Badnavirus) are non-enveloped bacilliform DNA viruses with a monopartite genome containing about 7.2 to 9.2 kb of dsDNA with three to seven open reading frames. They are transmitted by mealybugs and a few species by aphids in a semi-persistent manner. They are one of the most important plant virus groups and have emerged as serious pathogens affecting the cultivation of several horticultural crops in the tropics, especially banana, black pepper, cocoa, citrus, sugarcane, taro, and yam. Some badnaviruses are also known as endogenous viruses integrated into their host genomes and a few such endogenous viruses can be awakened, e.g., through abiotic stress, giving rise to infective episomal forms. The presence of endogenous badnaviruses poses a new challenge for the fool-proof diagnosis, taxonomy, and management of the diseases. The present review aims to highlight emerging disease problems, virus characteristics, transmission, and diagnosis of badnaviruses.Badnaviruses (Family: Caulimoviridae; Genus:  Caulimoviridae) contain eight genera with two distinct virion morphologies: Caulimovirus (10 species), Soymovirus (one species), Solendovirus (two species), Cavemovirus (two species), Petuvirus (one species), and Rosadnavirus (one species) have isometric particles, whereas members of Badnavirus (32 species) and Tungrovirus (one species) have bacilliform ones. All pararetroviruses contain a double-stranded DNA genome and replicate through an RNA intermediate, like retroviruses. However, in contrast to those, pararetrovirus genome integration is not part of their standard replication cycle. Instead, they accumulate as minichromosomes in the host nucleus. Illegitimate and usually fragmented pararetroviral integration occurs in different plants, as reported (Tabac TVCV-like) [SotuEPRV) [LycEPRV) [et al. [Florendovirus, members of which have colonized the genomes of large diversity of flowering plants including apple, citrus, cacao, grape, cassava, rice, potato, maize, papaya, soybean, tomato, etc. Integration occurs on average every million years [Plant pararetroviruses (Family: imovirus 0 speciesnavirus 3 species CV-like) , (potatootuEPRV)  and (tomLycEPRV) . Recentl [et al.  describeon years .in vitro tissue culture process [Badnaviruses, the plant pararteroviruses of this review, infect a broad range of economically important crop plants all over the world ,10,11. T process ,16 and i process . The preCommelina yellow mottle virus (ComYMV), Kalanchoe top-spotting virus (KTSV), Piper yellow mottle virus (PYMoV), Cacao swollen shoot virus (CSSV), and Taro bacilliform virus (TaBV), are also known to be seed-transmitted (Rubus yellow net virus (RYNV), Gooseberry vein banding associated virus (GVBaV), and Spiraea yellow leaf spot virus (SYLSV) are transmitted semi-persistently by aphids. The first report of the occurrence of seed transmission (up to 40%) was in KTSV [Badnaviruses are known to infect both monocots and dicots, though most of the species have a limited host range. In general, symptoms caused by badnaviruses are variable depending on the host, its cultivars, virus species, and environmental conditions. In most cases symptoms are mild to moderate. They include chlorotic mottle or necrotic streaks, deformation of leaves, and reduced internode length leading to stunting of plants . Asymptonsmitted . The sec in KTSV . In CSSV in KTSV . Similar in KTSV ,21 and o in KTSV .Badnaviruses are distributed in tropical and temperate regions of Africa, Asia, Australia, Europe, and South and North America . The majCauliflower mosaic virus (CaMV) (met and of (+) sense DNA by purine-rich cleavage products left after RNase H digestion of the pregenomic RNA [Replication of badnaviruses is thought to be similar to that of s (CaMV) . The gens (CaMV)  includesomic RNA ,23.The ORF I of badnaviruses contain 399 to 927 bp depending on the species. In the type member ComYMV, ORF I is 602 bp long and translates into a 23 kD polypeptide, which was shown to be virion-associated . The ORFSome badnaviruses have additional ORFs: PYMoV, Bougainvillea spectabilis chlorotic vein banding virus(BsCVBV), Dioscorea bacilliform SN virus (DBSNV), Grapevine Roditis leaf discoloration-associated virus (GRLDaV), TaBV, and Yacon necrotic mottle virus (YNMoV) have one ,33,34,35Rice tungro bacilliform virus (RTBV) it was shown that some scanning ribosomes initiate translation at those weak start codons, while others pass them by leaky scanning, finally initiating translation at ORFs II and III [Most badnavirus RNAs include three ORFs positioned solely on the sense strand, and encoding proteins of about 23, 15, and 216 kD . How are and III .Banana streak virus (BSVs) and Sugarcane bacilliform virus (SCBVs) of clade 3 published [Gooseberry vein banding associated virus (GVBaV), RYNV, Grapevine vein clearing virus (GVCV), BsCVBV, YNMoV, DrMV, and Pelargonium vein banding virus (PVBV). The subgroup 1c comprised all the BSV species of clade 1 as per [Banana streak GF virus (BSGFV) were grouped together in subgroup 1d. The other members of subgroup 1c are Pineapple bacilliform CO virus (PBCOV), Mulberry badnavirus 1 (MBV-1), and Cycad necrotic leafspot virus (CyNLV). Group 2 was further divided into two subgroups. Sub-group 2a consisted of two species, viz. PYMoV and TaBCHV, and subgroup 2b consisted of members of BSVs, CSSV, and CYMV as reported in clade 2 of the previous study [Fig badnavirus 1 (FBV-1) and GRLDaV.At present, 70 complete genome sequences representing 28 recognized species and 20 complete genome sequences representing 16 putative species are available in the GenBank database . CSSV haublished ; and sub1 as per . In contus study . The othCavemovirus, Caulimovirus, Petuvirus, Solendovirus, and Tungrovirus, are also present as integrated fragmented sequences in some host plant genomes. These are referred to as endogenous pararetroviruses (EPRVs) [Florendovirus under the Caulimoviridae with 34 distinct species existing only as endogenous viral sequences, was reported [in situ hybridization of labeled viral probes to the chromosome, Southern hybridization of the viral probe to the digested DNA of the host, immunocapture (IC)-PCR, rolling circle amplification (RCA), and finally plant genome sequencing have been employed by different workers to differentiate episomal and integrated forms of the virus. There are two forms of integrants: those that can form episomal viral infections and those that so far cannot. Integrants of three badnaviruses, Banana streak OL virus (BSOLV), BSGFV, and Banana streak IM virus (BSIMV) [Tobacco vein clearing virus (genus: Cavemovirus) [Petunia vein clearing virus (genus: Petuvirus) [de novo infective particles possibly through a two-step intra-strand homologous recombination [Badnaviruses and other pararetroviruses, such as  (EPRVs) ,13,47,48 (BSIMV) , as wellmovirus)  and Petutuvirus) , are knobination , therebybination ,53. Intebination ,56,57,58The presence of endogenous viruses complicates approaches needed for diagnosis, taxonomy, safe movement of germplasm, and management of badnavirus diseases. Some endogenous viruses may just serve as components of plant genomes, while others could provide viral immunity ,53,59.et al. [The symptoms caused by badnavirus infections are variable, and transmission to indicator plants is difficult due to the narrow host range of these viruses . Visual et al.  reportedet al. ,65,66,67et al. ,69,70.in situ hybridizations, and complete sequencing of the virus genome may be required to elucidate the precise identification of the type of sequences present.As mentioned above, endogenous badnaviruses pose a serious challenge to diagnostic testing. An assay that will detect episomal virus sequences may also detect integrated sequences due to the similarity in the sequences used to design primers. Thus to allow detection of episomal virus DNA alone, IC-PCR or multiplex immuno-capture polymerase chain reaction (IC-PCR) was used ,71,72. RBanana streak virus (BSV), Cocoa swollen shoot virus (CSSV), Citrus yellow mosaic virus (CYMV), Dioscorea bacilliform virus (DBV), Piper yellow mottle virus (PYMoV), Sugarcane bacilliform virus (SCBV), and Taro bacilliform virus (TaBV). The International Committee on Taxonomy of Viruses (ICTV) [The genus contains several viruses of economic importance due to the severe diseases they cause: s (ICTV)  listed 3s (ICTV) .Banana streak virus (BSV) species. Since its first report in Morocco in 1974, BSV has been reported from numerous countries in Africa, South America, and the Pacific and probably occurs worldwide wherever bananas are grown [cv. Virupakshi, AAB) plants kept at 22 °C in an insect-proof glass house for six months to one year developed streak symptoms and BSGFV infection was confirmed by RCA and sequencing [M. acuminata × M. balbisiana genotypes, including many newly created hybrids, showed BSV-infected propagates from originally virus-free source plants propagated by tissue culture, probably generated from endogenous sequences [Banana streak disease of bananas and plantains worldwide is caused by a collection of re grown ,80,81. Sre grown ,83. Pottquencing . BSV infquencing . The disequences , and seePlanococcus citri is the most prevalent, and vertically by mass-micropropagation—the main way to propagate banana plants or suckers.BSV is horizontally transmitted to bananas in a semi-persistent manner by mealybugs, of which Banana streak OL virus). This was followed by complete genome sequencing of two Australian species, namely, BSMYV (Banana streak MY virus) and BSGFV (Banana streak GF virus) [et al. [Banana streak VN virus (BSVNV) Banana streak IM virus (BSIMV), and Banana streak Yunnan virus, have been reported [et al. [Musa endogenous badnaviruses without episomal counterparts (see below); and clade 3 consists of all BSV species from Uganda except BSUAV [Though initially all referred to as BSV , many diF virus) ,87. Harp [et al. ,89 reporreported ,90. Jame [et al.  proposed [et al. , namely, [et al. ,92. The pt BSUAV ,53.We constructed a phylogenetic tree using the maximum-likelihood method using the MEGA 5.0 software by aligning of amino acid sequences of ORF 3 from the 27 complete genome sequences of BSVs available in NCBI GenBank with RTBV as the outgroup member . DiffereMusa A (M. acuminata), B , and S (M. schizocarpa) species [M. balbisiana genome (B genome). The other endogenous badnavirus sequences described [et al. [Musa genomes [Several BSVs, called endogenous BSV (eBSV), became part of the genetic material of their host species by endogenization of ancestor badnaviruses. The hosts are several  species . At this species . These eescribed ,93, name genomes ,53,94,95in situ hybridization integration of BSOLV sequences into metaphase and pro-metaphase chromosomes of the banana cultivar, Obino l’Ewai at two loci. in situ hybridization to stretched DNA fibers indicated that one of them is approximately 150 kb long, whereas the other is estimated to be 50 kb long [Musa sequences. Sequence