Vector mosquitoes are responsible for transmission of the majority of arthropod-borne (arbo-) viruses. Virus replication in these vectors needs to be sufficiently high to permit efficient virus transfer to vertebrate hosts. The mosquito immune response therefore is a key determinant for arbovirus transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. Besides this well-established antiviral machinery, the PIWI-interacting RNA (piRNA) pathway processes viral RNA into piRNAs. In recent years, significant progress has been made in characterizing the biogenesis and function of these viral piRNAs. In this review, we discuss these developments, identify knowledge gaps, and suggest directions for future research. Bunyaviridae (-ssRNA), Flaviviridae (+ssRNA), Reoviridae (dsRNA), Rhabdoviridae (-ssRNA), and Togaviridae (+ssRNA) [Mosquitoes and other hematophagous arthropods transmit important human and animal viruses, some of which are responsible for debilitating diseases such as dengue, chikungunya, and Zika . Collect(+ssRNA) . Because(+ssRNA) ,3. Inter(+ssRNA) ,5. This (+ssRNA) .Whereas the evolutionary conserved Toll, Imd, and Jak-Stat signaling pathways are implied in antiviral defense , the corMicroRNAs comprise an independent class of small RNAs that may be involved in the cellular response to arboviral infections by regulation of host immune genes . They arAedes aegypti and Ae. albopictus and in cell lines derived from these animals [In this review, we will focus on the most enigmatic class of small silencing RNAs in the context of arbovirus–vector interactions: PIWI-interacting (pi)RNAs. piRNAs associate with the PIWI clade of the Argonaute protein superfamily, display a broad size range (24–30 nt), and are produced independently of Dicer . The can animals –39. BiogDrosophila melanogaster (Drosophila and other model organisms. This becomes apparent in many aspects: (i) The composition of piRNA pathway components differs between Drosophila and mosquitoes  Moc tissue , whereas tissues –47.  domain of PIWI proteins ,66. In cRecent studies have proposed that secondary piRNAs initiate Zuc-dependent production of phased piRNAs ,53. CleaDrosophila ovarian somatic sheet (OSS) cells persistently infected with several RNA viruses [Drosophila germ cells, does not give rise to vpiRNA production [Aedes cell lines and in somatic tissues of adult Aedes mosquitoes upon infection with several arboviruses, including members of the Togaviridae [Flaviviridae [Bunyaviridae [Reoviridae [Flavivirus genus, Flaviviridae family) and Sindbis virus  have been verified to be 2′-O methylated at the 3′ terminal nucleotide  [Initial evidence for vpiRNA production came from the analysis of small RNA deep-sequencing data of the  viruses . OSS celoduction , despiteaviridae –33, Flaviviridae –36, Bunyaviridae ,29,37–39cleotide , a modifcleotide . PIWI-de family) ,32,36 anO methylation marks at the first two nucleotides downstream of the cap structure [The substrate for the antiviral siRNA pathway, double-stranded RNA, is not abundant in the cytoplasm of healthy, uninfected cells and therefore serves as a danger signal indicating ongoing virus infection . In conttructure ,73. Additructure . In analDrosophila flamenco locus, which evokes piRNA biogenesis independent of its genomic context. However, whether this PTS is a structural motif or harbors a small, as-yet unrecognized sequence motif remains to be unraveled [cis-acting, 100-nt fragment in the 3′UTR of the piRNA-producing gene traffic jam that triggers piRNA production when expressed from unintegrated plasmid DNA. These plasmid-derived piRNAs were efficient in transcriptional silencing of endogenous genes [A clue that may help in understanding the mechanisms of target selection lies in the genomic distribution of vpiRNAs. While approximately equal levels of viral siRNAs (vsiRNAs) are produced along arbovirus genomes, vpiRNA production is mostly confined to specific hotspot regions. In alphaviruses such as Sindbis, chikungunya, and Semliki Forest virus, vpiRNAs are predominantly produced from a subgenomic RNA that is transcribed from an internal promoter sequence . This manraveled . Similarus genes . In lighpiRNA hotspots in flavivirus genomes, including dengue and cell fusing agent virus, differ considerably from those in alphaviruses. Flavivirus piRNAs mostly derive from few very discrete hotspots, sometimes representing single sequences . The natcis- and trans-acting factors required for the piRNA biogenesis from arboviruses of all families.Common to alphavirus and flavivirus piRNAs is their extreme strand bias towards sequences from the viral sense strands. In sharp contrast, bunyavirus piRNAs are produced from both antigenomic and genomic strands at a more equal ratio . It is cAedes PIWI proteins after gene duplication in combination with somatic expression are likely the main drivers of the expansion of piRNA substrates, including viral RNA. Ae. aegypti Piwi4, Piwi5, Piwi6, and Ago3 are abundantly expressed in somatic tissue of adult mosquitoes [Ae. aegypti Aag2 cells [Piwi5 and Ago3 as the main players for vpiRNA production from Sindbis virus. Piwi5 and Ago3 bind vpiRNAs from opposite strands and with distinct nucleotide biases. Whereas Piwi5 binds 1U-biased antisense piRNAs, Ago3 binds 10A-biased piRNAs derived from the viral sense strand [Functional diversification of squitoes  and Ae. g2 cells . In an Re strand . These oPiwi5 and Ago3—and to a lesser extent, Piwi6—results in reduced vpiRNA production from dengue virus serotype 2 in Aag2 cells [Aedes PIWI proteins have specialized in processing distinct RNA sources. This is further supported by the differential requirement of PIWI proteins for the processing of transposon-derived piRNAs that, in contrast to Sindbis virus–derived piRNAs, directly or indirectly relies on all somatic Aedes PIWI proteins [Knockdown of g2 cells . The addproteins  Fig 3)..Piwi5 anArboviruses establish persistent infections in mosquitoes without causing apparent fitness loss in their vectors, despite high viral load. Such a defense strategy in which high pathogen levels are tolerated and the focus lies on preventing infection-induced damage has been termed tolerance. In contrast, actively restricting virus growth and potentially clearing the infection is a defense strategy called resistance . AlthougPiwi4 in Ae. aegypti Aag2 cells, replication of Semliki Forest virus is strongly enhanced [Piwi5 and Ago3 in Aag2 cells results in profound decline in vpiRNA expression from Sindbis virus and dengue virus, but viral replication is not strongly affected [For example, it was shown that upon knockdown of enhanced . Yet, thenhanced . In lineenhanced . Therefoaffected . WhetherPhlebovirus genus, Bunyaviridae family), vpiRNAs are primarily detected late in infection following a first wave of vsiRNAs. The vpiRNAs vastly outnumber vsiRNAs at 72 hours postinfection [Ae. albopictus mosquitoes with chikungunya virus, yet that population is not seen at three days postinfection [Interestingly, in mosquito cells infected with Rift Valley fever virus  proteins to exogenous target sequences and as such provide heritable immunity against viruses and plasmids . The piRAedes mosquitoes and cell lines, vpiRNAs have thus far not been reported in important blood-feeding mosquito vectors from the Anopheles and Culex genera. The Anopheles gambiae genome encodes, like Drosophila, two orthologs of Piwi/Aub and one copy of Ago3. The Cx. quinquefasciatus PIWI gene family, however, has undergone expansion to seven members [Whereas vpiRNAs can be readily detected in  members ,41.An. gambiae with o’nyong-nyong virus  does not give rise to an abundant population of piRNA-sized small RNAs [Aedes mosquitoes, it would be interesting to investigate whether a ping-pong signature is also present for o’nyong-nyong piRNA-sized reads. This may also provide an explanation for the observed increase of o’nyong-nyong virus upon depletion of Ago3 in An. gambiae mosquitoes [Infection of all RNAs . Yet, insquitoes .Cx. pipiens mosquitoes infected with West Nile virus (WNV) or Usutu virus  did not uncover vpiRNAs, whereas vsiRNAs were readily detected [Cx. pipiens being unable to produce vpiRNAs or the inability of WNV to trigger vpiRNA production is unclear, especially as WNV also failed to induce vpiRNA production in Ae. albopictus C6/36 cells [Aedes cells gives rise to an abundant population of vpiRNAs [Culex mosquitoes  Which mosquito species are capable producing vpiRNAs and which viruses elicit a piRNA response? In relation to these questions, future research should investigate to what extent the piRNA pathway determines vector competence and the specificity of arboviruses for certain mosquito species. (ii) What is the composition of macromolecular complexes required for piRNA production from various RNA sources? It is of particular importance to investigate which PIWI proteins are required for piRNA production from different arboviruses as well as from transposons and other endogenous sources. Also, the contribution of additional proteins to piRNA biogenesis and function warrants investigation. (iii) What is the role of the mosquito piRNA pathway in mediating resistance to and tolerance for arbovirus infections? (iv) What is the contribution of endogenous viral elements to antiviral immunity and immune memory in mosquitoes? (v) Have arboviruses developed strategies to evade or interfere with the piRNA pathway? (vi) How widely do somatic piRNA pathways occur across the tree of life, and has piRNA-mediated gene silencing acquired additional functions beyond transposon control in other animal species? The mosquito piRNA pathway and in particular the production of vpiRNAs shows that the piRNA pathway is much more versatile than previously anticipated. It remains to be seen how many more surprises PIWI proteins have in store when we take a closer look at this fascinating pathway in other species.New small RNA sequencing data have been deposited in NCBI Sequence Read Archive (accession number SRA486748).S1 FigCx. pipiens mosquitoes were infected with 9,660 TCID50 Sindbis virus (pTE 2J 3′GFP) by intrathoracic injection. Two days postinfection, RNA was extracted from the mosquitoes using Isol-RNA lysis reagent. Small RNAs were isolated by gel-electrophoresis, and deep-sequencing libraries were prepared using Illumina’s Truseq small RNA preparation kit. Small RNA libraries were then sequenced on a Illumina Hiseq2500 system and mapped to the Sindbis virus genome. The size distribution of viral small RNAs derived from the sense strand (black) or antisense strand (grey) is depicted for sequencing reads that align to the genome with a maximum of one mismatch in the first 28 nt. The size profile suggests that Cx. pipiens does not produce vpiRNAs, but it cannot be excluded that vpiRNAs are found when using a different route of inoculation, at other time points, or in infections with other viruses. Deep-sequencing data have been deposited in the NCBI Sequence Read Archive under accession number SRA486748.(EPS)Click here for additional data file.


Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase  gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5′ untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5′-proximal codons of CHV1 as well as the 5′ UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in  Cryphonectria parasitica. Viruses identified as IRES positive include hypoviruses , totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5′ untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host,  Eachon codon . The sectrans-acting factors (ITAF) are required. Furthermore, there seem to be another class of IRESs containing unstructured sequences with shorter motifs that may be able to bind ITAF or 18S rRNA (An IRES was first identified in the 5′ untranslated regions (UTRs) of picornaviruses and later in other viruses, mostly positive-sense single-stranded RNA (ssRNA) viruses, as exemplified by the initially identified poliovirus (PV) and encephalomyocarditis virus (EMCV) , 4. A re7–18S rRNA , making 18S rRNA .Totivirus infecting Saccharomyces cerevisiae steal cap structures from cellular mRNAs via the cap-snatching activity of their capsid protein (CP)  viruses, positive-strand (+) and negative-strand (−) ssRNA viruses, and single-stranded DNA (ssDNA) viruses –16. Pred–ein (CP) , 20. Thiiviridae , 22. Howiviridae –25; thesCryphonectria parasitica, a phytopathogenic ascomycete, has been established as a model filamentous fungus for exploring virus-virus and virus-host interactions  and codon-optimized firefly and Renilla luciferase genes   (2 relative luminescence units [RLU]), whereas a relatively high level of background was observed for Renilla luciferase (~104 RLU) . Similarly, ORluc showed greater chemiluminescence than did the original Rluc (~103-fold), indicating that these codon optimizations were highly effective in C. parasitica (Renilla luciferase was negligible because of the high activity of ORluc (4-fold difference). Moreover, a preliminary experiment for the development of the dual-luciferase (DL) assay was carried out using mixtures of homogenates derived from independent OFluc and ORluc transformants and the WT strain  . Ten ind -OFluc) . The nonrasitica . ImportaT strain . Chemiluubstrate , step 3.10.1128/mBio.02350-17.1FIG S1 OFluc gene. The sequence was modified from the original Oluc gene  Sequence of the luc gene  such thaCH-DLst3 , are undCopyright © 2018 Chiba et al.2018Chiba et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ORluc and OFluc for the insertion of IRES candidates and a stop codon triplet for the translation termination of ORluc . Importantly, the difference in OFluc activities between pCH-DLst3 and pCH-DL, a variant without the stop codon triplet for ORluc producing the OFluc-ORluc fusion protein, was great enough to test IRES candidate viral sequences and identify IRESs  gene cassette was inserted between the HindIII and SphI recognition sites of pCPXHY3 to prepare a foundation construct, pCH-DLst3 . The plaof ORluc . It shoukgrounds . Consequfy IRESs .10.1128/mBio.02350-17.2FIG S2 C. parasitica. (A) Construction of pCPXHY3-based foundation vectors. The plasmid pCH-DL has no stop codon for ORluc, while pCH-DLst3 has triple stop codons (asterisks) for ORluc. The downstream MCS and OFluc regions are identical in these clones, and the ORluc and OFluc coding regions are in frame. (B) Comparison of mean chemiluminescence values for pCH-DL variants. C. parasitica strains transformed with two pCH-DL variants were subjected to the DL assay. The mean RLU  are presented. Although a detectable background of the OFluc RLU was recognized in pCH-DLst3, this was 103 lower than that of pCH-DL and small enough to test potential IRES sequences and measure IRES activities (OFluc RLU). Luciferase activities were measured for a nontransformed WT strain as a reference. Download FIG S2, TIF file, 0.7 MB.Toward the development of a dual-luciferase-based IRES detection system in Copyright © 2018 Chiba et al.2018Chiba et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. parasitica  (a chrysovirus), Helminthosporium victoriae virus 145S (HvV145S) (a chrysovirus), and Rosellinia necatrix quadrivirus 1 (RnQV1) (a quadrivirus)  , 30–33. irus) 30–, IRES acirus) 30–. Howeverirus) 30–.C. parasitica spheroplasts were transfected with in vitro-synthesized RNA from representative constructs. First, the optimal conditions were determined with the transcript carrying the single OFluc cistron. The highest OFluc activities were observed when spheroplasts were electroporated at 100 Ω, 2.4 μF, and 0.6 kV or 0.8 kV. These settings were applied to subsequent experiments with representative viral sequences. As shown in A transformation-based assay for IRES activities requires validation because IRES activities detected in such assays may represent those from spliced or degraded transcripts or transcripts from a cryptic promoter with the OFluc ORF situated at the most 5′-proximal end rather than from dicistronic dual-luciferase transcripts , 34. To Another data set for CHV1 and deletion mutant UTRs shown in These combined results with RNA transcripts validate the results shown in Chrysoviridae and Hypoviridae. Also, this study establishes a solid foundation for future mechanistic explorations of required initiation factors, including ITAF and viral cis elements.This study has unambiguously demonstrated IRES activities in the 5′ UTRs of diverse fungal RNA viruses, spanning from (+) RNA viruses to dsRNA viruses. This study represents the first report on IRESs originating from filamentous fungi as well as the first report on IRESs from members of certain virus families with RNA genomes such as S. cerevisiae (S. cerevisiae such as p150 (eIF4G) and TFIID mRNAs were found to be IRES positive (in vitro and/or in vivo tests with bicistronic transforming or transfecting DNA or transfecting RNA are conducted to identify IRESs (http://iresite.org/) (http://bioinfo.net.in/IRESPred/)   and IRESESPred/) . NeverthESPred/) . In factTotiviridae, which also includes filamentous fungus-infecting victoriviruses and yeast-infecting totiviruses. Viral strains of members in the genus Totivirus utilize canonical cap-dependent translation with the cap structures snatched from cellular mRNAs  ssRNA genomes were historically discovered as the biological control agents of chestnut blight caused by the chestnut blight fungus, rasitica . There arasitica , and sevrasitica –48. In te domain . Suzuki e domain –53. AnotAnother interesting observation about hypovirus IRESs was that CHV1 and CHV2 had much higher IRES activities than CHV3. There are a few possibilities to account for this difference. It should be noted first that CHV1 and CHV2 are more closely related to each other in their molecular phylogeny and genome organization than to CHV3. CHV1 and CHV2 have double-ORF genomes while the other one has a single-ORF genome. In the two-ORF-type hypoviruses, replication-related proteins encoded on the 3′-proximal portion of the downstream large ORF A are expected to be expressed at relatively lower levels than the ORF A-encoded proteins, because ORF B is translated by the stop/restart mechanism . This iscis elements. Other IRES-positive RNA viruses not discussed above include chrysoviruses. It will be interesting to investigate possible roles of the CAA repeats in the chrysovirus and partitivirus 5′ UTRs, long presumed to be translation enhancers. There are also many other fungal viruses predicted to employ IRES-mediated translation initiation. Such single-stranded, positive-sense fungal viruses include fusariviruses and yadokariviruses, while such dsRNA viruses include megabirnaviruses, yadonushiviruses, botybirnaviruses, and members of the unclassified dsRNA virus groups such as Phlebiopsis gigantea large virus 1 (PgLV1), Circulifer tenellus virus 1 (CiTV1), and Sclerotinia sclerotiorum nonsegmented virus L (SsNsV-L)  –56. All erfamily . Many oferfamily . ConfirmC. parasitica was used as an experimental platform for reporter assays. To obtain viral sequences, virus-carrying fungal strains were used: C. parasitica strains EP713 (CHV1) (Cryphonectria nitschkei OB5-11 (CnCV1) (Rosellinia necatrix strains W8 (RnPV1) (Helminthosporium victoriae strain A-9 (HvV190S and HvV145S) (The standard strain EP155 of the chestnut blight fungus 3 (CHV1) , NB58 (C3 (CHV1) , and 9B23 (CHV1) , 32; Cry (CnCV1) ; Roselli (RnPV1) , W57 (Rn (RnPV1) , W1029 ( (RnPV1) , and W10 (RnPV1) ; and HelHvV145S) . Full naOluc), designed for optimal expression in the model filamentous ascomycete Neurospora crassa, was kindly provided by J. C. Dunlap (Oluc plasmid by the overlap-PCR method to obtain a modified version of codon-optimized firefly luciferase (OFluc). The Renilla luciferase (Rluc) gene was similarly codon optimized (ORluc) by in vitro nucleotide synthesis and cloned into pUC57-Kan . The original firefly luciferase (Fluc) and Rluc and codon-optimized OFluc and ORluc gene fragments were introduced into an expression vector, pCPXHY3, a derivative of pCPXHY1 (ORluc and OFluc genes were independently PCR amplified and cloned in HindIII and SphI sites of pCPXHY3. A triplet of stop codons (TAATAGTGA) and a multiple cloning site  were designed to be included between 5′-proximal ORluc and 3′-proximal OFluc (pCH-DLst3)   and B. Ain vitro runoff