Closterovirus and Ampelovirus with monopartite genomes transmitted respectively by aphids and mealybugs and the Crinivirus with bipartite genomes transmitted by whiteflies. The Closteroviridae consists of more than 30 virus species, which differ considerably in their phytopathological significance. Some, like beet yellows virus and citrus tristeza virus (CTV) were associated for many decades with their respective hosts, sugar beets and citrus. Others, like the grapevine leafroll-associated ampeloviruses 1, and 3 were also associated with their grapevine hosts for long periods; however, difficulties in virus isolation hampered their molecular characterization. The majority of the recently identified Closteroviridae were probably associated with their vegetative propagated host plants for long periods and only detected through the considerable advances in dsRNA isolation and sequencing of PCR amplified replicons. Molecular characterization of CTV and several other Closteroviridae revealed that, in addition to genomic and subgenomic RNAs, infected plants contain several different subviral defective RNAs (dRNAs). The roles and biological functions of dRNAs associated with Closteroviridae remain terra incognita.The family Closteroviridae consists of two genera,  Despite the sophisticated replication machinery that viruses have developed for their survival, it is difficult to understand how some large, single-stranded, single-component RNA genomes, such as the ~30-kb genome of animal-infecting coronaviruses and most of the 15- to 20-kb genomes of Closteroviridae members, manage to survive in the face of incidental degradation and targeted dicing by the active host defense silencing machinery within their host cells and/or as they are carried by phloem-feeding aphid vectors. The biological cost of such a situation, in which every single fracture at each of the c. 20,000 possible fragile targets could lead to RNA disruption and to total genetic and energetic loss, could have been detrimental to the continuous survival of such big genomes. In addition, CTV must also contend with the grave consequences of high error rates of viral RNA replication , which fd genome . Furtherd genome , especiad genome . The gend genome . Geneticd genome .turnip crinkle virus (TCV) that reduces virus accumulation causes increased rather than decreased severity of disease symptoms  satellite RNAs, with sequences that are mostly or completely unrelated to their “helper” viruses and (ii) dRNAs that do not interfere with their helpers, or defective interfering RNAs (DI-RNAs) whose presence results in less virus accumulation and often in milder symptoms. Interestingly, however, the DI-RNA of symptoms . These dcis-acting elements necessary for replication of the parent virus and all are missing some of the genetic elements necessary for some essential virus functions, such as replication, encapsidation, or the ability to spread within a host.Only the replication of satellite RNAs and some dRNAs and DI-RNAs is dependent upon enzymes encoded by their helper viruses. However, a few other dRNAs are able to replicate autonomously in inoculated cells, although their ability to spread and move is restricted in intact plants. DI-RNAs and dRNAs have been reported for many animal and plant viruses and the characterization of their sequences has revealed a mosaic of truncated forms, suggesting a variety of situations that could have lead to their emergence. All dRNAs possess some of the beet yellows virus  woody hosts that are polyphenol rich handicapped purification of many of these viruses and delayed their characterization until finding that Closteroviridae infections are associated with large amounts of dsRNA  have bipartite genomes, although a virus with a tripartite genome, potato yellow vein virus (PYVV), was assigned to this genus  RNA molecules that consist of full-length plus and minus strands and a large number (up to 30 or more) of 5′- and 3′-sgRNAs. Interestingly, while the 5′-sgRNAs are all single-stranded and positive, the 3′-sgRNAs include both positive- and minus-strand molecules. Readers are referred to several other chapters in this series, which provide considerably more detailed discussions of the CTV genome and the replication strategies of this virus.A schematic presentation of the CTV genomic RNA is shown in One of the most prominent features of CTV, which was revealed soon after its genome was first characterized , is thatMany CTV isolates contain one or more dRNAs of various sizes. Most of these dRNA molecules consist of two genomic termini, with extensive internal deletions . It is iFigure 1).For convenience, CTV-defective RNAs (dRNAs) can be grouped into different classes. These classes are described below  that have been reported in CTV-infected plants (Class 3 CTV-dRNAs possess large (~12 kb) encapsidated dRNA molecules that are infectious when mechanically transmitted to citrus plants and d plants . The 3′ d plants . Two of in planta and there is no evidence for their ability to spread systemically within inoculated plants, they have become useful genetic platforms for the study of sgRNA transcription  can be transmitted to citrus plants by mechanical inoculation. However, the transmission of LD-RNA2 to protoplasts has been shown to be limited and cannot be detected by RT-PCR until 4 days after inoculation , 2003.Class 5: CTV-dRNAs vary in size (1.7–5.1 kb) and contain sequences that point to double-recombination events (DR). These sequences are comprised of two termini and a non-contiguous internal sequence from ORF2. Interestingly, LD-RNA2 and DR-dRNAs from three different CTV isolates all contain an identical 948-nt 5′ region.Class 6: CTV-dRNAs have variable regions between the 5′ and 3′ termini and inserts of short (14–17 nt) sequences that have no homology with the CTV genome . The quelettuce infectious yellows virus . Components of the replication-associated machinery were found to be involved in the evolution of other viruses and intergenic regions are known to be the preferred crossover sites for brome mosaic virus (BMV) recombinants  and Class 4 dRNAs harboring the entire battery of ORFs analogous to the RNA 2 of criniviruses suggests that similar forms of dRNA may have led to the evolution of the bipartite criniviruses from a monopartite velarivirus parent. In addition, dsRNA molecules corresponding to one or more major CTV-dRNAs have been found to account for substantial amounts of the total dsRNA found in CTV-infected plants. Since dsRNA molecules can be considered the dead ends of the viral replication process, their abundance in Closteroviridae  naturallterra incognita.In our early work, we could not associate any specific biological phenomena with the 2.4-kb dRNA or some of the other dRNA molecules. After two decades of continuous transmission of the VT isolate in Mexican lime and Alemow seedlings, the symptoms observed on the Alemow plants remained unchanged. However, among the SO (sour orange) plants that used to test for SY (seedling yellows), we noticed that some failed to exhibit the typical SY symptoms. The examination of the dRNAs of Alemow plants infected with CTV-VT subisolates that exhibited SY and non-SY reactions revealed the presence of one major dRNA population. The main difference between both subisolate groups was the association of 4.5- and 2.4-kb dRNAs with non-SY- and SY-reacting VT isolates, respectively. Furthermore, VT isolate #12, which contains a large dRNA with a complete 5′ moiety of the gRNA, did not induce the stem-pitting symptoms typically observed in infected Alemow plants. However, the possibility of differences between SY and non-SY, or stem pitting(SP) and non-SP, isolates in other genomic regions was not ruled out and the considerable progress that has been made recently in the field of sequencing techniques is expected to clarify this issue. In conclusion, the roles and biological functions of the numerous dRNAs associated with Closteroviridae remain The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pomegranate juice and its by-products substantially reduced macrophage cholesterol and oxidized lipids accumulation, and foam cell formation , leading to attenuation of atherosclerosis development, and its consequent cardiovascular events.The current paper summarizes the antioxidative and antiatherogenic effects of pomegranate polyphenols on serum lipoproteins and on arterial macrophages (two major components of the atherosclerotic lesion), using both  In urine, the main metabolites observed were the 6H-dibenzo  pyran-6-one derivatives, and were present as aglycones or as glucuronides. It was concluded that since only 3%–6% of the ingested punicalagin was detected as such or as metabolites in urine and feces, the majority of this ellagitannin has to be converted to undetectable metabolites or accumulated in nonanalysed tissues. Only traces of punicalagin metabolites were detected in liver or kidney. In humans, following consumption of PJ (180 mL) containing 25 mg of ellagic acid and 318 mg of hydrolysable ellagitannins , ellagic acid was detected in human plasma 1 hour after ingestion at a maximum concentration of 32 ng/mL, and by 4 hours it was completely eliminated . CIn vitro studies clearly show that PJ exhibit direct antiatherogenic effects on macrophages. Preincubation of J774A.1 macrophages with PJ resulted in a significant (P < 0.01) reduction in Ox-LDL degradation by 40% [n by 40% . On the μM), a dose-dependently decrease (by up to 30%) in cellular triglyceride content  MPM PON2 mRNA expression, and PON2 was previously shown to inhibit DGAT1 activity [μmol/L) to microsomes from PON2KO MPM still resulted in a significant reduction in DGAT1 activity, by ~50%, suggesting a direct, non-PON2-mediated effect of PJ on macrophage triglycerides [in vitro study, we have recently demonstrated that PJ consumption by C57BL/6 mice, by PON1KO mice, or by PON2KO mice resulted in a significant reduction in MPM triglyceride content by 17%, 16%, and 23%, respectively , a d This study demonstrates that PJ beneficial effects on macrophage triglyceride metabolism are direct effect of PJ, which are not mediated via PJ-induced stimulation of