Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes  are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals.Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization,  MAT) locus of pathogenic Cryptococcus species . The fungal MAT locus is a model for gene cluster evolution and shares features with sex chromosomes of plants/animals. In previous studies with C. neoformans/C. gattii, a model for this unusual locus was proposed in which two ancestral unlinked loci were fused into one contiguous locus. C. heveanensis represents an evolutionary window to test this model, since it is related to but distinct from pathogenic Cryptococcus species. The organization of MAT in two probably unlinked loci in C. heveanensis provides evidence supporting the model and additional novel insights into how serial gene acquisitions created evolutionary strata. We also discovered an extant sexual cycle for C. heveanensis and correlated fertility with MAT alleles. These findings provide direct experimental validation of the central tenants of sex-chromosome evolution originally proposed by Ohno involving sex determinants arising on autosomes, formation of gene clusters with coherent functions in sex, and mechanisms by which specialized recombinationally suppressed genomic regions expand to capture an entire chromosome as a sex chromosome.Comparative genomics provides an approach to understand evolution of sex-determining regions in animals and fungi. One example is the unusual mating-type ( Although many organisms, in particular microorganisms, can reproduce both asexually and sexually, the vast majority appears to undergo sexual reproduction during their life cycles. The hypotheses advanced as to why sex is so ubiquitous are myriad, but center on several recurrent themes S. cerevisiae have provided direct experimental evidence for a benefit of sexual reproduction. Isogenic diploid strains were engineered that are either capable of sporulation with meiotic recombination  or were able to sporulate but not undergo meiotic recombination  S. cerevisiae and the model alga Chlamydomonas provide additional support for a benefit conferred by sex in response to novel or challenging environments Until recently it has been difficult to test these models for the possible benefits of sex in experimentally tractable model organisms. However, recent studies with the model yeast MAT) occurs in two alternate forms, known as idiomorphs , and these govern the identity of the cell While the vast majority of sexually reproducing organisms occur as just two sexes or mating types, transitions in sexuality from two to multiple mating types, and vice versa, have occurred in the fungal kingdom. Sexual reproduction is common in fungi, and mating type occurs in two general patterns: bipolar and tetrapolar mating type , C. neoformans var. neoformans (serotype D), and C. gattii revealed that the MAT locus is composed of four gene strata of distinct evolutionary ages, including an ancient set of ancestral genes, two gene strata of more intermediate evolutionary origin, and a set of apparently recently acquired genes that are species- but not mating type-specific Cryptococcus bipolar MAT locus contains all of the genes normally found in the two unlinked tetrapolar loci suggests that a fusion of the two loci might have been involved in the origins of this unusual gene cluster linked to virulence and differentiation. The hypothesized evolutionary model posits that an ancestral tetrapolar mating system expanded by a series of acquisitions of genes of related function to form two large gene clusters that subsequently underwent fusion via a chromosomal translocation to form an unstable tripolar intermediate stage that subsequently underwent recombination or gene conversion to form the bipolar state. A series of more recent gene conversions and inversions then occurred, giving rise to the extant bipolar mating type alleles and resetting the molecular clock for some genes within distinct strata and even evicting genes from MAT in other examples MAT locus is flanked by and contains recombination hotspots provides evidence that the expansion and fusion of these loci may have been driven by activation of recombination Cryptococcus can be engineered to complete a tetrapolar sexual cycle and that the tripolar intermediate may have conferred an evolutionary disadvantage and therefore have been transitory MAT evolutionary model by comparative studies with related but divergent fungal species.Molecular analysis of the Cryptococcus species complex, which includes C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattiiCryptococcus amylolentus, Tsuchiyaea wingfieldii, and Filobasidiella depauperata, and was termed the sensu stricto clade. The other clade encompasses yeasts more distantly related to the pathogenic species complex, and was termed the sensu lato group. C. heveanensis lies in the sensu lato clade, together with Bullera dendrophila, Cryptococcus bestiolae, Cryptococcus dejecticola, and a recently described sexual species, Kwoniella mangroviensisC. heveanensis is located in a sister clade to the pathogenic Cryptococcus species complex makes it an excellent candidate to provide insights into the evolution of the MAT locus and sexual reproduction in pathogenic Cryptoccocus species.In a recent study, a multi-locus gene phylogeny was developed with species closely related to the pathogenic C. heveanensis is K. mangroviensis, which is also located in the sensu lato clade containing yeasts more distantly related to the pathogenic Cryptococcus species K. mangroviensis differs from that in C. neoformans and C. gattii and contains septa (phragmobasidia) whereas the pathogenic Cryptococcus species have nonseptate basidia (holobasidia) K. mangroviensis strains are co-cultured on CMA medium, dikaryotic hyphae with clamp connections and basidia are produced K. mangroviensis strains isolated from the Bahamas, 7 were fertile (37%) and 12 were sterile (63%). Among the seven fertile isolates, six were assigned to the α mating type and one to the A mating type. From these results, the authors proposed that the mating system appeared to be bipolar and biallelic. The nature of the K. mangroviensis mating system will be further clarified by analysis of additional fertile strains, progeny from genetic crosses, and the MAT locus.The closest yeast with a defined sexual cycle to MAT locus structure of C. heveanensis. First, we discovered an extant sexual cycle for C. heveanensis involving dikaryotic hyphae with clamp connections and basidia with cruciate septa and termed this teleomorph Kwoniella heveanensis. Second, we determined the structure of the MAT locus involving two probably unlinked gene clusters corresponding to the homeodomain locus and the pheromone/receptor locus. The