transcription using the RiboMAX kit (Promega).To create the DL transcription vector, pBluescript SK II(+) (pBS) was used. The 3′-terminal sequence of the CHV1 genome containing a poly(A) tail (22 residues) was PCR amplified  and inserted in the MCS of pBS vector in the sense orientation to the T7 promoter. The DL cassette from pCH-DLst3 was transferred to this plasmid by using HindIII and SphI recognition sites (pBS-DLst3). Viral sequences were then inserted in pBS-DLst3 using NotI and BstEII or HpaI. These clones were linearized by SpeI and served as the templates for OFluc and ORluc genes are appended as supplemental material . Oligonucleotide primers used in this study are available upon request. Sequences of rial see .C. parasitica EP155 were performed as described by Faruk et al.  in the same tube. Ten microliters of the homogenate was mixed with 100 μl of firefly or Renilla luciferase substrate solution in a round-bottom tube . Luminescence strength was then measured for 1 to 12 s using a MiniLumat LB 9506 luminometer (Berthold) or a GloMax 20/20 luminometer (Promega). The dual-luciferase reporter assays were conducted by following the manufacturer’s instructions. At least 5 to 10 biological replicates (independent transformants) were analyzed.Protoplast isolation, transformation, and regeneration of k et al. . The lucin vitro runoff transcription using the RiboMAX kit in the presence of the cap analog . In vitro-transcribed DLst3 fragments were introduced into C. parasitica protoplasts  by electroporation at 0.65 kV, 2.5 μF, and 200 Ω . Liquid RG medium was immediately added to the treated cells, which were placed on ice for 10 min before incubation for 4 h on the bench at 27°C in the dark. Cells were centrifuged  and resuspended twice in 1 M sorbitol before final collection and cracking by vortexing in 20 μl of solution . The DL assay was performed as described above with a time count of 12 s. Three independent assays were conducted and statistically analyzed.The dual-luciferase reporter assays with the transfection-based transient expression method were conducted as follows. pBS-based bicistronic constructs were linearized by SpeI and served as the templates for


Starting in 2015, microcephaly associated with Zika virus emerged as a public health emergency of international concern. Initial cases in the United States were travel-associated; however, there are increasing reports of local transmission in pockets of the country, and therefore public concerns may escalate.Fetal microcephaly and other congenital malformations are devastating consequences of Zika virus infection.Ultrasound findings in fetuses with congenital Zika virus infection include microcephaly, intracranial calcifications, ventriculomegaly and arthrogyposis, as well as abnormalities of the corpus callosum, cerebrum/cerebellum and eyes  and 2.4,5While the detection of specific point-of-care fetal ultrasound findings associated with Zika virus disease is beyond the scope of knowledge expected for EPs, any unusual appearance should prompt an assessment of the patient’s risk factors for the infection. This is particularly critical as the vast majority of patients will be asymptomatic. Through knowledge of US findings indicative of Zika-virus related congenital malformations, EPs will be empowered to facilitate timely referrals for concerned or possibly exposed pregnant patients who may otherwise only present to the ED for routine POCUS testing.


Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally “cloned” by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis. Establishing reference genomes are essential for characterisation of pathogens, their interaction with the host, development of vaccines, and responses to therapies including antimicrobial resistance. Betaherpesviruses are a classic example of effects of laboratory passage of viruses distorting understanding of natural transmission. In this case, betaherpesvirus human cytomegalovirus (HCMV) was originally cultured in human fibroblasts, the reference AD169 strain, with derivation of its genome being the largest at the time . SubsequgQ1 and gQ2 genes are in the homologous pathogenicity region to that in HCMV. Polymorphisms in gQ2 have similarly been detected in tissue culture passaged virus as well as in a BACmid clone of human herpesvirus 6A (HHV-6A) strain U1102 [Similar analyses of polymorphisms in the reference human betaherpesvirus 6, HHV-6A, have not yet been conducted. However, isolated reports indicate homologous regions are targeted and may also occur in virus replicated in vivo. HHV-6A also encodes a gH/gL complex central to infectious entry, as well as alternate multimeric complexes affecting cellular tropism; these are a related trimeric complex gH/gL/gO and a quadrameric complex gH/gL/gQ1/gQ2 ,9,10,11.in U1102 . Furtherin U1102 . Polymorin U1102 ,14. In ain U1102 , GG insein U1102 , and nucin U1102 . Two disin U1102 . A systeHuman herpesviruses cause a range of pathologies from oral diseases, incurable genital infections, to cancers. HHV-6A is linked with neurological and cardiac disorders in immune suppressed or naive patients , includiOur studies using deep sequencing showed we could distinguish between known circulating HHV-6A viruses and those that are integrated in our genomes, CiHHV-6A ,24. WithThis methodology was applied here to redefine the Roseolovirus type reference, HHV-6A strain U1102, using an early isolate and to characterise population polymorphisms and minor variants arising from effects of tissue culture passage of virus and in BACmid virus reconstruction. This was done in order to test whether the same mutagenic effects on the virus entry complex as shown for HCMV were also affected in HHV-6A. We further validate these polymorphisms by reference to in vivo virus “cloned” by integration in the germline. This not only defines the reference but also the changes critical to virus spread during cell culture that are relevant to general understanding of infection mechanisms.HHV-6A strain U1102 low passage from the original isolate, P3, was grown in JJhan cells as described . The conThe five virus-infected cell DNA samples were extracted  and CiHHV6A DNAs were prepared for target enrichment amplification and deep sequencing as described. These were HHV-6A U1102, bU1102, BAC-U1102, K21-HHV-6A-pU1102, and K21 BAC-U1102. For the specific amplification, 36 overlapping primer pairs were used which amplified overlapping PCR products across the complete genome as described, with all predicted sizes amplified -mem alignment algorithm by mapping to reference genomes using the HHV-6A U1102 reference genome (updated from denovo assemblies) as described . CoveragThe rapid annotations tool Rapid Annotation Transfer Tool (RATT) was used to transfer annotations and syntenic coordinates from the reference genome  (HHV-6 UComparative models were generated for HCMV gB from strain Merlin (UniProtKB/Swiss-Prot: F5HB53) and for HHV-6A U1102 gB from wt and passaged strains in the post-fusion conformation using Modeller9v7 . The modIllumina deep sequencing was conducted deriving HHV-6A strain U1102 genomes between 2000–10,000 times per group. These included three versions of HHV-6A strain U1102: low passage (P3), continuous cell passage, and BAC reconstructed virus. In addition, sequences were derived for the continuous passage virus (pU1102) and the BAC virus (BAC-U1102). The Miseq run was derived at the same time as resequencing CiHHV6A strains 2284, 5055, and 5814 used to confirm SNPs. Consensus sequences were determined and minor variants tabulated and quantified.The first analyses compared the NCBI Genbank reference genome HHV-6A strain U1102 genome to the deep sequenced early passage of the original isolate of strain U1102. In order to identify corrections to this reference sequence, all polymorphisms subsequently identified in the passaged strains were removed. Newly identified changes were in genes U40, U57 (major capsid protein), U58, and U100 (gQ1 glycoprotein). It is possible these were mixtures of minor variants, below <1%, not detected in the original sequence. Therefore, these were also compared to genomes from cultured passaged virus, as well as from artificial cloned virus genomes using BACmid technology or naturally cloned virus genome present in the germline of people with chromosomally integrated virus genomes, CiHHV6. These were all re-sequenced at the same Illumina run, and none of these genomes showed evidence for these SNPs screened down to 1% sensitivity by deep sequencing.Only five further SNPs were identified from a potential mixed original population. Two were previously described in U83 and gQ2 ,14,27, bHaving established a new reference genome sequence for HHV-6A strain U1102, this was then compared to the whole genome sequence of BACmid cloned virus. This had been cloned as BACmid using a BACmid cassette inserted in-between genes U53 and U54 , then thIn total, 16 SNPs were identified including two indels (one in the U86 immendiate early 2 (IE2) repeat and one in an intron of U100 gQ1). Six of those were non-synonymous changes in structural coding genes U31 tegument, U33 capsid, U39 gB, U50 capsid, U86 IE2, and U100 gQ2 . The SNPThe only major rearrangement observed was at the site of the BAC cassette