and Chinook salmon [IHNV is an enveloped, single-stranded, negative-sense RNA virus  of each IHNV strain by batch immersion, only the L strain caused significant mortality compared with mock-challenged groups. Infection frequency seven days after challenge was significantly higher for L than for some U and M strains, and there was a trend of higher infectivity for U strains relative to M strains. In addition, CRB Chinook salmon appeared to be generally more susceptible to infection than the Puget Sound population [In a recent study by Hernandez et al. , both inpulation . These oHere we conducted controlled laboratory exposures using diverse populations of CRB Chinook salmon to elucidate possible intraspecific variation in host susceptibility to three strains of IHNV. In Waples et al. , four ma50) with each virus strain were determined as a metric of virus infectivity. The influence of the host population, viral strain, and exposure dose on the outcomes of infection  were also evaluated using fixed-effects logistic regression. Finally, we compared the results of our controlled laboratory studies with field surveillance data to more comprehensively understand the host-pathogen interactions of CRB Chinook salmon with UC and MD IHNV. To do this, we assessed field patterns of IHNV infection in different life-history phenotypes of CRB Chinook salmon from virus surveillance data and genetic typing information. This multifaceted investigation is the first to assess intraspecies variation in susceptibility of diverse populations of CRB Chinook salmon to infectivity and mortality caused by different strains  of IHNV.In all experimental challenge studies, mortality and infection outcomes were assessed from both the virus and host perspectives. Variation in IHNV virulence and host susceptibility to disease was assessed by monitoring mortality over a 30-day time course following immersion exposure to two different doses of each virus strain. Variation in virus infectivity and host susceptibility to infection was assessed by quantifying infection frequency and magnitude of viral load three days after exposure to the virus. For infectivity assays, each experimental host population was exposed by immersion to four different concentrations of virus, and the dosages needed to infect 50 percent of each population  using standard biosecurity procedures recommended for control of IHNV [Chinook salmon of the Methow River  and North Santiam River  were selected to represent upper and lower CRB spring-run populations a, design of IHNV , incubatThree viral strains were included in this investigation b. Strain3 plaque-forming units (PFU) mL−1 (referred to as ‘moderate dose’) or 2 × 105 PFU mL−1 (referred to as ‘high dose’). These doses allow us to assess response to a standard high dose used in previous work [To assess IHNV virulence, triplicate groups of twenty fish were exposed to each viral strain by static immersion for 1 h in 1 L of water containing the virus at a concentration either of 2 × 10ous work ,28, and At the end of the 30-day observation period, all surviving fish were euthanized, and eight fish from each high dose treatment group were saved for a preliminary analysis of viral persistence. These fish were stored at −80 °C for later RNA extraction and quantification of viral RNA.2, 2 × 103, 2 × 104, and 2 × 105 PFU mL−1. These doses span from low to high infection probabilities for IHNV, as observed previously [−1. Three days post exposure (dpe), each set of eight fish per treatment group were euthanized and individually stored at −80 °C for RNA extraction.To characterize the infectivity of L, UC, and MD virus types in diverse populations of CRB Chinook salmon, twenty additional fish from each experimental population were exposed to each virus strain at four virus concentrations: 2 × 10eviously . Viral i−1 of fish of a guanidinium thiocyanate-based denaturation solution was added to each fish individually stored in a Whirl-Pak® bag . Each fish was homogenized using a Stomacher® 80 Biomaster , after which the homogenate was centrifuged for 30 min (2200 rpm). RNA was extracted from 1 mL of the homogenate with phenol-chloroform, precipitated, and resuspended in 50 µL of RNase-free water. RNA was assessed for quality and concentration by spectrophotometry, yielding a mean ± SE total RNA concentration of 671.4 ± 39.4 ng µL−1 (range 23.2–2475.5 ng µL−1). Complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase with random hexamer primers [Total RNA was extracted from whole fish as previously described . Briefly® FAM-labeled probe for the IHNV N gene, VIC®-labeled probe for the artificial positive control (APC) and amplified on an Applied Biosystems ViiA7™ real-time PCR machine . APC plasmid DNA was linearized and used to construct a standard curve (ten-fold dilutions from 5 × 107 to 5 DNA copies per RT-rPCR reaction) to quantify the absolute copy number of viral RNA [y = mx + b). The reaction efficiencies varied from −3.4 to −3.5 , and the y-intercept values varied from 39.8 to 40.9 (b), indicating similar limits of detection across the IHNV RT-rPCR assays. The detection limit of the IHNV N gene RT-rPCR assay was 2512.9 viral RNA copies per gram of fish tissue (3.40 log10 RNA copies g−1). This assay detects both genomic and messenger RNA, and this combined quantity will be referred to hereafter as viral load.Viral RNA was quantified using the universal IHNV N gene reverse transcriptase real-time PCR (RT-rPCR) assay as previously described . Brieflyiral RNA . Each saTo assess the influence of viral treatment on host survival, Kaplan–Meier survival analysis with a Mantel-Cox Log-rank test was performed using SigmaPlot v. 13 , as previously described [10 viral load (log10 RNA copies g−1), indicating early virus replication capability once inside the host.To characterize differences in host susceptibility to infection with L, UC, and MD genogroup viruses, binary infection data and viral load data were first analyzed using descriptive statistics. For each host population and virus strain, infection prevalence at 3 dpe is reported as an indicator of virus infectivity. For virus positive fish, the quantities of IHN virus detected at 3 dpe are reported as the mean log50) with each IHNV strain. As previously described in Breyta et al. [Stats package. For ten of the twelve models (three virus strains in four host populations), the binomial error family was used for the logistic regression. For the remaining two models, Up-Spring and Low-Spring Chinook salmon populations exposed to the L virus, a quasibinomial error family was favored because of apparent overdispersion [50 were determined using the dose.p function of the Mass package in R [50 values between fish populations and virus strains were assessed using the Welch-Satterthwaite 2-tailed t-test [50 estimates were evaluated between viral strains within host populations , or between host populations within viral strains .Exposure of juvenile Chinook salmon to a range of virus concentrations allowed estimation of the dosages needed to infect 50 percent of a host population , which decreases as the model’s out-of-sample predictive accuracy improves . To idenThe influence of exposure dose, viral strain, and host population on viral load was evaluated using a generalized linear model (GLM) in R. The form of the full model closely resembles Equation (1), with the exception that the dependent variable was  the AIC . As befoSurveillance records were reviewed to characterize the field occurrence patterns of UC and MD IHNV infection in spring-, summer- and fall-run CRB Chinook salmon , based oThe relative abundances of spring-, summer- and fall-run Chinook salmon across the CRB were estimated based on the average number of adult fish migrating past the Bonneville Dam between the years 2000–2012 . Host ab5 PFU mL−1, the upper CRB spring-run (Up-Spring) population had the lowest 30-day survival (p < 0.001) (p < 0.001)  after exposure to L, UC, or MD virus strains are illustrated in survival a with ansurvival c with an< 0.001) . Suscept< 0.001) c.p < 0.001). Comparing all results for L virus exposures, the only consistent differences observed among host populations involved the Low-Spring population, which was significantly less susceptible to mortality than all other populations at the high exposure dose, or to one other population at the moderate dose. The virus was recovered from 5/5 Low-Spring and 11/11 High-Spring fish that died after exposure to L virus, indicating that the lower susceptibility to mortality in Low-Spring fish was not due to lack of infection.Cumulative percent survival was higher in all host populations exposed to the L genogroup virus at a moderate rather than a high concentration . Among tp < 0.001) in all host populations exposed to UC and MD strains of IHNV relative to the L virus. This was also observed for the Up-Fall and Low-Fall populations at the moderate dose. Taken together, the positive control L virus strain was significantly higher in virulence than the UC and MD virus strains. Between the UC and MD virus strains tested, virulence was low and not observed to differ within or across host populations.Across all experimental host populations exposed to moderate or high concentrations of the UC virus, the average CPS ranged from 92% to 98%. Similarly, for all host populations exposed to MD virus at moderate or high concentrations, the average CPS ranged from 92% to 100%. Multiple pairwise comparisons of survival curves within and across Chinook salmon populations showed no significant differences in survival following exposure with the UC and MD viral treatments, and neither differed from the virus negative control treatments . After h10 viral RNA copies g−1 of fish) were used to evaluate differences in the infectivity and early in-host replication, respectively, for the three strains of IHNV. Three days following exposure, the proportion of fish from each experimental host population infected with each of the virus strains was determined  are reported for virus positive fish within each viral treatment at 3 dpe  relative to upper CRB  fish.As an indication of the ability to replicate early in infection, mean logat 3 dpe , with inpulation . Therefopulation a. On a wOf the three viral strains tested, the positive control L virus strain infected more fish than the UC and MD virus strains b. Howeve50) with each virus type were generated using binary infection outcomes for fish at 3 dpe. The ID50 values are reported in log10 PFU mL−1, where lower ID50 estimates represent higher virus infectivity because a lower concentration is sufficient to infect 50 percent of a population. For all virus strains in all host populations, ID50 estimates varied within a range of approximately 1.5 logs, from 3.46 to 4.96 log10 PFU mL−1  than the UC virus strain  when compared to Low-Spring fish , but not on viral load (p = 0.115)  and UC replicating to higher viral load (p = 0.042). This is consistent with general observations in The influence of virus strain, and exposure dose on the infection status and viral load was assessed using logistic regression (parameter estimates in = 0.115) . Consist= 0.115) . Virus s= 0.115) . Howeverp values in p < 0.001)  . Consist< 0.001) a.p < 0.005)  or lower CRB . Across all three contrasting population pairs, the lower CRB populations were observed to have higher total quantities of the detectable virus at 3 dpe  . However< 0.005) a, greate< 0.005) b. Nevertf levels . Each ofat 3 dpe a. Togeth10 viral loads (log10 RNA copies g−1 of fish) were determined for a subset of fish that survived exposure to the high concentration of each viral strain. Thirty days following exposure, detectable infections with L, UC, and MD IHNV were observed to persist in a proportion of fish from each experimental host population (n = 8) for each treatment group, the following trends were observed. Persistent infection prevalence was highest among fish exposed to the positive control L virus, relative to the UC and MD viral treatments. Similarly, mean log10 viral loads were generally highest among fish infected with the L IHNV strain. The total quantity of L IHNV detected at 30 dpe in all host populations combined was more than two logs, higher than the total quantities of the UC or MD viruses. While fewer fish were observed to be infected with the UC and MD IHNV strains, the mean log10 viral loads of fish infected at 30 dpe with the UC and MD viral strains were not inconsequential. Among Chinook salmon persistently infected with the UC IHNV strain, mean log10 viral loads ranged between 4.38–5.83 log10 RNA copies g−1 of fish. Among fish persistently infected with the MD IHNV strain, mean log10 viral loads ranged between 5.18–5.87 log10 RNA copies g−1 of fish. Using our previously published ratio for detection of viral RNA by RT-qPCR versus detection of infectious virus by plaque assay [−1 of fish. Overall, higher numbers of virus positive fish were observed with the UC IHNV strain relative to the MD strain. Further, higher total virus quantities were observed with the UC IHNV strain relative to the MD viral strain.To provide a preliminary indication of variation in viral persistence, infection prevalence and mean logpulation . Althougn = 10 out of 24), whereas the Low-Spring population had the lowest total number of persistently infected fish (n = 4 out of 24) at 30 dpe. Again, statistical analyses were not conducted, due to the small numbers of fish tested, but consistent with infection frequencies reported at 3 dpe, infection persistence was lower in the Low-Spring population than in the contrasting three host populations.The total numbers of virus positive fish at 30 dpe from each experimental host population ranged between 4–10 fish or 16.7–41.7% of fish tested, respectively . Among t10 total viral RNA copies. Total virus quantities were approximately 1–2 logs higher in the two upper CRB Chinook salmon populations than in the two lower CRB populations. As suggested by generalized linear models, that evaluated the influence of the host population on early viral load, these data support a non-significant observation that the geographic distribution of a host population may influence viral load outcome. In general, viral persistence and infection prevalence at 30 dpe are consistent with other quantitative observations of intraspecies variation in CRB Chinook salmon susceptibility to IHNV infection, as described above.The total quantities of virus detected at 30 dpe among the four host populations ranged between 6.89–9.15 logOf the three migratory run-types of adult CRB Chinook salmon, fall-run fish are most abundant (60%), followed by spring-run fish (27%), and summer-run fish comprise a minority (13%), based on avg. annual returns for 2000–2012 . The preThe specific prevalence of UC and MD IHNV infection across Chinook salmon populations of the CRB was determined as the proportion of UC or MD IHNV positive cohorts relative to the total number of IHNV positive cohorts for which genotype data were available . Among aThis investigation examined the host-pathogen interactions that drive the ecology of IHNV in the CRB, where Chinook salmon are by far the most abundant virus host. We, therefore, compared four CRB Chinook salmon populations selected to represent the phenotypic and genetic diversity of Chinook salmon in the CRB, using UC and MD subgroup IHNV strains that occur in the CRB. We also included an L group strain as a positive control known to be highly adapted and virulent in Chinook salmon. This differs conceptually from our previous work  by focusIn our results, disease in the host was measured as mortality after exposure to either high or moderate doses of virus in immersion challenges, and viral infection was assessed by several measures, including the frequency of initial infection, viral loads at 3 days post exposure, and persistence of detectable infection through 30 days. The quantification of multiple traits for three divergent virus strains in four diverse host populations generated an extensive and complex data set. Our analysis of those data was designed in terms of testing two null hypotheses: (1) There is no variation among the four host populations, and (2) there is no variation among the three virus strains. Our results do not support either of these hypotheses, but rather suggest a “general rule” in each case, with some exceptions.50 values in the four host populations provided 18 possible comparisons where a pair of hosts could differ , and only one of the 18 comparisons found a significant difference. To summarize what might be learned from the exceptions, specific cases where variation was observed among host populations are shown in From the host perspective, the majority of tests described here demonstrate no significant difference between the four populations tested. For example, measurements of mortality among the four host populations provided 36 possible comparisons where a pair of hosts could differ , and only five significant differences were found, all for L virus. Similarly, analyses of IDThus, while our data do not completely support the null hypothesis of no variation in IHNV susceptibility among host populations along every dimension, there is very little significant variation between them overall. Based on the finding that the Low-Spring population is somewhat less susceptible to both mortality and infection, while the Up-Spring is more susceptible, and both Fall