presence of two divergently transcribed homeodomain genes in the homeodomain locus suggests this MAT locus arrangement is ancestral to the pathogenic Cryptococcus species, and one or the other homeodomain gene has been lost recently from MAT in C. neoformans and C. gattii. In addition, the presence of numerous other genes around the C. heveanensis MAT locus that correspond to genes located on the same chromosome but far away from MAT in C. neoformans suggests intrachromosomal rearrangements occurred during the evolution of the C. neoformans MAT locus and flanking regions. Characterizing MAT alleles from other C. heveanensis isolates suggests that the homeodomain locus is multiallelic and the pheromone/receptor locus is at least biallelic. Additionally, the MAT-specific region is likely to be restricted to the SXI1 and SXI2 genes in the homeodomain locus and spans at least the STE3, STE12, MFA1/2, CNB00600, and CNG04540 genes in the pheromone/receptor locus, consistent with a tetrapolar mating system. This analysis reveals an evolutionary intermediate with two apparently unlinked MAT loci into which genes have been acquired, lost, and translocated, providing insights into the events that punctuated the evolution of a contiguous sex determining gene cluster in C. neoformans and C. gattii. These studies serve as an evolutionary window both for the evolution of mating type loci in fungi, and also into similar events that have driven the evolution of sex chromosomes in fungi, plants, and animals including insects, fish, and mammals In this study, we analyzed the mating behavior and MAT locus of C. heveanensis, the isolates  appear to represent related, but distinct species based on a higher level of divergence in the D1/D2 and ITS sequences  or MS media alone and in every pair-wise combination and incubated at room temperature in the dark or light. Five strains were self-filamentous when incubated alone while the other strains were not . The filenterica . Basidioenterica  and wereenterica . Fecundienterica ). These C. heveanensis MAT locus, probes to MAT-specific genes of C. neoformans and C. gattii were generated by degenerate PCR. These probes  were used to screen a fosmid library for the type strain C. heveanensis CBS 569. Sequencing of positive fosmids and assembly of the sequences resulted in two large gene clusters: a region containing the homeodomain locus and flanking regions spanning ∼80 kb and a region containing the pheromone/receptor locus and flanking regions spanning ∼180 kb (C. heveanensis contains two divergent homeodomain genes (SXI1 and SXI2) encoding an HD1 and an HD2 class product, similar to other basidiomycete MAT loci MAT locus alleles in C. neoformans and C. gattii contain only SXI1α or SXI2a but not both, this suggests that one or the other homeodomain gene has been lost recently in these pathogenic species.To elucidate the structure of the  ∼180 kb . The homC. heveanensis also contains the FAO1, FCY1, and UAP1 genes, which form the left boundary of the pathogenic Cryptococcus species MAT locus  locus of AT locus . Also inenterica .MAT locus of pathogenic Cryptococcus species are present in the region containing the pheromone/receptor (PR) locus of C. heveanensis . This region also contains the LPD1, GEF1, CID1, RPO41, and BSP2 genes, which were hypothesized to be present at the boundaries of the ancestral sex determining regions in the proposed evolutionary model  and to have been entrapped in MAT when the HD and PR loci were brought together via a chromosomal translocation C. heveanensis and T. mesenterica implies that these genes were likely to have all been linked to the ancestral PR locus prior to fusion to a contiguous bipolar sex-determinant.The remaining genes found in the eanensis . These iC. heveanensis have numerous other linked genes that are not present in the MAT locus of C. neoformans or C. gattii. Most of these genes  are also present in the T. mesenterica PR locus region suggesting these genes have recently been lost from the MAT locus and relocated to other genomic locations in C. neoformans and C. gattii . Strains BCC 8396 and BCC 11754 also appeared identical. Therefore, in these cases, one isolate from each strain pair was selected for further studies. Sequencing of an ∼12 kb region, spanning SXI1 and SXI2, from seven strains provided evidence that the locus is multiallelic with 6 different alleles of SXI1 and SXI2  were found in HD1 and this region targeted a reporter protein to the nucleus C. cinerea HD2 protein and HD2-reporter fusion proteins were not nuclear localized. Similarly in U. maydis, a bipartite NLS in bE (HD1) was predicted In RPL22 and CAP1 genes are part of the MAT locus in C. neoformans and C. gattiiRPL22 gene shares 88–90% identity among mating types while the 5′ and 3′ regions of the CAP1 gene show different profiles. For C. neoformans var. neoformans and C. neoformans var. grubii, the identity is 90–92% for the 5′ region of CAP1 and 84–85% for the 3′ region. For C. gattii, the identity is 88% for the 5′ region of CAP1 and 93% for the 3′ region. Sequences from RPL22 and CAP1 were analyzed from C. heveanensis to determine if the HD locus encompasses these genes. When 530 bp of RPL22 gene sequence from all 7 unique strains was analyzed, 97 to 99% sequence identity was observed. Similarly, comparison of 212 bp of CAP1 gene sequence from strains CBS 569, BCC 8313, and BCC 15000 resulted in 99.5–100% identity. Based on this analysis revealing the RPL22 and CAP1 genes are more similar between C. heveanensis isolates than the a and α alleles of C. neoformans, it is likely that the HD locus of C. hevenensis only includes the homeodomain genes, SXI1 and SXI2, although other linked genes should also be examined to further support this conclusion.The C. heveanensis is multiallelic with at least 6 different alleles determined among 7 strains. Thus, additional extant HD locus alleles likely remain to be discovered. In addition, the MAT-specific region is likely to be limited most prominently to the 5′ end of the SXI1 and SXI2 genes coding for the N-terminal regions of the proteins known to be involved in the specificity of heterodimer formation.These results show that the HD locus of STE3, STE12, CNB00600, and CNG04540 genes and the gaps between these genes were closed by primer walking. This sequence analysis resulted in an ∼9 kb sequence spanning the 5′ end of STE3, STE12, a pheromone gene, which was named MFA2, and the CNG04540 and CNB00600 genes. All of these genes were divergent between BCC 8398 and CBS 569 except CNB00600. Sequence identity values are 54% for STE3, 53% for STE12, 75% for MFA1/2, and 63% for CNG04540. In addition, the gene order also differed as indicated in the comparison and the dot plot (CNB00600 gene. The majority of this gene