For over a century, viruses have left a long trail of evidence implicating them as frequent suspects in the development of type 1 diabetes. Through vigorous interrogation of viral infections in individuals with islet autoimmunity and type 1 diabetes using serological and molecular virus detection methods, as well as mechanistic studies of virus-infected human pancreatic β-cells, the prime suspects have been narrowed down to predominantly human enteroviruses. Here, we provide a comprehensive overview of evidence supporting the hypothesised role of enteroviruses in the development of islet autoimmunity and type 1 diabetes. We also discuss concerns over the historical focus and investigation bias toward enteroviruses and summarise current unbiased efforts aimed at characterising the complete population of viruses (the “virome”) contributing early in life to the development of islet autoimmunity and type 1 diabetes. Finally, we review the range of vaccine and antiviral drug candidates currently being evaluated in clinical trials for the prevention and potential treatment of type 1 diabetes. Type 1 diabetes (T1D) is characterised by the chronic immune-mediated destruction of pancreatic β-cells, with affected individuals requiring lifelong exogenous insulin . GloballT1D is categorised into four stages: (1) presymptomatic T1D with the presence of multiple islet autoantibodies (type 1a) but normoglycemia; (2) presymptomatic T1D with progression to dysglycaemia; (3) dysglycaemia and clinical symptoms such as polyuria, polydipsia, polyphagia, weight loss, fatigue and diabetic ketoacidosis (DKA)); (4) long-standing T1D ,6. AcuteMost T1D is preceded by the development of islet autoimmunity (IA), serologically confirmed by the presence of at least one diabetes-associated islet autoantibody to insulin (IAA), glutamic acid decarboxylase 65 (GADA), protein tyrosine kinase-related islet antigen 2 (IA-2A) and zinc transporter 8 (ZnT8A). IA can appear from around six months of age with incidence peaking prior to two years of age in the genetically at risk but will be generally present months to years before symptomatic onset, reinforcing the need for early-stage interventions and increased monitoring of presymptomatic T1D. These autoantibodies typically appear sequentially rather than simultaneously, making it unclear whether multiple or single events precipitate seroconversion and eventual T1D development ,11. The + T cells, directly contrasted by the action of regulatory T cells. Dendritic cells and even mast cells  have also been implicated in T1D pathogenesis, as they present islet autoantigens to autoreactive T-cells, resulting in dysregulated peripheral immune tolerance . T. T238]. ple size . The recple size . In contThe most critical limitation of conventional NGS for virome applications is its poor sensitivity for eukaryotic viruses, due to signals drowned out by noise from an overwhelming background of non-viral or bacteriophage nucleic acid. In an attempt to boost the sensitivity specifically for vertebrate-infecting viruses, we and others have applied VirCapSeq-VERT to characterise the gut and blood virome of children with IA/T1D ,205. Thrp = 0.01) [Our recent systematic review and meta-analysis of the above observational studies (published by June 2020) that applied NGS to investigate potential associations between early life virome and IA/T1D reported a small, but significant association between IA and consecutive EV positivity in longitudinal samples  . This wa = 0.01) .Overall, inconsistencies in results across existing T1D virome studies  could beDriven by the strong rationale for developing antiviral therapeutics to prevent T1D ,248, theTo overcome such limitations, an alternative vaccine strategy has been explored for CVB1, 3 and 4, involving the use of empty VLPs as antigens ,253,254.Broadly speaking, antivirals can be divided into two categories: (i) those directly targeting viral proteins (reviewed for EVs in ); and . This represents the first randomised clinical trial with antiviral drugs to test the hypothesis that a six-month-long treatment with pleconaril–ribavirin combination can eliminate persistent EV infection in the pancreas.Altogether, a plethora of molecular and epidemiological evidence supports a strong rationale for the development of antiviral vaccines for T1D prevention. However, unanswered questions remain regarding which genotypes to target and whether other important viruses have been missed due to the substantial investigation bias towards EVs in previous studies using targeted virus detection methods. To address such concerns, a growing number of studies are applying unbiased NGS approaches to characterise the virome in diverse at-risk populations. One key area that is unknown is whether maternal virus exposure during pregnancy significantly influences the risk of virus exposure and IA development in the offspring, which may be elucidated by studies such as ENDIA and DIPP-novum.


Aedes aegypti, the yellow fever mosquito, and Aedes albopictus, the Asian tiger mosquito, are the most significant vectors of dengue, Zika, and Chikungunya viruses globally. Studies examining host factors that control arbovirus transmission demonstrate that insect-specific viruses (ISVs) can modulate mosquitoes’ susceptibility to arbovirus infection in both in vivo and in vitro co-infection models. While research is ongoing to implicate individual ISVs as proviral or antiviral factors, we have a limited understanding of the composition and diversity of the Aedes virome. To address this gap, we used a meta-analysis approach to uncover virome diversity by analysing ~3000 available RNA sequencing libraries representing a worldwide geographic range for both mosquitoes. We identified ten novel viruses and previously characterised viruses, including mononegaviruses, orthomyxoviruses, negeviruses, and a novel bi-segmented negev-like group. Phylogenetic analysis suggests close relatedness to mosquito viruses implying likely insect host range except for one arbovirus, the multi-segmented Jingmen tick virus (Flaviviridae) in an Italian colony of Ae. albopictus. Individual mosquito transcriptomes revealed remarkable inter-host variation of ISVs within individuals from the same colony and heterogeneity between different laboratory strains. Additionally, we identified striking virus diversity in Wolbachia infected Aedes cell lines. This study expands our understanding of the virome of these important vectors. It provides a resource for further assessing the ecology, evolution, and interaction of ISVs with their mosquito hosts and the arboviruses they transmit. Aedes aegypti, and the Asian tiger mosquito Aedes albopictus, are invasive hematophagous insects distributed worldwide within the tropical and subtropical zones  OR “Aedes”[All Fields]) AND (RNA-Seq[Title] OR transcriptomic[Title] OR transcriptome[Title] OR sequencing[Title]). We cross-referenced these publications with raw data deposited in Sequence Read Archive (SRA) hosted by NCBI. We excluded libraries and publications where metadata was not usable, conflicted with methods described within the publication or if uploaders of data were uncontactable. Original catch locations of colonies latitude and longitude positions were approximated from metadata and plotted using ggplot2  and the package “maps”  in the RStudio environment (Version 0.99.491). Fastq files from associated SRA accession numbers were downloaded using a Perl script written by Michael Gerth (Oxford Brookes University) and available from GitHub . To identify publications utilising high-throughput RNA-Seq of https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 20 April 2020) was used to identify adapters. Adapters and low-quality regions of sequencing were trimmed using the CLC in-house trimming tool  or Trimmomatic  . . Ae. aegsquitoes C. While four of the AOLV segments had detectable homologs from other insect reo-like viruses, three out of the seven incriminated segments of AOLV had no detectable homology with any known virus from this group or RNA viruses , suggestParvoviridae family known to infect both Ae. aegypti and Ae. albopictus  of these putative MDV strains. Phylogenetic placement of the NS1/2 and VP genes of these contigs (Parvoviridae (<80% amino acid identity from the replicase protein), they are not sufficiently divergent to be separate species of MDV.Mosquito densoviruses (MDVs) are single-stranded DNA viruses of the iewed in ). MDVs ius genus . We iden contigs  suggesteAe. albopictus U4.4 cells sequenced as part of the Arthropod Cell Line RNA-Seq initiative, Broad Institute, we examined the incidence of this MDV sequence in U4.4 cells and could not identify reads mapping to this MDV from any other U4.4 datasets . We were even able to partially assemble the virus genome from this data [Aedes cells, we identified MDV infections in Ae. albopictus RNA-Seq data from Manassas, USA [Ae. albopictus Foshan colony (BioProject: PRJNA275727) is most closely related to Aedes albopictus densovirus 7 isolate GZ07  [In 2014, Jingmen tick virus (JMTV), a segmented positive-sense RNA virus, was reported following isolation from e, China . The genll lines ,139. Sinll lines  and Chin7907936) . Phyloge7907936)  and also7907936)  , it appears unlikely that the introduction of JMTV into these samples are due to recent blood-feeding for maintenance. However, given that the Rimini colony was established in 2004 and sampled in November 2017, we cannot rule out the possibility that JMTV may have been introduced from the previous blood-feeding to maintain the colony. Further examination of the incidence and persistence of JMTV in Aedes ISVs, we mapped reads from small RNA sequencing libraries against our virus database, examining the size distribution of the virus fragments originating from these genomes and the mapping profiles of Aedes viruses  length small RNAs dominant in the population of small RNAs consistent with other studies examining the RNAi response of mononegaviruses ,144. Ae. aegypti colony metatranscriptomics [Ae. albopictus mosquitoes from several libraries [We could identify the vsiRNA profile for both segments of the Verdadero virus, a partiti-like virus previously identified in iptomics . We alsoibraries ,87. We cibraries   and 10 nt complementary overlapping reads. For the San Gabriel virus and all segments of Aedes phasma virus [1-A10) and over-represented complementary 10 nt overlapping pairs of sRNA from reads of 24–29 nt from these viruses  as bearing >85% nucleotide identity over a minimum of 500 bp for each virus. It is essential to appreciate that the RNA samples processing, sequencing library preparation, instruments used, and read length of all data used between studies are vastly different. As such, it is challenging to normalise and draw quantitative comparisons between studies. We believe, however, that the analysis presented here reasonably incriminates samples as positively infected. However, we concede that given the limitations of the library preparations, viruses not containing poly-A tails or in huge abundance may be incorrectly characterised as “negative”. Given this limitation, we recommend screening mosquito samples using RT-PCR or RT-qPCR methods. A summary of common ISV infections and their associated BioProject is available in To understand the composition and incidence of ISVs between different laboratory colonies, we created a database of representative genomes of novel viruses identified here and previously identified viruses known to infect these mosquitoes (n = 94). Libraries originating from one biological sample or group were trimmed, pooled, and mapped to these genomes. Previous studies have used an arbitrary cut-off of 100 mapped reads per million using a total library sub-sample. Some of the viruses presented here either do not have poly-A tails or are unlikely to have poly-A tails. As most of the data examined were produced from transcriptome studies using a poly-A enrichment step, we felt this cut-off might be too stringent. Instead, we used the complete libraries and visually inspected mapping coverage to exclude false-positive mapping. To additionally implicate viruses as being present, we queried our Ae. aegypti and Ae. albopictus suggests that ISV compositions are entirely separate between mosquitoes, with very few examples of potential contamination between laboratory colonies , almost all BioProjects (57/60) had sufficient ISV RNA identifiable in these libraries, with some harbouring up to 11 individual ISVs. While recently adapted colonies of mosquitoes had the most diverse viromes, there were some Ae. aegypti colonies that we could not detect any virus RNA in their corresponding data and, based on our analysis, are potentially “virus sterile”. The ROCK or Rockefeller strain, which is of Caribbean origin established in 1930 [In laboratory strains of  in 1930 , as well in 1930 , both apAe. aegypti genome and maintained at the Liverpool School of Tropical Medicine since 1936 , was idiewed in . This suAe. albopictus, which has been in culture since 1981 and used to assemble the nuclear genome [The reduction in virome diversity was similarly observed in the Foshan or Guangdong strain of r genome . While Fr genome ,86.Ae. aegypti samples recently adapted to the laboratory (within ten generations). The first study sequenced the midguts from individual mosquitoes from Thep Na Korn, Thailand midguts [Transcriptomic studies are increasingly using libraries prepared from single individuals rather than pooled mosquitoes as biological replicates. We set out to analyze individual datasets to explore the heterogeneity of viruses within individual mosquitoes. For this, we used two studies using individual  midguts . The sec midguts .Ae. aegypti with clinical isolates of dengue virus (DENV-1) [Ae. aegypti Cairns libraries [Both publications experimentally infected (DENV-1) , and (DE(DENV-1) . As the ibraries  were lowal. 