populations were intermediate, we did not find any generalized correlation of IHNV susceptibility with the spring- or fall-run life-history phenotypes. Thus, we do not confirm the trend suggested in Hernandez et al. , where t50 values for each virus strain provided 12 possible comparisons where two virus strains might differ , and only one comparison had a significant difference. From the virus perspective, there were some clear differences in virulence measures, specifically for the L virus, but the great majority of infectivity measures did not show significant differences among the three virus strains. For example, IDhttp://gis.nacse.org, accessed on 8 March 2019) [For the experiments described here, we used a single virus isolate to represent each of three IHNV genetic subgroups. The isolates used were chosen from major IHNV epidemic events and are the index isolates of the most dominant IHNV genotypes circulating broadly in Chinook salmon of the CRB of Washington, Idaho, and Oregon , and the Sacramento River watershed in California (mG011L for L). Genetic surveillance of 771 IHNV isolates from CRB Chinook salmon has found 199 viruses with genotype mG001U collected between 1985–2010, and 27 viruses with genotype mG110M collected between 2007–2013 (Molecular Epidemiology of Aquatic Pathogens IHNV database at ch 2019) . Similar50 values without extrapolation beyond the results, as is sometimes required in similar studies. Finally, the determination of infection status at a single sampling time point 3 dpe generated a “snap-shot” of relative infection levels that may not capture all relevant differences if virus isolates vary in their speed of replication in different Chinook salmon hosts. The choice of 3 dpe as an optimal first sampling time point to test was based on previously published time course studies of U and M virus replication in both rainbow trout [The experimental studies presented here involve some caveats that may impact the interpretation of the results. Although the virus isolates used for the work were selected as optimal representatives of the three major IHNV genogroups in North America , they were not obtained directly from Chinook salmon in the CRB b. Therefow trout  and sockow trout . In thosThe data from controlled laboratory infection experiments are interesting in comparison to the field occurrence patterns presented in While virus strain infectivity quantified in these controlled laboratory exposures does not explain the disproportionately higher prevalence of UC versus MD IHNV detected in surveillance of CRB Chinook salmon, in-host replication may instead influence field occurrence patterns. The total number of fish infected with UC virus was lower than observed for the MD and L viruses at 3 dpe, but those fish infected with the UC virus constituted the highest total viral quantities observed across the three virus types . Thus, UAlthough there was little intra-specific variation in host susceptibility observed here, it is interesting to consider possible explanations for the exceptions observed as lower susceptibility of the Low-Spring population to both infection and disease, and higher susceptibility of the Up-Spring population to infection. One explanatory factor to consider is genetic differences in the four host populations and the phylogenetic lineages that they represent. Phylogenies of large numbers of CRB Chinook salmon populations are consistent in finding that upper CRB Spring-run Chinook salmon populations form a monophyletic lineage that is genetically highly divergent from all other lineages ,9. This Another factor that may be relevant to the variations observed in host susceptibility is differential historical exposure of the four Chinook salmon populations to IHNV. Although the specific experimental fish groups tested here were all hatched from disinfected eggs and reared under controlled conditions to ensure they were IHNV-free, the four Chinook salmon populations they represent all originated from fish hatcheries that have had historical exposure to IHNV at different levels. These hatcheries are all located in sub-regions of the CRB that have frequent UC virus detections, but only the lower CRB populations have had frequent MD virus exposure . Based oAs potential broader impacts of the findings reported here, the Chinook salmon populations tested in these experiments represent four of the seven distinct Evolutionarily Significant Units designated for CRB Chinook salmon management and conservation . Three oIn summary, this investigation comprehensively examined intraspecific variation in CRB Chinook salmon susceptibility to infection and mortality following exposure to U, M, and L strains of IHNV. Results of this investigation generally supported our null hypothesis of no variation among host populations, but we did identify some intraspecific variation in CRB Chinook salmon susceptibility to infection in specific cases. Because infectivity assays demonstrated few statistically significant differences between the UC, MD, and L IHNV, we conclude that the low virulence of UC and MD IHNV in CRB Chinook salmon is not driven by the inability of these viruses to enter juvenile fish, but rather the ability of juvenile fish to control viral infections. When considering differences in UC and MD IHNV prevalence in Chinook salmon across the Columbia River watershed, we conclude that virus infectivity does not appear to be driving the asymmetric field occurrence patterns reported for Chinook salmon. Other factors that were not measured here, such as longer-term in-host virus persistence or viral shedding, may be influencing the large-scale epidemiological patterns observed for IHNV in the CRB. Additional studies are ongoing to better understand the host-pathogen dynamics of CRB Chinook salmon and UC and MD IHNV.


Hepadnaviridae, Papillomaviridae, and Alloherpesviridae, confirming long-term virus–host codivergence relationships for these virus groups. Collectively, our results revealed reliable and sufficient information within metatranscriptomics sequencing to characterize not only RNA viruses, but also DNA and retro-transcribing viruses, and therefore established a key methodology that will help us to understand the composition and evolution of the total “infectome” within a diverse range of vertebrate hosts.In a previous study, a metatranscriptomics survey of RNA viruses in several important lower vertebrate host groups revealed huge viral diversity, transforming the understanding of the evolution of vertebrate-associated RNA virus groups. However, the diversity of the DNA and retro-transcribing viruses in these host groups was left uncharacterized. Given that RNA sequencing is capable of revealing viruses undergoing active transcription and replication, we collected previously generated datasets associated with lower vertebrate hosts, and searched them for DNA and retro-transcribing viruses. Our results revealed the complete genome, or “core gene sets”, of 18 vertebrate-associated DNA and retro-transcribing viruses in cartilaginous fishes, ray-finned fishes, and amphibians, many of which had high abundance levels, and some of which showed systemic infections in multiple organs, suggesting active transcription or acute infection within the host. Furthermore, these new findings recharacterized the evolutionary history in the families  The development of metagenomics and next-generation sequencing technologies has revolutionized the way in which we discover and characterize viruses. These methods provide an unbiased view of the virome within a host, expand our knowledge of viral diversity, and fill in the “gaps” between many higher virus taxa ,2,3,4. AHepadnaviridae—a group of retro-transcribing (RT) viruses whose divergence from the newly established nackednaviruses (NDV) can be traced back to ~400 million years ago, well before the rise of tetrapods [Parvoviridae [Iridoviridae [Herpesvirales [Adenoviridae [Papillomaviridae [Polyomaviridae [Circoviridae [Herpesvirales shows the widest host range and a relatively straightforward virus–host codivergence relationship [Lower vertebrates represent important groups of hosts whose viral diversity was only recently expanded thanks to the metatranscriptomics approach ,12. Specetrapods . As for oviridae ,15, Iridoviridae ,17,18, Hsvirales ,20,21, Aoviridae ,23,24, Paviridae ,26, Polyaviridae ,28,29, aoviridae . Among ttionship , whereastionship . FurtherGiven that (1) RNA sequencing is capable of detecting viruses belonging to DNA and RT virus categories, and that (2) there are rich metatranscriptomics and transcriptomics sequencing results available in the public database that cover several key taxa in the lower vertebrates, we carried out a large-scale