corresponding to the 5′ portion (1285 bases) is 98% identical, while for a small portion at the 3′ end (139 bases for CBS 569 and 124 bases for BCC 8398), the sequence identity is 53% suggesting this gene may have represented the border of MAT or have been subject to more recent gene conversion.When the pheromone and the pheromone receptor gene sequences were amplified by PCR with CBS 569 specific primers, PCR fragments of the expected sizes were obtained for all strains except BCC 8398. Sequencing of these PCR products showed that the pheromone and receptor gene sequences were identical for all of the strains that yielded PCR products (data not shown). For the atypical strain BCC 8398, degenerate primers were used to amplify the dot plot . InteresC. heveanensis, MFA1 (from CBS 569) and MFA2 (from BCC 8398), encode 39 amino acid-long pheromone precursors , STE11 (99% in 470 bases), ZNF1 (100% in 410 bases) or IKS1 (99% in 1740 bases) genes. Also, Southern blot analysis using STE11 and MYO2 gene fragments as probes showed the same hybridization profile among all strains tested  from C. heveanensis, T. mesenterica, C. neoformans var. neoformans, C. neoformans var. grubii and C. gattii  appear to represent isolates of closely related cryptic species  and the second to the pheromone/pheromone receptor gene locus (A) (B locus (HD) includes a pair of divergently oriented genes encoding homeodomain proteins related to Sxi1/Sxi2 from C. neoformans and bE/bW from U. maydis. The A locus (PR) includes genes encoding a Ste3 pheromone receptor homolog and a mating pheromone. Both loci are embedded in larger gene clusters, including some previously identified in the C. neoformans MAT locus and novel ones. To establish which genes in the ∼80 and ∼180 kb assemblies are mating type specific, representative regions were isolated by PCR and sequenced from fertile and sterile isolates. This analysis revealed striking diversity in the intergenic region and 5′ coding regions of the SXI1/SXI2 genes, flanked by a less divergent region (∼80% identity) and followed by nearly identical 3′ coding regions , suggesting these genes are outside the mating-type specific region. Similar to other basidiomycetes, including U. maydis, S. commune, and C. cinerea, we suggest dimerization occurs between HD1 and an HD2 class homeodomain proteins encoded by different mating type locus alleles Analysis of the mating type locus for ocus (A) . Notably regions . The divA locus (PR) of C. heveanensis encodes a Ste3 pheromone receptor homolog, a mating pheromone, and other genes involved in pheromone sensing  are present or linked. Thus far, our sequence analysis for this large gene cluster spans ∼180 kb for the CBS 569 type strain and representative sequence from other isolates. This analysis allows us to conclude that the gene sequences, and the gene organization, differ substantially between two different alleles present in the population . Thus far we have not been successful in isolating basidiospores by micromanipulation, and this is a challenge as these are often embedded in a matrix together with basidia. Moreover, in our experience attempts to purify progeny from fungal mating mixtures often leads to a high rate of contamination from the parental isolates, confounding analysis. Future studies could approach this using genetically marked strains to enable selection of meiotic progeny to determine their mating type. A further implication of our findings and conclusions is that the mating type determination system for the closely related species K. mangroviensis, currently classified as a bipolar species with α and A mating types, may also turn out to be tetrapolar with further detailed analysis.Second, additional genetic evidence involving the isolation and analysis of meiotic progeny could further establish this species as tetrapolar. The sine qua non of a tetrapolar mating system is the production of four types of meiotic progeny , the most parsimonious model is that the organization of MAT in C. heveanensis represents the ancestral state prior to fusion, rather than the result of a recent translocation that separated a contiguous MAT allele. Third, the strata of most recently acquired genes  are all present in the right gene cluster linked to the pheromone and pheromone receptor genes, and thus were already present prior to linkage of the two regions. This differs in detail from the originally proposed model in which RPO41 and BSP2 had been depicted as linked to the homeodomain gene cluster, and GEF1, CID1, and LPD1 as linked to the pheromone/receptor locus MAT function, rather than being simply entrapped when the two unlinked MAT loci were fused by translocation. We note that the organization of the two gene clusters in C. heveanensis represents a juxtapositioning of MAT-specific genes  with genes that are part of the MAT locus in C. neoformans/C. gattii but not yet MAT-specific in C. heveanensis. Similarly, for the pheromone/receptor gene cluster, sequences that are not mating type-specific are embedded between mating type-specific sequences. These gene clusters therefore represent transitional intermediates representing MAT loci to which genes of related function have been recruited, but not yet captured into the mating type-specific regions of the genome or begun to diverge into mating type-specific alleles.Our analysis of the ry model . First, C. neoformans and C. gattii MAT alleles reflect episodic gene conversion events that occurred after the two ancestral MAT loci fused STE12 gene linked to the PR locus, and gene conversion events between alleles both prior to and following linkage of the HD and PR loci that influenced the evolutionary trajectory and plasticity of this dynamic region of the genome.The model presented in MAT locus of C. heveanensis, whereas only one or the other homeodomain gene is present in the a or α MAT alleles of C. neoformans and C. gattii. This provides evidence that, like other tetrapolar basidiomycete fungi such as U. maydis, Schizophyllum commune, and C. cinerea, the ancestral Cryptococcal MAT locus contained two paired, divergent homeodomain genes and one or the other was lost recently in the C. neoformans/C. gattii lineage. Examples of homeodomain gene loss are also seen in MAT of pathogenic Candida species Candida lusitaniae and Candida guillieromondii, both of which have retained complete sexual cycles, and in addition C. guilliermondii has also lost the a1 homeodomain gene. As discussed elsewhere MAT encoded homeodomain protein have only one chance to form a productive heterodimer required for completion of sexual reproduction. A second key finding is that the C. heveanensis A locus (PR) is at least biallelic; when additional isolates become available it will be possible to test if other extant alleles are