Few case reports have documented the use of topical cidofovir for refractory genital herpes simplex virus (HSV) ulcers in human immunodeficiency virus (HIV) infected patients. This drug formulation lacks a standardized concentration or even a procedural outline as to how it should be compounded. We aim to discuss the utilization of topical cidofovir in addition to presenting a procedural means of compounding it for treatment of refractory genital HSV ulcers. Our patient completed 21 days of intravenous foscarnet and 13 days of topical cidofovir with clinical improvement in the penile and scrotal ulcers. Genital herpes is a concern in patients with HIV because it generally manifests as a persistent infection. Physicians should be aware that when patients fail to respond to the conventional treatment regimens for genital HSV in a timely manner, other options are available, such as topical cidofovir as an adjuvant to systemic antivirals. But in individuals with HIV, mucocutaneous infections are generally longer lasting with increased rates of recurrence and severity.3 The conventional treatment for genital HSV includes antiviral medications such as acyclovir, famiciclovir, and valacyclovir, which have similar efficacy in suppressing recurrent infections. The goal of therapy is to induce remission. Eradication of the virus is rare as HSV establishes itself as a chronic infection within the ganglion of sensory neurons during the primary infection.2 Intravenous acyclovir may be required for patients exhibiting severe or persistent infections but some may still experience poor clinical response. Here we report a case of chronic refractory genital HSV ulcers treated successfully with intravenous foscarnet and topical cidofovir. Written consent was obtained to document and submit for publication all information related to this case report.In the United States, 1 in 6 people ages 14 to 49 have genital herpes.A 48-year-old incarcerated male presented to the hospital with chronic penile and scrotal ulcers . He had Complete blood counts and serum chemistries remained stable on treatment except for magnesium requiring daily replacement, allowing foscarnet to be titrated up to 90 mg/kg twice daily. Attempts were made to isolate HSV for resistance testing, but repeat tissue and swab cultures were negative. The genital ulcers were deemed to be secondary to HSV as evidenced by clinical response to foscarnet and positive immunohistochemistry staining despite negative viral cultures.36 Cidofovir is a nucleotide analogue that does not require enzyme activation for activity, unlike acyclovir and foscarnet. Imiquimod’s method of activity is not fully understood but believed to be related to stimulating the immune response through activation of toll-like receptors. Various concentrations of topical cidofovir have been cited, but a treatment algorithm for HSV genital ulcers recommends a 1% to 3% concentration.79 A request was made for the hospital pharmacy to compound topical cidofovir 1%, and while waiting for the pharmacy to approve and process the request, the patient was started on imiquimod 5% cream, which was commercially available and on the hospital formulary. On approval to compound topical cidofovir, the patient was transitioned to topical cidofovir 1%. The compounded formulation involved 375 mg of cidofovir in 37.5 g of an emollient base. Both imiquimod and cidofovir topical ointments were applied over the penile lesions with an overlying nonadherent Tefla pad, and secured with a Kerlix sterile gauze roll and tape. Dressing changes with topical medication was conducted by the patient, with nursing assistance, daily in the evening.The penile ulcers began to show improvement after 7 days of foscarnet, but there still appeared to be areas of activity so additional treatment measures were considered. Literature review revealed several reports that outlined successful use of topical cidofovir and topical imiquimod for treatment of HSV cutaneous infections.7 In addition, due to formulary constraints within the department of corrections, topical cidofovir was transitioned to topical imiquimod 5% thrice weekly for 1 month.The patient completed 21 days of intravenous foscarnet, 1 day of topical imiquimod 5%, and 13 days of topical cidofovir with significant clinical improvement in the penile and scrotal ulcers . Subsequ3 It was assumed that our patient was infected with an acyclovir-resistant strain of HSV despite failed attempts to isolate the organism with viral tissue culture resulting in our inability to pursue resistance testing. This could be explained by an inadequate tissue biopsy sample or an inadequate preservation of the tissue specimen en route to the microbiology laboratory. Nonetheless, we approached the patient’s genital ulcers as acyclovir-resistant due to the chronicity of his infection despite suppressive acyclovir therapy.Immunocompromised persons who are infected with genital HSV tend to exhibit persistent and extensive ulcers, which commonly fail to respond to conventional antiviral regimens. The failed response is usually related to the infectious strain of HSV being resistant to conventional modalities such as acyclovir.Even though our patient received multiple concomitant treatments, significant clinical improvement was only observed after several days on combined intravenous foscarnet and topical cidofovir. Initial intravenous foscarnet monotherapy was only partially effective. Imiquimod was given briefly during induction treatment, and antiretroviral therapy did not lead to a robust CD4 recovery, indicating that a direct contribution of these treatments was less likely.Genital herpes is a concern in patients with HIV infection because it generally manifests as a persistent infection, which, if not treated appropriately, can result in severe and disabling local disease. Physicians should be aware that when patients fail to respond to the conventional treatment regimens for genital HSV in a timely manner, other options are available. One such option is the use of topical cidofovir as an adjuvant to systemic antivirals such as foscarnet. Currently, topical cidofovir is not Food and Drug Administration approved for use in genital HSV infections, but with continued documentation of successful treatment responses, it is possible that this may become an additional indication. The majority of the documented responses are from case reports warranting the need for further studies.


To the Editor: Zika virus belongs to the genus Flavivirus, family Flaviviridae, and is transmitted by Aedes spp. mosquitoes. Clinical signs and symptoms of human infection include fever, headache, malaise, maculopapular rash, and conjunctivitis. Zika virus was first isolated in 1947 from the blood of a febrile sentinel rhesus monkey during a study of yellow fever in the Zika Forest of Uganda , East African (MR766 prototype cluster), and Asian. It has been postulated that the virus originated in East Africa and then spread into both West Africa and Asia ≈50–100 years ago (http://www.megasoftware.net/) with all available full-length Zika virus sequences from GenBank representing the 3 genotypes. Nearly identical phylogenetic trees were generated by using several methods , and a neighbor-joining tree was generated and analyzed with 1,000 replicates for bootstrap testing  platform. The raw sequence reads were analyzed and assembled by using the CLC bio Genomics Workbench  and Lasergene NextGen . The complete genome sequences were aligned by using ClustalW ( testing . GenBankIn agreement with the initial sequencing of samples from Brazil conducted by Zanluca et al. (As reported by Musso et al. (


Nairovirus, one of five bunyaviral genera, includes seven species. Genomic sequence information is limited for members of the Dera Ghazi Khan, Hughes, Qalyub, Sakhalin, and Thiafora nairovirus species. We used next-generation sequencing and historical virus-culture samples to determine 14 complete and nine coding-complete nairoviral genome sequences to further characterize these species. Previously unsequenced viruses include Abu Mina, Clo Mor, Great Saltee, Hughes, Raza, Sakhalin, Soldado, and Tillamook viruses. In addition, we present genomic sequence information on additional isolates of previously sequenced Avalon, Dugbe, Sapphire II, and Zirqa viruses. Finally, we identify Tunis virus, previously thought to be a phlebovirus, as an isolate of Abu Hammad virus. Phylogenetic analyses indicate the need for reassignment of Sapphire II virus to Dera Ghazi Khan nairovirus and reassignment of Hazara, Tofla, and Nairobi sheep disease viruses to novel species. We also propose new species for the Kasokero group , the Ketarah group  and the Burana group . Our analyses emphasize the sister relationship of nairoviruses and arenaviruses, and indicate that several nairo-like viruses  require establishment of novel genera in a larger nairovirus-arenavirus supergroup. Bunyaviridae is one of the largest virus families [With over 530 members, families . Bunyavifamilies ,2. The Sfamilies . NonstruBunyaviridae currently includes five recognized genera: Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus [versus vertebrate excreta-driven) and, until recently, very limited genomic sequence information [The family spovirus . Family ormation .Nairovirus includes seven species that are accepted by the International Committee on Taxonomy of Viruses (ICTV) [The genus s (ICTV) . Most ofs (ICTV) . The moss (ICTV) .The typical nairovirus genome is approximately 18.8 kb in length  and characterized by the genus-specific 3′ segment terminus AGAGUUUCU and 5′ segment terminus AGAAACUCU. Classical nairovirions are enveloped spheres (80–120 nm in diameter) spiked with heterodimeric glycoprotein projections consisting of the cleavage products of the glycoprotein precursor (Gn and Gc) [Nairovirus have been identified as bona fide nairoviruses [et al. reported the coding-complete sequences of 11 nairoviruses (Abu Hammad virus (AHV), Avalon virus (AVAV), Bandia virus (BDAV), Dera Ghazi Khan virus (DGKV), Erve virus (ERVEV), Farallon virus (FARV), GANV, Punta Salinas virus (PSV), Qalyub virus (QYBV), Taggert virus (TAGV), and Zirqa virus (ZIRV)) [Next-generation sequencing followed by coding-complete or complete genomic sequence assembly ) . An overet al. [Dera Ghazi Khan nairovirus species. At least seven nairo-like viruses should be classified into novel genera, and these genera and all nairoviruses are more closely related to arenaviruses than to other bunyaviruses.As is evident from the table, genomic sequence information for nairoviruses is still limited. Here we report either the coding-complete or complete genomic sequences of 23 nairoviruses . Ten of et al. . Four ofThe viruses used in this study were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch, Galveston, TX, USA. All of these viruses have been described before. Viral stocks were obtained in TRIzol LS , and RNAs were extracted using the Direct-zol™ RNA MiniPrep kit . RNAs were converted to cDNAs and amplified using sequence-independent single primer amplification as described previously  with somA set of nairovirus sequences  comprising the majority of the nucleotide (nt) sequences from GenBank available on 1 March 2016, were aligned using the CLUSTAL algorithm. Because the nairovirus sequences of all analyzed nairoviruses were so different that the alignment reached substitution saturation , alignments were instead implemented at the amino acid (aa) level (using MEGA Version 5 ). Non-coSystematic screening for