macrophage PON2.We conclude that PJ protects the macrophages against oxidative stress by increasing PON2 expression, suppressing Ox-LDL uptake by macrophages, inhibiting cellular cholesterol and triglyceride biosynthesis, or stimulation of HDL-mediated cholesterol efflux from the cells. All these effects could lead to attenuation in cellular cholesterol and triglyceride accumulation, and foam cell formation.Pomegranate extract prepared from the whole fruit contains an approximately 20-fold increased antioxidant activity in comparison to the level in the pomegranate aril juice . The antμg/mL), TPT was more potent than vitamin E or PJ when analyzed as a free radical scavenger. TPT reduces the absorbance in the DPPH solution by 75% in comparison to 62% and 48% reduction obtained by a similar total polyphenol content of PJ or by vitamin E, respectively. LDL oxidation induced either by copper ions or AAPH, was dose-dependently inhibited by TPT, with an IC50 of 2.1 and 1.4 μg/mL, respectively. Macrophage oxidative status was also substantially decreased by 50% on using 80 μmol/mL of TPT polyphenols. At a similar concentration, punicalagin was found to be even more potent than TPT in all the above assays.A product of total pomegranate tannins (TPT) containing 85% punicalagin, 1.3% ellagic acid, and a small amount of ellagic acid glycosides was purified from the pomegranate husk extract prepared by Dr. Navindra P. Seeram from the laboratory of Dr. David Heber at UCLA, USA . On a siThe antioxidative properties of pomegranate polyphenol extract powder, as well as of pomegranate fiber powder were also analyzed. The pomegranate fiber powder polyphenol concentration was 8-fold lower, as compared to the polyphenols in the pomegranate extract powder . The pomegranate extract was significantly more potent than the fiber powder in both scavenging of free radicals and in reducing macrophage oxidative stress. 0 mice were studied [in vitro. Treatment of J774A.1 macrophages with PBP , significantly decreased both cellular total peroxide content and Ox-LDL uptake. We thus conclude that PBP significantly attenuates atherosclerosis development by its antioxidant properties. The effects of a pomegranate liquid by-product , on atherosclerosis development in E studied . Consumpex vivo and in vitro studies, in which we compared the antiatherogenic properties of whole pomegranate fruit powder by- product (POMxp) to those of pomegranate by-product POMarils+seeds powder. Per mg powder, the POMxp contained 90-fold higher concentrations of polyphenols than the POMarils+seeds. Administration of 10 mg total polyphenols/kg/d of POMxp or of POMarils+seeds aqueous extract to E0 mice for 2 months, demonstrated that POMxp polyphenols are more potent than POMarils+seeds polyphenols in reducing oxidative stress. The amount of total peroxides in MPM from the mice that consumed POMarils+seeds, or POMxp was decreased by 14% or by 24%, respectively, as compared to MPM from placebo-treated mice  to J774A.1 macrophages, with 53% or 27% reduction in Ox-LDL uptake by the cells, respectively. Upon adding 41 μg of gallic acid equivalents/mL of POMxp or POMarils+seeds to the cells, HDL-mediated cholesterol efflux was significantly stimulated by 147% and by 52%, respectively. The extent of Ox-LDL uptake by MPM from mice that consumed POMxp, but not by MPM from the mice that consumed POMarils+seeds, was significantly reduced by 13%, as compared to Ox-LDL uptake by the placebo MPM . Furtherctively, , indicat0 mice , the atherosclerotic lesion area was significantly decreased by 44, 39, or by as much as 70%, respectively, as compared to lesion area observed in the placebo-treated group , arils (POMarils), and flowers (POMflower), in comparison to the whole pomegranate juice (PJ) . After ced group . POMariled group . POMflow content  and signt  to reduce macrophage oxidative stress and to attenuate macrophage cholesterol accumulation and atherosclerotic lesion development.PJ was shown to attenuate the accumulation of cholesterol in macrophages due to: (a) inhibition of cellular cholesterol biosynthesis, (b) inhibition of cellular Ox-LDL uptake, and (c) stimulation of HDL-mediated cholesterol efflux from macrophages. Furthermore, PJ also protects macrophages from triglyceride accumulation. Like PJ, pomegranate by-products were similarly shown  from the whole fruit. Since combination of antioxidants, as exist in PJ can provide a wider range of free radicals scavenging capacities than an individual antioxidant, clinical and nutritional studies in humans should be directed towards the use of combinations of several types of dietary antioxidants, including combinations of flavonoids together with other nutritional antioxidants, such as vitamin E and carotenoids. It is also important to use reliable biological markers of oxidative stress and to identify populations suitable for antioxidant treatment, as antioxidants treatment may be beneficial only in subjects which are under oxidative stress.All these antioxidative and antiatherogenic effects of pomegranate polyphenols were clearly demonstrated


Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E–like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus. Hepevirus. Its single-stranded, positive-polarity RNA genome of 6.6–7.3 kb harbors 3 major open reading frames (ORFs) flanked by a capped 5′ end and a poly A at the 3′ end. ORF1 at the 5′ end of the genome codes for several nonstructural proteins, ORF2 encodes the immunodominant capsid protein, and the partially overlapping ORF3 codes for a cytoskeleton-associated phosphoprotein with multiple functions (Hepatitis E virus (HEV) is a nonenveloped virus, diameter 30–34 nm, that belongs to the genus ,Rattus norvegicus, R. rattus, R. exulans) but also in some noncommensal wild rodent species (–R. norvegicus) trapped as part of the Rodent-borne Pathogens network (which coordinates activities with regard to rodent trapping during outbreaks) (,Hepatitis E, an acute self-limiting disease, occurs worldwide; large outbreaks have occurred in developing countries, as was recently reported from Uganda (During July 8–16, 2009, a total of 6 Norway rats, 3 male and 3 female, 65–432 g, were trapped in manholes of the sewer system of Hamburg, northern Germany, at the same locations where ≈12 months before HEV RNA had been detected in rat feces . Using pTo compare viral load in different tissues of the 2 HEV–positive rats, we developed a real-time RT-PCR selective for a region in the ORF2 of rat HEV. Parallel analysis of RNA isolated from 10 mg of each tissue or 10 µL of blood reproducibly showed the highest viral load to be in the liver; cycle threshold values for liver were 20.5 and 21.6 for each animal and lower for all other tissues . FurtherHepevirus no species demarcation criteria have been defined, the marked sequence diversities suggest that the rat HEV represents an additional virus species other than HEV-1, HEV-2, HEV-3, HEV-4, and the tentative species avian HEV, which are currently classified in this genus (Phylogenetic analyses and nucleotide and amino acid sequence comparisons demonstrated that the complete rat HEV genome sequences were consistently well separated from those of mammalian genotypes 1–4 and the tentative avian genotype. This finding suggests that these sequences represent an additional genotype , Table. Detection of rat HEV RNA and antigen in the liver cells of the infected Norway rats may indicate hepatotropism of this virus. Therefore, regarding its organ and cell-type tropism, this virus seems to be similar to the human and pig HEV genotypes (


Eucalyptus are the most planted for the fiber crop in the world. They are mainly utilized for timber, pulp and paper production. Brazil, helped by the favorable weather conditions, appears as a big producer and exporter of eucalyptus derivates. In 2002, the Brazilian network research of the Eucalyptus Genome (Genolyptus) was established with the goal of integrating several academic and private institutions currently working with eucalyptus genomics in Brazil. This project generated around 200.000 ESTs from several tissues and conditions. Consequently, several individual projects have been implemented generating other transcriptome databases, in special, using RNA-Seq technology. In 2010, a draft genome  of the specie E. grandis was produced by researches of the Joint Genome Institute (DOE-JGI) and the Eucalyptus Genome Network (EUCAGEN). The main goal of this work is to develop an Eucalyptusdatabase (http://www.lge.ibi.unicamp.br/genolyptus) integrating public and private data in a friendly and secure web interface with bioinformatics tools that allowing the users perform complex searches.The species of the genus First, the public and private ESTs  were assembled producing 48,760 unigenes . Basically, the bdtrimmer [The autofact pipeline  was usedhttp://www.lge.ibi.unicamp.br) sequenced ten RNA-Seq libraries from four species  using the Illumina/Solexa technology. Additionally, three RNA-seq libraries [The Genomic and Expression Laboratory at State University of Campinas (ibraries  were dowibraries  and TopHibraries  alignersIn order to perform a differential expression analysis between ESTs or RNA-seq libraries some normalization pipelines and statistical tests have been implemented.From ESTs, the differentially expressed genes between libraries were performed applying AC test  in assemTo integrate all data described above, we developed a web site Fig.  hosted i