present . Previous studies of U. maydis, which is tetrapolar with a biallelic pheromone/pheromone receptor locus Sporisorium reilianumA locus encoding pheromone and pheromone receptors may have similarly occurred in the lineages leading to C. heveanensis. The outcome for the species then in both cases is a restriction in outbreeding potential, which may serve to further promote inbreeding and clonality in the population, and this transition in sexuality may have contributed to the emergence of this clade of organisms as successful pathogens of animals, including humans.A key finding of this comparative analysis is that two divergently oriented homeodomain genes are present in the C. heveanensis type strain CBS 569, CBS 9459, and CBS 6097 were obtained from the Centraalbureau voor Schimmelcultures (CBS), Netherlands. Other strains of C. heveanensis were from the BIOTEC Culture Collection (BCC), Thailand Strains used in this study are listed in Kwoniella heveanensis Metin, Findley, & Heitman sp. nov.Standard description: Cryptococcus heveanensis (Groenewege) Baptist & Kurtzman comb. nov Etymol.: The epithet is chosen to be identical with that of Heterothallic fungus. Hyphae dikaryotic, clamp connections fused. Basidia clustered, submerged initially, finally on aerial hyphae, globose, 7.5–10.5 µm, cruciately septate, sterigmata not observed. Basidiospores subglobose, 2.3–3.3 µm, germinating by conidia.Kwoniella heveanensis Metin, Findley, & Heitman sp. nov.Latin description: Species heterothallica. Hyphae dikaryoticae, fibulae fusae. Basidia aggregata, primum submersa, deinde superficialia, globosa, 7.5–10.5 µm diam, septis cruciatis divisa, sterigmatibus carentia, Basidiosporae subglobosae, 2.3–3.3 µm diam, cito conidia proferentia.2, 0.1 mM CaCl2, 0.1 mM ZnCl2), they were resuspended in 180 µl NS buffer. Then, 20 µl RNase A  and 5 µl of propidium iodide  were added. After incubating at room temperature in the dark with mixing for at least 2 hours, 17.5 µl of the cells were mixed with 700 µl of 50 mM Tris-Cl pH 7.5 and the tubes were sonicated for 45 seconds. Flow cytometry was performed on 10,000 cells and analyzed on the FL1 channel with a Becton-Dickinson FACScan.The protocol for the preparation of cells for FACS was based on a previous protocol For microscopic examination of mating, either the mating mix was inoculated into V8 medium (pH 5) medium plates, and later a block of agar containing the hyphae and basidia was transferred to microscope slides, or the mating mix was directly inoculated onto slides containing a thin layer of mating medium.For staining, a modified version of a previously published protocol was used E. coli phage-resistant cells overnight at 37°C. Fosmid clones were then picked into 96 well plates and eventually transferred to 384 well plates, which were stored at −80°C.For library production, the CopyControl Fosmid Library Production Kit (Epicentre) was used according to the manufacturer's instructions. In each library, a total of ∼16,000 clones were picked into 96 well plates. Purified genomic DNA (∼40 kb fragments) was first sheared, end-repaired, and separated using contour-clamped homogenous electric field (CHEF) on a CHEF DR-II apparatus (Bio-Rad). The following conditions were used: 1- to 6-sec switch time, 6 V/cm, 14°C for 14 to 15 hrs. The size-fractionated DNA was recovered by gel extraction and precipitated. The insert DNA was then ligated into the CopyControl pCC1FOS cloning-ready vector and incubated overnight at room temperature. The ligated DNA was packaged and plated on For DNA isolation, strains were inoculated in 50 ml YPD medium, grown for two days at room temperature with shaking and centrifuged. After freezing at −80°C, the pellet was freeze-dried overnight and DNA was isolated as previously described LPD1, RPO41, CAP1, ZNF1, STE20, IKS1, FAO1 and NOG2 based on known gene sequences from C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii. These primers (C. heveanensis genomic DNA using Ex Taq polymerase (Takara) and the products obtained were gel extracted with QIAquick Gel Extraction Kit (Qiagen). The PCR fragments were cloned into pCR 2.1-TOPO using the TOPO TA Cloning Kit (Invitrogen). From the resulting colonies, plasmid isolation was performed with QIAprep Spin Miniprep Kit (Qiagen) and the plasmids were sequenced with M13F and M13R vector specific primers. After obtaining MAT gene sequences, specific primers were designed using a web-based program, Primer3 (http://frodo.wi.mit.edu/). After PCR with specific primers, the resulting gene fragments were gel-extracted as previously described and used as probes to screen the library.Degenerate primers were designed for  primers  were useFor library screening, colony lifts were performed on Hybond membranes (Amersham) using standard protocols Positive fosmids were isolated from 200 ml of overnight grown cultures using the Qiafilter plasmid midi kit (Qiagen). The fosmid DNA obtained was sheared using a hydroshear apparatus (Genomic Solutions) to 1.6–3 kb fragments. The gel extracted sheared DNA was then ligated to adapters. To prepare the vector, pUC18 plasmid DNA was cut with EcoRI and HindIII removing the polylinker site, and ligated to single stranded oligos that contain one end complementary to the restriction enzyme site, and the other non-complementary to itself to prevent self-ligation. The sheared and adapter-ligated DNA was then ligated to the pUC18-derived vector and the reaction was used to transform DH5α competent cells (Invitrogen). White colonies were inoculated onto 96 well plates and plasmid isolation was performed with DirectPrep 96 MiniPrep kit (Qiagen). For each positive fosmid, four to five 96 well plates were used.Taq polymerase (Takara).Sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on PE3700 96-capillary sequencer (Applied Biosystems) in the Sequencing Facility at Duke University. The sequence reads were assembled using Phred, Phrap, and Consed program packages C. heveanensis strains, 5 µg of genomic DNA from each strain was digested with EcoRV, electrophoresed on a 0.8% agarose gel, and blotted onto Hybond membranes (Amersham) using standard protocols MAT gene probes generated by PCR as described above.For Southern hybridization of C. heveanensis sequences with the most similar protein sequences identified by BLASTX. Genbank accession numbers are, GU129941 for CBS 569 homeodomain locus, GU205379 for CBS 569 pheromone/receptor locus, GU129940 for BCC 8398 pheromone/receptor locus, GU129942, GU129943, GU129944, GU129945, GU129946, and GU129947 for homeodomain loci of the strains BCC 8313, BCC 8384, BCC 8398, BCC 11754, BCC 11757, AND BCC 15000, respectively. Genbank accession numbers for D1/D2 and ITS regions are GU585738 and GU585749 for CBS 9459, GU585739 and GU585750 for CBS 6097, GU585740 