the presence of recombination patterns was pursued by using the nt alignments and the Recombination Detection Program (RDP ), BootscGeneious 4.8.3  was used for sequence assembly and analysis. Topology, sizes, and targeting predictions were generated by employing SignalP 4.1, NetOGlyc 4.0, NetNGlyc 1.0, Prop 1.0, tied mixture hidden Markov model (TMHMM) 2.0 , SnapGenA and TGAAACUCU). Likewise, the Qalyub virus (QYBV) genomic segments termini differ from the consensus sequences by one nt (AGAGAUUCU and AGAATCUCU).The results of phylogenetic analyses of the newly obtained L, M, and S segment sequences are shown in Consistent with the genomic organization characteristic for already sequenced nairoviral genomes, each of the 23 viral genomes sequenced during this study is comprised of three RNA segments including (a) a small (S) segment encoding the NP and, in an ambisense orientation, a non-structural protein (NSs); (b) a medium (M) segment encoding a GPC; and (c) a large (L) segment encoding an RNA-dependent RNA polymerase. Fourteen nairovirus genomes were completely characterized. The 3′ terminal sequences were obtained for 57 segments, and the 5′ terminal sequences were obtained for 51 segments . For mosCrimean-Congo hemorrhagic fever nairovirus. Likewise, both Kupe virus (KUPEV) and Nairobi sheep disease virus (NSDV) did not cluster with Dugbe virus (DUGV), and, therefore, should be removed from the species Dugbe nairovirus and re-assigned to new species ) and “Nairobi sheep disease virus” (NSDV), respectively). Ganjam virus (GANV) is clearly identified as an isolate of NSDV. Our analysis confirm that Leopards Hill virus (LPHV), Kasokero virus (KAS(O)V), and Yogue virus (YOGV) form a novel nairovirus genogroup (proposed species “Kasokero nairovirus”), as do Keterah virus (KRTV) and Issyk-kul virus (ISKV) (proposed species “Keterah virus”) [Dera Ghazi Khan nairovirus. Another new species, proposed to be named “Burana nairovirus” should be established for Wēnzhōu tick virus, Huángpí tick virus 1, and Tǎchéng tick virus 1. Finally, the phylogenetic trees demonstrate that several nairo-like viruses with three , Sānxiá water strider virus 1 (SWSV-1), South Bay virus (SBV)) or two genomic segments  should not be classified in the genus Nairovirus.The phylogenetic placement of the newly sequenced viruses is largely consistent with their previous serological classification, including recent amendments  Table S. However virus”) ,42. The  virus”)  is ident virus”) . The TUNAlthough genomic segment reassortment has been found very frequently among CCHFV strains and lineages ,86,87,88Nairoviral NPs may recognize specific nairoviral RNA sequences, bind non-specifically to single-stranded RNA, and form the ribonucleoprotein (RNP) complex ,92,93,94453(L/M), (L/F)HQ457). The other region is delineated by residues R134, R140, and Q468. The CCHFV NP stalk region also contains a positively charged region consisting of residues R195, H197, K222, R225, R282, and R286. Only three of the positively charged residues  are absolutely conserved among all nairovirus NPs, although most of the substitutions observed maintain the overall hydrophobicity profile.In the case of CCHFV NP, two positively charged regions are responsible for RNA binding . One regCCHFV NP interacts with the antiviral defense factor MxA  and the Because protein structure is evolutionarily conserved to a higher degree compared to the primary aa sequence, we used homology modeling principles and techniques to identify conserved structures among nairovirus proteins. We used the Phyre2  server to model the structure of the proteins and to align remotely related sequences based on hidden Markov models (HMMs) .In addition, upon identification of a conserved nairovirus protein structure, we performed mutational sensitivity analysis using the Disease-Susceptibility-based SAV Phenotype Prediction (SuSPect) tool , to predFinally, the mutational sensitivity score was plotted for each position against the average percent identity for that position in the nairovirus protein alignment. We assumed that if a mutation would have an influence on the structure of a conserved domain, the frequency of that mutation would be diminished. In summary, we expected that the positions with a higher score in the mutational sensitivity analysis should directly correlate with positions of higher conservation. The validity of this approach was tested initially with nairovirus NP sequences A,B. As eInterestingly, we identified two other nairovirus NP domains that were structurally similar to other known, non-viral structures. The first domain, at approximate position 150–200 of the nairovirus alignment, is similar to the globular tail of myosin-4 motor protein  ID: 3mmi). Myosin-4 motor protein is a monomeric myosin with motility uniquely adapted to transport mRNA . InteresBurana nairovirus” M segments). The predicted masses of the encoded unmodified polyproteins, the GPCs, ranged from 143 kDa (Thiafora virus (TFAV)) to 187 kDa (CCHFV). Each nairovirus polyprotein was approximated, via software modeling, to contain a signal peptide at the N-terminus. Experimental evidence garnered from CCHFV GPC processing [Qalyub nairovirus and “Keterah nairovirus”). The RRLL motif is the second most prevalent cleavage site with the exception of viruses belonging to “Keterah nairovirus” and Qalyub nairovirus, which do not contain the motif. The RKPL and RRLL motifs are also conserved across nairoviruses of many species.ORF analysis of 36 sequenced nairovirus M segments generally yielded single unit polyprotein-encoding ORFs in each case , the nairovirus GPs stemming from GPC processing were predicted and annotated numerically, starting at the N-termini and depicted as colored arrows . We predN- and O-linked glycosylations, were predicted for all analyzed nairoviruses using software modeling . Averages of N-linked glycosylation sites between viruses of different species ranged from seven to twenty sites along polyproteins. The most N-linked glycosylations were found for members of the “Keterah nairovirus” and Sakhalin nairovirus species, whereas the members of the Dugbe and Hazara nairovirus species had the fewest. The extent of nairovirus glycoprotein O-linked glycosylation ranged from four to over 130 sites , were predicted for viruses of the Crimean-Congo hemorrhagic fever nairovirus and “Keterah nairovirus” species. Relatively few sites were predicted towards the C-termini of nairovirus GPC, with the exception of the “Keterah nairovirus” species members, which were predicted to contain a small cluster of O-linked sites between residues 994 and 1033.Other post-translational modifications, such as, 30 sites . The fewO-glycosylated GPCs were typically characterized by site clustering towards the N-termini. O-glycosylated members of the Dugbe, “Keterah,” and Qalyub nairovirus species contain RKPL and RKLL proteolytic motifs in these areas. If used by proteases, these motifs could mediate the production of separate, stand-alone peptides that are O-glycosylated. Such peptides have been identified in the cases of CCHFV and DUGV as mucin-like domains (MLDs) [Leopards Hill nairovirus” species encode GPC with shorter regions of O-linked glycosylation clustered towards the N-termini in the vicinity of predicted proteolytic cleavage sites. Of nairovirus genus members containing regions of O-glycosylation/MLDs, unmodified averaged masses ranged from eight to 37 kDa. Notably, of the GPC of seven viruses belonging to the Hughes nairovirus species, none were predicted to be O-glycosylated.Heavily s (MLDs) . AdditioQalyub nairovirus species had the fewest transmembrane regions in their glycoprotein precursors . By comparison, CCHFV and DUGV GPCs have five TMDs. All nairovirus GPCs were predicted to have at least a single conserved C-terminal transmembrane region approximately 40–60 residues prior to the C-termini of the GPC . The number of TMDs varied between one and five domains. Interestingly, viruses of the  the GPC .et al. [Nairovirus RNA-dependent RNA polymerases (Ls) are substantially larger than other bunyavirus L homologs. All nairovirus L sequences maintain the characteristic RNA-dependent RNA polymerase core motifs described by Poch  and Mullet al.  comprisiMoreover, inter-motif regions are moderately conserved, suggesting structural constrains on their three-dimensional arrangements. The invariant sequences DXX KW and SDD of motifs A and C, respectively, may have metal-binding activities necessary for catalytic functions ,107 Fig. A phyloOur analysis demonstrates that the nairovirus and nairo-like virus RNA-dependent-RNA polymerase core domain is more closely related to the arenavirus domain than to any other bunyaviral domain, supporting the existence of an arenavirus-nairovirus supergroup.CCHFV encodes a deubiquitinase (DUB) of the OTU family, which unlike eukaryotic OTU DUBs, also targets interferon-stimulated gene 15 (ISG15) modifications ,109. TheThe majority of nairoviruses pathogenic to humans V, and NSDV) or other mammals (NSDV/GANV) contain an identifiable topoisomerase I active site motif, whereas most nairoviruses without that domain are only known to infect arthropods or to establish subclinical infections in vertebrates. The deubiquitinylation and deISGylation activities of CCHFV L have been proposed as a mechanism of virus evasion from the innate immune response via efficient interference with antiviral signaling pathways mediated by nuclear factor (NF)-kB, interferon regulatory factor 3 (IRF3), and type 1 interferon (IFN-α/β) ,109. The2H2-type zinc finger domain and several aa positions conserved among all RNA-dependent RNA polymerase modules of segmented negative-sense RNA viruses, was named domain B [Position-specific iterated (PSI)-BLAST searches had previously demonstrated that the region between the nairovirus OTU-like cysteine protease domain and the core region of the RNA-dependent polymerase (domain A) is conserved ,113 Fig. A regiodomain B ,107   novel species will have to be established; (b) that some nairoviruses will have to be moved between species; (c) that a long-thought phlebovirus is actually a nairovirus; and (d) that several newly discovered bunyaviruses do not directly fall into the nairovirus clade but are nevertheless more closely related to nairoviruses than to other bunyaviruses  have been determined but are not yet available for analysis ,32,33,34i.e., the relatively free swapping of S, M, and L segments, between taxonomically diverse bunyaviruses simultaneously infecting the same host and consequently resulting in novel viruses) has been described for orthobunyaviruses, phleboviruses, and tospoviruses [In vitro experiments should be performed to evaluate these hypotheses.Genomic reassortment  of bunyavirus sequences using the National Center for Biotechnology Information’s (NCBIs) PASC tool. Histograms from this analysis demonstrates the distribution of the number of virus genome pairs at each identity percentage. Histogram peaks and valleys can be used to differentiate taxon ranks and to establish taxon demarcation criteria using objective criteria [Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus genera (data not shown). Using these cut-offs, PASC confirms the monophyly of the genus Nairovirus as outlined in Bunyaviridae, are more related to these viruses than they are to other bunyaviruses, suggesting the need to establish an arenavirus/nairovirus supergroup within the family  that appeared to be closely related to nairoviruses ,41. Intecriteria ,129. A pe family .