data revealed disrupted and truncated eBSVs with deletions, insertions, duplications, or inversions. Analysis of badnavirus integrants in M. acuminata showed their distribution among all 11 chromosomes [M. balbisiana are infective [It was demonstrated by double target  kb long ,96. At eomosomes . All theomosomes  reportednfective ,49,97 annfective ,97. A honfective . HoweverInfective eBSVs constitute an extreme case of parasitism, as well as a newly described strategy for virus vertical transmission . The actet al. [et al. [cv. Williams , where excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The frequency of BSV elimination was 90% through cryopreservation, compared to 76% obtained through conventional meristem culture.Banana is vegetatively propagated either by tissue culture or by production of suckers. Hence accurate diagnostic methods are necessary for identification of virus-free material. The presence of high levels of serological variability between BSV species and the presence of eBSV sequences make serological and PCR-based diagnosis difficult. For banana plants that contain ‘B’ genome and harbor both episomal and activatable endogenous BSVs , use of RCA and PCR are needed, while for other banana plants, only RCA could be used to distinguish between endogenous and free viral genomes. Development of real-time PCR for the rapid detection of episomal BSOLVand BSMYV was reported ,102,103.et al.  develope [et al.  reportedBougainvillea spectabilis chlorotic vein-banding virus (BsCVBV) was first reported in Brazil in 2001 and subsequently from Taiwan and India [B. spectabilis in two different locations of India and with distinct symptoms  could be distinguished by sequencing of the RT/RNaseH region [Bougainvillea chlorotic vein-banding disease caused by nd India ,107,108.nd India . The virnd India  , which infects cocoa (Theobroma cacao), was first discovered in the eastern region of Ghana in 1936. It causes a devastating disease affecting cacao cultivation of most West African countries [Planococcoides njalensis, Planococus citri, and at least 12 other species of mealybugs [ountries . The disountries . Both viountries . The recealybugs . The disealybugs .et al. [et al. [Florendovirus in the cacao plant. Jacquot et al. [et al. [The first complete genome sequencing of CSSV showed that it contains 7161 bp with five ORFs namely ORF I, II, III, X, and Y encoding proteins of 16.7, 14.4,211, 13, and 14 KDs, respectively, and an intergenic region  Figure . ORF II et al.  reportedet al. . ORF X det al. . In addit et al.  reported [et al.  reportedCanna yellow mottle virus (CaYMV) causes yellow mottle disease of canna (Canna indica), characterized by vein necrosis and mottling combined with streaking of stem and flowers. The disease was originally reported in Minnesota, Florida, and Washington, USA [Cucumber mosaic virus and an unidentified flexuous rod-shaped virus was reported in a few cases. The causal virus was identified based on electron microscopy, ISEM, PCR, and sequence analysis of a portion of the RT/RNaseH region [ton, USA ,120 and ton, USA ,122. A mH region . RecentlH region .et al. [Citrus yellow mosaic virus, based on particle morphology, serology, and partial nucleotide sequence of the viral genome. Citrus yellow mosaic (or citrus mosaic) disease has been reported so far only from India. It is common and especially severe in sweet orange (Citrus sinensis), with an incidence rate ranging from 10% to 70%. The reduction in fruit yield was 77%, and fruits from affected trees had 10% less juice and ascorbic acid [Citrus decumana, Satgudi sweet orange, and pumelo. The first complete genome sequence of the virus from sweet orange and construction of an infective clone of CYMV for agroinfection was reported [C. sinensis (sweet orange), C. jambhiri (rough lemon), C. aurantifolia (acid lime), and C. grandis (pumelo) revealed high homology among all virus isolates [et al. [Florendovirus in citrus plant.Ahlawat et al.  first rebic acid . The mosreported . The CYMreported ,127. Theisolates ,128. GeeCommelina yellow mottle virus (ComYMV) is the type member of the genus Badnavirus. It infects the monocot weed, Commelina diffusa (day flower). The complete genome of the virus consists of 7489 bp with the three typical badnavirus ORFs capable of encoding proteins of 23, 15, and 216 kD [et al. [C. diffusa through Agrobacterium-mediated infection. Analysis of the viral transcript indicated that the virus encodes a single terminally redundant genome-length-plus-120-nucleotide transcript [d 216 kD . Antibodd 216 kD . Through [et al.  demonstranscript ,129. Theanscript , as repoanscript .et al. [et al. [Medberry et al.  reportedet al. . Deletioet al. . Matsuda [et al.  reportedDioscorea bacilliform virus (DBV) occurs commonly in several species of Dioscorea (Yam) in the southern hemisphere. The disease symptoms include chlorotic-mosaic on leaves, leading to reduced sugar formation and minimal starch storage, thus causing a significant reduction in tuber yield and quality. The virus is transmitted vegetatively and through mealybugs (Planococcus citri). Badnaviruses were first reported in the yam in association with a flexuous virus, causing internal brown spot disease in D. alata and D. cayenensis-rotundata in the Caribbean [D. alata were named “Dioscorea alata bacilliform virus” (DBALV). The complete genome sequencing of two isolates of DBALV from Nigeria comprised 7413 bp and 7415 bp each with three standard badnavirus ORFs potentially coding for proteins of 29.5, 25.2, and 228.3 kD [D. sansibarensis from Benin with only 62% identity with DBALV, thus representing a new badnavirus species termed Dioscorea sansibarensis bacilliform virus (DBSNV). The two isolates of DBSNV are 7262 and 7276 bp long, with four ORFs each coding for proteins of 143, 126, 1887, and 95 amino acid [et al. [D. rotundata, but not in D. praehensilis, D. abyssinica, D. alata, and D. Trifida [et al. [D. cayenesis–rotundata complex that showed sequence originating from various parts of badnavirus genomes, resulting in a mosaic structure. They reported that eDBVs belong to at least four distinct badnaviruses indicating multiple, independent endogenization events. So far there is no report on whether eDBV can give rise to infective virus particles under abiotic stress conditions.aribbean . The bad228.3 kD . Later, 228.3 kD  reportedino acid . In addiino acid . Compari [et al.  proposin [et al. . Five of Trifida . Umber e [et al.  reportedFig badnavirus 1(FBV-1), causing mosaic disease in figs (Ficus carica), has been detected by PCR and RCA in 19 countries in Europe, Australia, Africa, and South and North America [ America ,142. The America . Based oRibes in Europe and North America. The diseased leaves show chlorotic laminar tissue adjacent to the central veins [Gooseberry vein banding associated virus (GVBaV), is transmitted in a semi-persistent manner by different aphid species . Ribes spp. is vegetatively propagated, and hence the primary spread of the disease is by vegetative means. GVBD could be transmitted mechanically from infected gooseberry to Nicotiana occidentalis [et al. [Gooseberry vein banding disease (GVBD) occurs in cultivars of al veins . The caudentalis . Xu et a [et al.  reported [et al. .Grapevine vein clearing virus (GVCV) is associated with severe vein-clearing and vine decline syndrome on grapevines (Vitis vinifera) in the Midwest region of the United States. The typical symptoms are translucent vein clearing on young leaves, short internodes, and decline of vine vigor. The disease could be transmitted through grafting. Sequencing of the complete virus genome revealed a 7753 bp sequence, coding for the three standard badnavirus ORFs [et al. [Florendovirus in grape. Further, they reported that in grapes, 9% of the endogenous florendovirus loci are located within introns and therefore may influence host gene expression.rus ORFs . StudiesKalanchoë blossfeldiana) is characterized by the appearance of numerous yellow spots on the leaves of affected plants. The disease occurs in many commercially traded cultivars of K. blossfeldiana in Europe and North America and can be economically damaging. The disease was shown to be graft-, seed-, and pollen-transmitted. It can also be transmitted by mechanical inoculation and by the citrus mealybug, Planococcus citri. In commercially grown kalanchoë, virus spread occurs primarily by vegetative propagation of infected stock plants [The top-spotting disease of kalanchoë  was reported for the first time in China. It caused yellow mosaic disease of pagoda trees (Styphnolobium japonicum) in 2013. The complete genome of the virus determined by high throughput sequencing of siRNAs comprised 7424 bp, with five ORFs sharing 40%–45% identity with other badnaviruses [aviruses . PYMAV tPineapple bacilliform virus was first detected in pineapple hybrids in Australia during 1995. It is transmitted by the gray pineapple mealybug, Dysmicoccus neobrevipes, and serologically related to SCBV [Pineapple bacilliform comosus virus (PBCOV) and Pineapple bacilliform erectifolius virus (PBERV), were reported from Australia [Anannus metavirus (AMtV) were found to be associated with pineapple mealybug wilt disease. The complete genome sequences of isolates of the virus both from China and from Hawaii comprised 7451 bp with the three standard badnavirus ORFs [By using ISEM,  to SCBV . An unid to SCBV . Because to SCBV . Two badustralia . In addirus ORFs . Putativrus ORFs .Piper yellow mottle virus (PYMoV) was first reported in 1997 in black pepper and betel vine in Malaysia, the Philippines, Sri Lanka, and Thailand [Piper species [Thailand . SymptomThailand . The occThailand ,154,155  species .Planococcus citri and Ferrisia virgata [Diconocoris distanti) [The primary spread of the virus occurs through vegetative means and seeds ,21, whil virgata ,153, andistanti) . The firistanti) . ORFs I,istanti) . PYMoV-fistanti) ,70,157.Rubus yellow net virus (RYNV), infecting Rubus species and its cultivars mainly in North America and Europe, is an essential component of the raspberry vein banding mosaic disease complex, which causes a serious decline in plant vigor and productivity. The virus is transmitted in a semi-persistent manner by the large raspberry aphid, Amphorophora idaei, in Europe and A. agathonica in North America. The virus-infected plants are either symptomless or show a very faint vein netting in the leaves. Virus-specific primers have been designed to detect the presence of the virus in infected plants [d plants . The comd plants  and thatd plants . Alternad plants ,161.Schefflera ringspot virus (SRV) infects Brassaia actinophylla (umbrella tree), Schefflera arboricola (dwarf umbrella tree) and Aralias  in Australia, Barbados, Cuba, Mauritius, Honduras, Taiwan, Thailand, and the USA . SRV cauSpiraea yellow leaf spot virus (SLSV) causes yellow leaf spot disease of spiraea in the mid-western USA. The virus has a 7.4 kb dsDNA genome and resembles badnaviruses