2017 , at 24hpWithin the recently colonised Thep Na Korn, Thailand strain , we idenAedes mosquitoes, most individual mosquitoes are infected on average with two to three ISVs and can be super-infected with up to five viruses in individual mosquitoes.Virus heterogeneity was similar in whole mosquitoes originating from Cairns, Australia. However, the most abundant identified virus was Aedes aegypti toti-like virus 1 (ATLV-1), which was almost fixed in the mosquitoes with ~90% of individuals infected with ATLV-1 (38/42 samples) B. The seAe. aegypti Aag2 cell line indicated a persistent infection with an insect-specific flavivirus CFAV [Phenuiviridae) [Ae. albopictus RML-12 cell line [Birnaviridae) is a common laboratory contaminant of Ae. albopictus U4.4 cell line [Ae. albopictus C7-10 cells and Ae. aegypti Aag2 cells [Cell lines are invaluable tools in arbovirus research, and numerous reports have demonstrated that most harbour persistent infections of ISVs . Early rrus CFAV ,153,154.viridae) ,155, andviridae) . PCLV isell line . It is aell line ,151 and g2 cells . In addition to RNA virus infection, mosquito cell lines are known to be persistently infected with densoviruses . Aedes aAedes cell lines were uncovered. For example, we identified Wenzhou Sobemo-like virus 4 (WSBLV-4) in Ae. albopictus C7-10 cells from total-RNA sequencing obtained as part of the Arthropod Cell Line RNA-Seq initiative, Broad Institute (broadinstitute.org), and three independent small RNA sequencing datasets [Previously reported ISVs were identified in this study . Howeverdatasets ,164. It datasets  suggestidatasets . The RNAWolbachia are super-infected with a range of viruses, including the positive-sense RNA virus WSBLV-4 and three negative-sense RNA viruses San Gabriel virus, PCLV, and AeAV  were more closely related to the cell line-identified San Gabriel virus strains (98–99% over 99% of the genome) than to the wild-derived San Gabriel virus from 95.86% over 99% of the genome of the Ae. albopictus colony from the USA. This observation indicates that these infections are likely to have originated from the parental RML-12 cell lines for reasons previously discussed [The super-infection of WSBLV-4, San Gabriel virus, PCLV, and AeAV was observed in two independent ed cells ,65 and Aed cells . San Gabiscussed .Ae. aegypti Aag2.wMelPop-CLA cell lines with up to 1.2 million out of ~22 million small RNA reads (~5.45%) mapping to AeAV [wMelPop-CLA cells . The RNAi profile indicates both the genome and replicative intermediate are targeted by the RNAi response, indicating active infection in these cells. By comparison, we could map no reads from the paired Aag2 cell line. We identified both WSBLV-4 and Saint Gabriel virus in the Ae. albopictus Aa23 cell line and a second Ae. aegypti cell line transcriptome that was stably transinfected with the Wolbachia wAlbB strain from Aa23 cells (Aag2.wAlbB), suggesting contamination of San Gabriel virus from the Aa23 cells during the production of the Aag2.wAlbB cell line [Wolbachia does not confer a refractory virus phenotype to negative-sense RNA viruses [Wolbachia and the positive sense WSBLV-4 deserve further experimentation.We have previously demonstrated that another mononegavirus, AeAV, persistently infects the RML-12 and  to AeAV . Reanalyell line . The pre viruses ,165,166.Ae. aegypti and Ae. albopictus colonies that infect colonies, wild-caught populations, and cell lines worldwide. Phylogenetic analysis of the viruses reported here suggests all these viruses are closely related to other virus families that have recently been described to infect arthropods except for one Ae. albopictus colony potentially infected with the vertebrate infecting Jingmen tick virus. While most of these viruses are likely to have an insect-restricted host range, experimental validation should be undertaken due to the extensive distribution of some of these viruses in Aedes populations. We show that recently established lab colonies harbour up to five viruses in individual mosquitoes and very few colonies of Aedes mosquitoes are “sterile” using our analysis. In addition to identifying novel viruses, we have explored the geographical distribution of previously known ISVs of Ae. aegypti and Ae. albopictus. As we establish the contribution of viruses associated with mosquitoes to the vector competence of arboviruses, understanding the prevalence and distribution of these viruses provides a resource for further assessment of the ecology, evolution, and interaction of ISVs with their mosquito hosts and arboviruses they transmit.We have identified up to ten novel viruses associated with


Buffalopox outbreaks caused by vaccinia virus were observed in villages of Tamil Nadu, India, among lactating buffaloes and cows. Milkers also had lesions on their fingers. Because vaccinia virus is known to have extended its host range in Brazil, we recommend continuous surveillance to understand cross-species transmission and to curtail disease effects. In India, sporadic outbreaks of buffalopox, which can be caused by vaccinia virus (VACV), have been reported among cattle and buffaloes , panel ATo investigate the causative agent, we used existing clinical samples. Scab samples were collected randomly from 20 affected animals . We examined scab suspensions under transmission electron microscope (EM) at 80 KV and inoculated the suspension in BHK21 cell line for virus isolation. We examined scab homogenates and cell culture fluid by PCR for differential diagnosis of cowpox virus and VACV infection , panel CDuring the global eradication of smallpox, strains of VACV were used as vaccine. VACV infection sometimes transmitted from the vasicular lesion of vaccinae to domestic animals, usually cattle; in turn, infected animals transmitted VACV to susceptible humans (The outbreaks resulted in financial loss to the farmers because of mastitis and loss of milk production. The outbreaks also created public health concern because of human infection. The source of infection could not be identified; it is possible that VACV could be lurking in rodents, as reported earlier (


S-locus F-box genes (SLFs) tightly linked to an S-RNase termed type-1. Multiple SLFs collaborate to detoxify nonself S-RNases while being unable to detoxify self S-RNases. However, it is unclear how such a system evolved, because in an ancestral system with a single SLF, many nonself S-RNases would not be detoxified, giving low cross-fertilization rates. In addition, how the system has been maintained in the face of whole-genome duplications (WGDs) or lost in other lineages remains unclear. Here we show that SLFs from a broad range of species can detoxify S-RNases from Petunia with a high detoxification probability, suggestive of an ancestral feature enabling cross-fertilization and subsequently modified as additional SLFs evolved. We further show, based on its genomic signatures, that type-1 was likely maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through S-locus deletions or by retaining duplications. Two deletion lineages regained SI through type-2 (Brassicaceae) or type-4 (Primulaceae), and one duplication lineage through type-3 (Papaveraceae) mechanisms. Thus, our results reveal a highly dynamic process behind the origin, maintenance, loss, and regain of SI. The self-incompatibility (SI) system with the broadest taxonomic distribution in angiosperms is based on multiple  Genetic, functional, phylogenomic, and ancestral state reconstruction studies reveal a highly dynamic process behind the origin, maintenance, loss, and regain of self-incompatibility in angiosperms. S-loci (single to multiple) (Self-incompatibility (SI), the inability of a fertile seed plant to produce a zygote after self-pollination, serves as a widely occurring outcrossing mechanism to prevent inbreeding in angiosperms. Approximately 40% of all angiosperm species possess SI. Various SI systems have evolved and are classified based on their associations with floral morphology , the genetic control of pollen SI phenotypes (sporophytic SI [SSI] or gametophytic SI [GSI]) or the number of ultiple) . SI has ultiple) . HoweverS components, corresponding to S-RNase and S-locus F-box (SLF), also named S-haplotype-specific F-box (SFB), respectively. So far, type-1 SI has been found in four eudicot families: Solanaceae, Plantaginaceae, Rosaceae, and Rutaceae, spanning two major clades  has been shown to carry the S-locus containing an S-RNase flanked by a cluster of 9–37 SLF genes  group into two other classes of T2 RNases (Classes I and II) compared to S-RNases (Class III) (Ananas comosus) was shown to be tightly linked to several genes encoding F-box family members  . However members , indicatS-locus contained a single SLF linked to an S-RNase  inactivation of the S-RNase; and (3) deletion of the entire S-locus. However, the relative contribution of these three mechanisms is not known. Moreover, it is unclear how type-1 SI was maintained in the face of whole-genome duplications (WGDs), which would have caused breakdown of SI via Route 1.Type I SI system has been lost in multiple lineages . Such loS-loci, reactivation of an S-RNase, or evolution of a new SI system. The latter process likely accounts for the other types of SI in eudicots (S-locus cysteine-rich (SCR) protein/S-locus protein 11 and a female S-locus receptor kinase  stigma S (PrsS) and P. rhoeas pollen S (PrpS) , anther position-controlling GLOBOSA (GLO), a functionally unknown Conserved Cysteine Motif (CCM), Pumilio-like RNA-binding protein (PUM) and a Kelch repeat F-Box  . Type 4 Box KFB; . HoweverPetunia with a high detoxification probability, suggesting a likely mechanism for how type-1 SI might have originated. We further show, based on the distribution of type-1 S-locus signatures in the genome, that a type-1 SI system was maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through deletion or duplications of the S-locus. Inactivation of the S-RNase was only detected in horticulturally selected lines, suggesting this route to self-compatibility (SC) is not favored in natural populations. Two deletion lineages regained SI through type-2 or type-4 mechanisms, while one duplication lineage regained SI through a type-3 mechanism.Here we use a combination of phylogenetic and functional approaches to reveal a highly dynamic picture of SI evolution. We show that SLFs from a broad range of species can detoxify S-RNases from S-locus. For example, Antirrhinum contained a Class III T2 RNase on chromosome 7, whereas its S-locus maps to chromosome 8 , two from monocot families (Poaceae and Bromeliaceae) and other angiosperms as well as gymnosperm species . Their TFigure  1;S-like-RNases in the eudicot exemplar species, or to Class I/II T2 RNases of monocot and other angiosperm species  repeat proteins encoded by those genes linked to  species . The FBAS-RNases, the SLF subfamily included paralogs outside the S-locus in exemplar species. These members typically belonged to the same clade as the SLFs from the same plant family, suggesting that they arose through duplications within the family lineage  closely linked to an S-like-RNase on chromosome 7, suggesting duplication of an ancestral S-locus region  that