The workshop took place at the Research Institute Aggeu Magalhaes (IAM), the state of Pernambuco research branch of Fiocruz, responsible for hosting the meeting and its organization. Representatives from several institutions working on Zika virus science attended, including researchers and academics from several other Fiocruz research entities , from major Brazilian Universities  and from other Brazilian research centers (such as the Evandro Chagas Institute—IEC). Foreign participants included representatives from the National Institute of Allergy and Infectious Diseases NIAID-USA and the University of Glasgow, among others. The Workshop brought together more than 600 participants and was accessible, live, through the web (over four thousand accessions from 26 countries). The purpose of the meeting was to provide an updated view on Zika virus in Brazil and to discuss recent advances and challenges on its research. With the recent Zika virus outbreak in Northeast Brazil and especially the remarkable increase in newborns suffering a congenital disorder termed microcephaly—a rare neurological condition in which an infant's head is significantly smaller than the heads of other children of the same age—recently linked to Zika virus infection during pregnancy , the BraArboviruses, Zika and vaccines–discussed data regarding different circulating arthropod borne viruses in Brazil (such as chikungunya and dengue), new data about Zika virus biology, in vitro models of infection and also strategies for vaccine development; Biology of the virus vector interaction–focused on new findings on virus vector studies, including an evaluation of potential vectors for Zika virus and countermeasures to mosquito control; Clinical–with the intention of discussing the last clinical findings on Zika virus infection and major birth congenital malformations; Diagnosis—plotted new strategies to improve laboratory tests, development of technological products and also a historical update of Zika virus identification in Brazil; Epidemiology—this session was designed to provide an update on the clinical epidemiological studies under development in Brazil, with a presentation of the data on a case-control investigation of microcephaly cases and cohort studies.During the meeting, special sessions were organized by experts in the following research fields: The first day the meeting opened with a talk from the IEC team on Zika virus infection and the global challenge to control its spread. Subsequent talks covered the clinical manifestations presented by Zika infected individuals, from a medical perspective. Medical doctors from Northeast Brazil reported that the major clinical findings on microcephaly confirmed cases were calcifications (approximately 98%), ventriculomegalia and disorder of cortical development, including simplification of gyrus and lissencephaly with a lower proportion of other abnormalities such as pachygyria. Official data from the Brazilian Ministry of Health confirmed that currently Zika virus is circulating in 22 of the 27 states in Brazil, with two confirmed deaths . By MarcOn the Diagnosis panel, the discussions were mainly on the development of new laboratory tools. Given the extensive cross reactivity with other circulating arboviruses in Brazil, the lack of reliable and specific serologic tests for Zika virus represents a major challenge. Currently, only two serological tests are licensed for Zika virus diagnosis in Brazil . In addition to serological tests, Fiocruz is providing a recently licensed multiplex Dengue/Zika and Chikungunya assay based on NAT technology (nucleic acid test or nucleic acid amplification test). Discussions about development and characterization of monoclonal antibodies and recombinant proteins, as biotechnological products to be applied on different platforms for Zika virus diagnostic, occurred during the event, however no new kits are being licensed at the moment, which could be explained, in part, by the lack of reliable serum panels for kits validation. Researchers from IAM investigating the microcephaly cases reported the detection of Zika virus specific IgM in roughly 90% of the samples of cerebrospinal fluid (CSF) tested from microcephalic newborns, which strongly correlates Zika virus infection with brain malformation. The team from the Oswaldo Cruz Institute (IOC—a Fiocruz branch from Rio de Janeiro) provided results on Zika virus identification from several body fluids such as urine, semen (up to 2 months after symptoms), saliva and CSF. The data showed that Zika virus found in unusual samples  can persist for longer time periods than in the blood. So far, however, only sexual, perinatal and vector transmissions have been documented for Zika virus. Therefore, is concerning the fact that Zika virus detection on semen for long periods after the initial infection has serious implications on sexual transmission. Although organ transplantation and blood transfusion are probable transmission routes, there are no reports yet in the literature –11. DespAedes  are the main species responsible for Zika virus transmission in Brazil [Culex quinquefasciatus 7 and 15 days post-feeding, confirming a high infection rate of 100% and 67%, respectively. In addition, advances and new approaches for mosquito control were presented in the session and challenging aspects for the implementation of control measures discussed, taking into account the socio-economical and environmental conditions of urban areas in Brazil.Mosquitoes from the genus n Brazil ,13. DuriSince its introduction in Brazil, Zika virus infection has spread rapidly through the tropical Americas and we hypothesize that sequential flaviviruses exposures can represent a complicating factor for its spread, especially in Brazil where rates for prior dengue infection are extremely high. Currently, a number of independent groups are developing projects on Zika virus investigation in Brazil. Recently, Fiocruz  established a National Network Zika Committee through the integration of efforts carried out by several of its research branches. These efforts are focusing on the development of better diagnostic tools, vaccine research, virus genetic studies, immunopathologic studies on Zika patients and research on vector populations and control. Currently, Zika virus is still expanding within the Brazilian territory and in other countries from Central and South America, probably due to the availability/abundance of vectors and the challenges in achieving efficient levels of vector reduction with a consequent impact on virus transmission. It is currently very difficult to determine how and to where will the virus spread over time. Assumptions by field specialists testify that when herd immunity reaches a level of ~80%, Zika virus transmission could then drop, however this is only speculative and based on previous data from recent outbreaks of dengue and chikungunya . Serious


Secoviridae family of viruses forms part of the Picornavirales order, an important group of non-enveloped viruses that infect vertebrates, arthropods, plants and algae. The impact of the secovirids on cultivated crops is significant, infecting a wide range of plants from grapevine to rice. The overwhelming majority are transmitted by ecdysozoan vectors such as nematodes, beetles and aphids. In this study, we have applied a variety of computational methods to examine the evolutionary traits of these viruses. Strong purifying selection pressures were calculated for the coat protein (CP) sequences of nine species, although for two species evidence of both codon specific and episodic diversifying selection were found. By using Bayesian phylogenetic reconstruction methods CP nucleotide substitution rates for four species were estimated to range from between 9.29×10−3 to 2.74×10−3 (subs/site/year), values which are comparable with the short-term estimates of other related plant- and animal-infecting virus species. From these data, we were able to construct a time-measured phylogeny of the subfamily Comovirinae that estimated divergence of ninety-four extant sequences occurred less than 1,000 years ago with present virus species diversifying between 50 and 250 years ago; a period coinciding with the intensification of agricultural practices in industrial societies. Although recombination (modularity) was limited to closely related taxa, significant and often unique similarities in the protein domains between secovirid and animal infecting picorna-like viruses, especially for the protease and coat protein, suggested a shared ancestry. We discuss our results in a wider context and find tentative evidence to indicate that some members of the Secoviridae might have their origins in insects, possibly colonizing plants in a number of founding events that have led to speciation. Such a scenario; virus infection between species of different taxonomic kingdoms, has significant implications for virus emergence.The plant-infecting  Picornavirales order contains viruses that infect a wide range of eukaryotic organisms including vertebrates (Picornaviridae), arthropods (Dicistroviridae), plants (Secoviridae), insects (Iflaviridae) and algae (Marnaviridae) Picornaviridae include rhinoviruses, poliovirus, Foot-and-mouth disease virus, and Hepatitis A virus. The plant-infecting members of the Picornavirales are in the Secoviridae family. Most secovirid species fall within the Comovirinae subfamily which contains the Nepovirus, Comovirus and Fabavirus genera Apple latent spherical virus (ALSV)Cherry rasp leaf virus (CRLV)Satsuma dwarf virus (SDV)Strawberry mottle virus (SMoV)Strawberry latent ringspot virus (SLRSV)Tomato torrado virus (ToTV)Secoviridae is significant: Grapevine fanleaf virus (GFLV) is the oldest and most widespread viral disease to affect grapevine, being first documented in 1865 Rice tungro spherical virus (RTSV) emerged in the 1960s to seriously disrupt rice production in Asia Torradovirus genus show signs of emergence in tomatoes Secoviridae from an evolutionary perspective will enhance our ability to develop adequate control strategies against present and future threats.The Picornavirales are all characterized as having a positive-sense single-stranded RNA genome with a conserved module incorporating a superfamily 3 helicase (HEL), a chymotrypsin-like protease (PRO) and RNA-dependent RNA polymerase (RdRp) functions. Large exons are proteolytic processed via post-translational cleavage into discrete structural and/or functional proteins. Virus particles are non-enveloped, icosahedrons of around 30 nm in diameter made up of 60 capsomers each containing three jelly-roll domains. Except for members of the Secoviridae, of which almost all are bipartite, viruses in the order Picornavirales have non-segmented genomes. In the vast majority of cases the viral RNA is polyadenylated at the 3’ terminus. A small virus-encoded protein (VPg), predicted for most of the species, has been shown to be covalently attached to the 5’ terminus Secoviridae consists of either one, two or three cleaved peptides, depending on the genus or species Nepovirus and Comovirus genera the functions of these regions remain unknown Members of the Secoviridae. High levels of purifying selection for the CP and MP have been calculated for GFLV strains isolated from California Broad bean wilt virus-2 (BBWV-2) found evidence of strong purifying selection exerted on four functional domains including the CP and MP Arabis mosaic virus (ArMV), at an interspecific level Grapevine deformation virus which appears to be a mosaic between GFLV and ArMV Bean pod mottle virus (BPMV) Zhang et al. Tobacco black ring virus (TBRV) and Grapevine chrome mosaic virus (GCMV) Comovirinae sequence analyses of certain functional domains have identified a chimeric-like composition to secovirid genomes that extends outside of the Secoviridae. The CPs of CRLV and ToTV are reported to have highest identities with non-plant infecting members of the PicornaviralesSecoviridae family. The results obtained help define the Secoviridae as an emerging group of viruses that we postulate in some cases originated in insects.Beyond a number of reports measuring selection pressures and detecting specific examples of recombination there is limited information on the evolution of the http://www.ncbi.nlm.nih.gov) (Raspberry ringspot virus). Full-length sequences were analyzed in Vector NTI (Invitrogen) with each open reading frame separated into its putative functional domains - Protease co-factor (ProCo), Helicase (HEL), Viral protein genome-linked (VPg), Protease (PRO), RNA-dependent RNA-polymerase (RdRp), Movement protein (MP) and Coat protein (CP) - as determined by reported cleavage sites, determined either experimentally or in silico. Since for some species there was functional ambiguity and/or additionally reported in-frame domains at the N-termini of ProCo and MP sequences, designation as the 1N(ProCo) and 2N(MP) domains, respectively, was more appropriate and adopted throughout this study (The full-length nucleotide sequences of 27 secovirids were downloaded from the NCBI GenBank database (nih.gov) . Selectiis study . BecauseN/dS) nucleotide substitutions using the Single-Likelihood