insertion. This cassette is derived from transfer cassette pHA2  and inclNext, using deep sequencing, the new reference HHV-6A strain U1102 genome sequence was compared to genomes derived from virus, which had been continually passaged (four passages) as infected cells. The results showed that the passaged virus consisted now of an expanded population of minor variants with the now expanded major variants emerging to dominate . InteresAlmost half of the SNPs, 11/24, were shared with the SNPs in the BACmid population of reconstructed virus genomes, therefore, indicating selection for tissue culture. These included the non-synonymous coding changes in U31, U33, U39 gB, U50 capsid, and U100 gQ2, which are mainly structural or envelope genes affecting infectivity. The new SNPs in this population included non-synonymous coding changes in U2, U41 DNA binding protein, U73 origin binding protein, U86 IE2, and three in U95. There were also two SNPs within the origin for replication. Of these, only three have been reported in another strain (GS). This strain is also a mixture from six original patients; two SNPs were reported for GS-KJ123690.1  and two SNPs for GS-KC465951.1 (U50 capsid and the ORI repeat). The other non-coding changes are uniquely reported here, U2, U73 origin binding protein, U86 IE2, and the three in U95, which are mainly non-structural genes affecting replication. These may represent low-level variants present in the original virus isolate expanded here or the effects of adaptation to tissue culture and improved replication. Notably, all the BACmid virus SNP coding changes were present as variants in the continually passaged virus population, identifying these as most likely tissue culture adaptations.Both the BACmid and cultured virus accumulated mutations in envelope glycoproteins of the entry complex, which mediate cellular fusion. This included glycoprotein gB, which has been characterised as a fusogen conserved in herpesvirus and also gQ1 and gQ2 which are part of the gH/gL/gQ1/gQ2 complex. Like gB, the gH/gL complexes are also conserved in herpesviruses, but there are additional trimer or quadrameric versions in Roseolovirus. The gH/gL/gQ1/gQ2 components are specific to Roseoloviruses; this complex interacts with a cellular receptor CD46, and is a target for a neutralising antibody, which inhibits cellular spread ,41,42. TThere are crystal structures available for homologous gB molecules from HCMV, HSV, and EBV  in the post-fusion conformation ,44. UsinThe passage mutation Thr193Ala is unlikely to affect the beta sheet prediction adjacent to FL2 (beta sheet 12 in HCMV domain I), but the reduced polar residues away from FL2 and increased hydrophobicity may promote membrane association via the Domain I structure as the mutation is within the FL2 adjacent beta-strand . HoweverWe used whole genome deep sequencing to re-define the reference genome of the Roseolovirus betaherpesvirus with unprecedented precision and further demonstrated emerging populations of minor variants from the effects of tissue culture passaging of virus and BACmid reconstructed virus. The results have implications for understanding cellular spread as well as clinical interpretation, analyses of drug resistance, and vaccine designs to inhibit virus entry. We analysed the original HHV-6A U1102 isolate in comparison to cell culture adapted virus and cell culture reconstructed BACmid virus. We also compared it to natural “clones” of HHV-6A in the integrated CiHHV-6A genomes we previously derived and re-sequenced here. Specifically, 32 SNP corrections were made to the reference genome sequence, which included nine corrections previously reported  giving aThe passaged stocks were shown comprised of complex polymorphic mixtures, with SNPs averaging 50% mixtures with the wild type reference virus genome. We used screens to the 85 defined coding genes as described for both virus and integrated genomes ,26. TherMixtures and variants of the strains of HHV-6A as well as related HHV-6B have been described previously. In these cases, the dominant SNP created the consensus sequence. The initial virus isolates from patients were not clonal and since the virus is cell associated, the virus strains were not clone purified previously. For example, there are at least two consensus genomes for HHV-6A strain GS  . This BAMH1 restriction enzyme profile most closely resembles that reported for the original isolate ,48. HoweRearrangements in the BACmid may also affect virus replication efficiency. The BAC virus genomes had deletion of the GFP gene. Only the recombined loxP site and associated sequences from the original vectors were maintained in both the parent BAC and the K21 treated BAC virus. Although this could be from homologous recombination mediated by the host, the efficient rearrangement in the entire population suggests a virus gene product is mediating this event. This would confound any selection process dependent on GFP, since mutations or rearrangements would have to occur to reduce this activity. Since GFP expression has been used to characterise genome integration, it is likely that selection for GFP may be artificially enhancing integration as demonstrated ,50,51. AThe SNPs in BACmid reconstructed virus are shared with those in the passaged virus, showing effects of cell culture passage, and all are in structural genes. In comparison, the virus-passaged genomes had only three additional SNPs, all related to replication, in the origin binding protein, and the immediate early genes IE2 and U95 “IE3”. There were five shared structural changes, two in capsid and tegument genes, U31 and U33, with the remaining three in envelope glycoprotein genes affecting virus cellular entry by cell fusion. These included gB and the gH/gL complex components gQ1 and gQ2. These may be culture adapted changes for improved growth in leukemic cell lines JJhan, SupT1, and HSB2, which may affect the selection. In contrast, clinical isolates were prepared on primary cord blood stem cells. The population of BACmid SNPs were in structural genes and the virus envelope and these, including gB Thr193Ala, were all contained in the passaged population of SNPs, which also included SNPs in essential replication genes. Of particular relevance, we showed treatment of BAC virus with a novel antiviral affected membranes, reverted the population to wild type, and eliminated the cell culture passage SNPs in the envelope glycoproteins .In HCMV, cell culture causes changes in the virus entry complex, specifically in components of the pentameric complex of gH/gL, in UL128, UL130, and UL131. In HHV-6A, the genes in the homologous genomic position also encode a fusion complex alternate to the trimeric gH/gL/gO. This is the quadrameric complex of gH/gL including components gQ1 and gQ2. In addition, there is a chemokine gene, a virokine, U83A, which is the positional homologue of UL128, but appears to independently direct cellular recruitment for dissemination or immune evasion. There is polymeric DNA editing polymorphism in this gene disrupting expression, the full-length gene is only rarely identified in culture, but has been identified in “cloned” integrated genomes . Therefo1,2 glycoprotein, which increases virus cellular spread. This is also in the fusion loop adjacent region, a Thr544Ile mutation, which also increases hydrophobicity [HHV-6A gB is closely related to HCMV gB, which enabled modelling of the ectodomain in the post-fusion conformation with high confidence. This demonstrated the mutation, Thr193Ala, was in Domain I, which mediates cell fusion, specifically in proximity to the fusion loop 2 (FL2) adjacent region within a conserved beta sheet. Interestingly, recent studies identified a cell culture induced mutation in the Ebola virus GPhobicity . The modhobicity ,29.The cell culture passage mutation in the FL2 proximal region is within domain I, the site of effects by gH/gL based on experimental results, although no stable intermediate has been identified . In HSV Taken together, our overall studies have redefined the reference genome and identified SNPs associated with cell culture and BAC virus passage, which can affect virus infection by cell fusion as similarly demonstrated in the related betaherpesvirus HCMV. Characterisation of host interactions, genome integration, immune interactions, and antiviral resistance all need to take account of the effects of a diverse mixed genome population present in laboratory-passaged virus and highlight the components of the virus entry complex affecting cellular spread.The reference virus genome was re-defined by deep sequencing of the early clinical isolate. Tissue culture passaged virus accumulated a population of genetic variants in genes affecting infection and replication. Virus culture of BACmid virus accumulated a subset of these SNPs in genes affecting infection, including envelope genes gB and gQ1 and gQ2 of the gH/gL complex. Efforts to study virus properties need to focus on the newly defined reference strain. New BACs or different culturing protocols may be required to do this. Additional study of these cell associated mutations from BAC and passaged virus would provide further understanding of cell associated infection and regulation of virus production versus integration.