AbstractTrichosanthes, is the largest genus in the Cucurbitaceae family, with over 90 species. Recent molecular phylogenetic data have indicated that the genus Gymnopetalum is to be merged with Trichosanthes to maintain monophyly. A revised infrageneric classification of Trichosanthes including Gymnopetalum is proposed with two subgenera, (I) subg. Scotanthus comb. nov. and (II) subg. Trichosanthes, eleven sections, (i) sect. Asterospermae, (ii) sect. Cucumeroides, (iii) sect. Edulis, (iv) sect. Foliobracteola, (v) sect. Gymnopetalum, (vi) sect. Involucraria, (vii) sect. Pseudovariifera sect. nov., (viii) sect. Villosae stat. nov., (ix) sect. Trichosanthes, (x) sect. Tripodanthera, and (xi) sect. Truncata. A synopsis of Trichosanthes with the 91 species recognized here is presented, including four new combinations, Trichosanthes orientalis, Trichosanthes tubiflora, Trichosanthes scabra var. pectinata, Trichosanthes scabra var. penicaudii, and a clarified nomenclature of Trichosanthes costata and Trichosanthes scabra.The snake gourd genus,  Trichosanthes L. is the largest genus in the Cucurbitaceae family, with over 90 species. The genus has its center of diversity in Southeast Asia, but ranges from India throughout Asia east to Taiwan, the Philippines and Japan, and southeast to New Guinea, Australia, Fiji and Vanuatu (Trichosanthes cucumerina L.) is a popular vegetable in South and Southeast Asian cuisine and PageBreakcultivated in tropical and subtropical regions around the globe. Gymnopetalum Arn. includes four species , and incomplete herbarium collections, complicate taxonomical studies and have contributed to the description of nearly 300 taxa  sect. Cucumeroides (Gaertn.) Kitam. including subsect. Cucumeroides (Gaertn.) Kitam. and subsect. Tetragonosperma (C.Y.Cheng & Yueh) Rugayah, (iii) sect. Edulis Rugayah, (iv) sect. Foliobracteola C.Y.Cheng & Yueh, (v) sect. Involucraria (Ser.) Wight including subsect. Bracteatae C.Jeffrey ex S.K.Chen and subsect. Pedatae (C.Y.Cheng & Yueh) C.Jeffrey ex S.K.Chen, and (vi) sect. Asterosperma W.J.de Wilde & Duyfjes. However, both Infrageneric classifications of the genus  authors . The mos authors  propose Trichosanthes including 3(-4)-porate and 3(-4)-colporate pollen with psilate, perforate, verrucate, reticulate, and regulate ornamentation. Their study of pollen from 37 species distinguishes five pollen types, two of which are further divided into subtypes, and categorized these using exine ornamentation patterns for the major types and ectoaperture characters for the subtypes.Pollen morphology has also been used for infrageneric classification in the genus , but as PageBreakGymnopetalum strongly resembles certain Trichosanthes species, but lack thread-like fringes on the petals, and the overall shape of the folded petals in the mature bud is elongate (short and rounded in Trichosanthes)  sect. Gymnopetalum containing the type Gymnopetalum tubiflorum (Wight & Arn.) Cogn. from southern India and Sri Lanka, and (ii) sect. Tripodanthera (M.Roem.) Cogn. containing the Southeast Asian and Malesian species. Later authors did not follow this classification (santhes) . A revissanthes) . Cogniaufication .Trichosanthes by de Trichosanthes and Gymnopetalum are both non-monophyletic, but together form a clade with high support in the Bayesian tree and weak support in the ML tree (0.99/62). This indicates that Gymnopetalum should be merged with Trichosanthes, and that a revised infrageneric classification is necessary. Some previously recognized sections in Trichosanthes and Gymnopetalum are well supported, but others need to be described or redefined.The molecular phylogenetic study of Trichosanthes and Gymnopetalum by de Gymnopetalum and the infrageneric classification of Trichosanthes. The species of Gymnopetalum are placed in different clades within Trichosanthes, with the sect. Gymnopetalum, including the type Gymnopetalum tubiflorum, grouping in a well-supported clade (1.00/84) together with species of sect. Trichosanthes and sect. Cucumeroides. The sect. Tripodanthera, consisting of the three other species in the genus, Gymnopetalum orientale W.J.de Wilde & Duyfjes, Gymnopetalum chinense (Lour.) Merr., and Gymnopetalum scabrum (Lour.) W.J.de Wilde & Duyfjes, forms a well-supported clade (1.00/86) together with the taxa in Trichosanthes sect. Edulis.The molecular phylogeny of Trichosanthes the support for the two clades here defined as subgenera Trichosanthes (1.00/94) and Scotanthus (1.00/97) is high. However,splitting the genus into two genera corresponding to subg. Scotanthus and subg. Trichosanthes would not improve clarity, as both would consist of species with fringed and fringeless corollas. Maintaining a large Trichosanthes is in accordance with the recent taxonomic revisions of the genus  sect. Cucumeroides (1.00/93), including subsect. Cucumeroides (0.99/-) and subsect. Tetragonosperma (0.99/69), (ii) sect. Edulis (1.00/75), and (iii) sect. Asterosperma (1.00/100). The subsections of sect. Cucumeroides are statistically supported, but subsect. Cucumeroides consists solely of accessions of Trichosanthes pilosa Lour. and species that have been reduced to its synonymy (Involucraria is only weakly supported (0.83/65), primarily due to the low support for inclusion of its type, Trichosanthes wallichiana (Ser.) Wight. The subsections of Involucraria are not supported, and taxa belonging to subsect. Pedatae are nested in different locations within subsect. Bracteatae. Sect. Tripodanthera is not supported by the analysis, and could possibly form a grade at the base of sect. Edulis. However, morphological support for this section is strong as all taxa share characters of flower morphology, i.e. fringeless corollas. Sect. Foliobracteola Cheng & Yueh, which in its original sense included the species related to Trichosanthes kirilowii Maxim. and Trichosanthes villosa Blume . Section Truncata, in its original sense including Trichosanthes truncata C.B.Clarke, Trichosanthes kerrii Craib, Trichosanthes homophylla Hayata  proposed to place it in section Foliobracteola subsect. Villosae based on it pollen morphology. The current phylogenetic data place the accessions of Trichosanthes subvelutina as sister (1.00/91) to a well-supported clade (1.00/84) consisting of sections Gymnopetalum, Trichosanthes, and Cucumeroides, and the species is here placed in a separate section, sect. Villosae (Yueh & L.Q.Huang) H.J.de Boer.PageBreakTrichosanthes subg. Trichosanthes, one including sections Asterosperma, Pseudovariifera, Foliobracteola, and Truncata, and the other including sect. Villosae, with Trichosanthes subvelutina . The latter clade is sister to the clade consisting of sections Trichosanthes, Gymnopetalum and Cucumeroides (1.00/84). The remaining sections in subg. Trichosanthes have porate ectoapertures, and varying exine ornamentation including psilate, (micro-)reticulate, perforate, verrucate, and rugulate pollen .Species with colporate ectoaperturate pollen form two monophyletic groups in e pollen . Gymnopee pollen , whereasTrichosanthes, in the common ancestors of: 1) sect. Involucraria; 2) sect. Edulis; and 3) sections Trichosanthes, Gymnopetalum and Cucumeroides.The other genera in the tribe Sicyoeae have colpate-colporate pollen, similar to that in many other distantly related groups in Curcurbitaceae . In the A revision of the infrageneric classifications suggested by previous authors on the basis of morphological studies  is here PageBreakTrichosanthes L. (1753) Sp. Pl. 2: 1008 – Type: Trichosanthes anguina L. [= Trichosanthes cucumerina L.]Trichosanthes subg. Scotanthus (Kurz) H.J.de Boer, comb. nov. – Gymnopetalum subg. Scotanthus Kurz (1877) J. Asiat. Soc. Bengal, Pt. 2, Nat. Hist. 46: 99. – Lectotype, designated here: Bryonia cochinchinensis Lour. [ = Trichosanthes costata Blume]Scotanthus Naud., nom. illeg. (1862) Ann. Sci. Nat., Bot. sér. 4, 16: 172. – Type: Momordica tubiflora Roxb. [ = Trichosanthes costata Blume]Trichosanthes sect. EdulisRugayah (1999) Reinwardtia 11: 232. – Type: Trichosanthes edulis Ruguyah.Trichosanthes densiflora Rugayah (1999) Reinwardtia 11: 252Trichosanthes dentifera Rugayah (1999) Reinwardtia 11: 253Trichosanthes dieniensis Merr. & L.M.Perry (1949) J. Arnold Arbor. 30: 59Trichosanthes edulis Rugayah (1999) Reinwardtia 11: 254Trichosanthes hastata Harms (1925) Bot. Jahrb. Syst. 60: 160Trichosanthes laeoica C.Y.Cheng & Lu Q.Huang (1996) Bull. Bot. Res., Harbin 16: 503Trichosanthes odontosperma W.E.Cooper & A.J.Ford (2010) Austrobaileya 8: 126Trichosanthes pulleana Harms (1925) Bot. Jahrb. Syst. 60: 160Trichosanthes schlechteri Harms (1925) Bot. Jahrb. Syst. 60: 159Trichosanthes sect. Involucraria(Ser.) Wight (1840) Madras J. Lit. Sci. 12: 52. – Involucraria Ser. (1825) Mém. Soc. Phys. Genève 3: 27, t. 5. – Type: Involucraria wallichiana Ser. [ = Trichosanthes wallichiana (Ser.) Wight]Trichosanthes anamalaiensis Bedd. (1864) Madras J. Lit. Sci. III, 1: 47Trichosanthes borneensis Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 369Trichosanthes bracteata (Lam.) Voigt (1845) Hort. Suburb. Calcutt. 58Trichosanthes celebica Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 385Trichosanthes cordata Roxb. (1832) Fl. Ind. 3: 703Trichosanthes coriacea Blume (1826) Bijdr. Fl. Ned. Ind. 15: 935Trichosanthes dolichosperma Duyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 84Trichosanthes dunnianaH. Lév. (1911) Repert. Spec. Nov. Regni Veg. 10: 148Trichosanthes ellipsoidea Merr. (1918) Philipp. J. Sci., C 13: 332Trichosanthes elmeri Merr. (1929) Univ. Calif. Publ. Bot. 15: 299Trichosanthes emarginata Rugayah (1999) Reinwardtia 11: 258Trichosanthes erosaDuyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 85Trichosanthes fissibracteata C.Y.Wu ex C.Y.Cheng & C.H.Yueh (1974) Acta Phytotax. Sin. 12: 438Trichosanthes floresana Rugayah (1999) Reinwardtia 11: 260Trichosanthes globosa Blume (1826) Bijdr. Fl. Ned. Ind. 15: 936Trichosanthes intermedia W.J.de Wilde & Duyfjes (2004) Sandakania 14: 19Trichosanthes inthanonensis Duyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 86Trichosanthes khasiana Kundu (1939) J. Bot. 77: 11Trichosanthes kinabaluensis Rugayah (2000) Reinwardtia 11: 419Trichosanthes kostermansii Duyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 89PageBreakTrichosanthes laceribractea Hayata (1911) J. Coll. Sci. Imp. Univ. Tokyo 30. Art. 1: 117Trichosanthes lepiniana (Naud.) Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 377Trichosanthes leuserensis Rugayah (1999) Reinwardtia 11: 265Trichosanthes longispicata Rugayah (1999) Reinwardtia 11: 266Trichosanthes montana Rugayah (1998) Reinwardtia 11: 218Trichosanthes morrisii W.E.Cooper (2011) Austrobaileya 8: 381Trichosanthes obscura Rugayah (1999) Reinwardtia 11: 269Trichosanthes pallidaDuyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 90Trichosanthes papuana F.M.Bailey (1900) Queensland Agric. J. 7: 349Trichosanthes pedata Merr. & Chun (1934) Sunyatsenia 2: 20Trichosanthes pentaphylla F.Muell. in Benth. (1867) Fl. Austral. 