Ancestor Counting (SLAC) method of the HYPHY program http://www.datamonkey.org/). Both SLAC and FUBAR 2 distribution. For the latter, alignments that generated trees with a single branch where episodic diversifying selection was identified were only considered positive if the same branches were identified during a re-run with maximum likelihood input parameters. Nucleotide substitution rate estimates were determined using BEAST 1.7 Nucleotide sequences of the seven identified functional domains – 1N(ProCo), HEL, VPg, PRO, RdRp, 2N(MP) and CP were aligned using the MUSCLE Maximum clade credibility trees with statistical support in the form of Bayesian posterior probabilities at each node were generated using BEAST and processed in the programs TreeAnnotator and FigTree. For maximum likelihood (ML) trees the MUSCLE alignments of each of the seven identified functional domains – 1N(ProCo), HEL, VPg, PRO, RdRp, 2N(MP) and CP and their respective optimal substitution models, as determined by JModeltest, were analyzed in PAUP*Evidence for recombination prior to calculating selection pressures was carried out, as mentioned above, using GARD http://www.ncbi.nlm.nih.gov/taxonomy) and ICTV (http://ictvonline.org/virusTaxonomy.asp) databases. An e-value of 10 was used as the threshold if the hit was with a viral functional homolog.Protein sequences of one member of a monophyletic virus group that was consistently identified in three or more ML trees were used to search for the closest homologues within picorna-like viruses. A first screen was done using the Blast algorithm with setting to exclude the input virus species. A Position-Specific Scoring Matrix (PSSM) was then generated for each protein using PSI-BLAST  with no exclusion Secoviridae family we began by dividing our analyses into the two principal components of virus evolution, 1) mutation (or associated elements: selection pressures and substitution rates) and, 2) recombination (or modularity).In order to gain an understanding of the evolutionary tendencies of the N/dS ratios were not statistically significant , with evidence of strong purifying selection  (N/dS ratios were more variable (ranging from 0.014 to 0.088), but were in-line with the full-length ratios, averaging 0.045. The total percentage of negatively selected codons varied depended on the method used, SLAC or FUBAR, the latter identifying many more. FUBAR also identified some positively selected codons where SLAC found none. These variations are attributed to the documented power and sensitivities of the methods employed Blackcurrant reversion virus) BRV 49%, GFLV 71%, SMoV 30%, Tomato ringspot virus (ToRSV) 61%, Tobacco ringspot virus (TRSV) 15%). Lower values for BPMV, SMoV and TRSV were also found using SLAC, and are reflected in the lower diversities obtained. The shorter sequences used for SMoV and TRSV might also be a reason for these differences. In addition to a global and codon-based calculations of selection pressures, we tested the alignments for the possibility of lineage specific variations in selection pressure, and were able to find significant (p<0.05) evidence of episodic diversifying selection for two isolates of RTSV ((accession number underlined) U70989 p = 7.01×10−6, U71440 p = 7.93×10−3), and one isolate each for GFLV (EU702441 p = 6.10×10−3) and ToRSV (AF135414 p = 5.80×10−5). Removal of these isolates from the dN/dS analyses did not significantly alter the original values obtained. Significantly, FUBAR was also able to identify positively selected codons, one and two respectively, for both GFLV and RTSV.Prior to analysis all alignments were checked for recombination using the GARD algorithm ctively) . For par−5 for Broad bean wilt virus-2 (BBWV2); a value of ≤1×10−7 being proposed as an indicator of unreliability −2 – 1.21×10−3). However, the values for BRV and SMoV were taken to be unreliable because of the limited date range collection period (<10 years). The strength of the temporal signal in the sequences was tested by comparing ‘time-stamped’ and randomized datasets. In all cases, except TRSV, the 95% HPD range was greater in the randomized data than in the ‘time-stamped’ data; the former ranging from 3 to 5 orders of magnitude compared to 1 to 3 for the former. In six cases  the 95% HPD values of the randomized data fell outside the mean rates estimated for the ‘time-stamped’ data, suggesting the differences were significant and that the temporal structure was sufficient for reliable rate estimation. A temporal structure was also supported  for the majority of species in regression analyses  as the rate prior in time-stamped alignments. Clock priors were varied  with the optimum combination of priors being selected using Bayes factors in the BEAST-associated program Tracer. Our initial attempts to generate a full tree for all members of the Secoviridae were unsuccessful due to nucleotide saturation. As a result, by focusing specifically on the Comovirinae subfamily, we were able to generate a coalescent tree using both a lognormal relaxed  defining the calibration nodes using the pre-determined TMRCAs for each virus species in conjunction with a Yule process (a simple model of speciation more appropriate for sequences from different species) tree prior, and 2) using the most conservative reliable mean estimate of the nucleotide substitution rate calculated for the CP data  is external to the Secoviridae. Instead, what is seen are relatively consistent similarities throughout with monophylies for the non-Comovirinae species for at least three domains with the ALSV/CRLV (cheraviruses), BRNV/SMoV/SDV, ToTV/ToMarV (torradoviruses) and MCDV/RTSV (waikaviruses).The existence of potential chimeric-like sequences Using the interspecific domain alignments described above neither GARD nor RDP3 found significant evidence of recombination. We were also unable to demonstrate any significant change in phylogenetic similarities that might be indicative of recombination by comparing ML trees. Another approach to identifying recombinants, particularly for alignments of relatively divergent viruses is to reduce noise due to potential misalignment by aligning only those sequences with higher identities Comovirinae viruses, choosing a representative virus for each apparent monophyly (eg. CRLV (representing an ALSV and CRLV clade), SMoV , SLRSV alone, and ToTV  and RTSV ) to initiate the search and generate a first Position-Specific Scoring Matrix (PSSM). Use of another representative from the same clade consistently produced similar hits to the same viruses. We principally focused on the non-replication association genome domains as these were the most divergent. The results for the 1N(ProCo), PRO, 2N(MP) and CP showed strikingly different associations for each gene  of the SLRSV PRO to the NIa-Pro of Potato virus V. A similar case of associations is found for the CP  similarities to animal-infecting picorna-like viruses  that share a higher similarity to monopartite animal-infecting viruses  there are over five times as many animal virus species. For the Comovirinae type members  the distribution is varied, with not a single defined host population exceeding double that of the corresponding population in the other host.The homologies identified for the CP and PRO domains is evidence of an ancestral link with animal infecting picorna-like viruses. The existence within the predominantly bipartite  viruses  than the viruses  supports viruses . Such a  viruses  show at SecoviridaeN/dS ratios itself could also possibly overestimate the degree of purifying selection by failing to detect directional selection All coat protein domains tested were found to be under extreme purifying selection, an observation already reported for members of the −03 to 2.74×10−03. This compares with 1.61×10−3 to 5.73×10−3 for the VP1 of enteroviruses (Picornaviridae) −4 to 3.5×10−2 for the CP of the Luteoviridae−3 for the CP of Rice yellow mottle virus (RYMV) LuteoviridaeSecoviridae family proved inconsistent due to nucleotide saturation. However, by focusing only on members of the subfamily Comovirinae, using both lognormal relaxed and strict clock models with the most conservative substitution rate obtained at the species level (2.74×10−3), we were able to generate reliable (converged) trees rooted at 726 and 944 years ago, respectively. Species divergence of all extant members of the Comovirinae therefore is estimated to have occurred in the past 500 years. The first records of fanleaf, the oldest known grapevine virus disease, date back to 1865 Comovirinae genera coincide with limited resolution at the root of both trees for the whole subfamily. Aside from the effects of heterotachy, there is also the possibility of underestimating the age of the Comovirinae due to the replacement of older viral lineages by younger ones Luteoviridae family the TMRCA was estimated at around 2,000 years ago, with the Polerovirus and Luteovirus genera TMRCAs falling around 1,000 years ago Potyviridae family has been estimated around 6,600 years ago Sobemovirus genus diversification is calculated to have occurred some 3,000 years ago Luteoviridae genera, and all extant secovirid species show a recent (>500 years) emergence in-line with the above reports and coinciding with both modern agriculture and the escalation of marine trade.Calculations of the nucleotide substitution rates for the same species’ coat proteins obtained values ranging from 9.29×10Secoviridae family. As a result, we broadened our search using both BLAST and the PSI-BLAST algorithms; the latter looks for more distant protein homologies by pooling conserved three-dimensional protein structural information Secoviridae, but also detectable similarities with animal-infecting picorna-like viruses. Such an arrangement produces a mosaic of viral functional domains that fits neatly into the proposed hypothesis that the Pro and CP, and the HEL and RdRp, are respective paralogous duplications of ancient proto-CP and proto-Rep genes Maximum likelihood cladograms generated for each secovirid functional domain were able to identify a number of statistically unrelated lineages that pointed to a possible origin of these monophyletic groups that lay outside the Secoviridae, 2) a higher similarity of these monopartite viruses to monopartite animal-infecting viruses, and 3) evidence of positive selection in the CP of a monopartite virus, it seems the most parsimonious hypothesis for host-switching, if we consider the Secoviridae as a whole, is from animal-to-plant. In order to explore this hypothesis further we used the Genbank database to analyze the distribution of related virus species according to their host  on the simple assumption that the degree of speciation will be directly proportional to the age of the virus population. Clearly, there are number of caveats to this approach, including the artificial composition of the sampled population, and the possibility of different rates of evolution and extinction, and unrecognized selection pressures )-recognized virus species and grouped them according to their host; the assumption being that the number of animal virus species, and in particular vertebrate-infecting viruses would dominate and therefore bias any interpretation on specific distributions. Contrary to our assumption, the plant viruses made up 45% of the total number of eukaryote-infecting viruses, with vertebrate-infecting viruses and insect-infecting viruses at 39% and 12%, respectively ) and assumed narrow ranges , Black queen cell virus (Dicistroviridae)) Based on three lines of evidence, namely 1) the existence of a minority of monopartite viruses within the ectively  , there is no evidence of virus replication in the vector, although some nepoviruses and comoviruses are described as being persistently transmitted Rhopalosiphum padi virus  is transmitted horizontally via plants in which it is able spread to all parts without replication Flock house virus  Nicotiana benthamiana was complemented by the movement proteins of two unrelated plant viruses. Furthermore, FHV was shown to replicate in the inoculated leaves of six monocot and dicot species A unidirectionality to novel host colonization may be too simplistic, but the very tight relationship between plant viruses and their vectors and the presence of related viruses in ecdysozoans presents a plausible scenario wherein ecdysozoan-infecting viruses could have been introduced during feeding into plants . In thisde novo inoculation and colonization of insect cells by a plant virus is equally as difficult to imagine. Picornaviruses require cell-surface receptors that are involved in cell attachment, signaling and endocytosis, and in triggering of capsid structural alterations that are required for infectious entry The only salient theoretical example of ‘plant-to-animal’ host-switching also involves a calicivirus; where it was postulated that nanoviruses, which are plant single-stranded, circular DNA viruses must have switched to an animal host and then recombined in the C-terminal of the Rep protein with the RNA of a calicivirus prior to returning to plants Secoviridae, and provide the basis for further studies into these and related viruses and their ability to adapt to a huge variety of different hosts. Unanswered questions raised in the discussion here that could be addressed in future studies are: 1) Does an RdRp domain evolve rapidly within a founder population colonizing a markedly different host? 2) Is secovirid genome segmentation an unavoidable consequence of plant colonization, and under what conditions does it become selectively detrimental? 