Coquillettidia venezuelensis  mosquitoes collected ≈ 30 miles away in the Bush Bush Forest. The urban vector was later identified as the midge Culicoides paraensis, but the sylvatic vector remains unknown. Virus has been isolated from the three-toed sloth, which is believed to be involved in the sylvatic transmission cycle.In September 1955, a virus was isolated from a 24-year-old forest worker from the community of Vega de Oropouche, near the town of Sangre Grande, on the island of Trinidad (country: Trinidad and Tobago), who presented with fever, backache, and cough, which resolved spontaneously after 3 days. The virus was isolated from the patient’s blood by intracranial inoculation of suckling mice at the Trinidad Regional Virus Laboratory. Five years later, the virus was isolated from The virus was shown to be unique but antigenically related to Simbu virus, which had recently been described from South Africa. It therefore became a member of the large family of bunyaviruses.1The virus, Oropouche virus, named in keeping with the tradition of designating arboviruses by using local geographic names, stems from the name of the village, a nearby swamp (wetland), and river. It derives from an Amerindian word, but the ancient meaning of the word is not clear.Oropouche virus has since proven to be one of the most common arthropodborne viruses infecting humans in the tropics of the Western Hemisphere. Clinical signs of infection include headache, myalgia, arthralgia, and chills; no deaths have been reported. It has been estimated to have infected more than half a million persons in Brazil alone, and there have also been large outbreaks in Panama and Peru. In keeping with the recent emergence of other arboviruses such as West Nile, chikungunya, and Zika viruses, Oropouche virus is a candidate for possible further urban spread and therefore warrants increased surveillance and diagnostics.


Three feeding regimens—centrally produced ready-to-use therapeutic food, locally produced ready-to-use therapeutic food, and augmented, energy-dense, home-prepared food—were provided in a community setting for children with severe acute malnutrition (SAM) in the age group of 6–59 months in an individually randomised multicentre trial that enrolled 906 children. Foods, counselling, feeding support and treatment for mild illnesses were provided until recovery or 16 weeks.Costs were estimated for 371 children enrolled in Delhi in a semiurban location after active survey and identification, enrolment, diagnosis and treatment for mild illnesses, and finally treatment with one of the three regimens, both under the research and government setting. Direct costs were estimated for human resources using a price times quantity approach, based on their salaries and average time taken for each activity. The cost per week per child for food, medicines and other consumables was estimated based on the total expenditure over the period and children covered. Indirect costs for programme management including training, transport, non-consumables, infrastructure and equipment were estimated per week per child based on total expenditures for research study and making suitable adjustments for estimations under government setting.No significant difference in costs was found across the three regimens per covered or per treated child. The average cost per treated child in the government setting was estimated at US$56 (<3500 rupees).Home-based management of SAM with a locally produced ready-to-use therapeutic food is feasible, acceptable, affordable and very cost-effective in terms of the disability-adjusted life years saved and gross national income per capita of the country. The treatment of SAM at home needs serious attention and integration into the existing health system, along with actions to prevent SAM.NCT01705769; Pre-results. Evidence suggests community-based therapeutic care was more cost-effective as compared with costs for inpatient treatment.However, controversy prevails over the cost-effectiveness of domiciliary treatment, particularly the ready-to-use therapeutic food component of the management strategy, which is deemed to be expensive and logistically difficult to procure, distribute and sustain.The study provides an estimate of the costs per child treated (US$56) and overall costs required to cover a population with known incidence, for the first time in India for home-based management of uncomplicated SAM.A randomised multicenter trial shows no conclusive evidence to prefer one regimen over the other in terms of costs per child treated.Home based management of SAM requires approximately 20% in administrative costs,10% for the cost of screening and identification, 5% for the cost of peer support, and 60-65% for the cost of treatment.Different level of health system development, types of terrain and and socio-cultural settings impacts the costs of home-based management of uncomplicated SAM.Low costs of treatment of uncomplicated SAM in community based settings suggests shifting the treatment from institutional care to home-based care.Screening and identification can be introduced as a preventive care strategy at primary care settings.India accounts for over half the global burden of severe acute malnutrition (SAM) in the world.It is imperative therefore that adequate evidence is generated on the efficacy of home-based management of uncomplicated SAM and identify the most appropriate diet regimen and management strategy. Domiciliary treatment may be preferred by families over facility care due to reduced opportunity costs.Within this context, we conducted a randomised trial to compare the efficacy of a centrally produced RUTF (RUTF-C) and locally prepared RUTF (RUTF-L) for home-based management of children with uncomplicated SAM on recovery rates compared with micronutrient-enriched (augmented), energy-dense, home-prepared food (A-HPF), which was the comparison group.However, the question arises on the cost of treatment and the scale-up and policy implications for the government. Controversy prevails over the cost-effectiveness of domiciliary treatment, particularly the RUTF component of the management strategy, which is deemed to be expensive and logistically difficult to procure, distribute and sustain.We conducted costing analysis as one of the secondary objectives of the main trialThe main trial was an individually randomised multicentre trial conducted between October 2012 and April 2015, and enrolled 906 children aged 6–59 months with uncomplicated SAM. Foods, counselling and feeding support were provided until recovery or 16 weeks. Outcomes were measured weekly during the treatment phase. In the sustenance phase access to the government nutrition services was facilitated. The primary outcome of the main trial was recovery during treatment phase, weight for height (WFH) ≥−2 SD and absence of oedema in the feet. The trial was conducted in the urban slums and resettlement colonies in the national capital region of Delhi; rural and predominantly tribal areas in Udaipur, Rajasthan; and rural and semiurban areas in Vellore, Tamil Nadu, where the prevalence of SAM is above the national average of 6.4%. Children with WFH <−3 SD of the WHO standard, and/or oedema of both feet, or both were included. Children with complicated SAM were referred to a hospital. Complications included presence of signs of severe illness, lethargic or unconscious, unable to drink or breast feed, vomits everything, convulsions, bulging fontanelle or stiff neck, pneumonia defined as fast breathing, chest indrawing, stridor, crepitations or bronchial breathing on auscultation, diarrhoea with dehydration, severe anaemia defined as haemoglobin <6, and other IMNCI danger signs. The cost of inpatient treatment was excluded from this study. The costs of three feeding regimens—RUTF-C, RUTF-L and A-HPF—were determined for each activity undertaken for enrolled children only under the treatment phase for the Delhi site. For Udaipur and Vellore sites, detailed costs were not calculated and only the key differences were identified. Detailed methods, primary and secondary outcomes, sample size estimation, randomisation, allocation concealment, blinding, and statistical analyses for primary and secondary outcomes with additional analyses are described in the main paper.Survey and identification were undertaken in the community setting by Accredited Social Health Activist (ASHA)-like workers (ALW) in a research setting. The activities included moving from house to house, confirming date of birth, filling in the register, measuring mid-upper arm circumference (MUAC) following written informed consent, filling in and recording the data, providing Integrated Management of Neonatal and Childhood Illness (IMNCI) guidelines to children with MUAC >13 cm or for families who refuse treatment with MUAC <13 cm, and escorting children with MUAC <13 cm to clinics.Screening and enrolment were done at the community clinic, which would be equivalent to a primary healthcare centre in the government setting. Children were screened for SAM using the WFH criteria by the outcome measurement (OM) worker in a research setting, who is equivalent to the auxiliary nurse midwife (ANM) in the government setting. The physician/nutritionist (ANM in the government setting) administered