and GU585751 for BCC 8305, GU585741 and GU585752 for BCC 8313, GU585742 and GU585753 for BCC 8384, GU585743 and GU585754 for BCC 8396, GU585744 and GU585755 for BCC 8398, GU585745 and GU585756 for BCC 11754, GU585746 and GU585757 for BCC 11757, and GU585747 and GU585758 for BCC 15000, respectively. The accession number of the ITS region of CBS 569 is GU585748.Annotations of the genes were done manually by comparing the 3-frame translated Percent sequence identity plots and dot plots were generated using the MultiPipMaker The comparison plot between pheromone/receptor loci of strains CBS 569 and BCC 8398 was produced using Artemis Comparison Tool Release 8 Homeodomain regions of the proteins Sxi1 and Sxi2 were predicted by comparison to the previously characterized homedomain proteins. Helical content of the N-terminal domains of Sxi1 and Sxi2 were predicted with Jpred 3 Phylogenetic analysis was conducted on coding sequences using MEGA4 Figure S1C. heveanensis, while the strains CBS 9459 and CBS 6097 are closely related but different species. Eight isolates obtained from BCC have identical D1/D2 sequence as the C. heveanensis type strain CBS 569, while there are two differences between CBS 569 and CBS 9459, and three differences between CBS 569 and CBS 6097. Similarly, six of the eight BCC strains have identical ITS sequence as the type strain CBS 569, and there is only one difference between the type strain and the strains BCC 8396 and BCC 11754. However, there are eleven differences between CBS 569 and CBS 9459, and eight differences between CBS 569 and CBS 6097 in the ITS region.Eight BCC isolates are confirmed to be (0.82 MB PDF)Click here for additional data file.Figure S2C. heveanensis isolates are haploid based on FACS analysis. The type strain CBS 569 and eight BCC isolates were found to be haploid based on FACS analysis conducted with the haploid control C. neoformans JEC20 and the diploid control C. neoformans XL442.(0.24 MB PDF)Click here for additional data file.Figure S3Sequence alignment of homeodomain proteins. (A) Sxi1, and (B) Sxi2 sequence alignments. Purple boxes indicate the alpha helical regions at the N-terminal region of the proteins. Blue box shows the homeodomain region and green boxes indicate the predicted nuclear localization signals.(2.26 MB PDF)Click here for additional data file.Figure S4Percent sequence identity plots of the homeodomain region. An extended version of (0.12 MB PDF)Click here for additional data file.Figure S5CID1, GEF1, and LPD1, (B) mating type-specific pattern exhibited by BSP1, SPO14, ETF1, STE20 and STE11 from C. gattii and C. neoformans, but not from C. heveanensis or T. mesenterica, (C) mating type-specific profile demonstrated by STE3 and STE12, and (D) SXI1 and SXI2 sequence alignments from C. heveanensis strains, C. gattii and C. neoformans.Phylogenetic analysis of selected mating type genes. An extended version of (0.14 MB PDF)Click here for additional data file.Table S1T. mesenterica genes presented in JGI annotation IDs for (0.02 MB PDF)Click here for additional data file.Table S2Primer sequences.(0.06 MB XLS)Click here for additional data file.


The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas. Ixodid ticks and by the contact with blood or tissue from viremic livestock and human patients [Bioinformatics analysis of complete microbial genomes has led to advances in the development of novel diagnostic techniques, in the research of microbial pathogenesis, and in the control and prevention of infectious diseases. Until the year 2006, only 2 complete genomes of Crimean-Congo hemorrhagic fever virus (CCHFV) had been sequenced . CCHFV, patients ,4. The apatients ,5. Presupatients ,3,4. TheThe Balkan peninsula, and Kosovo in particular, is a well-known endemic region for CCHF, and epidemic outbreaks and sporadic cases have been frequently been recorded -8. Five th day after onset of symptoms. Results of the laboratory analysis showed the presence of IgM antibodies (titer 1:400) and the presence of viral RNA in the concentration of 1.08 × 1010 copies per mL of serum. Virus was grown on Vero E6 cells in BSL-3 laboratory. Viral RNA was extracted with the Trizol reagent from the second passage of the CCHFV in Vero E6 cells, and used for the direct sequencing of the complete genome of the virus. Amplicons of S, M and L full length segments were obtained by following the protocols described previously [DQ133507, EU037902 and EU044832, respectively. Sequence alignment of CCHFV Kosova Hoti strain complete genome with preexisting CCHFV genomes was performed using the CLUSTAL W algorithm of MegAlign module . Phylogenetic relationships of different CCHFV strains were established with a software package TREECON [The CCHFV Kosova Hoti strain was isolated from a blood of a female fatal case during the epidemic in Kosovo in 2001 . The bloeviously ,13. Brie TREECON . The phy TREECON ,16. The  TREECON ,18.The genome size of CCHFV, strain Kosova Hoti, was found to be approximately 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. The open reading frame (ORF) of S segment is 1449 nt in length, encoding a 482 aa (nt position 56 – 1504) nucleocapsid protein. The ORF lengths of M and L segments are 5067 nt/1688 aa (nt position 78–5144) and 11838 nt/3945 aa (nt position 78–11915), respectively.Three phylogenetic trees were constructed based on the ORF sequences of S, M and L segments of CCHFV Fig. . The genThe sequence differences between the CCHFV strains in the group V. are shown in Tables 251, RKLL523 and RKPL1043 were identified in Kosova Hoti and are identical among all strains in V. group. They represent the cleavage sites for GP38, Gn and Gc proteins, respectively [523 tetrapeptid of Kosova Hoti is typical for all strains in group V (Europe/Turkey) but it differs from RRLL tetrapeptid in all other CCHFV strains sequenced. However, both tetrapeptides constitute a cleavage recognition site for subtilase SKI-1 [The analysis of the Kosova Hoti strain M segment encoded polyprotein predicted the cleavage of the signal peptide to occur between aa 27 and 28 (AHG-QS). This site is identical to those described for Kosovo 9553-01 and Kashmanov but differs from other strains in group V.  was identical among all CCHFV strains used in the L segment alignment  declare that they have no competing interests.DD performed RNA extraction, qualitative and quantitative RT-PCR, analyzed the data and prepared the draft manuscript. MK and STN provided the complete M and L segment sequences and revised the draft manuscript. AS sequenced the complete S segment. IHB performed the protein analysis. MP, ID and SA collected the samples and clinical data. TAZ isolated the virus, supervised the study and revised the final draft. All authors read and approved the final manuscript.