in particle and genome properties. Exceptionally, this virus is transmitted by an aphid, Aphis spiraecola [iraecola .Sugarcane bacilliform virus (SCBV) was first observed in Cuba in 1985, and then in sugarcane in many countries around the world [Sacharicoccus sachhari. SCBV can be transmitted by S. sacchari from infected sugarcane to bananas, with SCBV-infected bananas developing symptoms indistinguishable from those described for BSV under laboratory conditions; however, natural infection of SCBV in bananas in field conditions has not been reported. A Moroccan strain of BSV is serologically indistinguishable from some isolates of SCBV. In laboratory experiments, SCBV (and BSV) can also be transmitted mechanically to healthy sugarcane and bananas [et al. [Saccharum officinarum), commercial hybrids, and clones within the ‘Saccharum complex’ including S. robustum, S. spontaneum, S. barberi, Erianthus arundinaceus, and E. ravennae. Singh et al. [he world . It caushe world . It is p bananas . Based o [et al.  reportedh et al.  found miThe first complete genome sequencing of the virus  revealed a genome of 7568 bp with the three standard ORFs and intergenic region . A constet al. [et al. [Based on partial SCBV sequences representing RT/RNase H domain of 35 isolates from Guadeloupe, Muller et al.  reportedet al. . Based oet al. . Based o [et al.  reported [et al. .Saccharum officinarum indicated that SCBV is not integrated into the sugarcane genome [et al. [Saccharum inter-specific hybrids (var ROC 205) grown in China. The variable region from SCBV was shown to serve as a promoter for high-level expression of foreign genes in both monocot and dicot transgenic plants [Southern analysis of infected plants of e genome . They re [et al.  reportedc plants ,175.et al. [Sweet potato badnavirus a (SPVa) and Sweet potato badnavirus b (SPVb), which are collectively called Sweet potato pakakuy virus (SPPV). The complete genome sequence of both viruses was reported after filling the gap left in the sequence  through PCR amplification. The complete genome of SPVa is 8082 bp and SPVb is 7961 bp long, both coding for four ORFs. Deep sequencing of small RNAs of symptomatic sweet potato plants from Honduras, Guatemala [Based on small RNA sequencing of a Peruvian sweet potato land race, Kreuze et al.  reporteduatemala , and Tanuatemala  revealedTaro bacilliform virus (TaBV) infecting taro (Colocasia esculenta) is distributed throughout the Pacific Islands. The disease is characterized by vein clearing, stunting, and downward curling of leaf blades. TaBV is transmitted by seeds and mealybugs (Pseudococcus solomonensis) [et al. [Taro bacilliform CH virus (TaBCHV). The complete genome of two isolates of TaBCHV was 7641 bp long with six ORFs encoding proteins of 16.8, 14.1, 206.4, 13.2, 11.9, and 12.4 kD. A region of TaBV genome was shown to have promoter activity in taro leaf, banana suspension cells, and tobacco callus. When these promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of a reporter gene in the stem (or pseudostem), leaves, and roots, with the strongest expression observed in the vascular tissue, indicating that TaBV-derived promoters will be useful for the high-level constitutive expression of transgenes in either dicotyledonous or monocotyledonous species [onensis) ,35,57. T [et al.  reported species .Ambrosia asymptomatic virus 2 and Ambrosia asymptomatic virus 4, was reported from the asymptomatic plants of Ambrosia psilostachya (western ragweed) from the USA. Nucleic acid isolated from virus-like particles prepared from asymptomatic plants, when amplified and sequenced, showed the presence of four viruses, including two badnaviruses [The occurrence of two distinct badnaviruses, namely aviruses .Aucuba bacilliform virus (AuBV) causes yellow ringspot or yellow mosaic in Aucuba japonica. The virus is transmitted by seeds and mealybugs.Cycad necrotic leafspot virus (CyLNV) causes leaf necrosis disease of several cycads , characterized by chlorotic and necrotic leaf lesions that expand and produce larger necrotic zones. Electron microscopy of partially purified extracts showed 30 × 150 nm bacilliform virions that contained a ds DNA genome of 9.5 kb [f 9.5 kb . The comDracaena is characterized by mottling and chlorotic patches on the leaves of Dracaena sanderiana (known as “Fortune Bamboo”). The viral genome consists of 7531 bp and possesses seven putative ORFs on the plus-strand that potentially encode proteins of 17.6, 14.9, 215.0, 11.9, 11.3, 16.1, and 11.0 kD. ORF III of DrMV corresponds to that of badnaviruses. However, the nucleotide sequence coding for the RT and RNase H domain of DrMV shares less than 68% homology with that of any other known badnavirus. Southern hybridization of DNA from the infected plant indicated that the DrMV sequence is also integrated into the D. sanderiana genome [The mottle disease of a genome .Grapevine Roditis leaf discoloration-associated virus (GRLDaV) caused “Roditis leaf discoloration” (RLD), of grape vine, cv. “Roditis” in central Greece in the early 1980s [ly 1980s . RLD symly 1980s .Mulberry badnavirus 1 (MBV-1) was reported to infect the mulberry  in Lebanon, showing symptoms of leaf mottling and vein yellowing. Typical badnavirus-like particles (150 × 30 nm) were seen in a partially purified preparation and tissue-thin sections of infected plants. The virus was also detected from mulberry samples collected from Turkey and Italy, though the majority of samples were free from symptoms. The complete genome of MBV-1, obtained through small RNA sequencing, contained all sequence features and the characteristic functional domains of the genus Badnavirus. The complete genome is 6945 bp long with two ORFs and shared high similarity (54%) with FBV-1. In contrast to badnaviruses, MBV-1 resembled the genome organization of PVCV (genus: Petuvirus), which has a single ORF [ngle ORF ,184.Red clover bacilliform virus (RCBV) reported from Trifolium pratense plants showing dwarfing and mosaic symptoms in the Czech Republic showed bacilliform virions in phloem tissues that measured 220–500 nm × 30–31.5 nm. The virus was mechanically transmitted to Nicotiana occidentalis Wheeler, accession 37 B. The partial nucleotide sequence with similarity to open reading frame III of the badnavirus genome amplified had 74.4% nucleotide identity to that of Ananas comosus endogenous virus in the polyprotein gene covering reverse transcriptase [criptase .Stilbocarpa mosaic bacilliform virus (SMBV) in Stilbocarpa polaris plants showing mild to severe yellow mosaic symptoms on subantarctic Macquarie island between Australia and Antarctica was observed in 1993 [The occurrence of  in 1993 . The cyt in 1993 .Yacon necrotic mottle virus (YNMoV) infecting yacon  in Korea was reported in 2013. The infected plants show necrosis, chlorosis, stunting, and malformation of leaves. Transcriptome sequencing of infected plants revealed the complete genome of the virus as 7661 bp in length with four ORFs, wherein ORF III showed 45% amino acid identity with that of FBV-1 [of FBV-1 .Yucca bacilliform virus (YBV) was first reported in 2001 in plants of Yucca elephantipes imported into New Zealand from Costa Rica and Guatemala [Y. elephantipes in New Zealand that were free of symptoms and virus particles, suggesting that YBV also occurs as an endogenous form in its host.uatemala . The virBadnaviruses are non-enveloped bacilliform DNA viruses with a monopartite genome. Currently, 32 species are confirmed, some of which are economically important in that they cause serious diseases in the tropics in crops such as banana, black pepper, cacao, citrus, sugarcane, taro, and yam. The majority of badnaviruses have a restricted host range. Many of the badnavirus-infected plants remain symptomless or show only mild symptoms. Masking of typical symptoms and their reappearance under abiotic stress are not uncommon. Hence the role of abiotic factors such as temperature, drought (soil moisture deficit stress), and nutrient stress in symptom development and the severity of the disease need to be studied in such badnaviruses. The possible role of biotic stress in badnavirus disease development and severity, as caused by other viral, fungal, bacterial, nematode, and insect pathogens, still needs to be studied. The genome length of badnaviruses varies between 7200 and 9200 bp with three to seven ORFs. Except for ORF III, which is a polyprotein, the functions of the other ORFs are still not known. Intergenic regions of the badnaviruses have the potential to be used as promoters and could be exploited in the expression of any foreign genes in plants.Florendovirus under the family Caulimoviridae has 34 distinct species known to exist only as endogenous viruses in a large number of plants. Of these, endogenous BSOLV, BSGFV, and BSIMV can give rise to episomal viruses in certain hybrids when subjected to one or the other abiotic stress, while endogenous viruses of other badnavirus reported so far are not known to give rise to infective episomal viruses under stress conditions.Several of the pararetroviruses including badnaviruses occur also as endogenous viruses. The genus PCR, IC-PCR, real-time PCR, LAMP, and RCA-based assays have been developed for the sensitive detection of a few badnaviruses. As PCR-based methods can give false positive results due to the presence of endogenous badnaviruses, there is an urgent need to develop other fool-proof methods to detect and distinguish episomal and endogenous badnaviruses in plants. Methods such as RCA and PCR are needed to detect plants that harbor BSV and eBSVs that can give rise to infective viruses, while RCA alone can identify virus-free plants that harbor non-infective endogenous viruses. This is important for the identification of virus-free plants to be used for propagation and also for international germplasm exchange. Though badnaviruses could be cured from the plant germplasm using cryopreservation, chemo-, and thermo-therapies combined with meristem tip culture, the success percentage is negligible. In the case of CSSV, somatic embryogenesis was used to obtain virus-free plants of cacao. These efforts must be pursued further for all badnaviruses for the production of certified virus-free plantlet production. Use of badnavirus-free planting material, providing required nutrients, cultural practices, and chemical measures for control of vector would help to manage the disease to a certain extent, but development of resistant cultivars would be the best option in developing IPM programs. So far those have not been reported. With the advent of genome editing tools like TALENS, Zinc finger nuclease, and the recent CRISPR-Cas9 with short guide RNA-based methods, it becomes possible to delete the unwanted integrated viral sequences, which can spontaneously give rise to episomal forms. Efforts to unravel the significance of integrated badnaviral sequences would lead to a better understanding of acquired immunity and natural RNAi in plant crops.