3: 314Trichosanthes philippinensis Rugayah (1999) Reinwardtia 11: 271Trichosanthes planiglans Rugayah (1999) Reinwardtia 11: 273Trichosanthes pubera Blume (1826) Bijdr. Fl. Ned. Ind. 15: 936Trichosanthes quinquangulata A. Gray (1854) U.S. Expl. Exped., Phan. 15: 645Trichosanthes quinquefolia C.Y.Wu ex C.Y.Cheng & C.H.Yueh (1980) Acta Phytotax. Sin. 18: 351Trichosanthes refracta C.H.Yueh (1996) Bull. Bot. Res., Harbin 10: 500Trichosanthes rugatisemina C.Y.Cheng & C.H.Yueh (1974) Acta Phytotax. Sin. 12: 440Trichosanthes sepilokensis Rugayah (1999) Reinwardtia 11: 275Trichosanthes subrosea C.Y.Cheng & C.H.Yueh (1980) Acta Phytotax. Sin. 18: 349Trichosanthes tricuspidata Lour. (1790) Fl. Cochinch. 2: 589Trichosanthes valida Rugayah (1999) Reinwardtia 11: 277Trichosanthes wallichiana (Ser.) Wight (1840) Madras J. Lit. Sci. 12: 52Trichosanthes wawrae Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 384Trichosanthes sect.Tripodanthera (M.Roem.) H.J.de Boer, comb. nov. – Tripodanthera M.Roem. (1846) Fam. Nat. Syn. Monogr. 2: 48. – Gymnopetalum sect. Tripodanthera (M.Roem.) Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 390. – Type: Bryonia cochinchinensis Lour. [ = Trichosanthes costata Blume]Trichosanthes costata Blume (1826) Bijdr. Fl. Ned. Ind. 15: 933. — Type: Blume s.n. barcode L0589632,  Fl. Cochinch. 1: 156. – Gymnopetalum chinense (Lour.) Merr. (1919) Philipp. J. Sci. 15: 256 – Type: Loureiro †. Bryonia cochinchinensis Lour. (1790) Fl. Cochinch. 1: 595. – Gymnopetalum cochinchinense (Lour.) Kurz (1871) J. Asiat. Soc. Bengal, Pt. 2, Nat. Hist. 40: 57 –Type: Loureiro s.n. (BM), Vietnam. Momordica tubiflora Roxb. (1832) Fl. Ind. 3: 711 – Type: Wallich Cat. 6749 (holotype? K-W), Dacca, Bangladesh. Note: The existence of Trichosanthes chinensis Ser. (1828) Prodr. [A.P. de Candolle] 3: 315 blocks the transfer of Gymnopetalum chinense, based on the basionym Evonymus chinensis, to Trichosanthes. The second name in line of priority would be Gymnopetalum cochinchinensis, based on the basionym Bryonia cochinchinensis. However, the combination Trichosanthes cochinchinensis M.Roem. (1846) Fam. Nat. Syn. Monogr. 2: 96, based on Trichosanthes cucumerina Lour. PageBreak(1790) 722 (non L.), blocks the transfer. The third name in line of priority is Trichosanthes costata Blume (1826) Bijdr. Fl. Ned. Ind. 15: 933, and this name is available for Gymnopetalum chinense.Trichosanthes scabra Lour. (1790) Fl. Cochinch. 2: 589. – Gymnopetalum scabrum (Lour.) W.J.de Wilde & Duyfjes (2008) Reinwardtia 12: 268. – Type: Poilane 11322  Roxb. (1832) Fl. Ind. 3: 724. – Gymnopetalum integrifolium (Roxb.) Kurz (1871) J. Asiat. Soc. Bengal, Pt. 2, Nat. Hist. 40: 58. –Type: Wallich Cat. 6730 (holotype? K-W), Burma.scabravar. pectinata (W.J.de Wilde & Duyfjes) H.J.de Boer, comb. nov. – Gymnopetalum scabrum (Lour.) W.J.de Wilde & Duyfjes var. pectinatum (W.J. de Wilde & Duyfjes) W.J.de Wilde & Duyfjes (2008) Reinwardtia 12: 268 – Gymnopetalum integrifolium (Roxb.) Kurz var. pectinatum W.J.de Wilde & Duyfjes (2006), Blumea 51: 287. – Type: W.J. de Wilde and Duyfjes 21692 (holotype L), Java, Indonesia.var. penicaudii (Gagnep.) H.J.de Boer, comb. nov. – Gymnopetalum penicaudii Gagnep. (1918) Bull. Mus. Natl. Hist. Nat. 24: 374. – Gymnopetalum scabrum (Lour.) W.J.de Wilde & Duyfjes var. penicaudii (Gagnep.) W.J.de Wilde & Duyfjes (2008) Reinwardtia 12: 268 – Type: Pénicaud 43 (lectotype P), Hainan, China.var. Trichosanthes orientalis (W.J.de Wilde & Duyfjes) H.J.de Boer, comb. nov. – Gymnopetalum orientaleW.J.de Wilde & Duyfjes (2006) Blumea 51: 290. – Type: De Wilde and Duyfjes 21937 (holotype L), Lombok, Indonesia.Trichosanthes subg. TrichosanthesUnresolved placement within this subgenus:Trichosanthes reticulinervis C.Y.Wu ex S.K.Trichosanthes smilacifolia C.Y.Wu ex C.H.Yueh & C.Y.Cheng (1980) Acta Phytotax. Sin. 18: 347Trichosanthes sect. Asterosperma W.J.de Wilde & Duyfjes (2004) Sandakania 14: 6. – Type: Trichosanthes postarii W.J.de Wilde & Duyfjes.Trichosanthes auriculata Rugayah (1998) Reinwardtia 11: 216Trichosanthes fusca W.J.de Wilde & Duyfjes (2004) Sandakania 14: 17Trichosanthes postarii W.J.de Wilde & Duyfjes (2004) Sandakania 14: 26Trichosanthes rotundifolia Rugayah (1998) Reinwardtia 11: 223Trichosanthes sect. Cucumeroides (Gaertn.) Kitam. (1943) J. Jap. Bot. 19: 35. – Cucumeroides Gaertn. (1791) Fruct. Sem. Pl. 2: 485, t. 180, t. 4. – Type: Trichosanthes cucumeroides (Ser.) Maxim. [ = Trichosanthes pilosa Lour.].Trichosanthes adhaerens W.J.de Wilde & Duyfjes (2004) Sandakania 14: 11Trichosanthes beccariana Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 380Trichosanthes mucronata Rugayah (1999) Reinwardtia 11: 268PageBreakTrichosanthes pendula Rugayah (1998) Reinwardtia 11 (3): 219Trichosanthes pilosa Lour. (1790) Fl. Cochinch. 2: 588. Heterotypic synonyms according to Trichosanthes baviensis Gagnep. (1918) Bull. Mus. Natl. Hist. Nat. 24: 379; Trichosanthes trichocarpa C.Y.Wu ex C.Y.Cheng & C.H.Yueh (1980) Acta Phytotax. Sin. 18: 340; Trichosanthes holtzei F.Muell. (1886) Australas. Journ. Pharm. 1: 447.Trichosanthes siamensis Duyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 97Trichosanthes tetragonosperma C.Y.Cheng & C.H.Yueh (1974) Acta Phytotax. Sin. 12: 425Trichosanthes sect. Foliobracteola C.Y.Cheng & C.H.Yueh (1974) Acta Phytotax. Sin. 12: 427. – Type: Trichosanthes kirilowii Maxim.Trichosanthes homophylla Hayata (1921) Icon. Pl. Formosan. 10: 8Trichosanthes hylonoma Hand.-Mazz. (1936) Symb. Sin. Pt. 7: 1066Trichosanthes ishigakiensis E.Walker (1971) J. Jap. Bot. 46: 71Trichosanthes jinggangshanica C.H.Yueh (1980) Acta Phytotax. Sin. 18: 342Trichosanthes kirilowii Maxim. (1859) Prim. Fl. Amur. 482Trichosanthes mianyangensis C.H.Yueh & R.G.Liao (1992) Bull. Bot. Res., Harbin 2: 115Trichosanthes miyagii Hayata (1921) Icon. Pl. Formosan. 10: 11Trichosanthes multiloba Miq. (1865) Ann. Mus. Bot. Lugd.-Bat. 2: 82Trichosanthes rosthornii Harms (1901) Bot. Jahrb. Syst. 29: 603Trichosanthes sect. Gymnopetalum (Arn.) H.J.de Boer, comb. et stat. nov. – Gymnopetalum Arn. (1840) Madras J. Lit. Sci. 12: 52. –Type: Bryonia tubiflora Wight & Arn. [ = Trichosanthes tubiflora (Wight & Arn.) H.J.de Boer].Trichosanthes tubiflora (Wight & Arn.) H.J.de Boer, comb. nov. – Bryonia tubiflora Wight & Arn. (1834) Prodr. Fl. Ind. Orient. 1: 347. – Gymnopetalum tubiflorum (Wight & Arn.) Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 388 –Type: Rottler s.n. ex Herb. Klein in Herb. Wight Cat. 1118, February 1796 , Trincomalee, Ceylon.Trichosanthes sect. PseudovariiferaH.J.de Boer, sect. nov. Diagnosis: Similar to sect. Foliobracteola, but male flowers with stamens inserted low in receptacle tube, with pseudo-ovary , and fruit with white pulp. Type: Trichosanthes villosa Blume.Trichosanthes kerrii Craib (1914) Bull. Misc. Inform. Kew: 7Trichosanthes phonsenae Duyfjes & Pruesapan (2004) Thai Forest Bull., Bot. 32: 9Trichosanthes sericeifolia C.Y.Cheng & C.H.Yueh (1980) Acta Phytotax. Sin. 18: 346Trichosanthes villosa Blume (1826) Bijdr. Fl. Ned. Ind. 15: 934Trichosanthes sect. TrichosanthesTrichosanthes cucumerina L. (1753) Sp. Pl.: 1008Trichosanthes dafangensis N.G.Ye & S.J.Li (1989) Acta Phytotax. Sin. 27: 153Trichosanthes dioica Roxb. (1832) Fl. Ind. 3: 701Trichosanthes integrifolia Thwaites (1859) Enum. Pl. Zeyl. [Thwaites]: 127PageBreakTrichosanthes lobata Roxb. (1832) Fl. Ind. 3: 703. – Heterotypic synonyms: Trichosanthes perrotetiana Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 362; Trichosanthes villosula Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 342Trichosanthes nervifolia L. (1753) Sp. Pl.: 1008Trichosanthes sect. Truncata C.Y.Cheng & C.H.Yueh (1974) Acta Phytotax. Sin. 12: 427. – Type: Trichosanthes truncata C.B.ClarkeTrichosanthes truncata C.B.Clarke (1879) Fl. Brit. India [J.D. Hooker] 2: 608. – Heterotypic synonym: Trichosanthes ovata Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 365Trichosanthes sect. Villosae (Yueh & L.Q.Huang) H.J.de Boer, stat. nov. – Trichosanthes subsect. Villosae Yueh & L.Q.Huang (1997) Act. Phytotax. Sinica 35: 127. – Type: Trichosanthes subvelutina F.Muell. ex Cogn.Trichosanthes subvelutina F.Muell. ex Cogn. (1881) Monogr. Phan. [A.DC. & C.DC.] 3: 366


In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens.Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted.Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range.Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia. Pongo pygmaeus and 1 Sumatran, Pongo abelii) orang utans, two  adults and two  juveniles, from a cohort of 26 in the Singapore Zoological Gardens died suddenly. Post-mortem examination of the four orang utans revealed pathology characterized by multifocal myocarditis, pulmonary congestion and edema, hydropericardium, hydrothorax and Ascites.Between July 2001 and January 2002, four  as the etiological agent of viral myocarditis in captive primates. These primates included semi-wild bonobos , baboonsAlthough the potential for EMCV to cross the species barrier has been demonstrated as seen from the various zoo outbreaks described above, human cases have fortunately been rare. Sporadic human EMCV infections and disease have been documented by virus isolation from different specimen types such as serum, stool samples, cerebrospinal fluid and throat washings ,21. A rePicornaviridae[Enterovirus, and EMCV, a Cardiovirus. The Picornaviridae family is one of the largest and most diverse families of RNA viruses, and includes etiological agents that are responsible for a wide variety of human and animal diseases [We hypothesized that the etiological agent behind the deaths of the orang utans in the Singapore Zoological Gardens was either a Coxsackie virus B, or more likely, an EMCV. Although both viruses are members of the same family, naviridae, they bediseases .The picornavirions are small, non-enveloped and spherical, with a diameter of about 20 to 30 nm. Picornaviruses have a single-stranded, positive sense RNA genome that is between 7.2 and 9 kb in length and contain a large opening reading frame (ORF). The ORF encodes for a polyprotein that comprises both