Antibodies play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies, the Fc- and complement-dependent activities of these multifunctional proteins may also be critical in their ability to provide protection against most viruses. Protection against viral pathogens in vivo is complex, and while virus neutralization—the ability of antibody to inactivate virus infectivity, often measured in vitro—is important, it is often only a partial contributor in protection. The rapid fluorescent focus inhibition test (RFFIT) remains the “gold standard” assay to measure rabies virus–neutralizing antibodies. In addition to neutralization, the rabies-specific antigen-binding activity of antibodies may be measured through enzyme-linked immunosorbent assays (ELISAs), as well as other available methods. For any disease, in selecting the appropriate assay(s) to use to assess antibody titers, assay validation and how they are interpreted are important considerations—but for a fatal disease like rabies, they are of paramount importance. The innate limitations of a one-dimensional laboratory test for rabies antibody measurement, as well as the validation of the method of choice, must be carefully considered in the selection of an assay method and for the interpretation of results that might be construed as a surrogate of protection. Whether an animal control worker wants to determine if a rabies vaccine booster is necessary to establish an acceptable pre-exposure status, or a physician is considering the causes of encephalitis in a child, or the owner of an immunologically compromised dog is worried that the dog's response to rabies vaccination will not be sufficient to pass a serological test allowing them to travel to a rabies-free area, or a researcher is trying to determine if the rabies vaccine-bait response is adequate in a raccoon population, or one needs to assign a potency value to a rabies immune globulin product, all demand an accurate assessment based on the measurement of circulating antibodies. In each of these situations the measurement or simply the detection of rabies-specific virus-neutralizing or other antibodies will help resolve the question. However, just as the circumstances in each of these scenarios are different, the specifics of the method chosen to measure antibodies, the regulatory requirements of the testing, and the purpose of testing in each of these situations are different. Antibodies arise from the humoral immune response to rabies antigens, the process of which is controlled by many factors, including the amount of antigen, route of delivery, the expression and involvement of major histocompatibility complex (MHC) genes, and the health status of the individual, among others. An understanding of the host immune response, including the immunoglobulin (Ig) subclass (type) and the kinetics and longevity of the response, is necessary to obtain the measure or degree of rabies immunity.Initially, measurement of rabies virus neutralizing antibodies (RVNA) was performed in vivo using the mouse neutralization test (MNT). Subsequently, the rapid fluorescent focus inhibition test (RFFIT) was established as the “gold standard” in vitro test A review of the literature was conducted using the online database PubMed from 1975 to 2008 with US National Library of Medicine medical subject headings (MeSH). Reference lists of selected articles and reviews were also individually researched. In addition, unpublished rabies serology data from the Kansas State University Rabies Laboratory were reviewed.2 portions of neutralizing antibodies Animal models of protection against rabies have demonstrated the essential role of RVNA + T lymphocyte response, which includes cytokine production. Each individual's polyclonal antibody response to rabies vaccination is a unique mix of specificities to rabies virus antigens. Although the majority of individuals develop a variety of measurable antibodies in response to pre- or postexposure vaccination, there may be substantial variation in the neutralizing activity and quantity of RVNA produced.The rabies virus is highly neurotropic. Within the CNS, rabies virus infection develops for the most part undetected by the adaptive immune responses of the host until the infection is in its final stages. In addition to an infection that develops within this immune-privileged site, the rabies virus is able to subvert any host immune response that is mounted and thus the outcome of infection is fatal in nearly 100% of cases. In contrast, a rabies vaccine is able to stimulate high levels of circulating RVNA. This is why immunity following exposure to rabies virus in an unvaccinated individual, initiated with prompt wound cleansing to reduce viral load at the site of exposure and administration of rabies-specific Ig followed by a series of rabies vaccinations, is virtually 100% effective in preventing infection. Passive protection with rabies Ig (RIG) is critical to immediately neutralize a majority of infectious virions, keeping the virus from spreading while waiting for induction of the host's adaptive immune response. In general, rabies vaccines historically are killed whole-virus vaccines, which are expected to promote the development of anti-rabies virus-specific antibodies along with a CD4Antibodies develop out of intrinsic host responses and in response to extrinsic factors such as the amount of antigen given, type of antigen, and route of exposure or vaccination. For example, it has been demonstrated that the more potent the vaccine the higher the levels of RVNA that are induced in human subjects Methods available for the detection and measurement of rabies virus–specific antibodies are either antigen-binding assays or virus-neutralization assays. In antigen-binding assays, antibodies in serum or cerebrospinal fluid (CSF) are detected, quantified, and characterized by their ability to bind to various rabies virus antigens, from whole virus, purified subunits, or specific peptides that mimic epitopes. Such assays determine the affinity, avidity, and specificity of binding antibodies. Commonly, these methods involve fixing the antigen to a solid surface, i.e., tube, plate, or bead. The interaction between the antibody and the antigen is then visualized and quantitated by various detection systems that involve color development through an enzyme–substrate reaction or binding of a secondary antibody, conjugated to a fluorescent marker or staphylococcal protein A/G, to the primary antibody bound to the antigen. These assays can be used to measure antibodies that react to internal viral proteins, either structural or enzymatic, as well as to estimate levels of antibodies that bind to the external proteins against which neutralizing antibodies are directed. In contrast, modern virus neutralization assays are cell-based assays that detect the functional activity of antibodies in the serum or CSF against live virus. The mechanism of virus neutralization in cell culture (an in vitro assay) depends upon the interaction between the virion epitope and the paratope (the specific counteracting site) of the antibody, and may also be influenced by receptor characteristics of the cells upon which an assay is performed By changing various components, such as virus strain, detection system, etc., of the assay, it is possible to custom design a “fit-for-purpose” assay. For example, detection of RVNA in a particular colony of bat species associated with a rabies virus variant may be “specifically” measured using, in the in vitro assay, the suspected rabies virus variant in circulation among the bats as the challenge virus. The sensitivity of a test method may be adjusted by varying the amount of antigen or challenge virus dose to more precisely define and determine low levels of antibodies or the presence of nonspecific or cross-reacting