survey of DNA and RT viruses based on 711 RNA sequencing data that were generated from the previous studies. The results revealed divergent novel viruses, expanding the diversity of existing virus groups while providing key evidence that these are actively transcribing viruses, shedding important new insights on the virus–host relationship for these important virus groups.ftp://ftp.ddbj.nig.ac.jp/ddbj_database/tsa/, Our study contained two datasets—(1) the metatranscriptomics dataset (n = 125), which included the libraries used in the study that described the RNA viral diversity in lower vertebrate hosts  Table S; and (2)For each library, we compared the assembled metatranscriptomics and transcriptomics contigs against the GenBank non-redundant (nr) database using Diamond version 0.9.25.126 . The taxFor each newly discovered virus genome, open reading frames (ORFs) were predicted using TransDecoder and annotated based on those from the related reference virus genomes. Conserved domains were predicted by comparing the sequence against the conserved domain database (CDD) with an e-value threshold of 1. For large, complex DNA viruses, their genomes were further characterized and visualized using Easyfig v2.2.5 . Other vHerpesvirales, Parvoviridae, Papillomaviridae, Circoviridae, Hepadnaviridae, and Retroviridae. In addition to those revealed by this study, reference virus genome sequences representative of the diversity in each family were downloaded from the GenBank database. The family-level trees were reconstructed based on one or several conserved protein alignments—namely, the major capsid protein (capsid), ATPase subunit of terminase, and protein kinases for the order Herpesvirales , the non-structural protein (NS1) for the family Parvoviridae (n = 51), the viral replicase initiation protein (Rep) for the genus Circovirus (n = 29), the late protein (L1) for Papillomaviridae (n = 54), the polymerase protein for Hepadnaviridae and related viruses (n = 38) [Retroviridae (n = 70). Amino acid sequence alignment of these viruses was performed in MAFFT (version 7) [The phylogenetic trees were reconstructed for each vertebrate-associated DNA and RT virus family/order, including n = 38) , and thersion 7) , and ali , and thrsion 7) , employihttp://www.timetree.org/, accessed on 15 March 2021). We used the program Jane v4 [For those confirmed vertebrate-associated viruses identified in this study, we reconciled the phylogeny of each virus family/order and that of their host for the examination of the codivergence hypothesis. The virus phylogenies were based on the family/gene-level maximum likelihood phylogenies estimated in this study, whereas the related host topologies were inferred and downloaded from the TIMETREE website ( Jane v4  which isAll virus genome sequences generated in this study have been deposited in GenBank under the accession numbers MZ244208-MZ244223 (pending release) and in Figshare (doi:10.6084/m9.figshare.14569476).Circoviridae, Herpesvirales, Pappillomaviridae, and Hepadnaviridae , including Agnatha (jawless fishes), Chondrichthyes (cartilaginous fishes), Actinopterygii (ray-finned fishes), Dipnomorpha (lungfishes), Amphibia, and Reptilia. From these host groups, four vertebrate-associated DNA and RT virus families were identified—namely, aviridae A. In comaviridae A. Among aviridae B. On theaviridae B. Despitaviridae C. On theaviridae C. Generaaviridae D,E, and aviridae F.Hepadnaviridae, Parvoviridae, Circoviridae, Herpesviridae, Polymaviridae, and Papillomaviridae. Among these, Hepadnaviridae had the highest positive rates (For transcriptomics (poly-A tailed enrichment) sequencing data, we examined a total of 586 public transcriptomics libraries, which included 352 species of low vertebrate hosts, and detected members of ve rates G. The ovHoplobatrachus rugulosus)—HWWF (lung tissues) and HWWGP (liver and spleen tissues)—to which we temporarily assigned the species name ranid herpesvirus 4 (RHV4). Based on metatranscriptomics sequencing, we were able to recover 6 major contigs, which totaled 85,404 bp in length (non-repeat) and approximately 36.8% of the expected genome length  , which iHerpesvirales, we reconstructed phylogenetic trees based on three conserved proteins within the order—namely, capsid maturational protease, ATPase subunit of terminase, and protein kinase. The phylogenies showed that the RHV4 was consistently clustered with RHV2, which in turn belonged to the genus Batrachovirus of the family Alloherpesviridae (In order to place the newly identified RHV4 within the context of the order sviridae C.Parvoviridae (n = 2), Circoviridae (n = 1), and Papillomaviridae (n = 2) (Parargyrops edita (standing fish) and Schizothorax prenanti (Ya-fish), respectively, and we temporarily named them Parargyrops edita parvovirus 1 (PaePV1) and Schizothorax prenanti parvovirus 1 (ScpPV1), respectively. The two viruses were highly divergent from the other parvovirus strains. For PaePV1, we revealed the partial genome (1221 bp) with the NS1 gene, and it did not belong to any of the existing parvovirus genera (Dependoparvovirus sp.) shared only 35.4% amino acid identity. The second virus, ScpPV1, which had a 3741-bp-long contig covering both the NS1 and VP proteins, was clustered with the Syngnathus scovelli chapparvovirus (51.9%), and belonged to the genus Ichthamaparvovirus, subfamily Hamaparvovirinae  . The twos genera A, while ovirinae A.Anguilla anguilla). This virus contained the predicted ORF, replicase, and shared 95.8% and 96.2% of its amino acid identity with Anguilla anguilla circovirus and Anguilla anguilla circovirus 2 [Anguilla anguilla. Therefore, we temporarily named this virus Anguilla anguilla circovirus 3 (AACV 3). Furthermore, phylogenetic analyses revealed that fish-related viruses, including Anguilla anguilla circovirus [Anguilla anguilla circovirus 2 [Anguilla anguilla circovirus 3, and Silurus glanis circovirus isolate H5 [One circovirus-related contig (1757 bp) was identified from a transcriptomics sequencing of liver and brain tissues collected from European eel  and Urolophus aurantiaus papillomavirus 2 (UaurPV 2), respectively. Among these, the contigs associated with UaurPV 1 had only L1 genes, whereas those of UaurPV 2 had E1 and L1 genes. The two viruses were closely related to one another, but were extremely divergent (<47.3% amino acid identity) from the existing member of the family Papillomaviridae. Phylogenetic analyses revealed that UaurPV 1 and 2 formed a highly divergent clade in the papillomavirus tree, basal to all of the papillomaviruses identified from Tetrapoda as well as ray-finned fishes  and rat fish (Chimaera phantasma)—which were all recovered from the liver samples, suggesting that the associations between HBV and vertebrates are now extended to cartilaginous fishes. Among them, the complete genome (3435 bp) of hepadnavirus was identified from ringstreaked guitarfish, which was named as Rhinobatos hynnicephalus hepatitis B virus . The average coverage of RHHBV was 802 folds (407.5 RPM), which encoded the polymerase, core, and surface proteins, and shared an amino acid similarity of 37.2% with the roundleaf bat hepatitis B virus , and together they formed a fish-associated cluster , sister to the mammalian HBVs; Anguilliformes HBV 3 was clustered with the white sucker hepadnavirus to form a second fish-associated HBV cluster  enveloped within the hepadnaviruses clade; the three viruses identified from amphibians  formed a cluster with another amphibian virus, Tibetan frog HBV, and together they formed a sister clade  to HBV circulating in birds , ray-finned fishes (n = 2), amphibians (n = 46), and reptiles (n = 25), and some of them had been described in previous studies [Our BLAST search also revealed a number of contigs carrying reverse transcriptase (RT), which were related to those from the family  studies ,48. DespHerpesvirales (vertebrate-associated clade), Papillomaviridae, Hepadnaviridae, and Parvoviridae (vertebrate-associated clade). With the exception of Parvoviridae, the rest of the virus families/orders showed striking similarity between virus and host trees (p < 0.01), there were in some cases relatively more frequent host switch events for parvoviruses and papillomaviruses  can have complete genome coverage through RNA sequencing, DNA viruses often have disjointed or partial genome coverage, even with high viral abundance levels. This is expected given the presence of non-coding (and hence non-transcribing) regions, as well as the huge variation in the expression levels of different genes in the DNA virus genomes. Despite that, metatranscriptomics is able to reveal the core set of functionally conserved viral genes in DNA viruses A, which With