was not expressed  (5-SLF5ShS or 5-SLF6ShS) (Petunia hybrida genotype 3S3LS (3S3LPhS)   under thPhS3S3L) . The traained SC . Self-prained SC . S-haploS3-RNase . The tra3L-RNase .SLFs across a broader taxonomic distance, we introduced three Antirrhinum SLFs, one S-like SLF (Antirrhinum majus Chr1-FBX6 (AmChr1-FBX6) that does not belong to the SLF subfamily, under the control of strong pollen promoters into the 3S3LPhS background. Aniline blue staining showed that SI breaks down in transgenic plants expressing the SLFs and S-like SLF genes but not for the FBA/FBK-encoding gene  caused breakdown of SI by inactivating the PhS3L-RNase .In summary, the ability to inactivate S-RNases is a feature of the S-locus of P. avium  contains two types of SLF genes (SLF and SFB types)  and PaSFBs (SFB type) into the 3S3LPhS background  . Both tybed here . To testckground . PaS4-SLS-RNases .SLF from another member of the Rosaceae, apple , into P. hybrida. Its S-locus only contains SLF-type SLFs named S-locus F-box brothers (9MdSFBBβ-S)   and was S-RNase and SLF in Colorado blue columbine (Aquilegia coerulea) in the Ranunculaceae  into the 3S3LPhS background  = 7.38, P = 0.0066).To further explore the detoxification range, we annotated homologs of culaceae . Aquileg than SI . We detewn of SI  and prog Petunia  (assuminA. comosus), indicating that it may have the potential to produce type-1 SI  and pollen-specific (SLFs) expression may have evolved later to control eudicot type-1 SI. In the absence of genetic data, it is unclear whether the linked Class I T2 RNase and FBK genes found in monocots can confer type-1 SI. Ancestral state analysis showed that the common ancestor of angiosperms possessed linked Class I/II T2 RNases and FBA/FBK genes. Whether the eudicot MRCA had linked Class I/II T2 RNases/FBA/FBK genes or whether a subfamily evolved toward Class III T2 RNases/FBA/FBK genes could not be determined with certainty  tightly linked to several F-box family member genes was identified in pineapple  Deletion or inactivation of components of the S-locus (e.g. S-RNase); or (3) Deletion of the entire S-locus  deletion of duplicates; (3) reactivation of the S-RNase; or (4) evolution of a new SI system   would have led to duplications of the S-locus, causing loss of SI via Route 1  and clementine (Citrus × clementina) also had duplicate loci, one of which may have been degenerated in a similar manner. Solanum habrochaites, C. maxima, olive (Olea europaea), red date (Ziziphus jujube), and castor bean (Ricinus communis) had only one copy of the type-1 S-locus, indicating regain of SI via deletion of duplicates (Route b)  .A. majus and S. lycopersicum) showed either deletion (A. majus) or inactivation . Both species are horticultural varieties that have been selected for SC. Therefore, Route 2 for type-1 SI appears to have been favored in domestication but not in natural populations. Three other SC species, diploid cotton (Gossypium raimondii), common bean (Phaseolus vulgaris), and A. coerulea  or type-4 (cowslip [Primula veris]) SI  and regain of SI through Route d (We found no evidence of type-1  Route d .SRKs) and pollen (SCRs) components in a total of 17 species from 15 genera of the Brassicaceae as well as in spider flower (Cleome hassleriana) of the Cleomaceae  in upland cotton (Gosssypium hirsutum) and cacao (Theobroma cacao) of the Malvaceae and S. pennellii of the Solanaceae, but their linkage to SRK-like genes was absent . The clustering of genes in the S-locus supergene was found in P. vulgaris of the Primulaceae but not in species from twelve other families representing monocots and eudicots, including neighboring families . These findings suggested that the type-4 S-locus supergene evolved only in the Primulaceae , which exhibits type-3 SI, contained four type-1 S-like-loci, indicating ancestral loss of type-1 SI through duplication (Route 1) and subsequent regain of SI via Route d , indicating that type-3 SI evolved in an ancestor of the Papaveroideae , which exhibits GSI, possessed two copies of type-1 S-loci, suggesting its regain of SI via either Route a or d (Helianthus annuus), which exhibits SSI, had no type-1 S-like locus, indicating regain of SI via Route d  SLFs must not detoxify S-RNases from the same haplotype to maintain SI and (2) SLFs must be able to detoxify S-RNases from other haplotypes, allowing cross-compatibility. If the ancestral  S-RNase , the firf styles . It is dPetunia S-RNases in 22 out of 26 cases, corresponding to a detoxification probability of 0.85. With such a probability, pollen would be accepted by 72% of styles for a single SLF linked to an S-RNase , which may act within each species to reduce target range over evolutionary time, resulting in the currently observed intraspecific detoxification probability of 0.5.Once such a system was established, duplications of the  one SLF . Thus, sFBA/FBK with T2 RNases likely arose in the common ancestor of monocots and eudicots  does not support the self-recognition hypothesis. A further observation supporting nonself-recognition rather than self-recognition is competitive interaction between two functional S-haplotypes of tetraploid Chinese sour cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi) leading to SC . In the case of type-3 SI, four type-1 S-like-loci were found in the genome, indicating loss of type-1 SI via duplication. For all SI types, we found that ancestral pistil  and pollen  factors, predate the formation of linked SI components. SI may therefore have evolved by duplications and rearrangements bringing the male and female components in tight linkage followed by neofunctionalization or subfunctionalization (Type-1 SI has been maintained in many species in the face of WGD through deletion or inactivation of duplicate copies of the lization .RNase) and male (FBK/FBA) component coming into tight linkage. The ancestral single SLF had a high detoxification probability to allow type-1 SI to become established with sufficient cross-compatibility. Selection for further increase in cross-compatibility led to duplication and divergence of the SLF within the S-locus  was obtained from Tomato Genetic Resource Center  and M. domestica and P. avium from Dr Tianzhong Li’s lab at China Agricultural University (CAU). The partial self-compatible line of A. coerulea was from Hongzhi Kong’s lab at the Institute of Botany, the Chinese Academy of Sciences (CAS). All materials were planted in a greenhouse at the Institute of Genetics and Developmental Biology, CAS, except M. domestica and P. avium, which were planted in a CAU nursery.Self-incompatible lines of escribed . SolanumAhSLFs, PaSLFLs, and PaSFBs were described previously  strain LBA4404  by electroporation and transformed into leaf disks of P. 3S3Lhybrida PhS as previously described (http://www.gelifesciences.com/). Neomycin Phosphotransferase 2 (NPTII) probes were labeled with 32P using the Prime-a-Gene labeling system . The pre-hybridization, hybridization and membrane washing steps were based on the operation manual  solution for at least 8 h. Aniline blue staining of pollen tubes was then performed as described previously (About 48 h after self-pollination of transgenic plants and the wild-type eviously  and the http://phylodiversity.net/phylomatic/) with the divergence times in those trees derived from TimeTree (http://www.timetree.org/) and WGD/WGTs from previous studies  . BLAST was run using default settings with the expected threshold cut-off of 10−5 , 10−6 , 0.05 (SCR/SCR-likes and PrpS/PrpS-likes), and 0 (SRK/SRK-likes), respectively. In addition, BLAST for FBAs/FBKs was only performed in 33 species containing Class III T2 RNases except for O. sativa. Protein sequences obtained from BLAST were screened by InterProScan version 5.36 (FBAs/FBKs located near Class III T2 RNases (upstream or downstream within 3 Mb) were obtained, except for species without such FBAs/FBKs. The full-length protein sequences of T2 RNases and FBAs/FBKs were aligned using L-INS-i method in MAFFT version 7.407  was used for visualization, annotation and management of the trees.We obtained amino acid sequences corresponding to on 7.407   using RNAprep Pure Plant Kit . RNA purity and integrity were assessed using a NanoPhotometer® spectrophotometer  and RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system , respectively. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina . All libraries were sequenced using Illumina HiSeq 2000 (2 × 100 bp). Raw reads were quality-checked with FastQC96 (version 0.11.8)  accession GWHBFSA00000000, BioProject ID PRJCA006945) using STAR (version 2.7.1a)   with par 2.7.1a) . The genJEB33121 , respectS-locus, five discrete traits were selected and codified as: possessing Class I/II T2 RNases with no linked FBA/FBK genes; linked Class I/II T2 RNases and FBA/FBK genes; linked Class III T2/S-/S-like-RNases and SLFs/S-like SLFs; S-like-RNases with no linked SLFs/S-like SLFs; and absence of both S-/S-like-RNases and SLFs/S-like SLFs. Three additional traits  were selected to reconstruct the evolution of these three types of S-loci as well as the evolutionary relationship of the four types of S-loci. Ancestral state reconstruction was conducted using PastML  was used for visualization and annotation of the results.To evaluate the evolution of type-1 nP), we applied the equation “nP = 1 - (1 - RP) n” reported in RP. According to previous studies, a single SLF can recognize about 50% (i.e. RP = 0.5) (nP (n = 1) = 0.5) or less  (i.e. RP = 0.85) and pollen with a single SLF can detoxify 85% of S-RNases (i.e. nP (n = 1) = 0.85) (2) of styles . Likewise, pollen with two SLFs, each with 0.85 (RP) recognition probability, would have a detoxification probability of 0.98 (i.e. nP (n = 2) = 0.98) (2) of females.To estimate the detoxification probability of n SLF types (R = 0.5)  or less R = 0.5)  S-RNases or less . As SLFs = 0.85) , thus ac = 0.98)  and woulhttps://ngdc.cncb.ac.cn/gsa/browse; A. hispanicum in the Genome Warehouse database .All sequence data generated in the context of this manuscript have been deposited in the China National Center for Bioinformation Genome Sequence Archive database (The following materials are available in the online version of this article. ML tree of the T2 RNase superfamily of seed plants. ML tree of the FBA/FBKs of seed plants. SLFs of S. habrochaites function as pollen S factors. Both SLFs from the S-locus of A. hispanicum and an SLFL from an S-like-locus of A. majus function as the pollen S factors. Southern blot analysis of self-progeny plants of 4PaS-3S3LSLFL1 PhS, 4PaS-3S3LSLFL2 PhS, 3S3LPaSFB1 PhS, and 3S3LPaSFB4 PhS. Phylogenetic analyses of the S genes of A. coerulea. Transcript profiles of T2 RNases and their linked FBA/FBK genes in O. sativa and A. comosus. Evolution of SI systems and their S genes in a family-level phylogenetic tree of the seed plants. The evolution of type-2 SI system in angiosperms. Phylogenetic analyses of the S genes of type-2 SI. The evolution of type-4 SI system in angiosperms. The evolution of type-3 SI system in angiosperms. Phylogenetic analyses of S genes of type-3 SI. Pollination and genotype analyses of transgenic plants of ShSLFs 3S3LPhS. Pollination and genotype analyses of transgenic plants of AhSLFs 3S3LPhS and 3S3LAmSLFL PhS. Pollination and genotype analyses of transgenic plants of PaSLFLs 3S3LPhS, PaSFBs 3S3LPhS, and 3S3LMdSFBB PhS. Pollination and genotype analyses of transgenic plants of AcSLFs 3S3LPhS. Accession numbers of T2 RNase genes used in this study. Accession numbers of FBX/FBA/FBK genes used in this study. Summary of species, SLFs/SLFLs/SFBs used for transformation, and S haplotypes of compatible transgenic pollen caused by competitive interaction. RNA-seq data of the S-locus in four tissues of A. hispanicum used in this study. RNA-seq data of the S-like-locus in four tissues of A. hispanicum used in this study. Species annotated with linked pistil-pollen gene pairs in this study. Accession numbers of SRK/SRKL, genes used in this study. Accession numbers of SCR/annotated SCR genes used in this study. Accession numbers of PrsS/annotated PrsS genes used in this study. Accession numbers of PrpS/annotated PrpS genes used in this study. Accession numbers of transgenic FBX/SLF genes used in this study. Primer list. Genome databases used in this study. Fasta files of the sequence alignments corresponding to the phylogenetic analyses of the type-1–4 S genes. Tree text files corresponding to the phylogenetic analyses in Supplemental FiguresS8–koab266_Supplementary_DataClick here for additional data file.