non-structural and structural elements divided into three primary precursor molecules, namely P1, P2 and P3, encoding for 11 distinct proteins. The structural proteins VP4, VP2, VP3 and VP1 make up the viral capsid and are encoded in the P1 region towards the 5′-end of the genome. Non-structural proteins are derived from the P2 and P3 regions and are encoded towards the 3′-end of the genome, the largest of which is the RNA-dependent RNA polymerase (3Dpol). In addition, cardioviruses code for an L (Leader) protein at the N-terminus of their polyproteins. The genomic RNA also contains a highly structured 5′-UTR (untranslated region) that includes an internal ribosome entry site (IRES) from which viral protein translation is initiated in a cap-independent manner. The shorter 3′-UTR terminates with a heterogeneous poly(A) tail that is known to be involved in the binding process of the viral replicase complex.In this paper, we described the cell culture isolation and identification of two viral isolates obtained post-mortem from the two juvenile Bornean orang utans. The viral genomes of the two isolates were >95% sequenced, with the complete genome sequence lacking about 200 nucleotides (nt) from the 5′ end. Phylogenetic and sequence analyses suggested that the newly isolated viruses are highly divergent variants of EMCV and possibly a new serotype of the virus. This is the first report of EMCV infection in Singapore and South East Asia and its molecular characterization.Virus isolates, designated Sing-M105-02 and Sing-M100-02, were successfully isolated in African Green Monkey kidney (Vero) cells from the heart tissues of the first juvenile orang utan, and lung tissues of the second autopsied juvenile orang utan, respectively. Cytopathic effect (CPE) was observed in Vero cells inoculated with the heart homogenate, at 2 days post-infection (pi). Infected-Vero cells were distorted from its normal shape, became granular and finally rounded-up (results not shown). Similar observations of CPE were made with the inoculation of lung homogenate at 3 days pi. Uninfected Vero cells did not show any signs of CPE. Both virus isolates were also inoculated into Mardin Darby Canine Kidney (MDCK) cells (data not shown). At 1 to 2 days pi, CPE in virus infected-MDCK cells was characterized by rounded and wrinkled appearances.Picornaviridae[The viruses in infected-Vero tissue culture fluid were negatively stained and examined with electron microscopy. The electron micrographs revealed numerous small round virus particles with smooth appearances in the range of 20 to 30 nm for both Sing-M105-02 and Sing-M100-02 isolates  of both Sing-M105-02 and Sing-M100-02 isolates by primer walking Table  and 4, iCardiovirus strains. In the conserved 3Dpol region, the extent of sequence identities between the Sing-M100-02 and Sing-M105-02 isolates and other EMCV strains ranged from 76.4% to 80.9% (nt level) and 79.4% to 90.5% (aa level). The lowest sequence relationships were observed in the variable VP1 capsid region, with percent identities ranging from 61.2% to 73.6% (nt level) and 66.1% to 87.0% (aa level). Table To investigate the genetic relationship of Sing-M105-02 and Sing-M100-02 isolates with existing cardioviruses (EMCV and Theiloviruses), we performed sequence comparison for the entire ORF, VP1 capsid and 3Dpol gene regions. Table The aa sequence alignment of the P1 structural region was next examined and we observed significant differences in the BC-loop and loop I regions in the VP1 capsid of the Sing-M105-02 and Sing-M100-02 isolates with other EMCV strains . A selection of capybaras (80%), chimpanzees (31%) and orang utans (50%) exhibited neutralizing antibody titers to Sing-M105-02 virus, strongly indicating that they had been exposed to the virus  and lung (designated Sing-M100-02) homogenates in Vero cells. Virus isolation was not performed on the two autopsied adult orang utans. The viral agents isolated in Vero cells were examined under transmission electron microscopy and the viruses showed similar morphology to the picornaviruses  afforded us increased confidence that the Sing-M105-02 and Sing-M100-02 isolates constituted a divergent group of EMCV variants, as they clustered distinctly away from the other lineages of EMCV strains Figure . This hiOur serological survey of the animal population further suggested that there were at least two virus strains circulating in the Singapore Zoo; the first, an EMCV(Aust)-like virus, and the second, a variant of EMCV. High seropositive rates for the EMCV variant was found in capybaras, chimpanzees, orang utans and two rats caught in baited traps. In addition, neutralizing antibodies to EMCV(Aust) were also found in all except one of the chimpanzee, one orang utan and one of the rats, indicating prior exposure to single or dual infections with the viruses. Although no attempt was made to recover the virus from these animals, we postulated that the EMCV(Aust)-like virus could have been the parental virus from which Sing-M105-02 and Sing-M100-02 viruses gradually evolved. Rats have been considered to be the primary host reservoir and disseminators of EMCV, and they have been implicated in other zoological outbreaks of EMCV ,7,9. TheThe etiological agent responsible for the outbreak for 2 of the 4 cases of fatal myocarditis in orang utans at the Singapore Zoological Gardens was an EMCV. High divergence in nt sequences of the VP1 capsid and 3Dpol genes as compared to other known EMCV strains, indicated that the outbreak was caused by a highly divergent variant of EMCV and possibly a new serotype of the virus. This is the first report of an EMCV infection in Singapore and South East Asia.Post-mortem heart and lung tissues were obtained from the two juvenile orang utans. In addition, a total of 122 serum samples from various zoo animals were collected for sero-epidemiological study. These consisted of 6 agile gibbons, 1 cape hunting dog, 1 capuchin monkey, 10 capybaras, 13 chimpanzees, 3 colobus monkeys, 1 coloque monkey, 21 gibbons, 3 howler monkeys, 2 Japanese monkeys, 2 komodo dragons, 19 macaques, 2 mandrills, 14 orang utans, 1 patas monkey, 2 pig-tailed macaques, 1 raccoon, 2 lemurs, 3 siamangs, 1 slow loris, 5 spider monkeys and 4 white nose guenons. Two rats were also captured in a baited trap.x g. The homogenates were filtered with a 45 μm pore size and the clarified homogenate inoculated onto monolayers of African Green Monkey kidney (Vero) cells (ATCC-CCL81). The infection was carried out at 37°C and infected cells were observed for cytopathic cell effect (CPE) under a light microscope. The viruses isolated from the heart and lung tissues were designated as Sing-M105-02 and Sing-M100-02, respectively. Sing-M105-02, Sing-M100-02 and EMCV(Aust) were propagated in Vero cells in minimal Eagle’s medium (MEM) (Invitrogen) supplemented with 5% fetal calf serum (FCS)  at 37°C, 5% CO2 for 3 days. The culture supernatant was then harvested, centrifuged at 4,000 x g and stored at -80°C. EMCV(Aust) strain was imported into Singapore from Dr Peter Kirkland, MacArthur Institute, Australia. The EMCV(Aust) strain was isolated from a rodent in an Australian zoo as reported in Reddacliff et al., [Heart and lung tissues were collected from the post-mortem analyses of two juvenile orang utans. The tissues were homogenised in Hank’s balanced salt solution and the cellular debris cleared by low-speed centrifugation at about 3,000 to 4,000 The antiserum was serially diluted in two-folds and mixed with a known titer of either Sing-M105-02 or EMCV(Aust) in equal volume for 1 hr at 37°C. The antiserum-virus mixtures were next inoculated onto Vero cells seeded in a 96 well formant and examined daily for signs or absence of CPE.x g for 10 min. The virus was pelleted from the supernatant by ultracentrifugation at 159,000 x g for 1.5 h at 4°C and resuspended in PBS [Virus-infected tissue culture fluid was clarified by low-speed centrifugation at 2,000 to 3,000 d in PBS . Drops o2 tissue culture flask at 24 h pi and washed in phosphate-buffered saline (PBS), pH 7.4, by low speed centrifugation at 2,000 to 3,000 x g for 10 min. The cell pellet was resuspended in 1 ml of PBS and 5 μl of cell suspension was distributed into each well of a Teflon-coated slide and air-dried. Cells were fixed in cold acetone for 10 min and virus antigens detected by immunofluorescence using polyclonal anti-EMCV raised in pig as the primary antibody . The secondary antibody was goat anti-porcine IgG, conjugated to fluorescein isothiocynate (Chemicon). Evans Blue (Sigma Diagnostics) was used as counter-stain and the slide examined with a Olympus DX51 microscope.Virus-infected cells were scraped off from a 25 cmFor the initial determination of the virus genus and species, we used a total of 4 sets of PCR primers. Two published primer sets, RotbartFor/RotbartRev and EV1/EV2, both for the detection of enterovirus ,25, and Viral RNA was extracted from 100 μl of virus-infected Vero cell culture supernatant using the RNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. RNA was eluted in 50 μl of RNase-free water. For initial virus identification, the first strand cDNA was synthesized with gene-specific primers 2 μM) . Thermal cycling conditions included initial denaturation at 95°C for 5 min; followed by 45 cycles of 95°C for 30 s, 45°C for 30 s, 68°C for 2 min; and a final extension step of 68°C for 10 min. Touchdown PCR was also performed in several instances to improve PCR amplification specificity.For PCR, 2 μl of each viral cDNA sample was amplified in a 50 μl reaction mixture  PCR Buffer, 5 μl 2 μM dNTPs, 2 μl 50 mM MgSOTo obtain the 3′ end sequence of the virus, the GeneRacer kit and core module primers  were employed as per the manufacturer’s instructions. We were unable to obtain about 200 nt of the virus 5′ UTR sequence despite numerous attempts using the GeneRacer kit, other reverse transcriptases, DNA polymerases, PCR additives (such as DMSO and betaine), and various PCR thermocycling conditions.PCR products were resolved on 1-2% agarose gels (depending on amplicon size), and the subsequent PCR clean-up and sequencing of the amplicons were performed by AITbiotech (Singapore) using BigDye