metatranscriptomics sequencing, the pool discovery rate for vertebrate-associated DNA viruses (10.4%) is much lower in comparison to that for RNA viruses (84.2%). This is expected, because with RNA sequencing, it can only reveal DNA viruses that undergo active transcription, and therefore is not an exhaustive collection of all DNA viruses present within the sample. Indeed, a study that compares metatranscriptomics and viral particle enrichment approaches ,50,51,52To avoid false positives in virus discovery and incorrect inferences on virus–host associations, we limited the scope of our search to vertebrate-associated viruses, which excluded the majority of the viruses that are associated with reagent contamination ,55, undiRana pipiens [Bufo bufo [Our study revealed a novel alloherpesvirus (RHV4) in Chinese tiger frogs. The virus had relatively high abundance levels (up to 559 and 224 RPM) in pooled  lung and liver tissue samples, suggesting active infection that potentially involves multiple individuals within the population. Before this study, four herpesviruses had been identified in amphibians—namely, ranid herpesvirus 1, 2, and 3  and bufonid herpesvirus 1 ,41,57. A pipiens , and bufFurthermore, it is striking that metatranscriptomics alone can reveal more than one-third of the expected genome length for large DNA viruses. Indeed, the RHV4 genome structures revealed by metatranscriptomics largely followed those of its closest relative, RHV2 A. HoweveOur study also revealed a total of 12 divergent hepadnaviruses from Chondrichthyes, Ostrichthyes, and Amphibians—among which 5 had complete genomes—and therefore greatly expanded the diversity and host range of this family. In a previous study, Lauber et al.  discoverParvoviridae, and to cartilaginous fishes for Papillomaviridae; the latter is represented as the most divergent cluster in the tree (Recent pathogen discovery works have also witnessed an expansion of the host range for small DNA viruses—such as papillomaviruses, polyomaviruses, circoviruses, and parvoviruses—which was previously dominated by mammalian viruses, but now includes increasing numbers of lower vertebrate hosts ,45,64,65the tree , an obsethe tree . Neverth


In contrast, ZIKV seroprevalence was 22.2%  in West African chimpanzees (Pan troglodytes verus) and 11.1%  in king colobus (Colobus polycomos). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013–2018 in Brazil and 2006–2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire ( Macaca mulatta) in Uganda during the 1950s and later reported in native non-human primate (NHP) species and in forest-dwelling Aedes mosquitos in Africa and therefore transmission in parts of Africa is thought to be maintained through interepidemic sylvatic cycles [The Zika virus (ZIKV) was first isolated from a sentinel rhesus monkey  and tamarins (Saguinus labiatus) were susceptible to ZIKV in vivo [Sylvatic transmission cycles may permit ZIKV maintenance until the pool of susceptible humans is replenished by birth and migration, potentially contributing to a resurge of intense urban ZIKV transmission cycles. Whether animal reservoirs play a role in ZIKV transmission in the Americas remains unknown. The available data on ZIKV infection of NHP in the Americas are inconclusive. Some South American NHP species, such as marmosets  and Japanese Encephalitis virus (JEV) in cats and dogs  and of Win vivo , and altin vivo , only febopictus . In Afrivitattus . Whereasand dogs . Those aHere we investigated ZIKV spread in pets (i.e. dogs and cats) and peri-domestic animals  sampled in northeastern Brazil, the 2015–2016 outbreak’s epicentre, and in wild NHP, pets and peri-domestic animals sampled in Côte d’Ivoire, where ZIKV has been documented in forest-dwelling mosquitos .Leishmania spp. serosurvey [Colobus polycomos) and western red colobus (Piliocolobus badius) were sampled in the Taï National Park during 2006 and 2016 [Cercocebus atys) that has been under observation since 2012 [Pan troglodytes verus) were included; this population of chimpanzees has been followed daily since 1979 and necropsy samples have been systematically performed on all dead individuals recovered, since the inception of the veterinary programme in 2002 [In Brazil, equid, cattle, sheep, goat, dog and cat samples were collected in the northeastern state of Bahia during 2013–2018 within state routine veterinary surveillance activities or from dogs and cats presenting clinical signs, and in 2015 from dogs in the neighbouring state of Pernambuco for a rosurvey . In Côteand 2016 . Samplesnce 2012 . Additio in 2002 .90) to identify anti-ZIKV neutralizing antibodies . Because cross-reacting antibodies against co-circulating flaviviruses can yield false-positive ZIKV test results [90 endpoint titres with endpoint titres against co-circulating flaviviruses representing diverse serocomplexes, including Rocio virus , Saint Louis Encephalitis virus , Bussuquara virus , Wesselsbron virus , Spondweni virus , Dengue virus 2 , Yellow Fever virus , and West Nile virus . We used antigenic cartography to discern neutralizing antibody reactivity patterns [90 and not by ECLIA.A total of 1416 sera of pets and peri-domestic animals from Brazil and 298 sera of dogs, peri-domestic animals and NHP from Côte d'Ivoire were screened for ZIKV antibodies using the NS1-based Electrochemiluminescence immunoassay (ECLIA) Elecsys Zika IgG . This double-antigen test enables multispecies testing because it does not require species-specific secondary antibodies, but it was only validated for human sera . ECLIA-p results , we comppatterns . Due to 90 was conducted in 12-well plates seeded 1 day before the infection with 1.6×105 Vero FM cells for ZIKV, SPOV, YFV, WSLV, WNV and BSQV, 1.6×105 Vero B4 cells for DENV-2 and ROCV, and 1.2×105 BHK-21 cells for SLEV. Forty plaque-forming units were incubated with respective serum dilutions of 1 : 40, 1:80, 1:160, 1:320, 1:640 and 1:1280 for 1 h, added onto the cell monolayer, and incubated for 1 h before adding an overlayer containing DMEM with 2% FCS and 1.25% carboxymethyl cellulose. Cells were incubated for 3 days for WNV and SLEV, 4 days for ZIKV, 5 days for SPOV, DENV-2 and BSQV, 6 days for ROCV and WSLV and 7 days for YFV. The overlayer medium was removed, and cells were fixated with 6% paraformaldehyde and stained with crystal violet. PRNT90 endpoint reciprocal titres were calculated using a logistic regression function in GraphPad Prism 6 .PRNT90 fourfold titre differences, commonly considered decisive in flavivirus serology [90 endpoint titres were fourfold or higher for WNV in five (7.7%), YFV in three (4.6%), SPOV in one (1.5%) and WSLV in one serum (1.5%). A total of 31 ECLIA-positive sera  were negative for all flaviviruses tested by PRNT90, likely due to differential sensitivity of those tests. Among the eight ECLIA-reactive sera from pets and peri-domestic animals from Côte d'Ivoire, we found a monotypic reaction to YFV in one serum and to SPOV in another serum .The initial ECLIA-based ZIKV reactivity rate among pets and peri-domestic animals was 4.6%  in Brazil and 3.6%  in Côte d’Ivoire , Table 1serology , ZIKV-spCulex quinquefasciatus in Haiti [90 titres as decisive serological support [Antigenic cartography did not provide a robust separation of flavivirus serocomplexes in pets and peri-domestic animals from either Côte d`Ivoire or Brazil . The detin Haiti  it is un support . This in support .AF using PRNT90. Fourfold or higher ZIKV PRNT90 titres compared to other flaviviruses were found in 11.1%  of king colobus and in 22.2%  of chimpanzees  are predominantly found in the tree canopy [Erythrocebus patas) and Vervet (Chlorocebus pygerythrus) monkeys in Senegal [Cercopithecus ascanius), grey-cheeked mangabey , mantled guereza (Colobus guereza) in Uganda [Chlorocebus tantalus) and Mona (Cercopithecus mona) monkey in Nigeria [In NHP, ZIKV ECLIA reactivity was observed in 23.1%  of red colobus, 66.7%  of king colobus and in none of the 17 sooty mangabeys, while 88.9%  of the chimpanzees tested positive for ZIKVmpanzees  and in ne canopy . Notably Senegal  and by tn Uganda , and tan Nigeria , could cOur study was limited by the lack of longitudinal sampling from the different animal species in both locations in Brazil and Côte d’Ivoire, since long-term titre comparison would be more effective for understanding antibody responses after multiple flavivirus infections. We also did not test other synanthropic animals such as marsupials, known to be relevant arbovirus hosts, e.g. for the alphavirus Ross River virus  and whicIn brief, our data confirm that co-circulating flaviviruses challenge unambiguous ZIKV antibody test results in hyper-endemic areas . DespiteClick here for additional data file.


Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain . The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background.Cucumber vein yellowing virus (CVYV) is a member of the genus The online version contains supplementary material available at 10.1007/s00705-021-05248-y. Ipomovirus in the family Potyviridae. This genus includes seven species (ICTV Master Species List 2020.v1 [MSL36]). The first description of CVYV was published by Cohen and Nitzany in 1960 in Israel ), 6K1, CI , 6K2, NIa-VPg , NIa-Pro (nuclear inclusion protein a–protease), NIb  and CP (coat protein) [Cucumber vein yellowing virus (CVYV) is a member of the genus 20.v1 MSL6). The fn Israel , Cyprus n Israel , and Iran Israel , Spain [n Israel , and Fran Israel  and Tunin Israel ). CVYV ilanatus)  as well lanatus) . Symptom20.v1 MSL6). The flanatus) . Other einfected . In studeraceae) . In natuanically . Like otanically . This geanically  and tomaanically  and coccanically  encode aanically , 24. Othprotein) . To dateIn this study, the complete genome sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) was determined, and an infectious full-length cDNA clone was constructed. Sequence analysis revealed a high level of sequence identity (99.7%) when its nucleotide and encoded amino acid sequence were compared to those of CVYV from Spain (NC_006941.1).C. sativus cv. ‘Vorgebirgstraube’) that were naturally infected with CVYV PV-0776 served as the source of CVYV. The virus was further propagated in cucumber, and freshly infected plants were used to prepare dsRNA extracts at 15 days post-inoculation (dpi) [7 and ends at position 3228 with UAA, resulting in a protein of 77 amino acids (~9 kDa). When compared to the other available complete sequences, percent identity values for the complete genome/polyprotein are 99.7%/99.7% (vs. Spain), 94.3%/96.1% (vs. Israel), and 94.4%/96.1% (vs. Jordan), indicating the closest relationship to the isolate from Spain. In total, there are 36 nucleotide substitutions, resulting in eleven amino acid changes when compared to the isolate from Spain. The amino acid changes are located in P1a (four exchanges), P1b (two exchanges), NIa-Pro (two exchanges), and NIb (three exchanges). For detailed information, see Supplementary Table S2. A schematic representation of the genome of CVYV Portugal is shown in Figure At the Leibniz Institute DSMZ Plant Virus Department, cucumber plantlets (on (dpi) . Primerson (dpi) , the genRhizobium radiobacter GV2260 by electroporation and infiltrated into the lower surface of the cotyledons of four C. sativus cv. ‘Vorgebirgstraube’ plantlets using a needleless syringe. The OD600 in the inoculation buffer was adjusted to 1.0 ± 0.2. In all plants, symptoms occurred 19-21 dpi resembling those of the wild type  for different lengths of time to determine the acquisition access and inoculation time. The CVYV full-length clone was readily transmitted. Efficient virus transmission was observed when whiteflies were kept overnight on infected plants. However, a two-hour acquisition/inoculation period was found to be sufficient for transmission of the CVYV full-length clone. Virus symptoms became noticeable 14 days after whitefly transmission and were similar to those of the wild-type virus isolate from Portugal.Semi-persistent transmission of the CVYV full-length clone by 1 pol 63  was confCucumber vein yellowing virus. This now provides the possibility to introduce genome modifications to study vital biological virus traits, transmission, symptomatology, and host range.CVYV Portugal is the first reported infectious and transmissible full-length cDNA clone of a virus isolate of the species Supplementary file1 (FASTA 10 KB)Supplementary file2 (FASTA 3 KB)Supplementary file3 (PDF 350 KB)Below is the link to the electronic supplementary material.


In this study, oil-in-water Pickering emulsions of uniform micron droplet sizes were formulated using 20% red palm olein and 0.5% (w/v) cellulose nanocrystals-soy protein isolate (CNC/SPI) complex followed by encapsulation within beads. FT-IR was used to characterize the bonding between the alginate, chitosan, and Pickering emulsion. 2% (w/v) alginate-1% (w/v) chitosan hydrogel beads were found to be spherical with higher stability against structural deformation. The alginate-chitosan beads displayed excellent stability in simulated gastric fluid (SGF) with a low water uptake of ~19%. The hydrogel beads demonstrated a high swelling degree (85%) with a superior water uptake capacity of ~593% during intestinal digestion in simulated intestinal fluid (SIF). After exposure to SIF, the microstructure transformation was observed, causing erosion and degradation of alginate/chitosan wall materials. The release profile of TQ up to 83% was achieved in intestinal digestion, and the release behavior was dominated by diffusion via the bead swelling process. These results provided useful insight into the design of food-grade colloidal delivery systems using protein-polysaccharide complex-stabilized Pickering emulsions incorporated in alginate-chitosan hydrogel beads.The present work aimed to investigate the swelling behavior,  Nigella sativa seeds, has attracted significant attention in recent years to be used as an alternative to chemical drugs. TQ has been known to possess therapeutic benefits such as anti-oxidant, anti-inflammatory, and anti-cancer with minimal adverse effects and no severe toxicity (Thymoquinone (TQ), a major essential oil component of toxicity , 2. Furttoxicity  and inhitoxicity , 5. Thestoxicity . In additoxicity . These l2+  of alginate, forming crosslinked alginate-chitosan complexes, providing an enhanced protective coating for the encapsulated moieties  hydrogel beads with cellulose nanocrystals-soy protein isolate (CNC/SPI)-stabilized Pickering emulsion to develop a new delivery formulation for the loading of the hydrophobic bioactive model, TQ. The CNC/SPI-stabilized Pickering emulsion (TPE) was prepared by ultrasonication using a probe based on a previous study  and an i−1) was obtained from the Milli-Q® Plus apparatus  and was used in all the experiments. All other chemicals and reagents used were of analytical grade.Cellulose nanocrystals (CNC) (freeze-dried) were procured from the University of Maine, United States. Soy protein isolate (SPI) was provided by Shandong Wonderful Industrial Group Co., Ltd. . Sodium alginate, calcium chloride and low molecular weight chitosan (50–190 kDa) were purchased from Sigma Chemicals Co. . Red palm superolein  was acquired from Sime Darby Jomalina Sdn. Bhd., Malaysia. Ultrapure water  as the oil phase and deionized water as the continuous aqueous phase. Thymoquinone (TQ) was firstly dissolved in the oil phase with magnetic stirring at 500 rpm at room temperature for 1 h. Next, emulsions with a fixed oil content were prepared by mixing 20% (v/v) palm olein with aqueous suspensions of 0.5% (w/v) CNC/SPI complex at pH 7, adjusted by 1 M HCl and 1 M NaOH. Subsequently, the mixture was emulsified for 5 min using the ultrasonic horn at 60 W, and the emulsion was stored in glass vials for further usage. The measured average droplet size of formulated TPE was found to be ~23.5 ± 2.2 μm with a zeta potential of 34.3 ± 1.4 mV . PickeriAccording to a previously described method , alginatThe shape and average size of freshly formed Alg-Chi-TPE hydrogel beads and dried Alg-Chi-TPE hydrogel beads were investigated using a Stemi microscope . The size of the beads was measured using the ZEN2012 Blue software and was averaged based on eight replicates. The shrinking ratio of freshly prepared and dried alginate-chitosan beads was calculated using the following Equation (1).f is the average size of freshly prepared beads and Sd is the average size of dried beads.Where S−1 were recorded using the attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectrometer .Alg-Chi-TPE hydrogel beads were crushed using mortar and pestle before analysis. The FTIR spectra over the wavelength range of 400–4,000 cme) was calculated based on a standard calibrated curve of thymoquinone based on the following Equation (2).According to the previously described method for O/W emulsions , the enc2 and chitosan) and the washing solution was measured at the wavelength of 290 nm by a UV-Vis spectrophotometer . The encapsulation efficiency of thymoquinone in Alg-Chi-TPE hydrogel beads (EEb) was calculated using the following Equation (3).According to the previously described method for O/W emulsion-filled alginate-chitosan (Alg-Chi-TPE) hydrogel beads, the encapsulation efficiency was calculated by measuring the leakage during the crosslinking process . After t2 in phosphate buffer solution (pH 7.5). The mixture was stirred until homogenized for 1 h before use. Pepsin is responsible for breaking down protein during gastric digestion, while pancreatic lipase is important in the dietary triacylglycerol breakdown during intestinal digestion.Simulated digestion of Alg-Chi-TPE hydrogel beads in gastric and intestinal phases was performed according to a previously described method  with modThe water uptake of the Alg-Chi-TPE hydrogel beads was performed in two different digestive media: SGF and SIF. Accurately weighed beads were immersed in 20 mL of respective medium in a sealed conical flask and placed in an incubator shaker (100 rpm) at 37°C. The beads were separated from the medium at specific time intervals, wiped gently with filter paper and weighed. The weight change of the beads to time was determined using the following Equation (4).s is the weight of the beads in swollen