3) Are there ecdysozoan vectors harboring viruses that more closely resemble secovirids? 4) Are there emergent secovirid-like viruses that could threaten future agricultural production? 5) What are the characteristics of picorna-like viruses that favor host-switching? In answering these questions we hope to better predict the sources of emergent viruses and how to control them.Taken together, the results provide evidence for the recent evolutionary tendencies and putative ancestral origins of members of the Figure S1Comovirinae strict tree.(TIF)Click here for additional data file.Figure S2protein alignments.(TIF)Click here for additional data file.Figure S3Insect virus trees.(TIFF)Click here for additional data file.Table S1Recombination check(XLSX)Click here for additional data file.Table S2Secovirus CP Accessions.(XLSX)Click here for additional data file.Table S3R-squared values Time-stamped vs. Random.(XLSX)Click here for additional data file.Table S4PSI-BLAST.(DOCX)Click here for additional data file.Table S5HEL RdRp BLASTs.(XLSX)Click here for additional data file.Table S6Virus ICTV Species BLAST.(XLSX)Click here for additional data file.Table S7ICTV recognized species.(XLS)Click here for additional data file.File S1CP alignment dNdS.(MSF)Click here for additional data file.File S2(MSF)Click here for additional data file.File S3(MSF)Click here for additional data file.File S4(MSF)Click here for additional data file.File S5(MSF)Click here for additional data file.File S6(MSF)Click here for additional data file.File S7(MSF)Click here for additional data file.File S8(MSF)Click here for additional data file.File S9(MSF)Click here for additional data file.


Alzheimer’s disease (AD) is associated with considerable costs and has a significant impact on health and social care systems.This study assessed whether baseline comorbidities present in 2,594 patients with AD participating in two semagacestat randomized placebo-controlled trials (RCTs) would significantly impact overall costs.Resource utilization was captured using the Resource Utilization in Dementia Scale-Lite. Comorbidities and concomitant medications were tabulated via patient and caregiver reports. Only baseline data were analyzed. Direct and indirect costs per month were calculated per patient. The relationship between cost and explanatory variables was explored in a regression model.p ≤ 0.0001). The cost ratio was not impacted when the number of comorbidities was added to the model  or when combined with the number of concomitant medications . Inconsistent findings related to the impact of individual comorbidities on costs were noted in sensitivity analyses.The baseline monthly cost of care in this RCT population was £1,147 ± 2,483, with informal care costs accounting for 75 % of costs. Gender, age, and functional status were significant predictors of costs  are likely needed to better estimate the effects of disease and treatment on costs of illness captured in RCTs for AD. Alzheimer’s disease (AD) is progressive neurological disorder that is thought to be the causal pathology in roughly 70 % of dementia cases . AD is aPatients with AD incur far greater costs (inpatient and outpatient care and prescriptions) than their cohorts without AD. For patients with AD, inpatient stays and institutionalization account for the majority of direct care costs , 6. AddiCosts are influenced by a variety of factors, including severity of the illness , 9 and pConsiderable efforts are underway to find new treatments that will modify the underlying pathological process of AD . While rGiven the difficulties in capturing costs of care in parallel with clinical measures over a substantial period of time to test disease-modifying agents, previous cost-effectiveness analyses of AD therapies have relied on multiple data sources –18. CostThe present analyses used baseline data from a discontinued clinical trial to examine how comorbidities would impact costs of illness, as assessed in the trial. It was hypothesized that that the comorbidity profile would have a significant impact on overall costs, similar to patterns seen in observational and retrospective claims analyses. If the hypothesis is valid, evaluating the type of comorbidities at baseline and over time in clinical trials may be an important added covariate in assessing the overall impact of future pharmacotherapeutic candidates on costs.http://www.clinicaltrials.gov/ct2/show/NCT00594568?term=LFAN&rank=2 and http://www.clinicaltrials.gov/ct2/show/NCT00762411?term=LFAN&rank=3, respectively. The experimental treatment was discontinued early due to safety findings. The protocols were subsequently amended prior to completion in order to allow for continued safety monitoring of patients [The present analyses used baseline data from two similarly designed 88-week RCTs of semagacestat [H6L-MC-LFAN (IDENTITY) and H6L-MC-LFBC (IDENTITY-2)], a gamma-secretase inhibitor AD drug candidate previously in development, in patients with mild-to-moderate dementia due to AD . Furtherpatients . As a reCo-primary efficacy measures for the trial were the 11-item Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) , 23 and Resource utilization was assessed by the Resource Utilization in Dementia-Lite (RUD-Lite) scale , 32. ThiPatient comorbidities and concomitant medications at baseline were assessed primarily via patient and caregiver reports at study entry as well as via medical records, when available. Individual drugs were grouped according to drug class to facilitate reporting. Specific comorbidities were chosen for inclusion in the subsequent analyses that represented common age-related comorbidities in the study population most likely to be afflicted with AD; concomitant medications were selected based on the most frequently identified comorbid conditions.Only baseline data were analyzed and are presented. Summary statistics for the clinical scales and the profile of comorbidities and number of concomitant medications were generated. Although study inclusion criteria defined mild-to-moderate dementia severity by an MMSE score ranging from 16 to 26, the scores at baseline reflected a much broader range. Accordingly, dementia severity in the analyses herein was categorized as mild (20–30), moderate (10–19), and severe (0–9) using the standard convention. Mean resource utilization captured by the RUD-Lite, categorized by type of resource, was tabulated. Baseline report of caregiver time spent on personal ADLs, instrumental ADLs, and supervision in the past month is presented as mean hours per month and aggregated into a mean of total informal care time spent per month. The total caregiver time was capped at 540 h per month (18 h/day) to avoid overestimation. Each type of accommodation and hospitalization is presented as mean number of days per month, and community care is presented as hours per month.Direct and indirect costs per month were calculated for each patient in the study. All cost calculations were based multiplying RUD-Lite resource utilization unit estimates by published reimbursement costs in the United Kingdom (UK) Table  to allowt tests.Differences in mean costs between patient subgroups were tested for statistical significance using unpaired A cluster analysis using the Ward’s method of minimum variance to cluster countries with similar baseline healthcare costs was performed to examine potential differences in cost structures between countries. Due to the high number of countries missing at least one cost component, countries were clustered based on total monthly costs per patient. Results from an RCT do not need a cluster adjustment. However, the potential effect due to the clustering of patients within a site was considered. This, in combination with the small number of patients within each site, suggested that there was no need for a clustering adjustment.The relationship between cost of care and explanatory variables was explored in a regression model to determine the drivers of cost of care. Cost and resource data are usually of a distribution that constitutes of large number of zero observations, no negative observations, and a few very high observations. Cost functions are therefore estimated with a generalized linear model (GLM) . The GLMComorbidities, the number of comorbidities, and the number of concomitant medications, including AD medications, were the covariates to impact costs. Adjustment variables added to the costs model included demographics of the patient , cognitive (MMSE), functional (ADCS-ADL), behavioral (NPI), and global  disease indicators. The comorbidities selected as potential predictors included cancer, chronic obstructive pulmonary disease, depression, diabetes, hyperlipidemia, hypertension, cardiac ischemia, urinary tract infection, epilepsy, and stroke. Individual comorbidities were selected to represent common age-related comorbidities and to facilitate comparison to other samples .A total of 2,594 patients were included at baseline, 55 % were female with a mean age of 73 ± 8 years. As shown in Table The baseline monthly cost of care in this RCT population was £1,147 ± 2,483 for which informal care costs accounted for 75 % of costs (Table p ≤ 0.01). In this RCT population, age was a significant variable associated with baseline total costs of care. Patients <65 years old and older patients (>75 years) had significantly (p < 0.0001) greater costs compared to patients aged 65–75 (Table p = 0.003), whereas cardiac ischemia is significantly associated with lower total costs . In sensitivity analyses using US unit costs, the association between cancer and higher costs disappeared, but cardiac ischemia remained as a predictor of lower costs and hyperlipidemia was also associated with a lower cost ratio. The impact of gender, age, and functional status remained significant.The individual effects of patient demographics and disease severity on total costs of care were estimated in a cost model using gamma model distribution with a log link. Gender, age, and functional status were all significant predictors of costs . A more consistent impact of comorbidities on higher costs of care was hypothesized. Unexpectedly, cardiac ischemia was a predictor of lower costs, whereas in claims-based samples, patients with a history of cardiac disease had higher costs due to more frequent hospitalizations, preventable hospitalizations, etc. . This suBaseline rates of comorbid conditions were lower than those described in claims database studies  but consA challenge in self-reported methods for comorbidities in clinical trials is that it does not allow for an easy comparison with baseline rates of comorbid disorders within the healthcare system of interest. If access to actual medical records was included as part of trial consent, this would provide an opportunity to characterize the similarities and differences of a matched cohort within the same healthcare system, as well as help to understand baseline rates of diseases and acute events. Access to claims data may also eliminate some issues with proxy reporting of resource utilization, such as unawareness of care use or a too-long recall period , 8. In aFuture studies should consider evaluating the impact of comorbid conditions longitudinally within clinical trial populations. As demonstrated in claims databases or community-based studies, costs associated with comorbid conditions increase over time , 12. ColAlthough conclusive statements about the impact of comorbidities on costs could not be made with the present cross-sectional analyses, it is important to note that consistencies with the previous literature were noted, particularly around the association between greater functional impairment and costs . It is wThe present analyses offer an important step forward in understanding how clinical trials may help inform decisions about the overall impact of new therapeutics beyond traditional regulatory audiences. Notably, there is a paucity of published literature in AD on cost that has been directly garnered from clinical trials. Functional ability remains a consistent predictor of costs in the care of patients with AD. Comorbidities are likely less informative in determining variances in baseline costs if looking at clinical trial populations in isolation. Therefore, it is just as important for trial sponsors to include resource utilization measures in their trials as it is for decision makers to allow extrapolations beyond the clinical trial to best characterize and predict the full risk–benefit profile of a new therapeutic agent. Expanded precision and ease of diagnosis of AD pathology is also likely to move clinical trial populations closer to “typical” patient populations as the current paradigm shifts away from AD as a rule-out diagnosis.