Furthermore, vaginal medication (p = 0.0369), vaginal dryness (p = 0.0228), dyspareunia (p = 0.0234) and low frequency of sexual intercourse (p = 0.0022) were found to significantly correlate with the presence of genital lesions. According to our findings, standardized colposcopy in combination with toluidine blue dye facilitates accurate assessment of genital lesions. Genital trauma situated at another site than the posterior part of the vaginal introitus seems to be uncommon after consensual intercourse.This study aimed to assess the validity and efficacy of blue dye in colposcopic assessment of genital injury in pre- and postmenopausal women with and without history of consensual sexual intercourse. Two hundred women were prospectively enrolled and examined colposcopically with and without toluidine blue dye in order to detect and categorize genital lesions . Examination of genital trauma was accomplished in a standardized way and findings were photo documented. A wide range of influencing factors with a potential impact on prevalence and nature of genital injury was recorded beforehand using a questionnaire. The frequency of diagnostic injury differed substantially depending on the examination technique, ranging from 9% using colposcopic magnification only to 28% with the additional use of toluidine blue dye. A vertical laceration affecting the posterior fourchette was the most frequent lesion detected . Menopausal status seems to have significant impact on genital injury prevalence ( Differentiation between genital injury due to consensual penetrative vaginal intercourse and genital injury caused by non-consensual penetrative vaginal intercourse is a matter of ongoing scientific debate and obviously incorporates a fundamental issue in sexual assault medicine.The first hypothesis regarding this context rose in the mid-twentieth century as authors stated that physiologic changes (e.g. lubrication) and concurring interaction during consensual intercourse prevented genital trauma , 2. On tUntil now further genital injury research was undertaken with the vast majority of investigations concentrating on non-consensually caused trauma; most data were generated retrospectively after reviewing sexual assault records. However, the existing literature lacks primarily methodological consistency leading to inconclusive results; injury rates vary from 4 to 89%[Only a few investigations assessing genital trauma after consensual intercourse exist. Just as in the non-consent-publications, these studies vary in injury definitions, study protocols and investigative techniques. Two reviews of the literature were undertaken with the aim of locating existing evidence concerning genital trauma after consensual sex , 5. Due Due to the inconsistency of the existing literature and the fact that to the best of our knowledge there exists no information on genital injury with and without history of consensual intercourse including postmenopausal women, we designed this investigation. We hence deliberately chose to recruit women without scheduled consenting sexual intercourse in advance, in order to analyze genital trauma in a “real world setting” assessing possible influencing factors.This investigation was approved by the Ethics Committee II of the Medical Faculty Mannheim, Heidelberg University, Germany (2017-519 N-MA). A total of 200 women presenting at the Department of Gynaecology, University Medical Centre Mannheim between August 2017 and July 2018 were included in this prospective study after informed consent was obtained.Exclusion criteria were pregnancy (including puerperium), age below 16, prior extensive genital surgery  concomitant malign disease and/ or dermatological comorbidities . Written consent was obtained from all participants. Prior to the clinical investigation participating women were asked to fill in a questionnaire assessing behavioral and demographic parameters, which could potentially influence the appearance of their external genitals. Besides common demographic assessment, information on obstetrical history , prior/present diseases of the genitourinary system , topic genital medication , point in time of last menstrual bleeding as well as latter gynecological examination, was obtained. Postmenopausal status was defined as cessation of menstrual bleeding for at least 12 months. Furthermore, behavioral aspects were surveyed including measures of intimate hygiene , contraception, sexual life , genital piercings  and sports .Bruise: Located discoloration of intact epidermis due to an extravasation of blood.Abrasion: Areas of superficial epidermal disruption.Laceration: Discontinuity of epidermis and dermis.Clinical assessment including photo documentation was accomplished by one sole examiner, who was trained by forensic and gynecological physicians in visual inspection, colposcopy and toluidine-blue application prior to initiation of the investigation. Examination was accomplished in modified lithotomy position. Only the external genitals were assessed, slightly spreading and retracting labia majora. Colposcopy was accomplished with an ATMOS i View 31  colposcope system, using × 4 magnification, including an integrated digital camera, Sony Alpha 5000 . After macroscopic examination and annotation of findings colposcopy and photo documentation using a measuring tape were conducted. Findings were noted again. Then, toluidine blue was applied on external genitals and consecutively removed with 1% acetic acid. Colposcopy and photo documentation were repeated, and findings again noted. In line with previous publications the following categories of injuries were assessed as follows , 7:BruisAll data was stored in a Microsoft Excel sheet. After a thorough check for false data entry, the data was imported into SAS® . For normally distributed quantitative variables mean and standard deviation were calculated. For ordinally scaled data and quantitative discrete data, median values together with minimum and maximum are given. For qualitative data, absolute and relative frequencies are presented.p < 0.05 was considered as statistically significant.In order to compare two mean values a 2-sample t-test was performed for data approximately normally distributed. Otherwise a Mann Whitney U-test has been used instead. The association between two qualitative factors was investigated with Fisher’s exact test. A test result with A total of 200 participants were recruited; three questionnaires were returned incomplete and photo documentation of 6 women was rated as qualitatively insufficient. Hence, 191 observations were statistically assessed.The mean age of the study population was 41 (standard deviation ± 13) years. Table Macroscopically a total of 9 women (5%) were found to have at least one genital lesion, two (1%) had more than one injury. A total of 11 lesions  were identified.Using colposcopic examination only, 17 women (9%) were found to have at least one genital lesion. Seven (4%) of these had more than one lesion and a total of 25 lesions  were categorized. After toluidine blue dye, 55 women (28%) had at least one lesion, of these 14 (7%) had more than one and a total of 73 lesions were recorded respectively. Considering postmenopausal women only, the injury prevalence was found to be substantially higher  compared to premenopausal individuals .Using solely colposcopic magnification, of the 17 women with a lesion, 12 (71%) had at least one lesion affecting the posterior fourchette, 6 (35%) with lesions affecting the perineum or the labia minora. No further site was found to be injured.Subdividing the vaginal introitus into quadrants see Fig. , the mosAfter toluidine blue dye 63% of genital trauma was found at the “6 o’clock position” (n = 46/73), 16% in the lower right (n = 12/73) and 15% in the lower left quadrant (n = 11/73) respectively. Additionally, one lesion was found in the upper right  and three  in the upper left quadrant. Table p = 0.0165), as well as vaginal dryness (p = 0.0228), usage of topical vaginal medication (p = 0.0369) and dyspareunia (p = 0.0234). Furthermore, genital injury was also found to inversely correlate with the frequency of intercourse (p = 0.0022). Hence, lower genital injury rates were found in women with a higher intercourse frequency. Sub-classifying the entire collective into pre- and postmenopausal women we found this observation persistent for the premenopausal group only (p = 0.0052). As explained below, it has to be stressed that the majority of women (57%) in our collective did not have intercourse within seven days before examination. The remaining factors assessed, which were shown to have no influence on injury prevalence, are summarized in Table We identified several factors correlating with the prevalence of genital injuries. As expected, the menopausal status appeared to have a significant impact (p = 0.023. Furtherp = 0.0001). This staining was not detected in any participants without a history of vaginal intercourse. Of all women who reported to have had consenting intercourse, this staining was observed in 29% if intercourse took place within the past 2 days, and in 51% if intercourse took place within the last 3 to 7 days.Performing the toluidine blue dye, we observed a “U”-shaped see Fig.  stainingTo the best of our knowledge and in analogy with a review of the literature by Astrup et al., there is a total of 9 key publications assessing genital injuries after consenting sexual intercourse . While 7Also, the method of examination in the existing literature differs substantially, with some investigations using macroscopic examination only , 6. OtheThe first publications on genital trauma after consensual sexual intercourse were published by Laumer and McCauley et al. in the 1980s [Slaughter et al. were the first to use a colposcope and toluidine dye enhancement to analyze genital trauma in rape victims using the TEARS classification as stated above . FindingMcLean et al. assessed 68 women presenting for a routine cervical PAP-smear after consenting intercourse macroscopically, without using either a colposcope or dye. Injuries, categorized as bruise, abrasion and/or laceration, were observed in 5.9% [Similarly, Lincoln et al. examined a total of 81 women presenting for routine cervical screening or with sexual health concerns after consensual sex macroscopically . InjurieThe above stated investigations were comparative investigations that recruited women after consensual and non-consensual penetrative intercourse had occurred , 6, 8, 9Fraser et al. assessed 107 sexually active women two or three times over a 4- 6 month period using a consistent technique with colposcopic magnification and acetic acid . AuthorsFinally, Astrup et al. examined a total of 98 women within 48 h after consensual intercourse and found genital injuries  in 52% using colposcopy and toluidine dye [In the above stated review of the literature it is worth noting that if examination takes places after intercourse, prevalence ranges from 4 to 11%, while prevalence is much higher 25 to 55%) if clinical assessment is accomplished before scheduled intercourse  to 55% i, 10. HowIn the existing literature potential risk factors for genital trauma after consenting intercourse, analogously to injury prevalence, have to be considered with caution. However, reviewing the existing literature some findings of the above stated investigations are congruent. While existing data concerning skin color, penetrative use of finger(s) during intercourse and gynecological disease as potential influencing factors are controversial, findings are consistent concerning age, parity, use of lubricants and of tampons as well as contraceptives as the prevalence of injuries did not correlate with these factors . Furtherp = 0.0165): at least one injury was observed in 42.1% of postmenopausal versus 23.8% of premenopausal women. There exists some information about genital trauma after non-consensual intercourse [Our investigation additionally assessed postmenopausal women in this context and as expected menopause status had