the HemoCue and appetite tests and treated children for minor ailments. Additional research activities included obtaining written informed consents, obtaining mothers’ weight and height, escorting the child to the hospital and randomising the child to one of the three study regimens. In Delhi, 371 children were enrolled in the study and were randomised into one of the three feeding regimens, 124 each under RUTF-C and RUTF- L and 123 under A-HPF.Physicians or nurse managed minor illnesses. Cointerventions were administered; these were amoxicillin for 5 days for all children enrolled, mebendazole for children over 2 years for 3 days, and two bottles of iron to be consumed over 60 days for children over 2 years in the A-HPF group, with anaemia. Nutritionists counselled about the regimens, what to feed, how to feed, good hygiene and breastfeeding practices. In the research setting, home visits were also made for counselling, which may not happen in the government setting.During the first week, children randomised to one of the three regimens were provided specific food according to their weight, and the mothers were counselled on feeding by the nutritionists. Additional research activities included home visits to observe feeding, taking the dietary recall, filling in the baseline form, taking consent for assisted feeding and introducing peer counsellors to the families by nutritionists or ALW follow-up workers (ALW-FU). Under A-HPF it also included demonstrating recipes, and providing milk and egg vouchers.Weekly management under the three regimens for weeks 2–16 included recording weight, height and MUAC; checking oedema in the feet and skinfold thickness by the OM staff (ANM in the government setting); replenishing supplies by storekeeper, which would be equivalent to a pharmacist in the government setting; and counselling by nutritionists, who are equivalent to ANMs in the government setting. Additional research activities included travel to the house of the child for anthropometric measurements and visit by the ALW-FU worker to take dietary recall, observe feeding, collect used jars and replenish supplies.During weeks 2–16, children with illnesses were referred to the study clinic and treated by the study physician. For the research component of costing, the costs of visiting the households and costs of consultation over the phone were included. It also included emergency treatment and referral if required, assessment on completion of the treatment, and introducing to the Anganwadi worker.Costs for supervised feeding included costs paid to peer supporters and telephone reimbursement. The cost was incurred over 14 months for 182 enrolled children as this activity was initiated later into the study.Data on the number of covered and treated children for each activity were collected. Of the 48 634 covered children aged 6–59 months identified during survey, 371 children with SAM were treated. The number of children screened is higher than those identified under risk during survey as there were walk-ins as well.http://www.xe.com/currencyconverter/).Direct costs were estimated per week per child for human resources and consumables in rupees for the treatment under three regimens. The average exchange rate (US$1=62 rupees) of 2013 and 2014 was used to convert all the costs into dollars  per child.The average cost per week per child for the RUTF-C regimen (including eeZee Paste and containers given to 124 children over 25 months) was estimated at US$0.44 ((357110×12)/(25×52×124)=26.6 rupees) under research activity and US$0.40 (24.25 rupees) under the government programme (assuming no costs for containers).The costs of the RUTF-L regimen were calculated by adding the (1) costs of the consumables, (2) annualised costs (depreciated over 5 years) of equipment to prepare the paste and (3) costs of two staff for 27 months who prepared the paste and packaged them. Costs per week per child were estimated at US$1.4 (84 rupees) under research activity and US$0.7 (43 rupees) under the government programme (assuming lower costs of containers and staff).The costs per week per child under A-HPF, calculated by adding the costs of raw materials, Riconia LP bottle and jars provided to 123 children over 25 months, were estimated at US$0.9 (770 860×12/(25×52×123)= 57.9 rupees) under research and US$0.9 (56.8 rupees) under the government programme (assuming that the jars and utensils were not included).During the treatment phase, the costs of medicines were calculated for 1328 children and estimated at US$0.03 (1.85 rupees) per child per week for diagnosis and treatment. Further, under A-HPF, besides the regimen, iron supplementation was also given after the first 2 weeks of treatment to 102 children, and the cost per child per week was estimated at US$0.2 (14.32 rupees).Under the research programme US$11 (700 rupees) was planned per week for each peer supporter who was identified from the community to support the mother in feeding the child. The costs per week per child incurred for 182 children over 14 months were US$2.2 132 rupees) per treated child per week and US$0.01 (0.5 rupees) per covered child per week. Under the government programme, ASHA is given an incentive of US$2.5 (150 rupees) for each child with SAM treated or reaching an MUAC of 125 mm.2 rupees Personnel for administration, management and data support activities, which were computed from full-time equivalent (FTE) (based on the number of staff and the percentage of time each staff devoted) for the staff such as managers, supervisors, quality control officers, office staff and attendants for administration and management. The total administrative costs were estimated at US$11063 (686 000 rupees) for research and US$1246 (77300 rupees) for government programme. Government costs have lower salaries for the staff and lower estimated FTE.Training in MUAC measurement, field support, counselling and standardisation of anthropometry equipment. The total training costs were estimated at US$3717 (230 500 rupees) for research and US$1083 (67 200 rupees) for government programme .Transport, which included costs for one vehicle for the referred patients and for the survey team under research. For the government programme these costs were calculated based on 102 children who needed referral transport. It was assumed that each of these children was paid US$5 (300 rupees) . The total transport costs were estimated at US$4107 (254 700 rupees) for research and US$493 (30 600 rupees) for government programme.Stationary and computers included office supplies, field supplies, printing and computer supplies. Over 80% of these costs were considered for purely research purposes (not for programme activities) and not included. The total costs were estimated at US$3193 (198 000 rupees) for research and US$422 (26 100 rupees) for government programme (excluding computer supplies).Non-consumable goods, which included mainly anthropometry equipment, were calculated using annual 20% depreciation. The costs under both research and government programme were estimated at US$1330 (82 500 rupees).Infrastructure and equipment. Only 5% of the rental for the field office including security, water, electricity and office maintenance/repair costs were included under the research programme and were estimated at US$2344 (145 400 rupees). For the government programme these costs were considered to be a part of the health system.Communication, which included the costs of phones and net books for data collection, were calculated after depreciating the total expenditures for equipment at 33%. Only 40% of the total costs were included under the research programme and were estimated at US$1145 (71016 rupees). For government no expenditure was added under this head.Indirect costs such as administrative and programme costs cannot be allocated for specific child but are required for overall implementation of the programme. These costs were incurred over 33 months on the following:The programme management, administrative, training and operations costs were estimated at US$26935 (1670 000 rupees) for the research and US$4564 (283 000 rupees) for government programme over a period of 33 months. The costs were allocated equally over all the children covered and treated under the programme and were estimated at US$0.6 (34.3 rupees) for research and US$0.1 (5.8 rupees) for government per covered child per week, and at US$72 (4496 rupees) and US$12 (765 rupees) for research and government programme, respectively, per treated child per week.The average recovery rate for each of the three regimens was calculated by taking the weighted average of the number of weeks taken for child to recover where weights were the number of children recovered under different weeks of follow-up - from week one to week 16. The average weeks of treatment under the three regimens were 10.3 weeks for RUTF-C, 9.6 weeks for RUTF-L and 11 weeks for A-HPF.The baseline characteristics of enrolled children, the results of the primary and secondary outcomes, and additional analyses are described in the main paper.During week 1, the costs under each regimen included the cost of survey and identification, screening and enrolment, management through cointervention and counselling at time of enrolment, and management of each of the