Contemporary enthusiasm for the ownership of exotic animals and hobby livestock has created an opportunity for the movement of poxviruses—such as monkeypox, cowpox, and orf—outside their traditional geographic range bringing them into contact with atypical animal hosts and groups of people not normally considered at risk. It is important that pet owners and practitioners of human and animal medicine develop a heightened awareness for poxvirus infections and understand the risks that can be associated with companion animals and livestock. This article reviews the epidemiology and clinical features of zoonotic poxviruses that are most likely to affect companion animals.Understanding the zoonotic risk posed by poxviruses in companion animals is important for protecting both human and animal health. The outbreak of monkeypox in the United States, as well as current reports of cowpox in Europe, point to the fact that companion animals are increasingly serving as sources of poxvirus transmission to people. In addition, the trend among hobbyists to keep livestock (such as goats) in urban and semi-urban areas has contributed to increased parapoxvirus exposures among people not traditionally considered at high risk. Despite the historic notoriety of poxviruses and the diseases they cause, poxvirus infections are often missed. Delays in diagnosing poxvirus-associated infections in companion animals can lead to inadvertent human exposures. Delays in confirming human infections can result in inappropriate treatment or prolonged recovery. Early recognition of poxvirus-associated infections and application of appropriate preventive measures can reduce the spread of virus between companion animals and their owners. This review will discuss the epidemiology and clinical features associated with the zoonotic poxvirus infections most commonly associated with companion animals. Poxviruses have played a large role in human history. The most infamous of the poxviruses is variola, the causative agent of smallpox. Descriptions of smallpox can be found in Chinese texts from 4th Century AD and pox-like scars found on Egyptian mummies suggest the disease may have existed as early as 2nd millennium BC . SmallpoDespite their historic notoriety, infections caused by poxviruses are commonly missed in today's world. In Europe, humans, cats and pocket pets have fallen ill with cowpox, but rarely is poxvirus infection initially suspected by the patient or the clinician. In 2003, the United States experienced an outbreak of monkeypox involving both humans and animals. Eight people became symptomatic before the first case was reported to public health authorities [The term companion animal is often used synonymously with ‘pet’ which implies affection and frequent interaction. Traditionally this has included dogs and cats; however, rodents and other small mammals are also often kept as pets, and in the last few decades exotic animals and livestock have become more common as companion animals. These changes in pet demographics along with increased importation of exotic animals have resulted in poxviruses occurring in areas outside their normal geographic range and in certain high-risk human occupational groups. Therefore, it is important that pet owners and human and animal clinicians develop a heightened awareness for poxvirus infections and understand the risk associated with specific companion animals. Delays in diagnosing animal infections can lead to inadvertent human exposures and delays in human diagnosis could result in inappropriate treatment or delayed recovery. Also, early recognition of poxvirus infections is important for implementation of appropriate measures aimed at preventing virus spread. This review will focus on those zoonotic poxviruses most likely to be associated with companion animals and discuss what is currently known about poxvirus pathogenesis and diagnosis in general, as well as the epidemiology and clinical features associated with these zoonotic infections.The skin is the primary portal of entry for most poxviruses, however respiratory and mucosal routes have been associated with orthopoxvirus infections. Poxviruses are epitheliotropic; most infections are self-limiting and produce localized lesions that progress through macular, papular, vesicular and pustular stages over several weeks. Ultimately the lesion forms a crust and often leaves a characteristic “pock” scar for which the virus was originally named. Generalized infections occur depending on the species of virus and host. In generalized infections, virus spread is thought to occur through the regional lymphatics to the bloodstream, resulting in a primary viremia. The virus then multiplies in reticuloendothelial organs followed by a secondary viremia and seeding to other sites especially the skin ,5.The tropism of poxviruses is thought to be a function of host genes and immune response post cell entry, as opposed to virus binding and entry as is seen for most viruses . ZoonotiMany orthopoxviruses, including cowpox, monkeypox, and vaccinia, have a broad host range. Although the reservoir species for most of the zoonotic orthopoxviruses remain undetermined, the most likely candidates are sylvatic rodents which normally have little interaction with people, but may interact with animals commonly kept as pets. Domestic cats are efficient predators and though cowpox has been reported in a variety of species in Europe and Western Asia, clinical infections are primarily seen in cats. Rodents kept as pets have also been implicated in human disease outbreaks -11. In tToday cowpox is known to infect a broad range of species throughout Europe and Western Asia. Wild rodents are considered the reservoir, but the exact species is unknown. In the United Kingdom there is strong evidence supporting bank and field voles as reservoir species with wood mice and other rodents capable of maintaining the virus -21. AlthMost persons ultimately diagnosed with cowpox virus infection present to their physician with a single, painful pustular lesion located on the hands or face and complain of “flu-like” symptoms. The patient's usually have a history of owning a cat that ‘gifts’ them with dead rodents or they have had close contact with an ill cat or rodent. Cowpox lesions tend to be painful throughout the life of the lesion, are large, ulcerative with inflammation and edema and the crust it forms is thick, hard and black. Local lymphadenopathy and systemic symptoms such as pyrexia, lethargy, sore throat and general malaise are common and often severe enough for people to miss school or work. Infections are typically self-limiting as most people recover in 6–8 weeks ,14. MucoFelidae sp, especially the domestic cat, but has also been described in the domestic dog, rats, monkeys, elephants, and other animals [Panthera leo), anteaters (Mymecophaga tridactyla), ocelots , cheetahs (Acinonyx jubatus), monkeys and elephants [Infections in animals can be nearly asymptomatic or, at the other end of the spectrum, can result in death. Clinically significant cowpox has been recognized most often in  animals ,29,33-38 animals . Clinica animals ,39. Of t animals . Diseaselephants -37,40.Monkeypox is endemic to tropical rainforests of central and western Africa and rodents, possibly squirrels, are the likely reservoir candidates. The cumulative annual incidence of human disease is considered low and person-to-person transmission has been documented -46. WhenDuring the outbreak people presented with a generalized rash and lymphadenopathy which was proceeded by a 2–4 day prodrome of fever, headache, backache, lethargy and general malaise. Similar to what is seen in central and western Africa ,44,47. UDuring the US monkeypox outbreak pet prairie dogs presented with lethargy, anorexia, lymphadenopathy, blepharitis with ocular discharge, and upper respiratory signs such as cough, sneezing, and nasal discharge which eventually lead to pneumonia  as pets . These aOrf is also known as contagious pustular dermatitis, contagious ecthyma, soremouth, or scabby mouth in sheep and goats. It is considered enzootic wherever goats and/or sheep are raised and sporadic infections have occurred in cats and dogs ,63. TranPeople typically present with a solitary, non-healing lesion on their hands, occasionally on the face or axilla, and when asked report contact with sheep, goats, or associated husbandry items ,73,79-82In small ruminants, orf occurs most commonly in juvenile animals; although in a naïve herd, large numbers of adults can also be affected. Animals generally present with lesion around the commissures of the lips (Other recognized parapoxviruses known to cause human infection are found in cattle (bovine papular stomatitis virus and pseudocowpox virus), deer, seals and sea lions ,98-104. Diagnosis of some poxvirus infections can be made based on clinical features and case history alone. However, laboratory testing is needed to confirm infection. Because large quantities of virus can be found within lesions, vesicle fluid, crusts, or tissue biopsy are preferred as diagnostic samples. Electron microscopy and histopathology can also be helpful for confirming a diagnosis as poxviruses have distinctive morphology—large (approximately 300 nm diameter), box or ovoid shape, and outer membrane protrusions (creating a textured appearance)—and replicate in the cytoplasm of host cells ,115,116.Cowpox can sometimes be differentiated from other orthopoxvirus infections by the presence of large, cytoplasmic, eosinophilic A-Type inclusion bodies ,117. SerIn general, poxvirus infections are self-limiting and treatment is primarily supportive care. Antibacterial therapy is only warranted when secondary infection is present. Promising antivirals, developed under the auspices of bioterrorism preparedness, are currently under investigation, but there has been little consideration of their potential application to the prevention and treatment of poxvirus-associated zoonoses. Experimental evidence suggests that compounds with direct antiviral effects such as Cidofovir and CMX-001 and others that inhibit viral egress such as ST-246 may be effective in treating orthopoxvirus infections -128. CidVaccination is also available for prevention in some instances. Smallpox vaccine is considered protective against orthopoxvirus infections and is recommended for laboratory personnel working with monkeypox, cowpox, vaccinia, and variola . In animMany factors play into the emergence or reemergence of zoonotic disease and recent reports of poxvirus infections demonstrate that human behavior often plays a role. A plausible scenario for a future outbreak could involve transportation or abandonment of an infected companion animal(s) resulting in the release of poxvirus into a new environment. Our current understanding of poxvirus epidemiology suggests a need to more efficiently identify and control of these diseases in novel populations.Public health efforts that focus on increasing poxvirus awareness in animal owners could have a positive impact on behaviors that can reduce the risk of infection, including hand washing after handling an animal, applying quarantine when introducing new animals to a flock or herd, wearing gloves when manipulating an animal's oral cavity or when a cut is present on the hand, and seeking veterinary care for an ill pet. Early clinical recognition of poxvirus infections in animals can prevent virus spread, and heightened awareness in animal owners may lead to early health seeking behaviors, which will in turn optimize opportunities for clinical interventions.With increased human and animal movement across oceans the possibility for future outbreaks of zoonotic poxviruses is very real. This observation suggests a need for the development of therapeutics and biologics for both animals and humans. Antivirals are probably not a valid option in animals due to costs and concerns for resistance, but vaccines can play an important role. Research that focuses on vaccines that provide protection without contaminating the environment are key to protecting both human and animal health. Until approved therapies become available, early recognition of infection is important for preventing the spread of virus. Through heightened awareness and appropriate prevention measures, poxvirus infections can be minimized both in people and in their companion animals.