To the Editor: A recent article described the isolation and subsequent analysis of a tickborne phlebovirus: Hunter Island Group virus (HIGV), associated with an albatross disease event that occurred in 2002 on Albatross Island, 6 kilometers off the northwest coast of Tasmania, Australia  on Albatross Island, the only island inhabited by albatross within the Hunter Island Group Important Bird Area. At the time of collections, many immature albatross were dying. Records from this time indicate that postmortem blood samples were collected from the birds, and subsequent virus neutralization studies conducted soon after demonstrated that 50% of these samples were ABIV positive. Ensuing testing of samples collected in the next 2 years also identified a positive sample from a black noddy in Queensland (eensland . ABIV waWe recently sequenced the genome of ABIV by using high-throughput sequencing and have compiled near complete sequences for the large (L), medium (M), and small (S) segments (GenBank accession nos. KM198925–7). Overall, ABIV shares 99% nt identity with HIGV, and thus they can be considered isolates of the same virus. The translated nucleocapsid and S segment nonstructural proteins of both viruses are identical, and the polymerases and glycoproteins share 99% identity. There are 26 nt changes across the whole genome , but only 7 of these translate into an amino acid change . Predictive protein analysis indicates that at least 1 of the 3 aa changes occurs in the ectodomain of the Gn protein, which could affect virus–host interactions. Of the remaining changes, 14 are silent mutations and 5 occur in noncoding regions. In light of the genomic similarity of these 2 viruses, we suggest that the species name Albatross Island virus encompass both isolates, ABIV and HIGV, thereby representing the name of the original 1983 isolate and the location where both viruses were isolated. These 2 viruses are closely related to 2 tickborne phleboviruses: severe fever with thrombocytopenia syndrome virus, isolated in China  reportedly migrate from their breeding base in New Zealand as far as South America and eastward into shy albatross territory (Diomedea cauta), is controversial. The ease of albatross movement between vast geographic areas could provide an opportunity for intercontinental spread of emerging infectious diseases. Consequently, phleboviruses similar to ABIV may be present in bird populations in the southern areas of Africa and South America.Birds of the albatross family tend to fly long distances over open water. The geographic range of shy albatross extends from their breeding base in Tasmania to southern Africa (The need for intensified international investigations to identify genetically related tickborne phleboviruses with zoonotic potential is evident. The opportunity for the distribution of such viruses over a large global area is of concern to public health. Surveillance and investigation on an international level are needed.


C. neoformans and C. gattii, present eight major molecular types—VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII  prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and aMAT, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and aMAT were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and aMAT.Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents,  Cryptococcus neoformans/C. gattii species complex from the environment. Its most frequent clinical manifestation is meningoencephalitis [C. neoformans is cosmopolitan, affecting mainly immunocompromised individuals, especially patients with AIDS [C. gattii causes predominantly a primary infection in immunocompetent individuals, previously associated with tropical and subtropical climates, but now gaining prominence as an important cause of human and veterinary disease in temperate regions of North America [Cryptococcosis is a life-threatening systemic mycosis affecting humans and animals worldwide. The disease is acquired by inhalation of infectious propagules (desiccated yeasts cells or basidiospores) of the members of the phalitis . Cryptocith AIDS . On the  America –7.Cryptococcus neoformans/C. gattii species complex. C. neoformans is classified into the major molecular types VNI/AFLP1 and VNII/AFLP1A and AFLP1B (serotype A), VNIII/AFLP3 (serotype AD), and VNIV/AFLP2 (serotype D), while C. gattii correspond to VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7, all corresponding to serotypes B and/or C [C. neoformans/C. gattii species complex, a MLST scheme was established by the ISHAM working group “Genotyping C. neoformans and C. gattii” based on variable regions of seven independent genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region, taking advantage of the high discriminatory power and good reproducibility between different laboratories [http://mlst.mycologylab.org/.Molecular epidemiological studies have identified eight major molecular types within the and/or C . In addiand/or C ,10. To gratories . The allC. gattii in Brazil is endemic and shows a regional pattern, being more common in the North (N) and Northeast (NE) of the country [C. gattii in these regions is not well known and requires more specific environmental studies to detect possible outbreaks. Indeed, environmental sources related to trees colonized by the agents of cryptococcosis have been described in Roraima [Cryptococcosis by  country . One hun country –15. In t country , 15–16,  Roraima , Pará [1 Roraima  and Piau Roraima  states.C. neoformans and C. gattii in dwellings of the hinterland of the Brazilian Amazon, and characterized the molecular subtypes and mating types of the obtained isolates.Pioneering studies of cryptococcosis in AIDS in Central Africa and Brazil demonstrated the risk of those patients to acquire cryptococcosis from indoor dust , 21. TheThe study was conducted in the city of Santa Isabel do Rio Negro, Amazonas state, located 620 km straight or 772 km by waterway away from Manaus, the capital of the Amazonas state, and about 1,000 km far from the Atlantic Ocean coast of the Brazilian Northern region . The munC. neoformans and C. gattii. One sample per house was obtained combining dust from all rooms swept with a different broom per house to prevent cross contamination between houses, and the material was stored in sterile plastic bags (Nasco/WHIRL-PARK). The location of the selected households was obtained using a GPS (Garmin Etrex H). Cryptococcal cells were isolated as described previously [Dust samples were collected from 51 houses randomly chosen in different neighborhoods of the city of Santa Isabel do Rio Negro, to investigate the presence of the agents of cryptococcosis, eviously , 23. Brieviously . The plaC. neoformans and C. gattii were differentiated on canavanine-glycine-bromothymol blue (CGB) medium [Brown colonies were purified and tested for urease production on Christensen urea agar , and for) medium .URA5-RFLP analysis [URA5 gene was performed in a final volume of 50 μL. Each reaction contained 50 ng of DNA, 1X PCR buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl—Invitrogen), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen), 2 mM magnesium cloride, 1.5 U Taq DNA polymerase (Invitrogen), and 50 ng of each primer URA5 (5’ ATGTCCTCCCAAGCCCTCGACTCCG 3’) and SJ01 (5’ TTAAGACCTCTGAACACCGTACTC 3’). PCR was performed for 35 cycles in a Eppendorf gradient mastercycler , using the following cycling conditions: 94°C for 2 min initial denaturation, 45 s of denaturation at 94°C, 1 min annealing at 61°C, and 2 min extension at 72°C, followed by a final extension cycle for 10 min at 72°C. PCR products were double digested with Sau96I (10 U/μL) and HhaI (20 U/μl) for 3 h, and the DNA fragments were separated via 3% agarose gel electrophoresis at 100 V. URA5-RFLP patterns were assigned visually by comparing them with the patterns obtained from the standard strains WM 148 (VNI/AFLP1), WM 626 (VNII/AFLP1A), WM 628 (VNIII/AFLP2), WM 629 (VNIV/AFLP3), WM 179 (VGI/AFLP4), WM 178 (VGII/AFLP6), WM 175 (VGIII/AFLP5), and WM 779 (VGIV/AFLP7).After DNA extraction , the majanalysis . Amplifialpha  and JOHE9787 (5’ ACACCGCCTGTTACAATGGAC 3’); JOHE9788 (5’ CAGCGTTTGAAGATGGACTTT 3’) for mating type a (MATa) [The mating type was identified by PCR using specific primers for the pheromone genes: MFalfaU (5’ TTCACTGCCATCTTCACCACC 3’); MFalfaL (5’ TCTAGGCGATGACACAAAGGG 3’) for mating type C. neoformans and C. gattii [CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. All 7 loci were amplified as previously described [http://mlst.mycologylab.org/. The corrected sequences were aligned using the program MEGA version 5 [http://www.ub.edu/dnasp/). Novel genotypes have been submitted to C. gattii MLST database (http://mlst.mycologylab.org/).Sub-typing and molecular polymorphism analysis were performed according to the ISHAM consensus multi-locus sequence typing scheme for . gattii  includinescribed . The seqersion 5 . An un-rC. gattii  [C. neoformans was isolated from domestic dust in 13 (54%) out of 24 dwellings studied [C. neoformans in these studies was the presence of avians in the domestic environment or nearby the homes. The present study has no index-case and revealed the exclusive occurrence of C. gattii VGII in indoor dust obtained from dwellings in a remote human settlement formed in the heart of the Amazon rainforest in Brazil. A significant positivity, of 5.9%, in indoor dust samples was observed, thus suggesting daily human exposure to these potential disease agents inside those houses. On the contrary to previous reports, no domestic or caged birds were observed in the present study. The houses in Santa Isabel do Rio Negro are rustic, most of them built with planks of wood obtained from the extraction of native species of trees in the surrounding rainforest, especially the Brazil nut tree.Publications on the presence of agents of cryptococcosis in indoor dust are scarce. Two studies, originated from index-cases of aids-associated cryptococcosis in South America and Africa, showed similar results: in Rio de Janeiro, southeast of Brazil, the investigation of 77 dwellings revealed the isolation of  (14.3%) , and som studied . The priC. gattii VGII is widely distributed in Amazonia, e.g. this genotype has been isolated from several species of trees from Brazil and Colombia [Cryptococcus neoformans/C. gattii species complex are not associated with a particular tree, but with a specialized niche resulting from the natural biodegradation of wood [C. gattii, strain LMM645, by Fortes et al. 2001 from a native jungle tree (Guettarda acreana) on a wild island of the Amazon rainforest, suggests that wild tropical forests may harbour primary sources of this agent. This was recently again emphasised by a global study pointing to the same environmental isolate, LMM645, suggesting an ancient dispersal of the human fungal pathogen C. gattii from the Amazon rainforest [et al. in 1996 suggested already that hollows of living trees may provide environments that are sheltered, damp and probably less exposed to changes in climatic conditions, offering more suitable conditions for the survival, adaptation and reproduction of C. gattii [C. gattii VGII in the native rainforest, thus forming a microfocus in human dwellings.There is much evidence that Colombia . Previou of wood . Moreoveinforest . Lazéra . gattii . Likely,Cryptococcus neoformans/C. gattii species complex can help the understanding of the geographic expansion and the reproductive strategies of VGII adapted to human dwellings in the Amazon rainforest. The present