regimens. During the treatment phase, costs per week included diagnosis and treatment and weekly follow-up, including measurement of length/height and weight to ascertain recovery of children under each of the three regimens. We find the cost per week per covered child was lowest for RUTF-C and highest for A-HPF under the government programme. Under the research activity, during the first week A-HPF costs were higher than RUTF-L and RUTF-C, as more time was spent on demonstration of recipes, providing the feed and setting up the system of milk and egg vouchers with local persons. During weeks 2–16, the RUTF-L cost per week was higher under the research activity, as the cost of RUTF-L preparation was higher. Around US$66 (4100 rupees) was spent during week 1 per treated child under research activity, and between US$9 (550 rupees) and US$10 (600 rupees) under the government programme.The total costs for covered and treated children were derived based on the average number of weeks that a given regimen was required for the treatment. These were estimated at 10.3, 9.6 and 11 weeks for RUTF-C, RUTF-L and A-HPF, respectively, as shown in The average costs per treated child per episode under research were estimated between US$227 and US$238 (14 063–14 775 rupees) for each of the three regimens. Out of the total costs per treated child, about 30% of the costs were for administration under research activity. For the government programme, the costs per treated child were between US$53 and US$61 (3307–3797 rupees) for each of the three regimens, with administrative costs at about 20% of the total costs. The research costs were higher due to higher salaries, more staff being used for the same activity and also more activities being included under those costs such as escorts to the hospital or taking informed consent. The total costs for treating 371 children were estimated at US$85 876 (5324 000 rupees) for research programme and US$20 892 (1295 000 rupees) for the government programme.et al.Our results show that the government will require just about US$0.4 (25 rupees) per covered child in the defined population or under US$61 (3782 rupees) per treated child per episode for children with SAM. Under the research activity with higher costs of human resources, training and administration, the costs of treatment per child with SAM in community setting were under US$ 238. The results of our analysis compare well with that found in other studies. In African studies the cost of ambulatory community-based treatment of SAM ranged between US$46 and US$ 453 per child.There is no conclusive evidence to prefer one regimen over the other. The costs of treating with RUTF-C and RUTF-L are almost similar at US$0.4 (23 rupees) per child covered or US$53 for RUTF-C and US$54 for RUTF-L (~3300 rupees) per child treated. The number of weeks of treatment is lowest for RUTF-L, so even with a higher cost of regimen per week per child, the costs for RUTF-L are lowest per covered child. However, the lower number of weeks for RUTF-L does not outweigh the higher regimen costs per week for the treated child, and therefore RUTF-L remains marginally higher than RUTF-C per treated child. In the A-HPF group, food was supplied at 1.5 times the assumed requirements as well as fuel. This also led to the highest cost (US$61) of the A-HPF regimen. This household food sharing can be considered as an additional benefit for a distressed family, leading to 15% higher costs per treated child under A-HPF. The administrative cost under the government programme is approximately 20%, the cost of survey and identification is 10%, the cost of peer support is about 5%, and the cost of treatment varies from 60% to 65% for the three regimens.Under research activity, the cost per covered child was estimated at US$1.6, and the cost per treated child per episode of treatment was US$227 for RUTF-C, US$229 for RUTF-L and US$238 for A-HPF. Out of these approximately 26%–27% were spent for identification and enrolment, approximately 30%–34% each for treatment and administration, and 9%–10% for peer support.The total cost for all the 371 treated children under the research programme was estimated at approximately US$85 876 (5324 000 rupees) and almost a quarter at US$20 892 (1295 000 rupees) under the government programme. The administrative costs were approximately 30% in research and around 20% under the government programme.We did not conduct a full economic analysis or cost-effectiveness analysis because the purpose of the costing analysis was to inform the government about the cost of domiciliary treatment of SAM with the aim of scaling it up. However, we have taken the best possible robust assumptions to derive at the research and government expenses. The strength of the study is in its randomised controlled design, the rigour of implementation of the study, training and standardisation of the staff collecting the costing data, and maintaining specific minute details while tracking costs. The human resource costs have been derived using a time motion type of study, which is more difficult to estimate in a government setting, where the providers are engaged in more than one activity. Further, the costs derived take into account all programmatic and administrative costs, which are more difficult to allocate in the government setting. The limitation of the analysis lies in the extrapolation of research costs to government costs. Ideally, implementing in a government setting would provide a more precise cost estimate.Second, based on discussions and observations from field visits in Udaipur, Rajasthan and Vellore, Tamil Nadu, we found the costs of interventions identified above varied according to geographical, cultural and programme setting. The differences that would affect the costs were primarily observed in terms of human resource cost and administrative costs to run the programme.In Udaipur, because of its rural, hilly and widespread (houses away from access roads) terrain, higher transport costs were incurred in actively finding the children with SAM, transporting supplies, and bringing the children to the clinic, further follow-up and referrals. Four-wheel vehicles had to be used to carry the supplies and equipment for weekly follow-up, and long waiting time or several visits had to be made due to unavailability of parents during a visit (as they would be working on fields). Even though salaries are lower in Udaipur, longer time taken by human resources led to higher human resource cost. Larger number of monitoring visits had to be made and ALWs were incentivised based on recoveries. Further the supplies such as ultra-high temperature pasteurised milk had to be arranged from the cities due to unavailability in villages for caste reasons. Connectivity problems led to use of paper forms instead of tablets for recording in Delhi. More intake of contaminated water with RUTF-L and RUTF-C led to diarrhoea in the first fortnight. Families did not tell the staff visiting about diarrhoea to avoid going to clinic. Doctors did not do telephone consultation. Home management was low and referrals were very high. Families were brought to clinics in groups because of large distances. Once brought to clinics, children had to be treated for other morbidities also for ethical reasons, which led to higher costs. Many cases required both home management and hospital treatment. Keeping feeding assistants was a problem due to social reasons.In Vellore, a semiurban area, the costs seem to be lower. Due to lower SAM prevalence, 55 000 babies were screened to find 282 children with SAM, of whom 252 were enrolled. The lower costs were due to lower salaries in Vellore and lower time taken for different activities. Most deliveries were institutional deliveries and people maintained good record of immunisation and age cards. The distance travelled was from 0.5 km to 35 km, much shorter than in Udaipur. Activities were streamlined and also a nurse was used for several activities, instead of a physician. The costs of RUTF-L preparation and medicines used also seemed to be lower. Medicine procurement was done from internal pharmacy, and mostly generic drugs were dispensed by the pharmacist from the central medical store. The total expenditure on medicine was US$2453 (152 061 rupees) in Vellore compared with US$4285 (265 699 rupees) in Delhi over the same period of 25 months. The costs of RUTF-L worked out lower at approximately US$0.8 (50 rupees) per child per week as compared with US$1.4 (84 rupees) estimated in Delhi for research activity. Home visits and phone consultation were not done and families visited the clinics themselves. Vellore seemed more similar to the government setting, and therefore the costs derived for the government setting in Delhi are likely to be the costs for running the programme efficiently.Our study provides evidence that home-based management of SAM with an RUTF-L is feasible, acceptable, affordable and efficacious. The average costs per treated child in the government setting were estimated at US$56 (~3500 rupees), which can be considered very cost-effective in terms of the DALY saved and GNI per capita of the country. The treatment of SAM at home needs serious attention and integration into the existing health system, along with actions to prevent SAM.