To the Editor: Viruses in the Simbu serogroup are arboviruses that cause abortion, stillbirth, and congenital abnormalities in domestic ruminants. Akabane virus (AKAV), Aino virus (AINV), and Schmallenberg virus are the most studied in this serogroup; Shuni, Sabo, Shamonda, and Sango viruses  from 15 sheep, goats, and cattle. Most samples were from the 2 affected flocks in the northern valley; a few were from ruminants in additional locations: Avadon, near Israel’s border with Lebanon; Ein Hachoresh, near central Israel; and Hura, close to the Negev desert  Figure 1Of the 27 samples, 23 (85%) were positive for Shuni virus (SHUV) by PCR . SHUV, wFor further testing, we inoculated homogenate material from 7 distinct malformations (samples 1, 2, 6, 8, 11, 12, and 15 in the Our results showed the presence of SHUV in sheep in Israel during the winter of 2014–15 and suggest a northward expansion of SHUV from sub-Saharan Africa. Although SHUV was first isolated in the 1960s (A serologic survey conducted in Israel during the 2001–2003 outbreaks of AHS showed reactivity of AINV to serum samples of ruminants in Israel’s southern regions (The emergence and reemergence of arboviruses should interest medical practitioners, particularly epidemiologists. The appearance of exotic viruses in unexpected locations might result in more severe pathology in newly invaded regions than in the original arbovirus-endemic areas. Furthermore, SHUV has been detected in a child with febrile illness (Technical Appendix. Map showing locations where Shuni viruses were detected in ruminants in Israel, 2014–15, and phylogenetic trees of Simbu serogroup viruses.


Shortened treatment regimens for tuberculosis are under development to improve treatment outcomes and reduce costs. We estimated potential savings from a societal perspective in Brazil following the introduction of a hypothetical four-month regimen for tuberculosis treatment.Data were gathered in ten randomly selected health facilities in Rio de Janeiro. Health service costs were estimated using an ingredient approach. Patient costs were estimated from a questionnaire administered to 126 patients. Costs per visits and per case treated were analysed according to the type of therapy: self-administered treatment (SAT), community- and facility-directly observed treatment .During the last 2 months of treatment, the largest savings could be expected for community-DOT; on average USD 17,351-18,203 and USD 43,660-45,856 (bottom-up and top-down estimates) per clinic. Savings to patients could also be expected as the median (interquartile range) patient-related costs during the two last months were USD 108 (13–291), USD 93 (36–239) and USD 11 (7–126), respectively for SAT, facility-DOT and community-DOT.Introducing a four-month regimen may result in significant cost savings for both the health service and patients, especially the poorest. In particular, a community-DOT strategy, including treatment at home, could maximise health services savings while limiting patient costs. Our cost estimates are likely to be conservative because a 4-month regimen could hypothetically increase the proportion of patients cured by reducing the number of patients defaulting and we did not include the possible cost benefits from the subsequent prevention of costs due to downstream transmission averted and rapid clinical improvement with less side effects in the last two months.The online version of this article (doi:10.1186/s12913-016-1269-x) contains supplementary material, which is available to authorized users. Globally, in 2013, there were nine million new cases and 1.5 million deaths from tuberculosis (TB), with 95 % of the deaths occurring in low- and middle-income countries  and an iBrazil has been an early adopter of new technologies in general and of new TB technologies in particular , 15. AccThe city of Rio de Janeiro has one of the highest TB incidence rates in Brazil , with 90To ensure a representative range of costs estimates from different settings, we purposively selected four geographical areas to obtain a balance between the municipality of Rio and peri-urban areas. We then randomly sampled 12 facilities from a census of all eligible facilities currently treating at least 50 TB patients , stratified by workload  and type of clinic .Costs from a healthcare provider’s perspective were estimated using an ingredient approach –25 for eWe used a combination of bottom-up and top-down approaches to calculate unit costs. In a bottom-up approach, we identified the resources used within a certain input type and after allocating a value to them, we added them to calculate unit costs; for the top-down approach, we divided the total expenditure for a given input type by the units of activity. We adopted a micro-costing approach that mixed both methods. Drug costs are only reported as bottom-up estimates because records for drug expenditures were not reliable at facility-level. In Brazil, drug expenditure is centralized; and therefore not accessible at the facility level. We verified the prices of drugs and consumables sourced from suppliers with international databases . CapitalWe calculated cost per visit and episode costs (cost per person treated) by treatment modality and site. We extracted information on the total number of non-multidrug resistant TB patients being treated for each treatment modality  from service statistics for the period covered. We estimated the episode costs by month by multiplying the number of visits in this period by the unit costs. To allocate costs to the last two months of treatment, we first estimated the number of visits using two methods: (i) assuming a 10 % default rate after the 4th month, we estimated that the number of monthly visits would be 45 % of the number of visits if guidelines were fully adhered to; (ii) we estimated the number of visits during the last two months as reported by patients in the questionnaires. We did not include programme costs, defined as the costs associated with general support to service delivery .Finally, we estimated the total potential annual savings due to the introduction of a shortened first-line TB regimen considering that 76,020 persons were treated with TB in 2013 [Critério Brasil [All TB patients attending  at a participating clinic on their 5th or 6th month of treatment were invited to participate. After obtaining informed consent, data on demographics, assets, type of TB treatment and both direct and indirect costs were gathered. The questionnaire used to collect patient-related costs was based on a validated tool for patient costs developed by USAID and available online . Direct o Brasil  divided o Brasil , estimato Brasil .Costs per visit were multiplied by the number of visits in the last 2 months to arrive at total costs. We calculated the ratio of total costs during the last 2 months of treatment to the estimated monthly income by estimation method. We considered total costs during the last 2 months to be catastrophic when they represented over 40 % of the monthly income .t-test.Data collection took place from June 2013 to May 2014. The average exchange rate used was USD 1 = BRL 2.16. All health service cost analyses were performed using EXCEL 2013 . Statistics analyses were performed in SPSS 21 . Mean costs and standard deviations (SD) were compared using independent Universidade Gama Filho (268.778) in Rio de Janeiro; the Comitê de Ética em Pesquisa, Prefeitura do Rio de Janeiro (164A/2013); and the Observational and Interventions Research Ethics Committee of the London School of Hygiene and Tropical Medicine, UK (6322).The study was approved by the Institutional Review Board at the The 12 randomly selected clinics provided services to neighbourhoods with a wide range of income  and TB burden . All facilities offered SAT and at least one type of DOT (9 offered facility- DOT while the other three offered community-DOT). The facilities presented a wide workload range . Of 12 clinics approached, ten (83.3 %) agreed to participate  were community visits. Only 4 % of all visits  were SAT visits.Service utilisation for TB is presented by facility in Additional file The unit and total costs by treatment type as well as drug and episode costs by treatment phase and type are presented in Additional file Table n = 77) were male, 78 % (n = 93) belonged to the three lowest socioeconomic classes. Participants’ characteristics are detailed in Additional file We interviewed a total of 126 patients in the ten participating clinics. The median age among our sample was 43.0 (interquartile range 27.8–55.4) years, 61 % (n = 49), the median declared monthly income was USD 370 (IQR 298–625). Using the shadow salary method, the median monthly income was USD 420 (IQR 405–435); while using the family income per capita, the median monthly income was estimated at USD 195 (IQR 133–359).The estimated value of a productive hour using the different methods was: (i) minimum wage, USD 1.4/h; (ii) family monthly income per capita, USD 1.0 (IQR 0.7–1.8)/h; (iii) shadow price, USD 2.1 (IQR 2.0–2.2)/h; and (iv) declared income, USD 1.9 (IQR 1.5–3.2)/h. Among patients declaring their income , and patients under SAT and facility-DOT regimens Table . HoweverWe present a comprehensive cost study describing detailed information on first-line regimen costs that can serve as an input for decision-making and budget preparation for NTPs when considering the introduction of new shortened regimens. We estimated the methods of delivery to be the main driver of overall costs. From a health service perspective, community-DOT was the main contributor to total costs for first-line TB treatment in these primary care facilities, mainly due to high service utilisation . SAT was estimated to be the treatment modality with the highest unit cost, even though it was the treatment model contributing the least to total costs, due to the least number of visits. From a patient perspective, facility-based services (both SAT and facility-DOT) were modalities representing the highest costs for the patients, mainly associated to transport and productivity loss costs. Therefore, while bringing services to the community might seem more costly from a health service perspective, it can lead to substantial savings from a societal perspective. Besides costs, decision-makers should take into account the potential improvement brought by community-based treatment regarding treatment outcomes and equity in the access to care for an already vulnerable population. While the patient-level benefits of community-DOT compared to facility-DOT have been suggested in three clinical trials, the benefits of DOT over SAT are still controversial , 33. AddIn terms of cost differences among models of service delivery, our results are consistent with two previous studies in the country , 36. HowWe found a large variation both across clinics and between the top-down and bottom-up approaches, mainly due to estimations of staff time. While a top-down method might be less precise when disaggregating costs per activity, especially for staff costs allocation, it might be a more accurate representation of total costs, including inefficiencies in service delivery. Compared to the USD 88,000,000 national budget for TB control, the potential estimated savings should a four-month regimen be introduced nationally could represent up to 9–18 % of the NTP budget. These savings could be realised in their totality if, for example, staff would be relocated to other programmes. As this might not be the case, we are offering a top end estimate of potential savings. On the other hand, our estimates of potential cost benefits are likely to be conservative because potential savings due to a possible reduction in transmission and the possible differential in health improvements, with less side effects and therefore service utilisation during the last months of treatment, were not considered.Costs of treatment for patients can be a significant barrier to access and utilisation of healthcare , 37–39, In our study, costs for patients were significantly higher for those in the lower socioeconomic classes. Because their productive hour is less valued, we would expect lower indirect costs. Our finding (higher indirect costs) suggests that, no matter which method is used to estimate them, more hours are spent in or traveling to the facilities and raises more concern about the added vulnerability to an already disadvantaged population. The introduction of a shortened regimen would then benefit lower socio-economic class patients the most.Our study presents several limitations. We collected data in one only city, including both urban and peri-urban but excluding rural settings. Therefore, the findings can only be generalised to similar settings within Brazil. Additionally, we present potential cost savings during the last 2 months of treatment only and patients incur substantial costs during the pre-diagnosis period –42. In oAltogether, our findings show that significant savings can be achieved for patients and the health system with the introduction of shortened regimens. This is an important finding that sustains the rationale for shorter regimens even with equivalent effectiveness to the currently recommended 6-month regimen. We also highlighted the importance of considering the variation among models of delivery when undertaking cost-effectiveness analyses of future drugs. As the numbers of confirmed TB patients may increase with the introduction of new diagnostics , potenti