a significant impact on injury prevalence . To the best of our knowledge this finding has not been described before and might be the result of blue coloration due to superficial epithelium desquamation. Future investigations assessing genital injury should focus on this observation.Interestingly, an increasing frequency of vaginal intercourse correlated inversely with genital injury. To what extent this finding is affected by an increased libido with corresponding lubrication is debatable. Furthermore, the majority of women 57%) in our collective did not have intercourse within seven days before examination. Fourteen percent stated they had had sex within 2 days and 24% within the last 3 to 7 days before being examined. In contrast to the findings of previous investigations, the prevalence of injury did not correlate with time passed since last intercourse: Anderson et al. found a significant reduction of injury size within 72 h after consensual intercourse. Astrup et al. found a “median survival time” of genital lesions after consenting sex of 24 h using the naked eye, 40 h using solely the colposcope, and 80 h using colposcope magnification and toluidine dye. What has to be considered in this context is the fact that in both investigations recruitment took place before examination and authors examined women only up to 3  or 7  days respectively % in our . Our datIn accordance with previous investigations potential influencing factors  colposcopically and 73  using colposcope and toluidine dye. Bruises were not found. These findings are in accordance with previous investigations, as lacerations were found to be the most common genital injury after consensual intercourse , 19, 24.In previous investigations all types of injury were observed, but despite methodical differences findings are highly consistent as most authors identified one single vertically aligned laceration affecting the posterior fourchette as the most frequent major type of injury . Our resFew investigations focused on vaginal and cervical trauma, observing no , 4, 24 oSubdividing the topography of the external genital into quadrants we found 94% of lesions located in the lower quadrants. Hence, this region should be of special focus with respect to future scientific and forensic investigations.Referring to the objective of this investigation, the findings of which have to be considered physiological, one can summarize that lacerations and/or abrasions of the lower quadrants , especially in postmenopausal women CAN be physiological.With and without history of consensual intercourse, using colposcope and toluidine blue dye during examinations, the prevalence of genital injury seems to range between 25 to 30%. By far the most frequent lesion that was observed was a vertically aligned laceration affecting the posterior fourchette. Generally, the posterior boundaries of the vaginal introitus seem to be predominantly affected if consenting or no sexual intercourse occurred. Genital trauma affecting the anterior boundaries of the introitus has to be considered unusual after consensual intercourse. This circumstance as well as the identified risk factors, particularly postmenopausal status, should be taken into account in legal proceedings of rape victims.The frequency of diagnostic injury differed substantially depending on the examination technique used, ranging from 9% using colposcopic magnification only to 28% with the additional use of toluidine blue dye.A vertical laceration affecting the posterior fourchette was the most frequent lesion detected .p = 0.0165), as 42% (16/38) of postmenopausal compared to 24% (36/151) of premenopausal women had at least one genital lesion.Menopausal status seems to have significant impact on genital injury prevalence (p = 0.0001). This staining was not detected in any participant without a history of vaginal intercourse.Performing the toluidine blue dye, we observed a “U”-shaped see Fig.  staining


Giardia intestinalis is a flagellated protozoan responsible for giardiosis , the most prevalent and widespread parasitic infection in humans and mammals worldwide. The intestinal microbiota is highly diverse and any alteration in its composition may impact on the health of the host. While studies on the mouse model of giardiosis described the role of the gut microbiota in host susceptibility to infection by the parasite, little is known about the gut microbiota during natural infections in dogs and particularly in puppies. In this study, we monitored naturally G. intestinalis-infected puppies for 3 months and quantified cyst excretion every 2 weeks. All puppies remained subclinically infected during the sampling period as confirmed by fecal examination. In parallel, we performed 16S Illumina sequencing of fecal samples from the different time points to assess the impact of G. intestinalis infection on gut microbiota development of the puppies, as well as gut health markers of immunity such as fecal IgA and calprotectin. Sequencing results revealed that the canine fecal microbiota of Giardia-infected puppies becomes more complex and less diverse with increasing age. In addition, significant differences in the structure of the microbiota were observed between puppies with high and low Giardia cyst excretion. Chronic subclinical G. intestinalis infection appears to be associated with some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species and opportunistic pathogens, as well as a reduction of Lactobacillus johnsonii at specific time points. Calprotectin levels increased with age, suggesting the establishment of chronic low-grade inflammation in puppies. Further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). Giardia intestinalis  is a flagellate protozoan responsible for giardiosis (or giardiasis in humans), one of the most prevalent and widespread parasitic infection in humans and mammals worldwide ) containing a protease inhibitor cocktail . After homogenization by vigorous shaking for 30 min at room temperature, the suspensions were centrifuged for 20 min at 2100× g at 4 °C. The supernatants were collected and centrifuged for 30 min at 10,600× g at 4 °C. The final supernatants  were stored frozen at −80 °C prior to ELISA analyses following the manufacturer’s instructions .For IgA quantification, frozen fecal samples were thawed and 0.2 g aliquots of feces were diluted in 2 mL PBS. After homogenization with 0.10 to 0.25 mm diameter glass beads (Fisher Scientific) using a Precellys Evolution homogenizer  for 40 s at 6400 rpm, suspensions were centrifuged for 5 min at 13,000× escribed . Brieflyg rpm at 4 °C. After recovery of the supernatant, 30 μL of Proteinase K  was added and the samples were incubated for 10 min at 70 °C at 250 rpm in Multi-Therm , then for 5 min at 95 °C for enzyme inactivation. The tubes were centrifuged for 5 min at 14,000× g rpm at 4 °C and the supernatant was transferred to a deepwell. The plate was transferred on the nucleic acid workstation Chemagic STAR  and the extraction protocol was performed with Chemagic STAR DNA BTS kit  according to the manufacturer’s instructions.A modified version of the protocol by Godon et al.  was usedThe V3-V4 hypervariable regions of the 16S rDNA gene were amplified from DNA extracts during the first PCR step using universal primers PCR1F_343 and PCR1_R784 . PCR wasThe library pool (see above) was denatured (NaOH 0.1 N) and diluted to 7 pM. Then, 15% PhiX Control v3  was added to the pool as described in the Illumina procedure. Afterwards, 600 μL of this pool and PhiX mix were loaded onto the Illumina MiSeq cartridge according to the manufacturer’s instructions using MiSeq Reagent Kit v3 . FastQ files were generated at the end of the run . The run quality was checked internally using PhiX control and each paired-end sequence was assigned to its sample using the multiplexing index. The sequences were processed using FROGS . The quap < 0.05 was considered statistically significant.For statistical analyses, sequences were rarefied to a uniform depth of 57,698 sequences per sample to account for unequal sequencing depth between samples. Based on rarefaction curves, samples with less than 10,000 sequence reads were excluded from the analysis. Alpha diversity indices were compared using the Mann–Whitney test (GraphPad Prism software version 9.00). The principal coordinates analysis (PCoA) was performed with FROGS to visualize the clustering of samples according to various parameters, using Jaccard and unweighted Unifrac distances. Permutational multivariate analysis of variance (PERMANOVA) was used to test for significant differences in bacterial community composition across samples. All statistical tests were two-sided, and G. intestinalis cysts were quantified in fecal samples at seven time points: 8 days before the start of the study (D−8) and every 14 days after the start until day 85 (D85). All animals remained infected at day 85  were monitored weekly for 93 days and all puppies remained asymptomatic throughout the 93-day experiment (data not shown). t day 85 , suggestG. intestinalis genotyping may reveal different results depending on the genetic locus sequenced and multiple gene loci should be investigated to increase the specificity of the results [G. intestinalis assemblages in samples D−8 and D85 by sequencing gdh, tpi, β-giardin, and SSU rRNA genes . Next, we investigated whether IgA variations could be related to G. intestinalis cysts shedding. Fecal IgA concentrations were positively correlated with G. intestinalis cysts shedding in D85 samples , whereas no significant correlation was observed in D−8 and D42 samples. Fecal calprotectin levels were also measured by ELISA. As shown in p = 0.0006). Fecal calprotectin concentrations correlate positively with G. intestinalis cysts shedding in D−8 samples .Further analyses focused on three time points: D−8, D42, and D85 to investigate the effects of age on IgA levels and calprotectin concentrations, as non-invasive markers of intestinal health. Fecal IgA levels were measured by ELISA. As shown in p < 0.0001) and 46.71% of the variance using Unifrac distance . PERMANOVA was also applied to assess the similarities of microbial communities as a function of gender, age, and box in both distance matrices. None of these other variables were significantly associated with microbiota composition , which is consistent with previous studies [To characterize the evolution of the canine gut microbiota over time in naturally-infected puppies, we used the Illumina MiSeq technology to sequence the V3-V4 region of 16S rDNA obtained from a total of 54 fecal samples (18 puppies at three different time points). Sequencing generated a total of 5.90 million reads with an average of 109,300 reads per sample. The quality and chimera filtering produced a total of 4,475,733 reads with an average of 82,883 filtered reads per sample. Beta diversity was analyzed based on Jaccard and unweighted Unifrac distance matrices and represented by PCoA. The predicted PCoA representation of Jaccard and unweighted Unifrac beta diversity  and samp studies ,20.p < 0.0001; p < 0.0001; p = 0.0002; p = 0.0085, respectively). In D85 samples, observed OTUs and Chao1 alpha diversity increase compared to D42 samples (ANOVA p < 0.0001), while no significant differences were observed for Shannon and Inverse Simpson measures, suggesting that microbiota richness tends to stabilize over time in an adult-like microbiota.Rarefaction curves generated with these reads revealed that the sequencing depth appropriately covered bacterial diversity in all samples A. Sample−4 ± 2 × 10−4%). The abundance of the four dominant phyla varies greatly with time. Bacteroidota and Actinobacteriota significantly increase in D42 samples  and in D85 samples  compared to the D−8 samples. In contrast, Firmicutes abundance decreased in D85 samples compared to the D−8 samples (p = 0.0447). Fusobacteriota abundance is highly variable in D−8 samples, and tends to stabilise with increasing age in D42 and D85 samples. At the genus level, the abundance of most dominant genera