state and Wi is the initial weight of the untreated beads.Where WThe swelling and erosion of the Alg-Chi-TPE hydrogel beads were performed in two different digestive media: SGF and SIF. Beads were immersed in 20 mL of respective medium in a sealed conical flask and placed in an incubator shaker (100 rpm) at 37°C. The beads were removed from the medium at specific time intervals, wiped gently with filter paper, and the diameter was measured. The swelling degree and erosion degree of the beads to time were determined using the following Equations (5) and (6), respectively.s is the diameter of the beads in the swollen state, Di is the initial diameter of the untreated beads, We is the weight of the dried beads in the swollen state, and Wi is the initial weight of the untreated beads.Where DThe microstructures of Alg-Chi-TPE hydrogel beads after gastric digestion and intestinal digestion were observed through a variable pressure scanning electron microscope  and an upright fluorescent microscope . For VP-SEM preparation, the samples were air-dried, sputter-coated with gold under a vacuum before analysis. The samples were observed at an accelerating voltage of 10 kV. For the fluorescent microscope, the lipid phase of the emulsions was stained with Nile red (0.01% w/v) during the preparation process. A bead was placed on a microscopic slide and gently covered with a coverslip. The edge of beads was observed.The release of thymoquinone (TQ) from Alg-Chi-TPE hydrogel beads in gastric digestion and intestinal digestion were evaluated according to the method described in section Measurement of Encapsulation Efficiency (EE). In brief, the hydrogel beads were immersed in 20 mL of respective medium in a sealed conical flask and placed in an incubator shaker (100 rpm) at 37°C. The hydrogel beads were treated with SGF for 120 min followed by SIF for another 240 min. Then, at specific time intervals, 2 mL of the medium was withdrawn from the conical flask and topped up with the fresh medium. The release profiles of TQ were fitted to the Peppas model by the following Equation (7) .Where Mt/Mi is the cumulative release ratio at time t, k is the kinetic constant, and n is the diffusional exponent.2/chitosan solution (Thymoquinone (TQ) was first dissolved in the red palm olein phase before the emulsification process. Next, the cellulose nanocrystals-soy protein isolate (CNC/SPI) complex was used as the Pickering stabilizer and dispersed in the aqueous phase. The alginate-chitosan (Alg-Chi) hydrogel beads were then prepared by external gelation method through dripping a mixture of TQ-loaded CNC/SPI-stabilized Pickering emulsion (TPE) with different alginate concentrations into a pre-mixed CaClsolution . The digsolution .−1 for the asymmetric and symmetric stretching vibrations of C-H (-CH2) and carbonyl group (C=O) from ester  group of alginate  (−1), denoting the electrostatic interaction of chitosan with alginate in the hydrogel beads. On the other hand, the FTIR spectra (FTIR spectra of thymoquinone-loaded Pickering emulsion (TPE), Alg, Alg-TPE, Chi, and Alg-Chi-TPE hydrogel beads are shown in om ester . The FTIalginate . The shi groups) . The C=O spectra  of Alg-Ce) of thymoquinone (TQ) in the freshly prepared Pickering emulsion system was determined to be >99%. This is reasonable since TQ first fully dissolves in the oil phase before turning into an O/W emulsion. Alternatively, based on the hydrogel shells formed by external gelation that immobilized the O/W emulsion loaded with TQ, the TQ's encapsulation efficiency (EEb) according to Equation (3) of the Alg-Chi-TPE beads was determined to be 89.18 ± 3.63%, revealing an excellent loading efficiency using hydrogel beads. A decrease in the encapsulation efficiency could be due to the release of TQ during the ionic gelation and the hardening process.According to Equation (2), the encapsulation efficiency  and continued to achieve 643 and 593%, respectively at 240 min, demonstrating substantial intestinal digestion of hydrogel beads in SIF. The destabilization of the alginate polymer network at intestinal pH is due to the ion exchange between Na+ ions in the intestinal digestion fluid and Ca2+ ions in the hydrogel beads  alginate were disintegrated and deformed, where the weight measurement of the swollen beads could not proceed . Generalel beads .2+ ions at low pH, the COO− groups become protonated, resulting in the formation of hydrogen bonds within the alginate chains . A significant reduction in the weight of hydrogel beads can be related to the syneresis effect in an acidic environment where shrinkage is favored  beads, chitosan-coated alginate (Alg-Chi-TPE) beads and Alg-Chi-TPE beads after specific digestion in SGF or SIF are illustrated in As shown in The micrographs of Alg-Chi-TPE hydrogel beads at a different time point of intestinal digestion are illustrated in p < 0.05), while TQ releases in the SGF medium is undetected. Only about 4% of TQ was detected after the end of gastric digestion (240 min). The total TQ release in SIF medium and SGF/SIF medium was 48 and 83%, respectively (p < 0.05), showing the extensive release of TQ when the hydrogel beads were pre-soaked in the SGF medium before introducing to the SIF medium. The introduction of intestinal fluid at an alkaline pH and the extensive water uptake properties of the hydrogel beads could account for the initial rapid release (The release of encapsulated thymoquinone (TQ) from the hydrogel beads was achieved during gastrointestinal digestion. The TQ release profiles from Alg-Chi-TPE hydrogel beads in three different digestion systems  are presented in  release . In addi release , 50, thuBy comparing the TQ release in the SIF medium alone and SGF/SIF medium, it can be denoted that two possible mechanisms regulate the bioactive release out of the hydrogel beads: the swelling process and the diffusion process . When thThe results were analyzed using the Peppas model to distinguish the release mechanisms of TQ from the alginate beads. For hydrogel beads, the diffusional exponent (n) specifies the mechanism of release. The calculation of the n value was measured up to the initial 60% release of the bioactive. If the values of n are equal or <0.43, the release is associated with Fickian diffusion. If n values are within 0.43 to 0.85, the release is indicated with Fickian diffusion and Case-II transport (caused by the swelling process). If the values of n are more than 0.85, the release is solely contributed by Case-II transport , 51. Thein vitro digestion and hydrophobic thymoquinone (TQ) release from alginate-chitosan hydrogel beads immobilized Pickering emulsion (Alg-Chi-TPE). 2.0% (w/v) alginate hydrogel beads with spherical shape demonstrated the highest stability against structural deformation during the drying process. The presence of chitosan in beads formation improved the wall materials properties, providing a lower swelling degree and a rougher microstructure. Alg-Chi-TPE hydrogel beads demonstrated good stability during gastric digestion, and the release of encapsulated TQ was observed during intestinal digestion. Up to 83% of total TQ was released from the hydrogel beads after 2 h gastric digestion followed by 4 h treatment in the simulated intestinal fluid. The bioactive release mechanisms were incorporated with the Peppas model, which exhibited a Case-II transport caused by the swelling process during gastric digestion and Fickian diffusion during intestinal digestion. This study contributes to a better understanding of the swelling and digestion behaviors of alginate-chitosan hydrogel beads immobilized with food-grade Pickering emulsions to release a hydrophobic bioactive compound. This provides valuable information about its potential application in developing the colloids-based nutraceutical delivery system.This work demonstrates The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.SKW: conceptualization, methodology, investigation, data curation, formal analysis, and original draft writing. DL and JS: writing-review and editing. CHP and SYT: funding acquisition and validation. BHG, SM, TWW, and CHP: resources and review. SYT: supervision and project administration. All authors contributed to the article and approved the submitted version.The authors gratefully express gratitude to all parties which have contributed toward the success of this project, both financially and technically, especially the S&T Innovation 2025 Major Special Programme (Grant No. 2018B10022) and the Ningbo Natural Science Foundation Programme (Grant No. 2018A610069) funded by the Ningbo Science and Technology Bureau, China, as well as the UNNC FoSE Faculty Inspiration Grant, China. The Zhejiang Provincial Department of Science and Technology is also acknowledged for this research under its Provincial Key Laboratory Programme (2020E10018). This project was supported by the Tropical Medicine and Biology Platform, School of Science and Advanced Engineering Platform, School of Engineering, Monash University Malaysia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Juvenile Idiopathic Arthritis (JIA) requires complex care that generate elevated costs, which results in a high economic impact for the family. The aim of this systematic review was to collect and cluster the information currently available on healthcare costs associated with JIA after the introduction of biological therapies.We comprehensively searched in MEDLINE, EMBASE, Web of Science, Scopus, and Cochrane Databases for studies from January 2000 to March 2021. Reviewers working independently and in duplicate