The increasing burden of dengue fever (DF) in the Americas, and the current epidemic in previously unaffected countries, generate major costs for national healthcare systems. There is a need to quantify the average cost per DF case. In Mexico, few data are available on costs, despite DF being endemic in some areas. Extrapolations from studies in other countries may prove unreliable and are complicated by the two main Mexican healthcare systems . The present study aimed to generate specific average DF cost-per-case data for Mexico using a micro-costing approach.Expected medical costs associated with an ideal management protocol for DF  were compared with the medical costs of current treatment practice  in 2012. Real cost data were derived from chart review of DF cases and interviews with patients and key personnel from 64 selected hospitals and ambulatory care units in 16 states for IMSS and SS. In both institutions, ideal and real costs were estimated using the program, actions, activities, tasks, inputs (PAATI) approach, a micro-costing technique developed by us.Clinical pathways were obtained for 1,168 patients following review of 1,293 charts. Ideal and real costs for SS patients were US$165.72 and US$32.60, respectively, in the outpatient setting, and US$587.77 and US$490.93, respectively, in the hospital setting. For IMSS patients, ideal and real costs were US$337.50 and US$92.03, respectively, in the outpatient setting, and US$2,042.54 and US$1,644.69 in the hospital setting.The markedly higher ideal versus real costs may indicate deficiencies in the actual care of patients with DF. It may be necessary to derive better estimates with micro-costing techniques and compare the ideal protocol with current practice when calculating these costs, as patients do not always receive optimal care. Aedes aegypti mosquito. Although the effects of DF are usually mild, in some cases serious illness and even death may result. The average costs per case when extrapolated to society may therefore be high, particularly given the large number of people infected during an endemic year. In Mexico, relatively little is known about the average cost per case . Such information is important to guide decisions about health policy, e.g. vaccination or public education. We aimed to quantify the average cost per case of DF using a micro-costing approach, both for DF treatment according to an ideal protocol for the management of the patient  and according to current treatment practice in the health services . Our results were largely consistent with findings from other international studies, but showed higher ideal costs compared with real costs. We think this may point to inadequate use of laboratory tests and treatments for patients with DF in Mexico. Our cost data will be used in a subsequent publication regarding the economic impact of DF in Mexico.Dengue fever (DF) is caused by infection with the dengue virus, which is spread by the  Flaviviridae family [Aedes aegypti, and humans are the only known reservoir for the virus [Dengue fever (DF) is caused by infection with the dengue virus, a single-stranded positive-sense RNA virus of the e family . The virhe virus , 3. Clinhe virus . Based ohe virus  estimatehe virus .As in most of Latin America, the disease is widespread in Mexico, although the incidence rate has varied since its reappearance in the 1970s. Peaks in the number of cases occurred in 1980, 1997, and 2009, when more than 130,000 cases were reported nationwide . BetweenWhen the present study was initiated, some studies of the economic consequences of dengue had been conducted in Latin American countries including Brazil , ColombiIn Mexico, available data on the costs of the disease are limited. In one of the most comprehensive international studies of the cost of DF, Shepard et al.  used speThe co-existence of two different main public healthcare delivery systems (IMSS and SS) is not the only factor that may hamper extrapolation of cost data. In Mexico, the effectiveness of the healthcare sectors has been questioned after multiple reforms have been enacted , with paThe main objective of this study was therefore to assess the associated medical costs and cost to the individual with DF using a micro-costing approach to overcome the lack of cost center data. Chart review and interviews with patients and key personnel from 64 medical units in 16 states with endemic DF provided data on costs associated with actual treatment of the disease . For further comparison we estimated the costs that would be incurred if an ideal treatment protocol were followed .The study was approved by the National Commission of Scientific Research for the IMSS, register number: R-2012 785–070. All participants provided written informed consent and all patient data were anonymized.The cost per case of DF was calculated by summing the following costs: (1) direct medical costs incurred by healthcare units ; (2) costs of dengue from the patient’s perspective ; and (3) indirect costs (to the patient and their family) resulting from loss of productivity and loss of earnings. The second and third components include medical and nonmedical direct costs and also indirect costs, such as loss of productivity, caregiver’s costs, etc. All costs were calculated in Mexican pesos and converted to US$ using the exchange rate on September 12, 2012 (1 US$ = 13.03 Mexican pesos) and adjusted for inflation to December 2014 when the analysis was finished.A hierarchical micro-costing approach was used to calculate direct costs incurred by the healthcare services  , 27. ThiThe tasks and inputs included in the analysis were defined in two ways. In one scenario, an ‘ideal’ cost was calculated according to the tasks and inputs included in an ideal management protocol for patients with DF. In the second scenario, treatment costs associated with current practice  in the healthcare services were determined by calculating costs for the tasks and inputs actually performed. These tasks and inputs were determined by chart review and patient interviews. Where data from the medical units were missing or incomplete, the information was supplemented as far as possible from interviews with treating physicians and hospital administrators. The PAATI approach is summarized in Given the different structures and attributes of the two principal sectors of the healthcare system, costs were calculated for both of them. In addition, unit costs in the private sector were used to estimate the cost of the ideal protocol only, as we had no way of evaluating current treatment practice in the private sector, given the difficulties of selecting a sample from the myriad of private healthcare units in the 16 states with endemic DF. Therefore, in the present study, we only assessed operational costs or variable costs, and no estimates were made of sunk costs or other costs, such as overhead costs .The indirect costs from the patient´s perspective were calculated based on information derived from interviews with patients. For these data, the methodology used took into account costs reported by the patients, and these were used to calculate the average cost per case, as well as confidence intervals. A bootstrap analysis was performed to assess variability. Where possible, the patients interviewed were the same patients whose charts were reviewed. Indirect cost was defined as loss of earnings associated with the disease (patient and/or caregiver), with information taken either from direct questions or an estimate based on the number of days of work lost multiplied by the net loss of earnings per day, separately for each institution. For example, in patients who reported being employed, the average length of hospital stay in days was multiplied by the average daily salary. The same approach was used for caregivers. A cost was not assigned if the patient or caregiver did not report any income or they were students. For patients under 16 years of age, only loss of earnings for the caregiver was calculated.We selected a non-probabilistic sample. The study sample was drawn from 32 hospitals and 32 ambulatory care units in 16 Mexican states with endemic DF. Within each state, the hospitals and ambulatory care units with the highest number of cases were selected and up to a maximum of 40 cases per hospital or unit were selected at random for chart review. To avoid selection bias reviewers were instructed to use random numbers to select records. For units with fewer than 40 cases, a census was carried out, and all cases were included in the review. Only patients with an infection occurring in 2012 were considered for chart review.A sample size sufficient to provide reasonably robust data was calculated using the formula:n = sample size, N = total population, σ = standard deviation, e = acceptable sampling error limit (0.05), and Z = 1.96 . With these parameters, a sample size of 1,440 subjects was obtained.Where As discussed above, costs were calculated for both the SS and IMSS systems, and so the sample size calculation was applied to both systems separately, resulting in double the overall number of patients. This sample would be representative of overall proportions in the SS and IMSS systems at a national level, although it does not take into account state-level stratification.For indirect costs to the patient, a bootstrap sensitivity analysis was conducted to assess the robustness of the estimation of the average input (based on the method proposed by Efron ). BootstData were collected from review of 1,293 charts . From these charts, it was possible to obtain clinical pathways for 1,168 (81% of the target). Some clinical pathways could not be generated because data were incomplete and could not be supplemented by interviews with treating physicians and hospital administrative personnel. In total, 1,168 patients were interviewed for assessment of out-of-pocket expenses and indirect costs. The same patients were interviewed as those with chart review in 80% of the cases. The remaining patients interviewed had no corresponding chart review.Overall, 53% of the patients with chart review were women. The mean age of the patient population was 27 years and 34.7% were younger than 18 years old.As shown in In both the ideal and real scenarios, the costs to the IMSS were greater (by a factor of 2–4) than the costs to the SS across all patient settings. As for differences between ideal and real costs, the main driver was the cost of professional services. Expenditure on medical consumables and drugs and related products showed very little variation between the two systems, while expenditure on laboratory tests was somewhat higher for the IMSS.The overall costs of the ideal management of patients in the private sector was higher than the corresponding ideal costs in the IMSS for ambulatory patients  and hospitalized patients , while costs for patients in the ICU were similar .Expenses from the patient’s perspective are presented in In the outpatient setting, the direct medical care costs are almost double for IMSS patients than for SS patients. It is important to remember that these costs are independent from what was spent by the healthcare unit in the treatment of the patient.We estimated the loss of productivity based on the number of days reported in the patient interviews for general hospitalization or ICU care. The average cost for a hospitalized patient is therefore not the same as for an ICU patient because, among other factors, the average length of stay is longer for a patient who receives ICU care. Data from the national census of population and national health surveys indicate that, on average, IMSS patients are more affluent than SS patients , 23. TheThe questionnaire did not address loss of income for outpatients and these data are indicated as not available in In general, indirect costs appeared to be higher for patients in the IMSS system than for those in the SS system, with the exception of costs for patients in the ICU, which tended to be higher for SS patients . The indDespite the fact that a non-probabilistic sampling method was employed in this analysis, the extensive chart review and direct interviewing techniques provided a robust estimation of the average cost of treatment of DF in Mexico. The study also provided information according to type of healthcare system used, thus enabling qualitative comparisons.In this study we tried to address the limitations of other estimations of average cost per case of DF. The available