chemistry . In brief, the genomes of EMCV isolates Sing-M100-02 and Sing-M105-02 were determined from a series of PCR amplicons that contained overlapping cDNA fragments. Each gene region was sequenced at least two times. Raw sequences were first assembled and aligned with the reference EMCV Mengo M virus genome (GenBank accession number L22089) using the SeqMan Pro software found in the Lasergene Version 8 .Final nt sequences were aligned against other strains of EMCV and picornaviruses using the Clustal W method as implemented in the MegAlign program . Percent identity and phylogenetic trees were constructed for the entire ORF as well as for individual genes coding for the various proteins at the nt level and neighbor-joining trees were constructed after alignment by the same program. The consistency and robustness of tree topology were bootstrapped using 1000 trials and a seed of 111. The TreeView X 1.6.6 program  was usedThe partial sequence data for the VP3/VP1 genes have been deposited in GenBank under the accession numbers AF525466 for Sing-M105-02 and AY162280 for Sing-M100-02. For the partial 3Dpol genes, the nt sequences were deposited under the accession numbers AF510055 for Sing-M105-02, and AY162279 for Sing-M100-02. The full virus genome sequences (excluding about 200 nt from the 5′ end) have been deposited in GenBank under accession numbers KC310737 for Sing-M100-02, and KC310738 for Sing-M105-02.The authors declare that they have no competing interests.DS-YY, JEL, JCWL, EASL and HCT performed the laboratory experiments. SO performed the clinical assessment. CJF performed the necropsy and sampling. CJF, Y-NL and M-LS performed the virus isolation. B-HT, M-LN, E-EO, K-PC, SO and CJF conceived the study. B-HT and DS-YY were involved in the data analysis and manuscript writing. All authors read and approved the final manuscript.


Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle. Astroviridae comprises 2 genera, Mamastrovirus and Avastrovirus, known to infect mammals and birds, respectively. Since the first description of human astrovirus (HAstV) in children with diarrhea in 1975  disease . In particular, BSE has become a major public health concern after recognition of the association between BSE and prion-associated disease in humans. Therefore, early and rapid recognition of the cause of neurologic disease is vital to the safety of the food chain. Etiologic diagnosis of CNS disease in cattle requires substantial effort; is costly; and usually presents a challenge because of the large number of pathogens or problems that can cause neurologic disease, including viruses, bacteria, parasites, prions, toxins, and metabolic disorders. Pathogens known to cause CNS disease in cattle include bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5), lyssavirus (rabies), ovine herpesvirus 2, L. monocytogenes, Histophilus somni, Escherichia coli, Salmonella spp., Chlamydia spp., Neospora caninum, amoebas, and prions  was detected in the brains of 3 of 32 other cattle with encephalitides of undetermined etiology. Virus was detected by RNA by in situ hybridization within neurons in the brainstem, cerebellum, and/or spinal cord in all PCR positive samples from the 4 animals in this study.To search for potential viral etiologic agents, we performed an unbiased metagenomic analysis   of the Veterinary Medical Teaching Hospital  and at the Davis, Tulare, and San Bernardino branches of the University of California, Davis–California Animal Health and Food Safety laboratory system. All animals had a histologic diagnosis of either nonsuppurative or pleocellular encephalitis, encephalomyelitis, or meningoencephalitis. Histology was reviewed, and nucleic acids were extracted from selected sections of formalin-fixed, paraffin-embedded affected brain tissue by Agencourt Formapure Kit   and random hexamer. The oligonucleotide primer sets used were as follows: AstV-sF1: 5′-ACCGCCTTTCTGATGATGTGC-3′; AstV-sR1: 5′-CTCATCAACAACCTGCCAAAT-3′; AstV-sF2: 5′-GACTCTGAGGGTCAAATAACC-3′; AstV-sR2: 5′-GCCAAATGGTTTCTCCAACAG-3′ ; AstV-sF3: 5′-ACCGCCTTTCTGATGATGTGC-3′; AstV-sR3: 5′-CTCATCAACAACCTGCCAAAT-3′; AstV-sF4: 5′-GACTCTGAGGGTCAAATAACC-3′; and AstV-sR4: 5′-GCCAAATGGTTTCTCCAACAG-3′ . The PCR conditions were 95°C for 2 min, followed by 39 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 15 sec, and a final extension at 72°C for 10 min. PCR products were checked by 2% agarose gel electrophoresis, and amplicons of the appropriate size were confirmed by sequencing. PCR-positive and -negative (control) samples were concurrently used for in situ hybridization analyses.L. monocytogenes, and Neospora spp. were also negative by immunohistochemical examination of sections. Samples were negative for rabies virus according to fluorescent antibody test. Virus isolation, done independently at Cornell University  and at the California Health and Food Safety Laboratory  from a brain pool was negative. The tissue sample was placed onto bovine turbinate and swine kidney primary cell cultures. Two passages were made. At the end of the second passage, the cells were stained with a polyvalent viral antiserum, a pseudorabies antiserum, and bovine enterovirus 1–7 antiserum in an indirect immunofluorescence assay. In the sentinel animal, serologic test results for BoHV-1 were negative and for BVDV types 1 and 2 were positive at a titer of 512. Ostertagia spp. were detected in the abomasal contents, and Trichostrongyle spp. eggs and Coccidia oocysts were found in feces. Hepatic levels of copper (5.8 ppm) and selenium (0.13 ppm) were interpreted as being low.The animal was submitted to the California Animal Health and Food Safety Laboratory in Davis, California, for euthanasia and postmortem examination. PCR tests for BoHV-1, bovine viral diarrhea virus (BVDV), West Nile virus, bluetongue virus, epizootic hemorrhagic disease virus, and ovine herpesvirus 2 were negative. BVDV, BoHV-1, Brains from all animals and various segments of the spinal cord from 3 animals were immersion-fixed in 10% buffered formalin, pH 7.2, for at least 48 h. Transverse sections of the brain, including cerebral cortex and corona radiata, basal nuclei, thalamus, midbrain, medulla oblongata, cerebellar peduncles, cerebellum and brainstem in all animals; the full length of the spinal cord in animals 1 and 2 ; and cervical cord segments only in animal 3 were processed by standard histologic techniques by using 4-µm thick sections stained with hematoxylin and eosin.Colorimetric in situ hybridization was performed on 4-μm thick tissue sections, mounted on 3-aminopropyltriethoxylane–coated slides  by using a 28-nt oligomer probe (5′-ACATGGCTGTAAGCATTGGTGTGAAGTA-3′) complementary to the capsid region of BoAstV-NeuroS1 and 3′-labeled with digoxigenin-II-dideoxy undine . Tissue sections were deparaffinized with d-limonene  and digested by incubation with 0.25% pepsin in Tris-buffered saline (pH 2.0) at 37°C for 10 min, followed by a 5-min incubation at 105°C to stop enzymatic activity. Nucleic acid denaturation was achieved by incubation in formamide (100%) at 105°C for 5 min. Hybridization was performed at 37°C in a 10-µmol solution of the digoxigenin-labeled probe in hybridization buffer . Sections were incubated in a digoxigenin antibody solution (500:1 dilution) containing 2.5 mL buffer 1  with 5 μL of antidigoxigenin Fab fragments conjugated with alkaline phosphatase (750 U/mL) . Sections were then washed and developed according to manufacturer instructions before counterstaining with 1% fast-green FCF for 5 min. After counterstaining, slides were coated with aqueous mounting media  and covered with SHUR/Mount mounting medium . There was no observed hybridization in replicate tissue sections incubated with another, unrelated digoxigenin-labeled probe with similar guanine-cytosine content, and brain tissues from astrovirus-negative cattle exhibited no staining.Selected pieces of wax-embedded spinal cord tissue from the sentinel animal were extracted and postfixed in 2.0% glutaraldehyde and then routinely processed and embedded in epoxy resin . Selected thick sections were stained with toluidine blue. Ultrathin sections were examined by using a Zeiss  906E transmission electron microscope.A 168-kg crossbreed yearling steer originating from Mexico was found on a property in northern California in lateral recumbency, with opisthotonus and limbs in extensor rigidity. After deep sequencing of enriched viral particles from homogenized diseased brain tissue, we identified 170 sequence reads related to astroviruses using BLASTx (E-score <10-5), which could be assembled into 17 contigs covering ≈45% of the viral genome.The complete genome of the new astrovirus (GenBank accession no. KF233994) was then generated by linking fragments by PCR and by using 5′ and 3′ RACE to yield a genome provisionally named bovine astrovirus NeuroS1 (BoAstV-NeuroS1).The resulting genome was 6,471 nt long, with a GC content of 48%. As typical mamastroviruses, BoAstV-NeuroS1 had 3 putative open reading frames (ORFs), encoding the protease with ORF1a (860 aa), RdRp with ORF1b (525 aa), and capsid protein with ORF2 (757 aa). An expected ribosomal frame-shift signal was found in the ORF1a/1b overlap region that consisted of the heptameric AAAAAAC sequence, followed by a potential 20-nt pseudo-knot sequence. The conserved protease motifs and RdRp motifs, such as the potential conserved proteolytic cleavage site (VHL/TNT) and the characteristic YGDD motif, were present. The amino-terminal half of the capsid protein was more conserved than the carboxy-terminal region.To determine the genetic relationship between BoAstV-NeuroS1 and other astroviruses, we performed sequence alignments of the protease (ORF1a), RdRp (ORF1b), and capsid (ORF2). BoAstV-NeuroS1 shared the highest identity of 56%, 70%, and 66% aa similarity with the ORF1a, ORF1b, and ORF2 encoded proteins of ovine astrovirus, its closest relative. Phylogenetic analysis was performed, and neighbor-joining trees were generated . All 3 tL. monocytogenes in animals 3 and 4 was negative; for animals 2 and 4, Neospora caninum, Sarcocystis spp., BSE, and pseudorabies histologic lesions were negative by immunohistochemistry; and