appraised the quality and included primary studies that report total, direct and/or indirect costs related to JIA for at least one year. The costs were converted to United States dollars and an inflationary adjustment was made.We found 18 eligible studies including data from 6,540 patients. Total costs were reported in 10 articles, ranging from $310 USD to $44,832 USD annually. Direct costs were reported in 16 articles , showing a proportion of 55 to 98 % of total costs. Those costs were mostly related to medications and medical appointments. Six studies reported indirect costs . Four studies reported costs according to JIA category observing the highest in polyarticular JIA. Total and direct costs increased up to three times after biological therapy initiation. A high risk of reporting bias and inconsistency of the methodology used were found.The costs of JIA are substantial, and the highest are derived from medication and medical appointments. Indirect costs of JIA are underrepresented in costs analysis.The online version contains supplementary material available at 10.1186/s12969-021-00641-y. Juvenile Idiopathic Arthritis (JIA) is a generic term, used to describe a group of heterogeneous diseases characterized by chronic arthritis with onset before the age of 16 .Patients with JIA have symptoms of joint inflammation, morning stiffness, pain, contractures, fatigue, abnormal growth, and functional limitation . The difThe treatment of JIA must be multilevel, with a pediatric rheumatologist, psychological support, physical therapy, nutrition, and family support. Within pharmacological treatment, a wide range of options with different costs and effectiveness could be found. Some studies have shown that the costs increase in relation to disease activity and progression to disability . With thThe costs can be dived in those directly related with healthcare services , and those not related to healthcare services  . TherefoThere have been some reports about the costs of illness for JIA, but its variability is important according to the region, social context, and healthcare system. The aim of this systematic review was to collect, and cluster the information currently available on healthcare costs associated with JIA in the world after the introduction of biological therapies.https://www.crd.york.ac.uk/prospero) with the code CRD42019135865.We performed a systematic review to estimate the direct and indirect costs in patients with JIA, regardless of the subtype or region. This report followed a rigorous systematic review protocol that adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyzes (PRISMA)  recommenWe included complete economic evaluations , partial economic evaluations , and individual studies with cost reporting  regardless of publication status, size, or language. Primary studies published after 2000 that report total, direct and/or indirect costs related to JIA for at least one year were included, since the aim was to analyze the costs after the introduction of biological therapies. Non-primary studies , studies published in a non-peer-reviewed source , and studies where information to determine eligibility was not available, were excluded.A comprehensive search was carried out by an experienced librarian, advised by the principal investigators. The databases consulted were Ovid MEDLINE, EMBASE, Web of Science, Scopus, Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials for studies from January 1, 2000 to the date of the search . A search update was performed on March 11, 2021. The search strategy is available as Studies were entered into systematic review software . To ensure the reliability of selection among investigators, a pilot test was performed with a random sample of 60 studies derived from the search; these were reviewed for inclusion criteria by means of title and abstract. The exercise was repeated until we achieved a kappa 0.7 between the reviewers.Reviewers  worked independently and in duplicate to evaluate titles and abstracts on the selection criteria. After abstract screening and retrieval of potentially eligible studies, full-text publications were assessed for eligibility, with adequate inter-reviewer agreement (kappa 0.61). Duplicate studies and studies with overlapping populations were excluded. Disagreements were reviewed by a third reviewer and their inclusion was subsequently decided by consensus.Independently and in duplicate using a standardized database, the reviewers collected the following information from eligible studies: (1) study general data , (2) participants characteristics , (3) total costs (TC), (4) direct costs (DC), and (5) indirect costs (IC).The DC included those derived from medical appointments, medications , laboratory tests, clinical imaging, surgeries, hospitalizations, physiotherapy, devices, alternative medicine, administration of medications, adverse events, and complications. Also included derivatives of transportation, home adequacy, caregiver accommodation, travel expenses, informal and formal patient care, and insurance payments.In the IC, those related to the loss of productivity of patients and caregivers were considered .https://www1.oanda.com/lang/es/currency/converter/) considering the data collection date of the study. An inflationary adjustment was made as of December 31, 2019, with the Inflation Tool 2020 (https://www.inflationtool.com/).The costs were converted to United States dollars (USD) using the OANDA’s currency calculator tool , North America , one included population from those two regions (5.5 %), and one from Asia (5.5 %). The countries included in the studies were the United Kingdom, Germany, Finland, Canada, United States of America (USA), Turkey, Italy, the Netherlands, Sweden, France, Bulgaria, and India , being oligoarticular the most frequent  in DC. On the other hand, IC were cut in half in one study  and incrOnly one study reported an analysis of costs by country of origin, including patients from six European countries . That stRegarding economic studies, we found that two of eleven articles was classified as low quality due to problems in the quality of the analysis, methodology and measurement. The three cross-sectional and observational articles were classified as low risk of bias. All four cohort articles were classified as of good quality , and the ways of reporting it. The need for a consensus report in costs of JIA, especially from the patient’s perspective, is essential. The wide variability in costs reflects the low recording of the real costs of those who suffer this disease, which is reinforced by the little information related to indirect costs. Furthermore, the lack of data on absenteeism and presenteeism of patients and their caregivers is notable, as well as information regarding the long-term economic impact on these families .Although we found some studies that addressed costs from a therapeutic perspective, only one type of biological therapy was studied, and they focused the measurement on DC. It is necessary to explore the variation in IC before and after the start of different biological therapies to establish the long-term economic benefits.On the other hand, current literature reports costs by generalizing those across the course of JIA, thus costs at different time points during the disease journey  cannot be identified. This could be an important approach to understand the most critical moments of need for financial support for those families.Finally, if the costs and the proportion of these in relation to family income are reported, a better comparison between different regions would be achieved.The extensive and rigorous search in different databases, without language restrictions and carried out by an expert medical librarian, minimizes the probability of losing information, however, it is possible that there are data on costs in sources not included in this work, such as thesis, technical reports, and conferences. The risk of reporting bias is high, particularly due to the lack of consistency in the economic aspects described, different definitions, and reports based on the perspective of health institutions and external payers. It was not possible to perform a meta-analysis of costs, which would have been of great relevance to identify the areas of greatest need for support for patients and their families. Regardless of these limitations, this review has important strengths due to the synthesis of all the available evidence following a pre-designed protocol, with reproducible judgments on the selection of studies, quality criteria and data analysis.This study synthesized the costs of JIA and highlights the financial risk that families could face during the disease trajectory. Most studies focus on total or direct costs, while indirect costs are underreported. Despite this, the information collected allows us to identify that the costs of JIA are substantial and probably the highest are derived from medication and medical appointments. Which evidences the great economic impact of JIA and how catastrophic it can be for a family.A high risk of cost reporting bias was found and the variability of costs and the way they are measured is high, which makes it impossible to generalize the findings, although the values were higher in Europe, as well as high in polyarticular JIA.It is necessary to standardize the reports and generate information from developing countries to obtain a more accurate analysis of the impact of the disease in the world.Additional file 1. Supplementary search strategy.Additional file 2. Supplementary tables.