cost estimates in the literature showed great variability in their methodology, using primary data, macro-costing data, patient questionnaires, administrative data, or most commonly, a combination of data sources including some primary data in a highly restricted population. In our study, the use of micro-costing provides a more detailed understanding of the direct medical costs of dengue, and that is one of its major strengths.Although we used average costs, we did not want to make the assumption that patients from a single medical unit reflect the whole experience of a country. Therefore, in our study the average costs per case were calculated from data collected from medical audits of 64 healthcare units  and 1,168 patient interviews in 16 states where DF is endemic.To compare our results with other studies, here we present the original costs and within brackets the dollars adjusted for inflation using the national consumer price index by country and the official exchange rate, annual average for The World Bank for 2014. For ambulatory cases, the direct real medical cost of 2012 US$32.60 (2014 US$35.93) in the SS was lower than the direct medical costs reported by Sheppard et al. for Brazil 2010 US$49 (2014 US$54.05) ), and moThe direct medical costs of hospitalized patients, in relative terms, are higher in our study at 2012 US$490 (2014 US$ 539.99) and 2012 US$1,644 (2014 US$1811.71) for the SS and IMSS, respectively, compared with the estimates for Brazil 2013 US$318 (2014 US$346.80) ), ColombAlthough comparisons with other countries may be illustrative, firm conclusions cannot be drawn given the differences in economic development, population size, and healthcare systems, as well as the methodology used for the estimates.In the case of hospitalized patients, comparisons are more difficult because no distinction is made between hospitalized and ICU settings in most Latin American studies. Interestingly, the ideal costs estimated in our study, 2012 US$587 (2014 US$646.88) for SS and US$2,042  for IMSS were closer to the extrapolated costs reported by Shepard et al.  for totaFinally, we are not interested in presenting the virtues or deficiencies of the Undurraga approach; we simply want to clarify the differences with our approach.Indeed, a particularly striking feature of our results are the differences between the direct medical costs of actual treatment in clinical practice and those that would be generated if an ideal protocol, validated by experts in the field, were followed. The difference was particularly marked for outpatients. The first implication is that cost studies based on an ideal treatment protocol may not reflect clinical reality, at least in Mexico. The difference in personnel costs may reflect systematic differences in either the productivity of the personnel, their training, and treatment standards among the medical units of the institutions included in the study. The second implication is that there may be shortcomings in the Mexican healthcare systems, particularly in the outpatient setting, despite extensive reform in recent years with a view to improving the quality of care , 21, 31.Another noteworthy feature of the results presented here is the higher direct medical costs incurred within the IMSS system compared with the SS system. Another Mexican study utilizing chart reviews and direct patient interviews estimated the direct medical costs in the IMSS system for patients with osteoporosis and hip fracture . CompareThe greater availability of treatments in the IMSS system may explain the slightly higher treatment costs in the outpatient and hospitalized settings, although the more fragmented nature of the SS system may increase procurement costs. There is evidence that the heterogeneity of the state healthcare services (SS system) may lead to small variations between areas, resulting in care below that recommended in clinical guidelines. This substandard care is associated with lower costs.Of note were the expenses reported by the patient beyond those incurred by the healthcare institutions for hospitalized patients and patients in the ICU. Expenses incurred by patients were higher within the SS system, suggesting that these patients had to supplement the care provided by the SS to a greater extent than IMSS patients. Any comparison of the costs in the IMSS and SS systems should bear in mind that the cost of each activity was calculated using the average use reported per input and the unit cost reported for every organization and state. In general, the unit costs in the IMSS system are more stable and better registered at the central level of the organization than costs within the SS system.A limitation of our study is that loss of earnings due to days off work was only registered for patients admitted to hospital or ICU and thus was not captured for outpatients.Indirect costs were greater for patients who attended through the IMSS, possibly reflecting their higher socioeconomic status. Interestingly, the difference was greater for hospitalized patients than those admitted to the ICU. This observation can be explained by the fact that family members may be allowed to stay with patients in a hospital ward but not with patients in an ICU setting and they will thus incur more expenses.A second limitation was that we do not include data on management, electricity, and other utilities in the cost estimation. However, it was considered that the relevant cost is variable cost, and given the lack of data on management, electricity and other utilities, we considered that it would be misleading and add more uncertainty to the cost estimation.As noted above, a principal weakness of the study is possible bias resulting from the non-probabilistic sampling method employed in this study. The states where DF is endemic were chosen, and, within those states, the centers with the highest number of cases were selected. Furthermore, patients requiring hospitalization and those with DHF would be more likely to attend larger reference centers, so this approach may lead to an over-representation of patients with more severe disease. A further bias may have been introduced by not ensuring that all patients interviewed corresponded to those whose charts were reviewed. However, given that 80% of the patients interviewed also had a chart review, differences would have limited impact on the final average results.Nonetheless, by concentrating on larger centers with a larger number of cases, it was possible to collect extensive primary data that would otherwise be hard to acquire from probabilistic samples. In essence, statistical robustness was sacrificed to enable the data collected to reflect more accurately clinical practice in the sample, in contrast to many studies of costs of DF in the Latin American region. Additionally, the data available from chart review were not complete (and may not have been entirely reliable) and had to be complemented with other data sources. While this may also compromise the integrity of the data, we believe that chart review, despite its inherent limitations, ultimately provides the best approximation to what actually happens in clinical practice. It is also possible that recall bias was present in the interviews, but for most patients, DF is an event that they are unlikely to forget.In conclusion, this study pointed to real costs associated with DF in line with those reported in other Latin American countries . Of particular note were the large differences in the real costs derived from patient records and the ideal cost calculated from an ideal treatment protocol. This difference perhaps points to deficiencies in the care of patients with DF in Mexico. It also indicates the importance of collecting primary data when calculating DF costs to guide health policy decisions in this and other diseases that lack an appropriate estimate of its cost to the system, which poses a major hurdle for healthcare planning.S1 Appendix(PDF)Click here for additional data file.


Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times. Endogenous retroviruses (ERVs) are retroviruses which have been integrated into the host genome of germ-line cells, comprising approximately 10% of the host genome in mammalsThe RD-114 virus is a replication-competent feline ERV57891112et al. identified thirteen RD-114 virus-related clones, consisting of conserved gag-pol and unrelated env regions from six domestic cat genomesgag-pol region of the clones was conserved, the env region displayed considerable divergence from the replication-competent RD-114 virus clone derived from human RD cells, both in size and sequence homology, coinciding with previous results. In their followup study, feline chromosomes which contain RD-114 virus-related segments were identified with variation of distinct viral segments among individual catsPstI-digested pol-env fragment in all cats genomic DNAs examinedBamHI-digested gag fragment2Felis catus endogenous retrovirus (FcEV) which consists of the RD-114 virus-related gag-pol gene and unrelated env gene was discovered in all domestic cats genomes examinedenv genes are more likely to be FcEV integrations16Several research groups have searched for the active RD-114 proviral loci214In this study, we searched for loci encoding infectious RD-114 virus. Contrary to our expectations, we could not find any RDRSs capable of producing infectious RD-114 virus, but discovered that a RD-114 virus-related virus (RDRV) nearly identical to the RD-114 virus can be produced by recombination between two defective RD-114 virus-related proviruses. We also noticed that most RDRSs have not been fixed in domestic cat genomes, suggesting multiple invasions of cat genomes by the ancient RDRV. Our study sheds light on host-virus interactions and provides novel genetic markers of domestic cats available for tracing footsteps of cats.et al. reported that there are twelve RD-114 virus-like elements with more than 50% sequence identity, which cover at least 50% of query sequences in domestic cat genomesHindIII and a gag probe, we detected a 2.6-kb fragment corresponding to the HindIII fragment of RD-114 virus molecular clones in RD-114 virus producer Crandell-Rees feline kidney (CRFK) cells and RD-114 virus non-producer cell lines such as G355-5 , Felis catus whole fetus-4 (fcwf-4) (a feline macrophage-like cell line) and FeT-J (an interleukin-2-independent feline T-cell line) cells  in CRFK cells (env and two of them (RDRS C1 and C2b) have intact ORFs for all viral genes   which have the intact ORF for env gene. RDRS C2a of CRFK, G355-5 cells and feline PBMCs (n = 2) have stop codons within all viral genes in the same fashion. Phylogenetic analysis of RDRSs and RD-114 virus clones indicated that RDRS C2a belongs to a separated group from the other RDRSs and infectious RD-114 virus clones  using the SSEARCH programe) cells . By usinll lines . In acco numbers . InverseFK cells . We alsoFK cells . Again, and env) . We founs clones . In felis clones . These ds clones .−8 substitutions/site/year in cats was adopted for calculating proviral insertion timePrionailurus bengalensis) (n = 4) which inhabits Vietnam. Instead, we found a 54-bp sequence within the same locus. Intriguingly, the 54-bp sequence is part of a SINE element (Repbase ID: SINEC_Fc2), predicted by the Repeatmasker program (http://www.repeatmasker.org/) , Snow leopard (Panthera uncia), and Tiger (Panthera tigris) , the full length SINE elements were maintained in their genomes at the same location as the leopard cat, suggesting that SINEC_Fc2 integrated into the genome of the common ancestor of Felidae species and then RDRS C2a integration occurred, leading to the disruption of a part of the SINE sequence 27We next estimated the integration time of RDRSs. The divergence accumulated between 5′ and 3′ LTRs over time was previously used as a molecular clock to investigate the integration time of ERVer.org/) . The SINsequence . Anai etenv on any loci from the defective C2a-type env  with infectious-type env are present in Scottish fold cats. Domestic cats (Felis silvestris catus) originate from the African wildcat (Felis silvestris lybica), a wild cat living in the Middle East, and then spread over the world to Europe or a different route to Asiai.e. RDRSs possessing infectious-type env) might have infected domestic cats after cats started moving from the Middle East. There is a possibility that some additional loci with the infectious-type env were acquired in cats through introgression of newer RDRSs into their genomes by crossbreeding between different feline species or cat breeds2129303233Because RDRS sequences vary depending on where cats live or breed, it is possible that unidentified RDRS loci are present in domestic cat genomes of breeds investigated above. We developed a PCR method to differentiate infectious-type type env . RDRS C2RDRS C2a . As a rea do not . Asian dgag, pol and env genes, and RDRS C1 was most similar to the sequences of infectious RD-114 virus clonespol