study revealed an unexpected high genetic diversity amongst the obtained VGII isolates, with eight MLST sequence types (ST20 (= VGIIa), ST7 (= VGIIb), ST5, ST264, ST265, ST266, ST267, and ST268) being present amongst 45 environmental strains from three houses, compared with Australia where only 6 MLST sequence types (ST20 (VGIIa), ST7 (VGIIb), ST5, ST21, ST33, ST38, and ST48) were found amongst 54 clinical, veterinary and environmental isolates from the entire Australian continent [Studies on the population structure of the ontinent , and in C. neoformans, where it ranged from 0.20 in Asia, 0.40 in South America, 0.46 in Italy, 0.75 in North America and 0.79 in Africa [C. gattii, no previous similar analysis has been reported. The VGII haplotype diversity index observed in the present study (HD = 0.695) is impressive, and was based on the analysis of all the seven studied MLST loci, amongst all 45 investigated strains, suggesting a high genetic diversity, especially when considering the very restricted area studied (4 km2). The major genotype found in Santa Isabel do Rio Negro, ST7 (= VGIIb), is also the most common one found around the world especially in Australia, Thailand, and caused the minority of the Vancouver Island outbreak in British Columbia, Canada and the Pacific Northwest of the USA (PNW) [The haplotype diversity has till now only been described for n Africa . ConcernSA (PNW) ,34. The C. gattii VGII.In the positive house SI443, approximately 1 km away from the house DW650, the occurrence and distribution of the identified subtypes was totally different. Here the sequence type ST20 (= VGIIa) was predominant, which corresponds to the main agent of the Vancouver Island and Pacific Northwest outbreaks [C. gattii propagules may lead to subclinical infection with regressive pulmonary lesions and acquired natural immunity to cryptococcal infection, warranting a detailed clinical surveillance in the region. Evidence for a direct impact of continuous exposure to infectious propagules from VGII strains in domestic environment is given by the previous reports of a high proportion of cryptococcosis cases due to C. gattii VGII in the Amazon region of Brazil, in HIV negative children [Our results emphasise that indoor exposure risks to the subtypes of VGII must be considered as a possible infection source, although in the current study no evidence of a disease outbreak, based on the data available, has been observed amongst the residents of Santa Isabel do Rio Negro. Suboptimal diagnosis of cryptococcosis and the absence of surveillance may contribute to the underreporting of cryptococcosis. On the other hand, the frequent exposure to children , 15.C. gattii VGII, which have affected hundreds of otherwise healthy humans and animals [et al. (2005) initially suggested same-sex mating between to MAT alpha strains involving a low virulent Australian strain (genotype ST7 (= VGIIb) leading to the raise of the high virulent ST20 (= VGIIa) genotype strains and the expansion of the ecological niche of this species. Later studies showed a high genetic diversity among strains from the Amazon rainforest and the possible occurrence of mating between strains of opposite mating types as the source of a global dispersal of this species [Over the past two decades, several cryptococcal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by  animals –7. To un species .C. gattii VGII.In conclusion, the herein described results provide evidence for the later perspective and showed clearly the presence of two different ways of propagation, clonal and recombination, which can lead to the establishment of environmental populations present in indoor dust in urban houses within remote settlements in the Amazon rainforest. These yeast populations are most likely associated with the original sources of the building materials obtained from the surrounding forests. The current study established the basis for future studies investigating the impact of early and continuous exposure to the development of infections and the prevalence of subclinical and clinical infections amongst the inhabitants of houses colonized by


E. coli bacteria as the host cell population, an RNA phage as the lethal virus and a filamentous phage as the therapeutic virus. Basic dynamic properties are established for each virus alone and then together. Observed dynamics broadly agree with those predicted by a computer simulation model, although some differences are noted. Two cases of dynamics are contrasted, differing in whether the therapeutic virus is introduced before the lethal virus or after the lethal virus. The therapeutic virus increases in both cases but by different mechanisms. With the therapeutic virus introduced first, it spreads infectiously without any appreciable change in host dynamics. With the therapeutic virus introduced second, host abundance is depressed at the time therapy is applied; following an initial period of therapeutic virus spread by infection, the subsequent rise of protection is through reproduction by hosts already protected. This latter outcome is due to inheritance of the therapeutic virus state when the protected cell divides. Overall, the work establishes the feasibility and robustness to details of a viral interference using a therapeutic virus.The failure of traditional interventions to block and cure HIV infections has led to novel proposals that involve treating infections with therapeutic viruses–infectious viruses that specifically inhibit HIV propagation in the host. Early efforts in evaluating these proposals have been limited chiefly to mathematical models of dynamics, for lack of suitable empirical systems. Here we propose, develop and analyze an empirical system of a therapeutic virus that protects a host cell population against a lethal virus. The empirical system uses  The following paragraphs describe the essential details of each entity.We introduce and analyze an empirical system of two bacteriophages and a host cell in which a non-lethal virus protects the host from the other virus. The elements of our system are: a single bacterial host , but to be infected by either phage, the host must harbor the F plasmid. We evaluated two different F-bearing E. coli for suitability as hosts for Qβ infection (A/λ and IJ338) and chose the former because it was killed by Qβ more rapidly and to lower levels than was IJ338. Even with A/λ, the dynamic profile exhibits a much higher minimum cell density than is typical or predicted in the presence of lytic phages (2–3 logs below the density in the absence of phage), a point we return to below. The ramifications of a high minimum cell density is that it limits the magnitude of any beneficial effect of an intervention.β is used here to cause a lethal infection of its bacterial host. This phage has a genome of 4,127 bases of linear, single-stranded RNA with four protein coding genes. For the purposes of this study, the important attributes of this phage are twofold: (i) It is obligately lytic, so that an infection invariably leads to lysis and thus death of the cell 40–50 min after infection (lysis enables release of the Qβ progeny) and (ii) it infects F-piliated E. coli; the side of the pilus is used as the receptor, and the phage genome enters through the pilus (β receptor (the F-pilus) will become apparent when discussing the therapeutic virus.The phage Qhe pilus , 2002. TE. coli; the tip of the pilus is used as the initial attachment site (its primary receptor), but retraction of the pilus brings it in contact with the secondary receptor, the tolA protein on the outer surface of the bacterium, from which infection occurs  also infects F-piliated n occurs , 1997. Tn occurs , 1969. Eλ . In theory, the mechanism of protection by f1 could be from retraction of the pilus or even from loss of the F plasmid. Use of a test using T7 (see Methods) on f1-infected A/λ grown overnight suggested that the F plasmid is retained in a large majority of cells, possibly all of them.An important property of infection by f1 is that within minutes of infection, the pilus is retracted or modified, so that infection by other filamentous phages is blocked , 1969. Tβ . As the Overall, the protection from f1 evident from Bacteria. E. coli A/λ . Plates contained LB with 15 g Bacto agar per liter. In the case of phage titers, soft agar (7 g Bacto agar per liter) was used to overlay LB plates.Phages and titer methods. A filamentous phage (f1) was used as the therapeutic virus , aliquoting and freezing in 20% LB-glycerol at –80 °C. Cells were thawed just before use and added to 10 mL LB broth in 125 mL flasks, grown with aeration (170 rpm) for 60 min to a density of ∼108 cells/ml, at which point phage were added. The volumes were diluted 10× when cell densities were high enough to inhibit further host growth (indicated in the figures). The time points for these dilutions were determined from preliminary trials based on the density of cells nearing 109/mL if the culture was not diluted. The drug kanamycin, to which f1-infected cells were resistant, was never used in dynamics assays.In all cases, cultures were incubated at 37 °C. Frozen cell stocks of A/Dynamics with both phages. Cells were always grown for at least 1 h at 37 °C before phage addition. The timing and numbers of phage added are given in figure legends accompanying the results illustrated. Bacteriophage samples were taken at times indicated and cell densities were measured via plating on agar plates and incubating overnight at 37 °C. Samples of suspended f1 were purified by incubating the sample at 65 °C for 30–60 min to kill bacteria and kill Qβ, spinning down debris and collecting the supernatant (tests confirmed that Qβ was killed to at least 6 orders of magnitude by this procedure). Qβ samples were purified by mixing the sample with chloroform to kill f1 phage and bacteria, centrifuging and collecting supernatant . Qβ densities were measured from the number of clear plaques. Bacteriophage f1 titer was measured as described above. To determine the fraction of cells infected with f1 at each time point, colonies grown in the absence of drug were stabbed onto agar plates containing kanamycin. When the density of accompanying f1 is high at the initial plating (104 or more per plate), this procedure allows some f1 infection of uninfected cells to occur during colony outgrowth, which would lead to an upward bias in the estimate of f1-infection and a downward bias in the estimate of uninfected cells. These rates were measured in controlled settings and found to have no meaningful effect on our dynamics estimates or conclusions.Dynamics with single phage. Cells were grown (1–2 h) to a density of ∼108 cells/ml and either f1 or Qβ was added at a density as indicated. Each hour, phage were collected for titering and cells were plated on LB agar for density. In the case of f1 dynamics, overnight colonies from the LB agar plates were stabbed onto plates containing corresponding antibiotics as described above. Phage titers were determined as described above.Testing for cells resistant to Qβ. Using the colony-stabbing method to detect fractions of bacterial cells infected with f1, we observed instances of colonies that were not infected with f1 plated from cultures that had such a high density of Qβ that all sensitive cells should have been killed  that, if unimpeded, will spread into the host population and cause high host mortality, (ii) a non-lethal, therapeutic virus (density V) that, upon infecting the host, protects it against subsequent infection or attack by the lethal