was significantly enriched in D85 samples compared to the D−8 samples, including Fusobacterium (p = 0.0410), Prevotella (p < 0.0001), Alloprevotella (p = 0.0032), Lactobacillus (p = 0.0048), Bacteroides (p = 0.0003), Peptostreptococcus (p < 0.0001), Ruminococcus gnavus group (p = 0.0321), Parasuterella (p < 0.0001), Faecalibacterium (p = 0.0002), Prevotella Ga6A1 group (p < 0.0001), Bifidobacterium (p < 0.0001), and Enterococcus (p = 0.0002).A total of eight phyla and 81 genera were identified A. High iG. intestinalis affects the richness and diversity of the canine gut microbiota. To determine the impact of G. intestinalis on the composition of the fecal microbiota, we classified the samples into three categories according to their parasite load: Low, intermediate, and high. The beta diversity of these three categories was then analyzed using Jaccard and unweighted Unifrac distance matrices and was represented through PCoA (p = 0.0404) and 23.18% of the variance using unweighted Unifrac distance (PERMANOVA p = 0.0152).ugh PCoA . In sampG. intestinalis cysts excretion at the three time points . A positive trend was also noted in the observed, Chao1, and Shannon richness but without reaching the significance threshold of 0.05. On the contrary, when puppies are older and have a more mature microbiota, cysts excretion correlates with a significant decrease in the observed and Chao1 richness . At D42, a slight decrease in the observed richness was noted but no significant richness difference, which is consistent with the intermediate stage.In addition, a significant correlation was observed between alpha diversity and e points . BacteriG. intestinalis cysts shedding at the three time points , Prevotella , Lachnoclostridium , an unclassified genus of Peptostreptococcaceae , Anaerovibrio , Megamonas , Catenibacterium , Veillonellaceae , an unclassified genus of Ruminococcaceae , Bacteroides caecigallinarum , Allobaculum stercoricanis , and Anaerobiospirullum thomasii . In D42 samples, G. intestinalis cysts shedding correlates positively with Anaerobiospillum succiniproducens  and an unclassified species of Suterella . In D85 samples, G. intestinalis cysts shedding correlates positively with Fournierella , Faecalibacterium prausnitzii , and unclassified species of Suterella  and Parasuterella . On the contrary, G. intestinalis cysts shedding correlates negatively with Lactobacillus johnsonii in D85 samples .The abundance of specific OTUs correlates significantly with e points . In D−8 G. intestinalis is one of the most common enteric parasites worldwide and is remarkable for its ability to cause an array of clinical phenotypes, ranging from asymptomatic infections to severe acute diarrheal disease to chronic gastrointestinal disease in human and animals. Acute giardiasis is auto-resolutive in 2 to 4 weeks in adults but could become chronic especially in young subjects, resulting in serious growth stunting and long-term health consequences [G. intestinalis remained infected at 21 weeks of age (D85) when no rescue treatment was administered, suggesting that G. intestinalis infection was not auto-resolutive in these animal hosts. In a murine model of infection, animals infected neonatally did not clear the parasite when they reached adulthood, although naive adult animals exposed to G. intestinalis were resistant to infection, suggesting that G. intestinalis manipulated the host during the postnatal period to favor its own persistence [equences ,41. In tsistence . Genotypsistence ,43,44.G. intestinalis clearance by targeting the parasite variant-specific surface proteins (VSPs) [G. intestinalis cysts shedding at day 85. No correlation was observed in earlier time points  ,46. We ds (VSPs) ,47,48. Fe points A. These der mice . HoweverG. intestinalis. Fecal calprotectin concentration was associated with G. intestinalis cysts shedding in 9-week-old (D−8) puppies. No correlation was observed in later time points . SCFAs display health properties that promote host intestinal health. For example, they regulate cell differentiation and proliferation, gut endocrine functions, immune response, and promote the intestinal epithelial barrier function. Finally, butyrate (one of the main SCFAs) is the primary source of energy for enterocytes [Bifidobacterium, Lactobacillus, and Enterococcus increased with age, contrasting with findings in humans [The canine fecal microbiota was investigated during 12 weeks and revealed a clear gradual shift from a simple and micro-aerobic microbiota in 9-week-old puppies (D−8) to a more complex, homogeneous and anaerobic microbiota in 21-week-old puppies (D85) . An incr in dogs ,60 and h in dogs ,62. Otheerocytes ,64. The n humans .Giardia impact on the gut microbiota focused on animals experimentally infected with laboratory adapted strains and little is known about the canine microbiota during natural infections, in particular in asymptomatic animals. Some report described altered composition [Giardia-positive to Giardia-negative individuals. In this study, all animals are infected by the parasite and the effects of the parasite load on the fecal microbiota composition were investigated instead. Results showed that the gut microbiota structure was dependent on the parasite load, as puppies with high cysts shedding displayed altered microbiota compared to puppies with a low cyst load , G. intestinalis was associated with increased bacterial richness, while in older animals with adult-like gut microbiota (D85), the parasite load correlated positively with decreased bacterial richness  after the clearance of the parasite [G. intestinalis infection in puppies are known to be associated with inflammatory bowel disease (IBD), including families Ruminococcaceae [Prevotella [Alloprevotella [Lachnoclostridium [Suterella [Parasuterella [Bacteroides [G. intestinalis infection might predispose puppies to later development of either post-infectious IBS or IBD similarly to humans.Epidemiological studies have linked parasite ,93,96. Iparasite . Finallyoccaceae , Peptostoccaceae ,100, andoccaceae ,102,103 evotella , Allopreevotella ,106,107,uterella ,108, Paruterella ,110, andteroides ,112,113.L. johnsonii correlates negatively with G. intestinalis in puppies at D85 (L. johnsonii is the most common species among the isolates from the pre-weaning dogs, suggesting that L. johnsonii might be a specific species of infant dogs [L. johnsonii species is known for its probiotic properties and probably plays an important role in the development of a healthy gut in puppies. Allain et al. have demonstrated that L. johnsonii La1 possess anti-giardial activity through the production of bile salt hydrolases in vitro and in vivo in a murine model of infection [G. intestinalis infection [s at D85 . L. johnant dogs . L. johnnfection . Thus, pnfection .G. intestinalis. 16S Illumina sequencing revealed that the canine fecal microbiota becomes more complex and less diverse with age. Although some studies have linked the presence of intestinal parasites to gut health [G. intestinalis infection in puppies appears to induce some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species, and opportunistic pathogens, as well as in a reduction of L. johnsonii at specific time points. These results do not resolve whether infection is responsible for these changes. It is possible that certain compositions of the gut microbiota confer susceptibility or resistance to colonization by the parasite, as has been suggested before [G. intestinalis infection is associated with electrolyte, water and nutrient malabsorption, arginine deprivation, brush-border microvilli shortening, and disaccharidase deficiencies [G. intestinalis infection. Finally, the increased calprotectin levels illustrate the establishment of a chronic low-grade inflammation in puppies. However, further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as IBS or IBD.The aim of this study was to describe the gut microbiota of growing puppies naturally infected with t health ,117, subd before ,25. On tciencies . By modu


Through improvements in trauma care there has been a decline in injury mortality, as more people survive severe trauma. Patients who survive severe trauma are at risk of long-term disabilities which may place a high economic burden on society. The purpose of this study was to estimate the health care and productivity costs of severe trauma patients up to 24 months after sustaining the injury. Furthermore, we investigated the impact of injury severity level on health care utilization and costs and determined predictors for health care and productivity costs.This prospective cohort study included adult trauma patients with severe injury (ISS≥16). Data on in-hospital health care use, 24-month post-hospital health care use and productivity loss were obtained from hospital registry data and collected with the iMTA Medical Consumption and Productivity Cost Questionnaire. The questionnaires were completed 1 week and 1, 3, 6, 12 and 24 months after injury. Log-linked gamma generalized linear models were used to investigate the drivers of health care and productivity costs.In total, 174 severe injury patients were included in this study. The median age of participants was 55 years and the majority were male (66.1%). The mean hospital stay was 14.2 (SD = 13.5) days. Patients with paid employment returned to work 21 weeks after injury. In total, the mean costs per patient were €24,760 with in-hospital costs of €11,930, post-hospital costs of €7,770 and productivity costs of €8,800. Having an ISS ≥25 and lower health status were predictors of high health care costs and male sex was associated with higher productivity costs.Both health care and productivity costs increased with injury severity, although large differences were observed between patients. It is important for decision-makers to consider not only in-hospital health care utilization but also the long-term consequences and associated costs related to rehabilitation and productivity loss. Injury is a leading cause of death and disability for both young and older adults . In 2017Apart from the burden on a person’s life, severe trauma is also putting a high burden on society regarding health care utilization and health care costs related to severe trauma. Although multiple studies have explored long-term consequences of trauma on health-related quality of life –10, few The assessment of the health care utilization and productivity loss and associated health care and productivity costs provides insight into the economic burden of severe trauma on society. In this study we aimed to determine the total long-term costs of severe trauma patients. We provide a detailed overview of health care utilization, total in-hospital and post-hospital medical costs and productivity costs of severe trauma patients up to 24 months after sustaining injury. We investigated the impact of injury severity level on health care utilization and costs and determined predictors for health care and productivity costs.This prospective cohort study, including severely injured patients, is part of the Brabant Injury Outcome Surveillance (BIOS) study . SeverelDuring a follow-up of 24 months, data was collected with repeated questionnaires at 1 week and 1, 3, 6, 12 and 24 months after injury. The first questionnaire included questions on sociodemographic characteristics including age, sex, comorbidities and health status. Health status was measured with the EQ-5D-3L at 1 week after injury. If patients did not fill in the 1 week questionnaire, the score from 1 month questionnaire was used. The Dutch value set was used to calculate the utility score ranging from 0 (death) to 1 . The utility score can also have a negative value for health states worse than death .Data on type of injury, mechanism of trauma  and Abbreviated Injury Scale (AIS)-codes were obtained from the Brabant Trauma Registry (BTR). AIS coding was done by personnel or medical professionals who received a training in injury coding. Each injury is attributed an Abbreviated Injury