Diabrotica virgifera virgifera. The assembled genome has 13,182 nucleotides with a 3′ polyadenylated tail. Two open reading frames are predicted to encode polyproteins of 2,838 and 1,073 amino acids.The genome sequence of a novel small RNA virus was assembled from the transcriptome of the western corn rootworm,  Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), causes significant yield losses in Zea mays L. in North America and Europe. In efforts to devise novel virus-based management approaches for this pest, we investigated the viral pathogens of WCR. Adult and larval WCR were collected from the United States and Europe, and RNA was isolated from ~100 whole insects per location, or from viruses purified from the WCR samples as described previously , an iflavirus (Diabrotica virgifera virgifera virus 2 (DvvV2), an unclassified novel ssRNA virus (Western corn rootworm (WCR), eviously  and annoeviously  against NA virus . Here, wDiabrotica virgifera virgifera virus 3 (DvvV3), has 13,182 nucleotides (nt), a polyA tail, and is AT-rich (66.24% AT). The genome contains two open reading frames (ORFs), ORF1 from nt 676 to 9189 and ORF2 from nt 9235 to 12453, as well as a 674-nt 5′ untranslated region (UTR) and 726-nt 3′ UTR. The intergenic region (IGR) is only 45 nt in length. The 5′ end rapid amplification of cDNA ends  did not result in identification of an additional sequence. The majority of the DvvV3 sequence was confirmed by reverse transcription-PCR using SuperScript Ш . Sequences of DvvV3 were detected in field-collected samples from the United States and Europe, and in the transcriptome of an inbred, nondiapausing laboratory culture of Diabrotica virgifera virgifera maintained at the USDA-ARS North Central Agricultural Research Laboratory, Brookings, South Dakota, USA.The genome of this virus, tentatively named Rosy apple aphid virus    between KY200663.The DvvV3 genome sequence was deposited in GenBank under accession number


Agrobacterium-mediated transformation in Nicotiana benthamiana, albeit with a significant delay compared to the wild type clone. The mutants also had a common spontaneous E96K mutation in the coat protein. These results indicated that the initial assembly site and the sequence of the adjacent region affected the infectivity of the virus and that E96 might play an essential role in this process. We constructed two single point mutants—E96A and E96K—and three double mutants—V94A-E96K, V97-E96K and T104A-E96K—to further understand the role of E96 in CGMMV pathogenesis. After inoculation in N. benthamiana, E96A showed delayed systemic symptoms, but the E96K and three double mutants exhibited typical symptoms of mosaic at seven days post-infection. Then, sap from CGMMV-infected N. benthamiana leaves was mechanically inoculated on watermelon plants. We confirmed that E96 affected CGMMV infection using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and sequencing, which further confirmed the successful infection of the related mutants, and that E96K can compensate the effect of the V94, V97, and T104 mutations on virus infectivity. In addition, Northern blotting showed that the accumulation of viral RNA corroborated the severity of the symptoms.Cucumber green mottle mosaic virus (CGMMV) is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein), followed by the construction of the V94A and T104A mutants. We observed that these three mutations caused mosaic after  Tobamovirus, is a devastating virus infecting plants of the family Cucurbitaceae. It was first reported as cucumber virus 3 by Ainsworth in 1935 [Chenopodium amaranticolor and/or Datura stramonium and cause mottling and mosaic in tobacco [Cucumber green mottle mosaic virus (CGMMV), a member of the genus  in 1935 . CGMMV i tobacco . The typ tobacco ,7. No in tobacco . CGMMV o tobacco . Thus, CBiological measures, seed treatment ,12, moleTobamovirus CP identifies the assembly initiation site located in the genomic RNA in the initial steps of virion assembly, followed by virus rod elongation in the 3′ and 5′ direction [The single-stranded, positive-sense RNA genome of CGMMV contains four open reading frames encoding 186, 129, 29, and 17.3 K proteins. The 186 K protein is the read-through product of the 129 K protein, both of which are viral replicases. The other two proteins correspond to the movement protein (MP) and coat protein (CP) . It is wirection . The assirection . TherefoCurrently, reports regarding the relationship of assembly initiation site with virus infectivity are limited. In this study, we mutated the assembly initiation site on the infectious cDNA clone of CGMMV to analyze this association. Results showed that the assembly initiation site and its adjacent region and the 96th amino acid of CP affected CGMMV infectivity.Nicotiana benthamiana) was used as the host plant in Agrobacterium-mediated infiltration of this study. Healthy or inoculated N. benthamiana plants were incubated in a growth chamber at 28 °C and under a 16 h light/10 h dark cycle. The watermelon (Citrullus lanatus) strain Zhengkang No. 2 was subjected to sap-mechanical inoculation. Escherichia coli strain TOP10 and Agrobacterium tumefaciens GV3101 stocks from our laboratory were used. Commercially available antibodies of CGMMV were obtained from ADGEN  for double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Roche’s  DIG northern starter kit and Hybond-N membranes from Amersham Biosciences  were used for Northern blot.Tobacco (All mutants in this study were constructed by site-directed mutagenesis and direct polymerase chain reaction (PCR) according to the instructions of the Fast Mutagenesis System . The detailed procedure of the construction of the CGMMV clones has been described in previous study . The wilA. tumefaciens strain GV3101 by the freeze–thaw method. Agroinoculation of N. benthamiana plants (6–8 leaf stage) was performed by infiltration of the top two or three fully-expanded leaves of 30-day old plants. Bacteria bearing the wild type (C284R) gene were inoculated as a positive control at the same time. For every inoculum, 12 individual N. benthamiana plants were inoculated and assayed thrice. The mutated plasmids were transferred into N. benthamiana leaves. N. benthamiana leaves infected with the mutant virus were ground in 0.01 M phosphate buffer (pH 7.4). Fine silicon dioxide powder was dusted on both cotyledons before mechanical inoculation and at the 1–2 fully expanded leaf stage. For every inoculum, six individual watermelon plants were mechanically inoculated. After inoculation, the plants were grown in a growth chamber at 26 °C with a 14 h light/10 h dark photoperiod.Watermelon leaves were mechanically inoculated with the sap of CGMMV-infected N. benthamiana and watermelon using the Trizol  reagent according to the manufacturer’s instructions. A pair of primers  were designed to synthesize the probe for detecting the CGMMV RNA. The probe was complementary to 6212–6423 nucleotides of the CGMMV genomic RNA. Then, the hybridization signal was imaged using the chemiluminescence imaging system . The instructions of the DIG Northern Starter kit (Roche) were followed for northern blotting. MP-SP (ATGTCTCTAAGTAAGGTGTCAG) and 3’UTR-AP (TGGGCCCCTACCCGGGGAA) were used as primers for RT-PCR (~1.4 Kb) according to manufacturer’s recommended procedure . The mutated sequences were confirmed by sequencing.CGMMV accumulation was determined using DAS-ELISA at 7, 14, 21 days post-inoculation (dpi) according to the method of Liming et al. . SimilarN. benthamiana. Plants inoculated with the WT infectious cDNA clone were the first to show mosaic symptoms at 7 dpi, which were prominent at 10 dpi. In contrast, no symptoms were observed on the upper uninoculated leaves of N. benthamiana inoculated with mutants V94A, V97, and T104A till 14 dpi or later , had an Asp-Val change at amino acid 19 which induced a yellow mosaic phenotype in N. tabacum instead of the light or dark green color [N. sylvestris [N. benthamiana and watermelon, and that these mutations affected the infectivity of CGMMV.On the basis of their ability to form ordered structures that encapsulate the viral nucleic acid, plant virus CPs possess multiple functions such as replication, virion formation, cell-to-cell movement, vector transmission, genome activation, and symptom modulation . The CP en color . A pointlvestris . Substitlvestris . SimilarN. benthamiana. Mutations of these CP residues are associated with virus infectivity and occurrence of host symptoms. These mutations in the gene encoding CP altered the sequence of both the RNA and amino acids of CP. However, we could not conclude whether these changes altered the pathogenesis of the virus. The initiation site for reconstitution of a watermelon strain of CGMMV was located approximately 320 nucleotides away from the 3′ terminus, and was within the CP cistron [To investigate the role of residue E96 in CGMMV pathogenicity, we established three double mutants in the background of the E96K mutation using the infectious cDNA clones of V94A, V97, and T104A. Subsequent experiments using an agroinfiltration assay and mechanical inoculation suggested that E96 was important for infecting  cistron . Further cistron ,27. Henc cistron . TherefoIn a previous study, a series of TMV mutants with insertions and deletions in the CP allowed cell-to-cell movement but destroyed the assembly function and limited the systemic spread of the virus . All ino−5 and 1.3 × 10−3 substitutions per site per year, which are similar to those seen in a broad spectrum of animal and plant RNA viruses [The occurrence of a spontaneous and non-anticipatory mutation in three different mutants is unlikely to be a random event. In our previous research, amino acid residue 284 of the replicase reversed Cys to Arg after inoculation. The reversions to wild type may play major roles in the stability of mutants . Accordi viruses . Amidst  viruses . The occ