gene  resulting in amino acid substitutions  had infectivity as an RD-114 virus clone   . Of the  region) . The 27-env region214PstI-digested fragment corresponding to a complete env region was detected in all cat DNAs examined as a single copy in the previous study; however, they did not isolate a clone containing a complete RD-114 provirusPstI-digested env fragments derived from RDRS C2a is similar to infectious RD-114 virus clones, pSc3c and pCRT1, in size (4.7 kb) with high sequence homology . These data suggest that the single locus in all cat genomes which Spodick et al. identified was in fact RDRS C2a and not the complete infectious RD-114 provirus. Another group identified eight RDRSs  in domestic cat genomesBamHI gag fragment specific to the RD-114 virus clones was present in cat genomes as a single copy on chromosome B3; however, domestic cats are polymorphic with respect to the presence or absence of this RD-114 virus-related segment2The RD-114 virus was originally isolated from human RD cells after passages in fetal cat brainsIn this study, we identified six RDRSs in domestic cats genomes. These proviruses are not identical to the sequences of infectious molecular clones of RD-114 virus reported previouslygag, pol and env) are disrupted in RDRS C2a. The other RDRSs have not yet been fixed in domestic cat genomes. Furthermore, we found that most Asian cats are free from new RDRSs harboring infectious-type env.Remarkably, number of RDRS proviral copies vary considerably with type of cell lines and cat breeds. All cats have RDRS C2a in common, but all proviral genes (Felis silvestris lybica)Because domestic cats originated from the African wildcat living in the Middle East (British Shorthair (BSH) was established in England from European Shorthair which first appeared in Sweden, and later American Shorthair (ASH) and American Curl (a variant of ASH) originated from BSHin vitro as observed in other gammaretroviruses such as porcine endogenous retrovirus44env and some other unidentified RDRSs or FcEV.Several feline cell lines including CRFK and FER cells produce RD-114 or RD-114-related viruses, but there are no proviruses identical to previously reported RD-114 viral sequences. Several novel retroviruses have been reported to be generated by the recombination of ERVs4142pol gene, compromising the ability of the Emv2 to produce infectious virus particlespol gene in vivo in aging C57BL micein vivo.Retroviral recombination events occur by template switching during reverse transcription464111235051env. Recently, it was reported that in immune-compromised mice, ERVs in the host genome were resurrected and induced lymphoma5454In the previous study, we revealed ubiquitous expression of the RD-114 viral receptor, termed ASCT2, in domestic cat tissuesAll animal care and procedures that were performed in this study conformed to the guidelines for animal experiments at Institute for Virus Research in Kyoto University (IVRKU), and all experimental protocols were approved by the Committee on the Ethics of Animal Experiments of IVRKU. Blood samples of domestic cats were collected for diagnostic purpose in veterinary clinics. Genomic DNAs of Leopard cats, serval and snow leopard were stored samples which had been used in our previous studies56Domestic cat blood samples for isolation of PBMCs were provided from Fujimura Animal Hospital , Kyoritsu Seiyaku Corporation  and Veterinary Clinics in Japan. The Snow leopard and Serval cat tissue samples were provided from Sapporo Maruyama Zoo . These animals died from feline parvovirus infectionFeT-J and 3201 cells were cultured in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 IU/ml) and streptomycin (100 μg/ml) . CRFK, G355-5, fcwf-4, FER, AH927 (feline embryonic fibroblast), HEK293T and TE671 (human rhabdomyosarcoma) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% FCS, penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Invitrogen).Genomic DNA was extracted from cell lines and PBMCs using a QIAamp DNA Blood Mini Kit . Genomic PCR was performed by using PrimeSTAR GXL polymerase  or Ex Taq polymerase (TaKaRa). The former was used for cloning RDRSs and the recombinant RDRV, and the later was used for screening for the presence of RDRSs. Amplicons were analyzed by electrophoresis in 0.8% or 2% agarose gels. Primers used in this study are listed in HindIII for detection of gag and env genes, SacII for detection of gag and pol genes, EcoRI for detection of env genes, or SphI for detection of gag and env genes. Digested fragments were separated by a 1.0% agarose gel and transferred to positive charged-nylon membrane . Hybridization was performed using digoxigenin (DIG)-labeled PCR probes and DIG easy Hyb (Roche) at appropriate temperatures  for 16 hours. After hybridization, the membrane was washed with a low stringency buffer (0.1% SDS and 2 × SSC) at room temperature followed by a high stringency buffer (0.1% SDS and 0.1 × SSC) at 68°C. The hybridized bands were visualized using CDP-Star reagent (Roche) following the manufacturer's instructions. Probes were prepared from an infectious molecular clone of RD-114 virus, termed pCRT1A total of 5 μg (or 3 μg) genomic DNA was isolated from PBMCs or cell lines, digested with EcoRI or SphI and 100 ng of digested genomes were ligated by T4 ligase  at 16°C overnight. Ligated DNA was amplified with specific primers listed in Genomic DNA was digested with Full-length RDRSs were amplified using specific primers (listed in 4 cells per well one day before infection and diluted supernatants of inoculated HEK293T(LacZ) cells were inoculated onto TE671 cells in the presence of 8 μg/ml polybrene. Two days after inoculation, the inoculated cells were stained with X-Gal, and virus titers, expressed as focus forming units (f.f.u.)/ml, were determined as described previously35HEK293T cells were transfected with 4 μg of RD-114 virus molecular clones or plasmids containing RDRSs using Lipofectamine2000 (Invitrogen). The pcDNA3.1 vector was used for mock transfection. Infectivities of RDRSs were examined by the LacZ marker rescue assayDpnI methodDpnI to eliminate the parental plasmid DNA. Digested DNA fragments were purified and transformed into Escherichia coli DH5 alpha.RDRS C1 mutants were made by 6 cells per well one day before infection and diluted supernatants of the inoculated HEK293T cells (36 d.p.i.) were inoculated onto TE671 cells in the presence of 8 μg/ml polybrene. Six hours after inoculation, genomic DNAs were extracted and proviruses were amplified by PCR. PCR was performed with PrimeSTAR GXL polymerase (TaKaRa) and primers listed in HEK293T cells were transfected with 4 μg of pCRT1 or plasmids containing RDRSs using Lipofectamine2000 (Invitrogen). pcDNA3.1 vector (Invitrogen) was used for mock transfection. Culture supernatants were collected 48 hours post-transfection and filtrated through 0.45 μm-pore-size filters and then inoculated into HEK293T cells with 8 μg/ml polybrene (Sigma-Aldrich). After inoculation, supernatants were collected and filtrated every four days. TE671 cells were seeded in 6-well plates at 1 × 10Sequencing was performed by a commercial DNA sequencing service . Alignment of nucleotide sequences was computed using L-INS-i program in MAFFT version 7RDRS A2, C2a, C1, E3, D4 and C2b are deposited in GenBank as LC005744- LC005749.S.S. and T.M. designed the experiments. S.S. performed the experiments. S.S., S.N. and T.M. analyzed data and wrote the manuscript. All authors read and approved the final manuscript.Supplementary Information


Microsatellite markers containing simple sequence repeats (SSRs) are a valuable tool for genetic analysis. Date palm is a dioecious and slow flowering and is very difficult to identify the gender of the trees until it reaches the reproductive age (5–10 years). A total of 12 microsatellite primers were used with 30 date palm samples, 14 parents  and 16 progeny (developed from parents breeding) which showed that microsatellites were highly polymorphic, having a great number of alleles. A total of 124 alleles were characterized in 12 SSR loci. On average, there are 9.08 alleles per locus, with a range from 5 to 16 alleles, for primers mpdCIR15 and mpdCIR57, respectively. These primers produced 15 polymorphic loci specifically in male date palm samples and the seedlings harboring the unique fragments were further characterized as male plants. Increasingly, 38.46 % of these loci were scored as homozygous alleles while 61.53 % heterozygous allelic loci were determined. Primer mpdCIR48 produced a specific locus (250/250) in all male samples whereas the same locus was absent in female samples. Similarly, a locus of 300/310 bp reoccurred in 5 date palm male samples using marker DP-168 which indicated that these are the promising candidate marker to detect the sex in date palm seedlings at early stage. The data resulted from combination of 12 primers enabled the 16 seedling samples progeny (developed from parents breeding) of date palm cultivars to divide into two groups i.e., male and female regarding their sex expression comparative to the parents  using the principle coordinate analysis. Amplification was carried out in veriti 96-well fast thermal cycler (Peq Lab) under the following conditions: initial DNA denaturation at 95 °C for 10 min, 35 cycles  and final extension at 72 °C for 7 min. Resolution of PCR products of 30 date palm parents and progeny amplified by 12 SSR primers were visualized by 6 % denaturing polyacrylamide (PAGE) gel followed by ethidium bromide.Polymerase chain reaction (PCR) was carried out using 20 µL of reaction mixture containing 2 µL (10 ng) of total genomic DNA, 2 µL of 10× buffer solution [ binary format  for allele(s) on respective locus. Efficacy and degree of polymorphism of reported SSR markers were assessed through power marker  in all male plants whereas the same locus was absent in female plants. Primer mpdCIR25 identified six male plants with a specific homozygous locus 270/270. Similarly primer mpdCIR10 amplified two different fragments, one homozygous locus (230/230 bp) in male-2, while one heterozygous locus (230/240) in male-4 and male-5. The marker mpdCIR16 scored one homozygous fragment (190/190 bp) in male-1 and another fragment of 180/180 bp was identified in male-3. Primer mpdCIR25 characterized six male plants out of eight and a locus of 270/270 bp was present in male samples of 1, 2, 4, 5, 7, 8. Likewise, mpdCIR32 amplified a specific fragment 290/294 bp in three male samples. Three heterozygous loci were scored in male-4, 5 and 6 while a single homozygous locus (235/235 bp) was only amplified in male-8.Great variations were observed in primer mpdCIR57, where a locus of 85/85 bp was found in male-3 and male-4 and a fragment of 295/295 bp was found in male-2 that is the most promising locus to determine the male plants from the progeny. Moreover, primer mpdCIR70 differentiated the two male plants with a heterozygous locus 300/310 bp and a similar locus was identified in male-2, 3, 4, 5 and male-6 with the primer DP-168. Following two allele sized 170/175 bp amplified with primer mpdCIR 93 and 160/162 bp (exhibited by primer mpdCIR93) were repeated twice in male 4 and 7, but these alleles were not observed in six female date palm trees. The data resulted from combination of 12 primers enabled the seedling samples of date palm cultivars to be divided into two groups regarding their sex expression  compared to their parents using the principle coordinate analysis (PCoA). Preferably Hamming distance was selected because, for shared characteristics it does not consider the common absence of alleles. For present study it was therefore considered to be most suitable, that included highly polymorphic data of microsatellite straddling policy at two levels. The PCoA suggested the total four broad groups comprising 8 male parents, 6 female parents, 11 male progenies and 5 female progenies. Trees are autonomous with 29 % variations explained by first axis and 14 % by the second axis. Figures In date palm, limited numbers of suckers are produced, i.e., 15–20 throughout the female tree life, difficult to handle the large population, tedious and costly, need space and resources, so farmers are currently facing the problem to propagate from seeds. But farmers are unable to identify the gender of the date palm trees until it reaches the reproductive age (5–10 years) as this species is very slow flowering. The use of the flowering and vegetative characters Rhouma  and isozPresent study is the first comprehensive investigation on the sex identification of date palm seedlings with SSR markers from Pakistan. The 1-year-old date palm seedlings were characterized as male and female, and identified candidate markers involved in sex determination. This study will provide a substantial resource for speeding up the date palm breeding programs. These results will be helpful for an efficient screening, management and use of date palm genetic resources in selection and breeding program.