Scale (AIS) score , allocated to one of six body regions . The ISSIn-hospital medical procedures  are obtained from the Brabant Trauma registry. Detailed information on intramural and extramural care are collected with the iMTA Medical Consumption Questionnaire (iMCQ), a self-reported questionnaire with questions related to non-disease specific healthcare consumption . The iMCAll unit costs were retrieved from a cost-reference manual , presentThe productivity costs were determined with the friction costs method. The friction costs method assumes that productivity loss is confined to the period needed to find replacement for the absent employee. We used a friction period of 85 days, in accordance with Dutch guidelines. Productivity loss longer than the friction period was valued equal to 85 days . If the Pearson’s chi-square, Fisher’s exact test and Mann-Whitney U test were used for comparisons between participants and non-participants. Descriptive statistics , median, and interquartile range (IQR)) were used to determine the health care utilization, in-hospital costs, post-hospital costs and productivity costs for patients with ISS 16–24 (severe) and ISS ≥25 (profound). Health care and productivity costs were rounded to the nearest 10 euro. An AIS score of ≥3 was considered as severe injured body region. ‘Head injury’ included head, face and neck injuries. ‘Extremity injury’ included upper and lower extremity injuries. ‘Thorax injury’ included thorax and abdomen injuries Predictors for health care costs and productivity costs were assessed with log-linked gamma generalized linear models (GLM). The relative difference in mean costs (exp[parameter estimate]) with 95% Confidence Interval (CI) were reported. For the GLM, only patients with available data on patient and injury characteristics and costs were included. Patients who died during the two-year follow-up were not included in our analysis. All statistical analyses were performed in IBM SPSS version 24 . A p-value of ≤0.05 was considered statistically significant.A total of 433 severely injured patients were asked to participate in this study and 239 (55.2%) signed an informed consent. There were 174 (40.2%) severe injury patients who completed the iMCQ and iPCQ . Of thesA detailed overview of health care utilization is shown in includes sample size and range). Both in-hospital and post-hospital costs were twice as high for patients with ISS≥25 compared to patients with ISS 16–24. Mean productivity costs per patient were €8,800. The mean costs of staying at a hospital ward were €7,620. The costs of stay at a rehabilitation centre and day treatments at a rehabilitation centre were three times higher for patients with an ISS≥25, respectively €5,360 and €4,610, compared to mean costs of €1,190 and €1,500 for patients with ISS 16–24.The total mean costs per patient were €24,760, with mean in-hospital costs of €11,930 and mean post-hospital costs of €7,770  and highest for patients with injuries in multiple body regions  . Post-hoIn general, patients with ISS ≥25 were on average 10 years younger than patients with ISS 16–24, respectively 47.3 (SD 17.6) years and 56.7 (SD 19.0) years. More than 50% of patients with ISS ≥25 (n = 23) had injuries in multiple body regions, compared to 20% of patients with ISS 16–24. A lot of variation can be observed between patients both for health care and productivity costs . Of the Lower health status, sex, higher ISS, extremity injury, spinal cord injury and having injuries in multiple bodies regions, showed significant associations with health care costs in the univariable model . In the Male sex, higher ISS, having injuries in multiple body regions, having extremity injuries and lower health status were found to be associated with higher productivity costs in the univariable model. In the multivariable model, only male sex was associated with higher costs with lower productivity costs for females.This study estimated the health care and productivity costs of severely injured patients up to two years after injury. The mean total costs of these patients were €24,760. Length of hospital stay was a key cost driver of total costs. Post-hospital costs where twice as high for patients with ISS ≥25 compared to patients with ISS 16–24, mainly due to costs of stay and day treatment at a rehabilitation centre. Higher ISS, spinal cord injury and lower health status at 1 week after injury were found to be associated with higher health care costs. Although the small sample size, male sex was found to be associated with higher productivity costs. On average, patients with paid employment returned to work 21 weeks after injury. The associated productivity costs had a considerable impact on the total costs. The present study showed that there was a large variety in health care utilization and health care and productivity costs between severely injured patients.Several other studies provided information on the cost of injury for the entire trauma population , 24, 42.Several longitudinal studies of the health status of severely injured patients showed a high level of functional limitation after 12–36 months. These studies also showed that recovery patterns varied widely between patients groups , 46, 47.A strength of this study was the consideration of long-term care and productivity loss in addition to in-hospital care. Therefore, this study presented a comprehensive overview of both the health care utilization and related costs for the severe injury population. Detailed information was collected at several different times and a combination of registry data and questionnaires was used.There are several limitations to this study. First, the relatively low inclusion rate of 40.2% reduces the generalizability of the study. Patients with a full recovery are probably more likely to have been lost to follow-up. The differences between participants and non-participants indicate that younger patients and patients with a higher injury severity were more likely to participate, suggesting a non-response bias. This could result in an overestimation of health care and productivity costs However, compared to other studies, our population was older and represented predominantly a less injured polytrauma patient cohort. Second, not all respondents filled out every questionnaire, resulting in missing data on health care utilization. Health care utilization and associated costs per service were based on the respondents with available data. Additionally, data on diagnostics were only available for a sub-group of the population. Finally, the sample sizes in our analysis were small, limiting the statistical power. Future research is needed to further explore the possible predictors of high health care and productivity costs in the severe injury population.To conclude, health care costs were increasing with injury severity and were especially high for those with spinal cord injury, extremity injury or multiple injuries. Additionally, there is high variety in health care utilization and associated costs in the severe trauma population. Only a part of the severe trauma population need long-term health care including rehabilitation and future research should further investigate which patients are at risk for a slow recovery and use of long-term care. It is important for decision-makers to consider not only in-hospital health care utilization but also the long-term consequences and associated costs related to rehabilitation and productivity loss, as post-hospital care and productivity costs comprise approximately 50% of total costs.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The virus load function, the target model, and the exponential approximations show excellent fits for the data considered. Our virus-load function offers a new way to analyze patient-specific virus load data, and it can be used as input for higher level models for the physiological effects of a virus infection, for models of tissue damage, and to estimate patient risks.We introduce an explicit function that describes virus-load curves on a patient-specific level. This function is based on simple and intuitive model parameters. It allows virus load analysis of acute viral infections without solving a full virus load dynamic model. We validate our model on data from mice influenza A, human rhinovirus data, human influenza A data, and monkey and human SARS-CoV-2 data. We find wide distributions for the model parameters, reflecting large variability in the disease outcomes between individuals. Further, we compare the virus load function to an established  The ongoing global SARS-CoV-2 pandemic has stimulated new research on viral infection and transmission. COVID-19, the disease caused by SARS-CoV-2, has found an abundantly susceptible population with no previous immunities. The disease has infected more than 150,000,000 people world wide, with about 3,000,000 deaths (as of May 2021). While progressing mildly in most cases, severe cases show respiratory symptoms followed by complications in other tissues, such as cardiac tissue, blood vessels, kidney, digestive system and nervous system [Baccam model [target model [For example, in a recent study of over 600 SARS-CoV-2 patients in France , a corream model , which iet model . It is aet model , we propWhile our research is certainly motivated by the current COVID-19 pandemic, similar dynamics are known for other acute viral infections, such as Influenza A  or MERS Virus load curves, as reported in ,10,11, hTo develop our virus load function below (see (1)), we consider Phase I of the sigmoid increase between time points d a2 see A,B. PhasBased on , we writThe specific form of sigmoid curves for atuyi in  in a canVirus load functions are in high demand in the virus modeling community. For example in , a largeWe use data from five different sources: mice influenza A from , human rIn , 120 micIn , 24 patiIn , six patIn , a cohorIn , nine rheffective virus load function as follows:We fit the virus load function (1) to the five data sets presented above. The virus load titer is measured as a relative RNA expression, as compared to a reference gene. Hence, the measurements have a significant measurement threshold LsqFit.jl package for Julia. The initial guess for To fit the data, we use the Levenberg–Marquard algorithm available in the n is the number of data points. This is equivalent to the likelihood intervals if we assume normally distributed errors with mean zero and variance When fitting the viral load function to each subject separately, we compute the set of parameter values that provide a similar residual sum of squares (RSS), compared to the best fit within relative tolerance SS(θ^)/n ,21. A 0.SS(θ^)/n . To compThe hyperbolic tangent function,B, the function has reached 99.5% of its saturation value. Indeed, if we set In Similarly, at point A at In , it was We conducted a number of numerical tests (examples in The straight lines with this slope are shown as red lines in This approximate slope is shown as a thin black line in This last formula (8) is a convenient way to estimate the exponential growth rate of Phase I. We just need the two time points of viral onset odel see C,D. We cThere is extensive mechanistic modeling of viral load curves based on ordinary differential equation models ,18,23,24target model with the eclipse phase comprises four ordinary differential equations (ODEs) for the target cells The k the rate at which infected cells become infectious, p the virus production rate, c the virus decay rate, Here, ic model  in whichnd decay . The basows (see ):(10)R0=Typical outcomes of the target model are shown in We fit our virus load function (1) to the virus load data of infection of influenza A for mice and humans, rhinovirus for humans, and in SARS-CoV-2 data for humans and Macaque monkeys. Details on these data sets and our data fitting procedure is explained in We begin with Influenza A virus load data from , as thesThe corresponding model parameters of the virus load function (1) are listed in imate in , using lThe estimated