immunohistochemical analysis results for West Nile virus, Chlamydia spp. and Toxoplasma spp. in animal 3 were negative.For 32 animals, formalin-fixed, paraffin-embedded tissues of bovine brain or spinal cord  were tested for BoAstV-NeuroS1 by specific reverse transcription nested PCR, and samples from 3 (9.4%) animals  were positive. Samples from these same animals also were positive by in situ hybridization. Animal 2 was a 178-kg, polled Hereford heifer from northern California that was found recumbent with intermittent seizures, unable to move the hind legs, and lacking hind limb pain withdrawal reflex. Animal 3 was a euthanized 3-year-old Angus cow that on the farm was unable to rise and exhibited occasional star gazing and unresponsiveness to thiamin therapy. Animal 4 was a euthanized 3-year-old Holstein cow with a history of circling and blindness. The laboratory diagnostic workup for CNS pathogens in these 3 additional animals was not consistent but included negative test results for BVDV, BoHV-1, and rabies virus. Culture for The anatomic pattern of lesion distribution was remarkably consistent and unusual in all 4 animals. The meningoencephalomyelitis was largely confined to gray matter in the brain and spinal cord, with most severe lesions in the cerebellar folia and brainstem and throughout all segments of the spinal cord gray matter. Rostrally, there was minimal involvement in the midbrain, thalamus, and basal nuclei, but the cerebral cortex and underlying corona radiata were devoid of inflammatory cell infiltrates  in the brain tissue of 4 cattle from ranches in California with a clinical neurologic disorder characterized histologically as a neurotropic meningoencephalomyelitis and ganglioneuritis. The virus was initially identified by viral metagenomics, and its presence was subsequently confirmed retrospectively in the CNS of 3 other cattle that had bovine encephalomyelitis of unknown etiology by PCR and in situ hybridization for viral RNA. Also, ultrastructure studies in 1 animal demonstrated intracytoplasmic particles in degenerating neurons tentatively considered morphologically compatible with an astrovirus. Our attempts to recover this virus in tissue culture have not been successful, and no concurrent intestinal infection was detected.,Other astroviruses have been identified and associated with neurologic signs in an immunodeficient child and in minks with a shaking syndrome in farm outbreaks reported in Denmark, Sweden, and Finland  fall into the same genetic clade of astroviruses, together with viruses of other species so far not associated with disease .Clostridium botulinum, Clostridium tetani, L. monocytogenes, Histophilus somni, Babesia bovis, BSE prion, and BoHV-1 and -5. Infection with BoAstV-NeuroS1 is a new addition to the differential diagnosis of neurologic disorders in cattle, and constituted ≈9% of the undiagnosed cases of encephalitis in our retrospective study. This finding probably underestimates the prevalence of BoAstV-NeuroS1 disease because detection in this retrospective study was limited to formalin-fixed, paraffin-embedded tissues from a single region of the brain. Our preliminary microscopic and in situ hybridization results suggest that the best target for virus detection would be the spinal cord. Rapid diagnosis of astrovirus RNA by PCR or in situ hybridization in the brain or spinal cord of cattle with neurologic signs may enable more rapid exclusion of infection with the BSE prion. The involvement of the cerebellum is distinctive and rare and has been reported only with the still enigmatic European sporadic bovine encephalitis. However, the neurotropic lesions in that disease also intensively involve the hippocampus. Arthropod-borne diseases, such as louping-ill in sheep and Russian tick-borne encephalitis in dogs and horses, share the features of neurotropism, Purkinje cell necrosis, and a similar anatomic distribution with BoAstV-NeuroS1 infection.Bovine neurologic diseases can be caused by bacteria, parasites, viruses, toxins, or nutritional disturbances (Further research will be required to determine whether development of the neurologic signs seen here required other factors, including co-infections and/or a genetic or acquired immunodeficiency. PCR testing and genomic analysis of bovine fecal isolates also may provide information about the incidence and duration of virus shedding, which—as for other asymptomatic intestinal astrovirus infections—is expected to be short. In our study, in situ hybridization was negative on intestinal sections of the affected cattle. Seroprevalence studies in healthy cattle of different ages could measure any prior exposure to BoAstV-NeuroS1 and determine how frequently such asymptomatic infections occur.


The International Committee on Taxonomy of Viruses authorizes and organizes the taxonomic classification of viruses. Thus far, the detailed classifications for all viruses are neither complete nor free from dispute. For example, the current missing label rates in GenBank are 12.1% for family label and 30.0% for genus label. Using the proposed Natural Vector representation, all 2,044 single-segment referenced viral genomes in GenBank can be embedded in  Characterizing genetic sequences and determining viral origins have always been important issues in virology Retroviridae family (RNA viruses) contain erroneous DNA label information. Additionally, there are 17 families containing both circular virus(es) and linear virus(es) (Table S2 in File SI). This is not possible based on the ICTV classification criteria The composition and structure of viral genomes is more varied than bacterial, plant, or animal kingdoms. The viral genomes may be single-stranded or double-stranded, linear or circular, and in single-segmented or multi-segmented configuration. There are 2,418 reference viral genomes in the current GenBank collection (up to 2012-4-6). In this study, we focus on the 2,044 single-segment viruses among them  by means of the natural vector mapping which is based on the quantity and global distribution of nucleotides in the sequence. Each sequence is uniquely represented by a single point in this subspace. The Euclidean distance between two points represents the biological distance of the corresponding two viruses. Using the natural vector representation we can perform phylogenetic and cluster analysis for all the existing viral genomes.To study virus classification and phylogeny rapidly and accurately, we construct a novel viral genome space as a subspace in N = 2). We emphasize that our natural vectors depend only on the numbers and distributions of nucleotides in the viral genome sequences. They do not rely on any model assumption. There are two reasons that the virus is represented as a point in the viral genome space without losing inherent biological information. First, the 12-dimensional natural vector mapping on all the viruses we examined is one-to-one. Second, we do not gain any more useful information for classification purposes using the 16-dimensional or higher natural vector mapping. Our new approach to classifying viral genomes is not a partial-sequence-based method. It is constructed based on the global sequence information of genomes.A key finding of this work is that this viral genome space is a 12-dimensional space  of a nucleotide sequence S is defined byThe natural vector A, C, G, T. That is, for k = A, C, G, T, let the weight k at position i. For example,In DNA/RNA sequencing data, the standard International Union for Pure and Applied Chemistry (IUPAC) nucleotide code is used to describe ambiguous sites, where a single character may represent more than one nucleotide (see Table S3 in File SI). Our natural vector defined above can be easily extended to handle the nucleotide sequences with ambiguous letters other than n, we can compute its (n+4)-dimensional natural vector as in (A1). A noteworthy contribution of this work is that we do not need to compute the central moments  is still one-to-one. Thus, we use the 12-dimensional natural vectorWe use this natural vector to construct a viral genome space, which is a moduli space of viral genomes. In this space each point corresponds to a viral genome. The distance between two viruses in the space reflects the biological distance between these two viruses. For a viral genome sequence of length Distance matrices or similarity matrices are used by many algorithms With the construction of a natural vector distance matrix we propose a natural graphical representation to overcome the disadvantages of existing methods for inferring phylogenies. Specifically, given a distance matrix of finite elements, the algorithm is as follows:A, find the closest point(s) B . We compute the distance matrix for these connected components. The distance between two components is defined as the minimum of all distances between an element in one component and an element in another component. We then obtain a new distance matrix, in which the elements are the connected graphs obtained in step (1).Repeat the process in steps (1) and (2) to obtain higher-level graphs until we get one connected component for all elements, which is the final graphical representation.A in graph 1 and element G in graph 2. So, we connect these two elements to get a connected graph as shown in For example, given the distance matrix of 10 elements in A, virologists would like to know which virus B is closest to A. An arrow from A to B in the graph represents this relation. Here we need to point out that the natural graphical representation is not necessarily a tree. As in the example, a cycle may exist in the graphical representation which may show interesting biological information.The direction in the graph shows the closest element(s) to each element based on their biological distances. For example, given a virus For each virus we find its nearest neighbor in the 12-dimensional NV genome space and check whether its label matches that of its nearest neighbor. If we have a complete genome space which contains all of the viruses it is reasonable to assume any virus must have a neighbor sharing the same label.Firstly, given a nucleotide sequence along with its