antibodies or substances. For binding assays, such as an enzyme-linked immunosorbent assay (ELISA), a labeled anti-IgM as the secondary antibody detects only IgM rabies virus antibodies as the primary antibody. The anti-IgM level can be compared to the anti-IgG level in the same sample by performing the same assay, but using a labeled anti-IgG as the secondary antibody. The competitive ELISA method utilizes a competing monoclonal antibody to a rabies protein epitope and measures the ability of antibodies in the test sample (of serum or CSF) to out-compete the monoclonal antibody for attachment to that particular epitope Determination of the cut-off value  is specific for each test method and is critical to how results are interpreted. In general, a serum neutralization titer of 1∶100 (90% endpoint titer) is acceptable as effective, even though antibody levels in tissue might actually be lower Standardization of rabies serologic test methods is essential to provide a meaningful comparison of antibody characteristics and potency in vaccinated or exposed individuals. This becomes especially relevant when rabies serology results are compared between different laboratories and different studies over time. The fluorescent antibody virus neutralization (FAVN) test was developed in part to address standardization of the various modifications of the RFFIT in use The international reference standards for RIG products in use today include of the WHO first International Standard for Rabies Immunoglobulin, the WHO second International Standard for Rabies Immunoglobulin, and the Office International des Épizooties (OIE) canine RIG reference serum. The potency of each of these products has been assigned a value by serum neutralization methods The use of potency values for RIG that are assigned by serum neutralization methods as opposed to those assigned by binding assays is inappropriate and problematic. “Method-of-result” calculation is an additional factor to consider in the standardization of an assay. For example, the use of either 50% or 100% endpoint titers, both of which can be calculated for serum neutralization assays, will yield different titer values. For any comparison of results, it is essential to know, and should always be stated, what calculation was used to generate titer values.It is important that characteristics and variability associated with different assay methods that define antibody titers continue to be defined as we advance our understanding of immunity and disease prevention. The measurement of rabies specific antibodies in vitro is essential and should be the first step taken to establish whether rabies immunity following vaccination is successful. Even so, in vitro measurements may never completely correlate with what may be regarded as protective in vivo. Even use of “gold standard” measurements of rabies virus neutralizing activity in vitro provides only an estimate of protection. Despite the long history of using virus neutralization tests, there are still no internationally recognized standard protocols for measuring rabies-specific antibodies, for specifically defining RIG potency for human rabies biologics formulation, or for assessing oral rabies vaccine uptake in wildlife. Method standardization requires the careful examination and evaluation of laboratory test performance, which may include audits of laboratory operations, the publication and sharing of standard operating procedures, and inter- and intralaboratory evaluation of proficiency in performing tests. Proficiency testing, training, and certification, as well as trends in assay performance are components of quality assurance programs to ensure continuing adherence to acknowledged and accepted standards. These efforts will require the collaboration of all organizations performing tests, including national laboratories, regulatory agencies, commercial companies, and rabies diagnostic and research laboratories.For a fatal disease such as rabies, where vaccination and passive immunity (use of HRIG) are absolutely required for protection upon exposure, verification of the accuracy and efficacy of every assay involved in predicting the relevant in vivo protection against the virus is vital. Moreover, there is a basic requirement to confirm the appropriateness and applicability of any neutralization test system or antigen binding assay used, as a predictor of in vivo protection, with each new type of prophylactic biologic, be it a vaccine, Ig, or monoclonal antibody formulation. In the case of vaccines, it is imperative to understand how antibodies neutralize viral infectivity in order to have input into the design and presentation of immunogens of the vaccine that elicit the specificity and isotype of antibody produced to confer the maximum neutralizing, and ultimately, protective activity.1. RVNA have been demonstrated to be critical for protection against rabies. Even so, in vitro measurements are only a partial determination of the degree of protection provided in vivo. Not all methods that measure rabies-specific antibodies will determine the neutralizing function of the antibodies.2. In the selection of the most appropriate assay for rabies antibody detection, consideration of the purpose and use of the results is as important as the established performance characteristic of the assay.3. Standardization of assays includes both assay components and test conditions. Alterations will cause variation in results; therefore use of a particular assay  does not guarantee comparable results if the assays have not been standardized.4. Because rabies is a fatal disease for which development of a sufficient RVNA response is paramount for protection, assay specificity, sensitivity, and accuracy must be verified for meaningful clinical decisions to be made based on the results.5. Steps toward better understanding and use of rabies serology assays will include collaboration of national laboratories, regulatory agencies, as well as commercial and research laboratories. Greater cooperation and standardization of rabies serology assays will lead to increased understanding of the relationship between in vitro measurement and in vivo protection.1. Smith JS, Yager PA, Baer GM (1973) A rapid reproducible test for determining rabies neutralizing antibody. Bull World Health Organ 48: 535–541.2. Cliquet F, Aubert M, Sagne L (1998) Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods 212: 79–87.3. Aubert MF (1992) Practical significance of rabies antibodies in cats and dogs. Rev Sci Tech 11: 735–760.4. Grassi M, Wandeler AI, Peterhans E (1989) Enzyme-linked immunosorbent assay for determination of antibodies to the envelope glycoprotein of rabies virus. J Clin Microbiol 27: 899–902.5. Irie T, Kawai A (2002) Studies on the different conditions for rabies virus neutralization by monoclonal antibodies #1-46-12 and #7-1-9. J Gen Virol 83: 3045–3053.Alternative Language Abstract S1French translation of the abstract by Celine Jiron Corrales.(0.03 MB DOC)Click here for additional data file.


We assessed the societal costs and benefits of a subtype-specific surveillance system for identifying outbreak-associated Escherichia coli O157:H7 infections. Using data from Colorado, we estimated that if it averted five cases annually, the system would recover all its costs.


The activities of certain enzymes in the tissus of mice infected with Friend virus have been studied. Progressive increases in LDH, G6PDH and 6PGDH have been observed in the spleen and liver concomitant with malignant transformation. Plasma LDH activity was also observed to increase over the period of study, but changes in the isoenzyme distribution probably resulted from contamination of the Friend virus preparation with lactic dehydrogenase virus. The tissue enzyme changes are similar to those observed in human malignancies.