parasite, (iii) uninfected hosts (density Hu), and (iv) hosts infected with therapeutic virus (density Hv). To specifically represent our empirical system, the model requires two additional properties of cells in this latter category: (v) cells transmit the therapeutic virus for life, and (vi) they transmit the infection to their progeny .One of our motivations is to test the predictability of the empirical system. As host-parasite dynamics are intrinsically difficult to grasp intuitively, we use a mathematical model. The variable entities of our system are (i) a lethal parasite  indicates the value T minutes in the past); because lethal infections have such a short lifespan, an equation for hosts infected with the lethal virus is omitted. Our model assumes dynamics occur within a closed system, so we included a logistic growth function (G) to limit host density to a carrying capacity. Since f1 infection affects maximal host density and maximal host growth rate, we include separate logistic functions and growth rates for infected and uninfected hosts . Our parameter values are within an order of magnitude of those of β burst size and lysis time approximate values from the literature  allow for rapid Qβ increase in the time frames and cell densities used here. Results illustrated in figures below that are sensitive to parameter values will be noted accordingly.Runs illustrated below used the parameter values from terature , 2009; iβ.It is easily appreciated that the simplest and most straightforward dynamics occur when only one virus infects the host population. This case is of interest at least to enable a clear determination of model fit for each virus but also to ascertain general behaviors. To facilitate comprehension, we henceforth use ‘therapeutic virus’ for f1 and ‘lethal virus’ for QWhen therapeutic virus alone is introduced to the population, the model predicts a rapid spread by infection and no decrease in host density (except through a reduction in carrying capacity). Virtually the entire population becomes infected within 3 h  and 2B. β, there is no recovery of the bacterial population.The dynamics are expected to follow standard models for lytic phages , 1977: iThe experimental dynamics when only the lethal virus is present exhibit a shallower decline of hosts than predicted; host density levels off and even starts to rise—in clear violation of the model compare  with 3B.Even for a single set of parameter values, there are myriad conditions to consider when both viruses are introduced, depending on initial abundances and the time when each virus is introduced. Yet the intuitive expectation in all cases is that the population should ultimately be dominated by a high density of hosts infected with the therapeutic virus, because (i) infection with the therapeutic virus protects the host and all descendants from being killed, and (ii) the lethal virus is never lost from the population, so sensitive hosts are always at risk. How this endpoint is reached depends on which virus is introduced first, however. This outcome also requires that intrinsic host resistance is rare or absent in the initial population.When the therapeutic virus invades first, the host population can be assumed to reside near its maximum density, because it has not been decimated by the lethal virus. Therapeutic virus spread will be largely infectious. In the extreme case, with initial host density near carrying capacity, the density of hosts may remain almost constant while their state changes from uninfected to infected by therapeutic virus, with little death and little reproduction by therapeutic-virus infected hosts. If protection against lethal virus is total and if the therapeutic virus is introduced well in advance of the lethal virus, this case reduces to that when only therapeutic virus is introduced, because the lethal virus has nothing to infect when finally introduced. Given that protected hosts support a low level of lethal virus growth , the letThe case when the therapeutic virus invades second is more complicated. To a suitable approximation for intuition, the effect of introducing the lethal virus first is to reduce host density prior to therapeutic virus introduction. Upon introduction of the therapeutic virus, there can be infectious spread, but the number of hosts gained through infection can be no more than the standing density of hosts–which will be several orders of magnitude less than carrying capacity. Henceforth, hosts infected with therapeutic virus will rise to carrying capacity by reproduction because both they and their progeny are protected from the lethal virus. This latter increase of therapeutic-virus infected hosts is slower than infectious transmission. Thus, we expect the infectious component (which is fast) to predominate when therapeutic virus is introduced first but the vertical component (which is slow) to predominate when therapeutic virus is introduced second. This latter expectation is strictly a consequence of the fact that protection is lifelong, and progeny of protected hosts are also protected.Numerical solutions of the system of differential equations The delay between introduction of therapeutic virus and lethal virus is expected to influence dynamics up to a point . In the Empirical results broadly agree with the numerical analyses . Introdu10/ml in culture compared to the predicted maximum of just over 106. This suggests either that the density of therapeutic-virus infected cells increased more slowly than expected or that protection from the therapeutic virus was not immediate or only partial; the latter is supported by the observations in One anomaly between the numerical analyses and empirical data was evident in the density of free lethal virus, which exceeded 10The preferred design for this experiment would be to add lethal virus to a host population near carrying capacity and let the population crash before adding therapeutic virus. A technical problem with this approach is the rapid selection and rise of resistant bacteria even before introduction of the therapeutic virus. Our initial conditions were thus established by adding a low density of hosts to a high density of lethal virus, approximating the equilibrium that would have resulted after a high density host population crashed. Therapeutic virus was introduced 30 min later.e, per hour, using the last three samples in The empirical dynamics are in reasonable agreement with numerical analyses . Within β . Those cells were plated and the resulting ∼18 h colonies were tested for lethal-virus sensitivity. It appeared that they were mostly resistant to the lethal virus, but a small subset appeared to be sensitive. However, these assays were difficult to interpret: the density of lethal virus was so high at plating that 18 h colonies commonly contained contaminating lethal virus. It is thus possible that colonies became exposed to lethal virus during their outgrowth and only then evolved resistance, in which case the count of resistance to the lethal virus was an overestimate. Even so, the presence of even a modest portion of sensitive cells in these populations is unexpected if they were fully susceptible to infection. We expect that they were partially resistant cells, as suggested previously for QrvGvHv term in the equation for A unique property of our therapeutic virus (f1) is that the protection afforded to the host is transmitted vertically to the offspring, which is not necessarily true of all possible therapeutic viruses. This property is what allows the density of protected hosts to increase by reproduction, and thus for the population to rebound moderately fast after being suppressed by the lethal virus. We thus considered the effect of abolishing vertical transmission; as there is no known way to abolish vertical transmission of f1, we did so by numerical analysis. Equations When therapeutic virus is introduced first  protects its host from a lethal virus. Protection is by conferring resistance against the lethal virus, by affecting host gene expression rather than by genetically converting its host to a resistance state, although the dynamics are the same for either mechanism of protection.Two motivations underlie our efforts. One is to suggest a new mechanism of dynamical suppression that, along with previously proposed mechanisms, might be used to assist the treatment of chronic infections such as HIV-1 , 2012. Oex vivo and then re-introduce the cells into the patient , the protection spreads infectiously, with little change in overall host density. The hosts are merely changing their status from sensitive to resistant.In contrast, when the lethal virus is introduced first, the population declines before protection is introduced. In this decimated host population, the therapeutic virus spreads infectiously at first. But as the available hosts become resistant to the lethal virus, the subsequent increase in protected hosts is by reproduction of the host population—a direct consequence of the therapeutic virus protecting not only the host cell that was infected but also protecting progeny. Simulations revealed that inheritance of the protected state is critically important to the success of the therapeutic virus when the therapeutic virus is introduced second to the lethal virus .β)  , 1988. SOther anomalies between model and observation were also noted, such as the therapeutic virus not providing immediate, full protection and the therapeutic virus having a mildly deleterious effect on the host. Those are perhaps best classified as minor deviations from ideal behavior, easily accommodated by changes in parameter values or additional equations.β-infected cells before they lyse, and (ii) delayed protection of f1-infected cells. Further extensions that could also be implemented include variation in sensitivity of the host, parameters dependent on host density and time since infection, reversible and irreversible adsorption steps, and loss of f1 infection. Many of these improvements would have quantitative but not qualitative effects, although variation in sensitivity of the host appears to have a significant effect on dynamics. The purpose of the paper was to start with a deliberately minimal model and see how well it matched empirical runs—as a test case for actual applications in the future. The simplified model appeared adequate to a first approximation in matching the data.These anomalies point to several modifications in the model that would potentially increase its accuracy if appropriately parameterized. Equations could be added or modified to include (i) QAlthough the mechanism of dynamical suppression studied here differs from those of other proposals, the overall success here of matching the models with empirical observations justifies continued consideration of dynamical suppression approaches. It is to be expected that every implementation will be unique in several ways and offer its own set of challenges. Yet there is reason to be confident that dynamical suppression mechanisms will be robust enough to lend themselves to broad classes of behaviors that can be captured with abstract models. There are even broader parallels, albeit shallow ones, between our models here and protection of a host population with a transmissible vaccine, a connection we develop elsewhere.10.7717/peerj.2166/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2166/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.2166/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.2166/supp-4Supplemental Information 4Click here for additional data file.