slopes are Now, we fit the virus load function (1) to the three groups of human rhinovirus data . The resailable) . We reitIn both previous cases, the virus load function was fitted to average data. We now fit data to individual subjects to show the benefits of patient specific fitting. The results of fitting the virus load function to human influenza A data are shown in Phase II A,C, (2) Phase II B,D, and We compare the approximate slope in Phase I, orted in . We repoorted in )”. For PWe fit our virus load function (1) to eight patients in this data set as shown in The shaded areas in the plots of For patient 907, the fit is exact, but it should be noted that in this case, we have more parameters than data points, and a good fit is not very meaningful. For Patient 930, we have very few data points, but they seem to be more scattered to obtain an exact fit. Patients 904, 930 and 942 show an extended initial Phase I, which spans over 8 days for Patients 904 and 930 and 10 days for Patient 942. This is, of course, related to the missing initial information about the early infection times.We see that the virus load function (1) describes all virus load curves very well. It is easily adapted for long and short virus infection periods, and it is fully flexible in detecting all three phases of the viral progression. Using the tolerance cloud, we can also visualize regions of greater uncertainty due to missing data points. We will see that the dynamics for macaque monkeys is rather similar.In see also C. IntereCompared to the human data, we notice that the virus half-life times in Phase II is see also ).In Overall, we see a large variability of the model parameters, in particular, the values For the decay rate α red in , we see As mentioned in the introduction, the virus load modeling with ordinary differential equations is a well-developed field ,18,23. HIn Fitting the virus-target model (9) to data constitutes statistical and numerical challenges. For instance, initial conditions are unknown in most cases, and estimating these values has shown to be impractical . HoweverThe explicit form of this new standard virus load function (1) is simple and convenient.The purpose of the function is to model a viral infection in an individual showing a fast exponential increase in the virus load, followed by an initial slow and later fast exponential decrease. Our virus load function is not intended to replace the Baccam or target model approach. Indeed, the target model has been used with great success in many applications and experiments ,34,35,36We have shown that this virus load function can replicate observed virus load titers from Influenza A in mice  and humately see C,D. FinaThe virus load function is able to fit data, having either a bi-phasic or tri-phasic profile, as seen in the human influenza A and monkey SARS-CoV-2 data. The bi-phasic infection is characterized by a fast decay in the viral load after reaching the peak. The tri-phasic profile is characterized by a slow decay in the viral load followed by a fast clearance of the virus. We believe that the distinction of the bi-phasic and tri-phasic is a first indication of disease severity. In , a correained in , where aained in , a measuOur model fitting to data shows a large patient-to-patient variability, as expected see . The likThe approximate slope of Phase I, We understand the large patient-to-patient variability as confirmation that patient-specific modeling is useful. The large variation in the values for keys see , for exa time b1 ,13. HoweThe virus load function is specifically designed for acute virus infections, such as influenza and corona viruses. It is not expected to be useful for viral infections with a different profile, such as HIV, for example .A few notes are worth considering when fitting the virus load function to individual data. Firstly, the measurement threshold for viral titer needs to be taken into account, e.g., by fitting to the effective virus load function (5). Secondly, an initial parameter estimate can be gained by a simple visual inspection of the viral load data. Then, the parameter space should be tested to guarantee a global minimum. Thirdly, the range of virus load functions obtained forms some parameter interval estimations , which should be computed to observe a family of possible virus load curves that reflect the uncertainty on the data. We find the 0.15 likelihood intervals (6) by computing the residual sum of squares within relative tolerance . AlternaThe virus load function (1) can be used in future work in many different ways. In , the tarhibitors , will altibodies , will taIf models for the immune response are considered explicitly, then the virus load function can be used as a model input, allowing us to correlate the time parameters


There has been considerable interest in the impact of short-chain fatty acids (SCFAs) on inflammatory effects related to the microbiome. Here, we present evidence that SCFAs may also be important in disease by providing an energy source or disease-associated cue for colonizing pathogens. Aggregatibacter actinomycetemcomitans (Aa) is both a common colonizer of the oral cavity and has been implicated in the pathogenesis of periodontal disease. Here, we present a whole-genome phylogenetic analysis of Aa isolates from humans and nonhuman primates that revealed an ancient origin for this species and a long history of association with the Catarrhini, the lineage that includes Old World monkeys (OWM) and humans. Further genomic analysis showed a strong association with the presence of a short-chain fatty acid (SCFA) catabolism locus (atoRDAEB) in many human isolates that was absent in almost all nonhuman OWM isolates. We show that this locus was likely acquired through horizontal gene transfer. When grown under conditions that are similar to those at the subgingival site of periodontitis , Aa strains with atoRDAEB formed robust biofilms and showed upregulation of genes involved in virulence, colonization, and immune evasion. Both an isogenic deletion mutant and nonhuman primate isolates lacking the ato locus failed to grow in a robust biofilm under these conditions, but grew well under the carbohydrate-rich conditions similar to those found above the gumline. We propose that the acquisition of the ato locus was a key evolutionary step allowing Aa to utilize SCFAs, adapt, and modulate subgingival disease.Like the bacterial residents of the human gut, it is likely that many of the species in the human oral microbiota have evolved to better occupy and persist in their niche.  The resident microbiota of mammalian species appears to diversify or “cospeciate” in parallel with the divergence of its hosts –5, which–Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative oral bacterium that colonizes tooth surfaces in both humans   , and it is (LAP) . Despite animals , 12, and animals  have not disease , scenariAa is thought to be transmitted horizontally by maternal transmission to infants  and contrasting with surveys of New World monkeys, the Platyrrhini  for 14 Aa isolates  of the Catarrhini approximately 32 million years ago (mya) , the divergence between clades I and II was at least 11,000 years old. A reasonably slower evolutionary rate of 1.19 × 10−9 showed the divergence time to be close to the MRCA of the Catarrhini clade, 32 mya  . To test, 32 mya , a time 10.1128/mBio.03581-20.1TEXT S1Text S1, DOCX file, 0.08 MB.Detailed methods and extended discussion. Download Copyright © 2021 Moustafa et al.2021Moustafa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ato locus (atoRDAEB), which is predicted to be involved in short-chain fatty acid (SCFA) catabolism. The absence of this locus in clade II suggested the possibility that it was acquired by horizontal gene transfer (HGT) in the MRCA of clade I, an event that took place at least 12.4 mya if we assume an Aa-Catarrhini codivergence  genomes, and 100% of encapsulated Hflu strains  of the assembled genomes were compared among strains using a pangenome approach. There were no genes that were uniquely found in isolates from any particular host species, reflecting the presence of some human and nonhuman isolates across the tree. However, there were a small number of unique and ubiquitous genes defining the two major clades (see to locus  (atoRDAEvergence . To testood tree . Interesemselves  showed t10.1128/mBio.03581-20.2FIG S1Aa isolates from primates. (A) A pie chart of the pangenome, breaking down the genes into core, soft core, shell, and cloud and showing the number of isolates they are present in. (B) A table showing the genes that are unique to the two major clades (Human and Monkey) in the tree of FIG S1, DOCX file, 0.3 MB.Pangenome analysis of 95 Copyright © 2021 Moustafa et al.2021Moustafa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03581-20.3FIG S2H. influenzae species showing the presence/absence of the atoRDAEB locus and capsule. A maximum likelihood phylogenetic tree was constructed with 755 genomes from GenBank. The first ring and second ring from inside to outside represent the atoRDAEB locus and capsule, respectively. All capsulated genomes have the atoRDAEB locus. The tree was edited using iTOL website (v4.2.3). The tree scale indicates the number of substitutions per site. Download FIG S2, DOCX file, 2.9 MB.Phylogenetic analysis of the Copyright © 2021 Moustafa et al.2021Moustafa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03581-20.4FIG S3atoDAEB locus in the Pasteurellaceae family with outgroup species from the Gammaproteobacteria. A maximum likelihood tree of the atoDAEB locus in the Pasteurellaceae family was created using RAxML with Gammaproteobacteria species Y. intermedia, M. morganii, and Erwinia teleogrylli included as outgroups. The root was determined on the branch that makes the Pasteurellaceae family an ingroup and other members from the Gammaproteobacteria an outgroup. Bootstrap values are reported on all branches. Species clades with more than four clusters were collapsed for easier visualization. The alignment was limited to atoDAEB, atoR was excluded as it is absent outside the Pasteurellaceae family. The sequences were clustered using CD-HIT (100% identity and coverage). All clusters were composed of individual species. The total number of genomes in each cluster and sequence length are shown, respectively, and separated by underscore in the nodes that were not collapsed. Download FIG S3, DOCX file, 0.02 MB.Phylogenetic analysis of the Copyright © 2021 Moustafa et al.2021Moustafa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia coli , we showed that Aa grown in butyrate, under anaerobic conditions, transcriptionally upregulated a leukotoxin (ltxA) gene that can kill activated neutrophils, the epithelial adhesin apiA gene, the biofilm/adherence gene flp-1, and the exopolysaccharide gene pgaC (Because the anaerobic subgingival pocket is the site of periodontitis, we hypothesized that ene pgaC , and F.Aa butyrate catabolism on disease modulation is unclear. Interestingly, the four Finnish strains from clade II that do not have the ato locus appear to have been obtained from “healthy or minimally diseased individuals”(–The anti-inflammatory properties of SCFAs in the gut are well documented , 42, but43–ividuals”–17.Aa than previously recognized and highlights a major clade dominated by isolates from rhesus macaques, raising the possibility of a long history of primate-Aa codiversification. Based on our molecular clock analysis, we favor the hypothesis that the MRCA of Aa was present in the MRCA of the Catarrhini about 32 mya. We propose that after this codivergence event, the lineage that would eventually dominate in humans acquired the ato locus, allowing it to more successfully colonize the periodontal pocket replete with butyrate and modulate the host response to disease.In conclusion, the present study shows a higher level of genomic diversity in The genome sequences and the reads have been submitted to GenBank and SRA under BioProject PRJNA641505. Accession numbers are available in