topological information , we can use our method to predict its Baltimore class label. For a single-stranded DNA sequence or double-stranded RNA sequence, there is no need to predict since it exactly belongs to class II (ssDNA) or III (dsRNA), respectively. For a double-stranded DNA sequence, it may belong to class I (dsDNA) or VII (dsDNA (RT)). For a single-stranded RNA sequence, it may belong to class IV (ssRNA (+)), V (ssRNA (−)), or VI (ssRNA (RT)). Here we use “#virus” to denote the number of viruses, and “#error” to denote the number of viruses with inconsistent nearest neighbor labels. Then the inconsistency rate is defined by #error/#virus ×100%. For classes I and VII, the inconsistency rate for linear viruses is 0/599 = 0% since all class VII viruses are circular, and the inconsistency rate for circular viruses is 3/(177+44) = 3/221 = 1.36%. For classes IV, V, and VI, the inconsistency rate for linear viruses is 45/(563+66+58) = 45/687 = 6.55%, and the inconsistency rate for circular viruses is 0/1 = 0%. Thus the overall inconsistency rate is (3+45)/(599+221+687+1) = 3.18%.The reason for the inconsistency is due to the sparsity of the reference dataset, i.e., the dataset in this study does not cover all of the viruses. In this case, prediction based on a distant neighbor is not reliable. For example, the distance between virus #170 (class I) and its nearest neighbor virus #62 (class VII) is 1265.021. To measure the relative magnitude of 1265.021, we collect all the distances D(A) between virus A in class VII and its nearest neighbor within class VII. Then (1265.021−maximum)/(Q3−Q1) = 2.651, where “maximum”, “Q3”, “Q1” are the maximum, .75th quantile, and .25th quantile of the collection of D(A)'s, and equal to 808.147, 320.170, 147.861, respectively. Such a big relative distance indicates that the distance from virus #170 to its nearest neighbor virus # 62 is much bigger than all the nearest distances of the viruses belonging to class VII. The prediction that virus #170 belongs to class VII based on the label of virus #62 is thus unreliable.In practice, for each class, we collect the nearest distance for each virus within the class, and get the .75 quantile of those nearest distances. To predict the class label of a virus, we first find its nearest neighbor which belongs to class I, for example, then compare its nearest distance with the .75th quantile of the nearest distances of class I. We make the prediction only if the nearest distance is less than the .75th quantile, called a prediction with .75-cutoff. Based on this cut-off setting, for classes I and VII, the inconsistency rate for circular viruses is updated to 2/(177+44) = 2/221 = 0.90%. For classes IV, V, and VI, the inconsistency rate for linear viruses is updated to 16/(563+66+58) = 16/687 = 2.33%. Thus the overall inconsistency rate is 18/(599+221+687+1) = 1.19%. Therefore, in this study we calculate a .75 cut-off for predicting Baltimore class, family, subfamily, and genus labels to avoid unreliable predictions. For cut-off quantile other than .75, see Tables S4–S5 in File SI.Secondly, given a virus with only sequence information, we can predict its Baltimore class label with exceptional results. As shown in We next go one level deeper and check the predictability of family labels given their Baltimore classification with the NV using the same nearest neighbor prediction framework. Given a virus with only sequence information, we can predict its family label according to Baltimore class as shown in Using our method we are also able to make predictions for viruses with no assigned family, subfamily, or genus labels. For any virus missing any of the above information we check its nearest-neighbor's label. If the distance is sufficiently small we say with confidence that the missing label is the same as that of its nearest-neighbor. Using this method we obtain the results presented in Tables S8–S10 in Hepadnaviridae and Caulimoviridae are clearly separated in the graph. In the Hepadnaviridae family there are two genera Avihepadnavirus and Orthohepadnavirus. Viruses #1476 (Ross's goose hepatitis B), #1529 (Sheldgoose hepatitis B), and #1583 (Snow goose hepatitis B) are not assigned ICTV genus labels. In the figure we can see their nearest neighbors are all in Avihepadnavirus genus, thus we predict that they belong to the genus Avihepadnavirus. These predictions are consistent with other researchers' work Badnavirus, Petuvirus, Caulimovirus, Cavemovirus, Soymovirus, and Tungrovirus) in the family Caulimoviridae. Virus #988 (Lucky bamboo bacilliform) has no genus label and its nearest neighbor is virus #482 (Dracaena mottle). The distance between the two viruses is only 14.52, far less than the other distances, thus we predict confidently virus #988 is also in the Badnavirus genus. This prediction is consistent with the result obtained by Chen et al. Badnaviruses by ICTV. However, in our genome space these two viruses are far away from all the other Badnaviruses. Similarly, viruses #325 (Cestrum yellow leaf curling), #616 (Eupatorium vein clearing), and #1481 (Rudbeckia flower distortion) are far away from all the other Caulimoviruses. Thus we question the ICTV genus classifications for these viruses as shown in the figure.In Endornaviridae, Hypoviridae, and Totiviridae are clearly separated in this graph. Virus #372 (Circulifer tenellus virus 1) and #1611 (Spissistilus festinus virus 1) are not assigned with ICTV family labels. They are very close together (distance 308.13), but far away from the other three families. Thus we predict these two viruses belong to a new family. This prediction is consistent with Spear et al.'s work Totiviridae family, however, in our 12-dimensional genome space it is far away from the majority of Totiviridae family. Thus we question the family classification for this virus. Similarly, Lim et al. Endornaviridae family and Endornavirus genus by Totiviridae family includes not only single-segment but also multi-segment genomes (Helicobasidium mompa No. 17 dsRNA virus). Here we only focus on our dataset of single-segment viruses. Further study of multi-segment viruses may provide an explanation for the scattering of the Totiviridae family members.In Filoviridae, Rhabdoviridae, Bornaviridae, and Paramyxoviridae are clearly separated in this graph. We predict that viruses #186 (Beilong virus) and #877 (J virus) form a new genus since these two close viruses are far away from other genera in Paramyxoviridae family. Actually, this prediction is precisely consistent with other researchers' work Jeilongvirus. Viruse #634 (Fer de lance virus) also belongs to a new genus according to our work, and other researchers Ferlavirus. Viruses #1202 (Nyamanini virus) and #1058 (Midway virus) form a new genus named Nyavirus according to the literature Morbillivirus II; viruses #875 (Iranian maize mosaic nucleorhabdovirus), #1011 (Maize mosaic virus), and #1778 (Taro vein chlorosis virus) form a new genus; virus #2011 (Wongabel virus) form a new genus, which is consistent with Gubala et al.'s work Ephemerovirus and Jeilongvirus, we question their family classifications provided by ICTV because they are closer to other families (Jeilongvirus to family Filoviridae and Ephemerovirus to family Paramyxoviridae) based on our graphical representation. For two Metapneumovirus memebers #109 (Avian metapneumovirus) and #814 (Human metapneumovirus), ICTV puts them into the Paramyxoviridae family, while our results show that they are closer to the Rhabdoviridae family. In addition, these two viruses are not connected in our graphical representation. One possible reason is that there might be some metapneumovirus viruses from animals other than human and avian that may be missing from our dataset.In For Baltimore classes I (776 viruses), II (328 viruses), IV (563 viruses), and VI (58 viruses), we provide complete nearest-neighbor relationships in the graph description of Baltimore classes at Supporting Information. It is computationally difficult, if not infeasible, for multiple sequence alignment to handle these large classes.Flaviviridae and genus Flavivirus by the ICTV. Recent reports of widespread transmission of this mosquito-borne virus in humans in the United States highlight this threat to public health. During August 2012, the Centers for Disease Control and Prevention reported 1,590 human cases and 65 deaths nationwide Flaviviridae family in our dataset. In Hepacivirus, Flavivirus, and Pestivirus are clearly separated. Viruses #1999 (NC_001563.2) and #2000 (NC_009942.1) are two West Nile viruses, and viruses #879  and #1962  are closest to them, respectively. This is consistent with Flaviviridae family revealed in our findings could provide more information helpful to public health officials for prevention and treatment.West Nile virus (WNv) is a single-stranded plus-sense RNA virus that is classified within the family As a comparison, we also do Multiple Sequence Alignment (MSA) analysis for three small Baltimore classes ; VII, dsDNA(RT)) to test if each virus' nearest neighbor belongs to the same family as itself. We use the ClustalW program from the MEGA 5.0 software k with respect to mutations involving k letters (k is negligible with respect to the length n). See Section 4 Simulated evaluation of  genome space including Table S11 in In conclusion, there are four major advantages to our method: (1) Once a virus' NV has been calculated, it can be stored in a database. It is unnecessary to recalculate the NV of a virus for any subsequent applications, whereas in multiple alignment methods realignment is necessary when additional sequences are added to the previously aligned group. (2) Our method is much faster than alignment methods and easier to manipulate. The complexity of our method is File SIThis file SI contains: (1) Dataset, including Table S1–S3; (2) Discussion on cut-off setting, including Table S4–S5; (3) Predictions by our method, including Table S6–S10; (4) Simulated evaluation of  genome space, including Table S11; (5) Graph descriptions of Baltimore I, II, IV, VI; (6) List of virus information used in this paper; (7) Supplementary references.(PDF)Click here for additional data file.