With the exception of the oyster herpesvirus OsHV-1, all herpesviruses characterized thus far infect only vertebrates. Some cause neurological disease in their hosts, while others replicate or become latent in neurological tissues. Recently a new herpesvirus causing ganglioneuritis in abalone, a gastropod, was discovered. Molecular analysis of new herpesviruses, such as this one and others, still to be discovered in invertebrates, will provide insight into the evolution of herpesviruses.We sequenced the genome of a neurotropic virus linked to a fatal ganglioneuritis devastating parts of a valuable wild abalone fishery in Australia. We show that the newly identified virus forms part of an ancient clade with its nearest relatives being a herpesvirus infecting bivalves (oyster) and, unexpectedly, one we identified, from published data, apparently integrated within the genome of amphioxus, an invertebrate chordate. Predicted protein sequences from the abalone virus genome have significant similarity to several herpesvirus proteins including the DNA packaging ATPase subunit of (putative) terminase and DNA polymerase. Conservation of amino acid sequences in the terminase across all herpesviruses and phylogenetic analysis using the DNA polymerase and terminase proteins demonstrate that the herpesviruses infecting the molluscs, oyster and abalone, are distantly related. The terminase and polymerase protein sequences from the putative amphioxus herpesvirus share more sequence similarity with those of the mollusc viruses than with sequences from any of the vertebrate herpesviruses analysed.Malacoherpesviridae, that was based on a single virus infecting oyster can now be further established by including a distantly related herpesvirus infecting abalone, which, like many vertebrate viruses is neurotropic. The genome of Branchiostoma floridae (amphioxus) provides evidence for the existence of a herpesvirus associated with this invertebrate chordate. The virus which likely infected amphioxus is, by molecular phylogenetic analysis, more closely related to the other 2 invertebrate viruses than to herpesviruses infecting vertebrates (ie chordates).A family of mollusc herpesviruses,  Haliotis spp[Haliotis diversicolor [In 2005 there was an outbreak of acute ganglioneuritis in an Australian population of the edible gastropod mollusc, abalone or iotis spp. Using tiotis spp. Potentirsicolor . Recentlrsicolor .Herpesvirales - the Herpesviridae which infect Mammalia, Aves and Reptilia, the Alloherpesviridae infecting Amphibia and Osteichthyes (bony fish), and the mollusc-infecting Malacoherpesviridae containing a single virus that infects an invertebrate class, Bivalvia [Herpesvirales order and provides two key links to understanding the nature of the ancient ancestors of mollusc and vertebrate herpesviruses. To understand the structural and evolutionary relationships of the abalone virus to other herpesviruses, we purified abalone virus particles and isolated and sequenced genomic DNA using methods previously described[Three herpesvirus families have been described in the order Bivalvia -8. The pBivalvia ,8. GastrBivalvia  combineddescribed,9. The Ddescribed and sequOstreid herpesvirus 1 , a virus infecting bivalve mollusc species[Based on the assembled DNA sequences of the abalone virus, several protein coding sequences predicted using Artemis showed vc species,13. BLASc species of assemBranchiostoma floridae (amphioxus) coding sequences with significant homology to some of those in AbHV-1. The genome of amphioxus has been recently sequenced[NC_005881), we identified 19 herpesvirus gene homologues. Consistent with this being an integrated virus, we found that 18 of these genes are clustered within a 150 kb region of a single amphioxus scaffold BRAFLscaffold_217, including the herpesvirus specific terminase gene[in situ hybridisation of chromosomes would confirm this. The 19 coding sequences identified are listed in Table During the search for homologues of predicted AbHV-1 proteins using BLAST we identified, in the non-redundant (nr) Genbank protein sequence database, sequenced althoughnase gene and all The putative terminase, or DNA packaging ATPase, appears specific to herpesviruses and some bacteriophages, such as T4 and is tHerpesvirales order. A common ancestral origin for the mollusc and amphioxus viruses is confirmed by the absence of introns in the terminase gene and the presence of additional amino acid loops  motif sequences from 20 of the 34 terminase proteins listed in Table s Figure . AlthougHerpesvirales order. The five Alloherpesviridae viruses are clustered together, with separate clades for frog and fish viruses as found previously [Herpesviridae are clustered into separate major clades reflecting their taxonomic groupings of alpha-, beta- and gammaherpesvirinae sub-families. The phylogenetic analysis confirms a relationship between the amphioxus virus and the abalone and oyster viruses in a deep invertebrate clade. The level of divergence makes estimation of the relative divergence times of the 3 herpesvirus families difficult. Interestingly, the amphioxus virus is in the clade with mollusc viruses, which may not have been expected given the amphioxus chordate host lineage is more aligned with vertebrates than molluscs.The phylogenetic analysis comparing concatenated polymerase and terminase full-length proteins Figure , Table 3eviously , and theHerpesviridae in vertebrates indicate a divergence of alpha-, beta- and gammaherpesviruses to over 400 Mya, and longer times are predicted for divergence of Alloherpesviridae and Malacoherpesviridae[Malacoherpesviridae may have the ability to infect across species, a feature not typically observed in vertebrate herpesviruses, although the infection observed is restricted to related mollusc species[The invertebrate herpesvirus clade provides a unique branching point to inform the metazoan diversification of the herpesviruses. It is thought that during the Cambrian era, the Bilaterial species diverged to generate the Protostomes  and the Deuterostomes (from which the chordates and then the vertebrates evolved),21. Mollesviridae. An altec species.Alloherpesviridae, Malacoherpesviridae and other invertebrate herpesviruses become available it will allow a more informative analysis of their evolution. Of particular interest will be new herpesviruses yet to be discovered in species which share bilateral symmetry such as amphioxus, sea squirts, flatworms or squid. Our discovery of clustered intact herpesvirus genes in amphioxus suggests an opportunistic integration has occurred in the amphioxus genome. This may not be a normal feature of infection and latency, but herpesviruses can occasionally integrate into the genome of their host[As more sequence data and gene structure for heir host. Surprisheir host. Furtherheir host and PCR-heir host based onHerpesviridae family. Convergent evolution may have given rise to the neurotropism seen in some members of the Herpesviridae and now the Malacoherpesviridae families. The rooting of a neurotropic invertebrate virus near or before the divergence of alpha-, beta-, and gammaherpesviruses, may also suggest that early mammalian herpesvirus precursors were neurotropic and that some have retained this feature over time. It is interesting to speculate as to the earliest functional interactions between sensory cells and viruses, as the first sign of neurons appeared over 600 million years ago in "cnidarians," (eg: hydra), but organisms basal to them like sponges do not have neurons or synapses[Cnidaria , thought to be related to the first species with sensory receptors[The neurotropism of certain herpesviruses is well documented but this behaviour is not known outside the families of herpesviruses infecting terrestrial vertebrates,28. The  synapses. Recent  synapses. Others[ synapses have spereceptors. FurtherHerpesvirales order, we suggest the creation of a new genus called Haliotivirus within the Malacoherpesviridae family and assignment of AbHV-1 as a species under Haliotivirus .To accommodate the new abalone virus, which we have suggested naming abalone herpesvirus or AbHV-1, within the Branchiostomid species chromosome as amphioxus-associated virus, AaHV-1. We suggest the species name Branchiostomid herpesvirus 1. Given the unique nature of the virus revealed by phylogenetic analysis and the unique evolutionary positioning of amphioxus as an invertebrate chordate, we suggest this virus, if classified, could be a member of a new family, Aspondyloherpesviridae (from the Greek for "no spine").We have referred to the putative virus genome integrated into the The authors declare that they have no competing interests.KWS, FW, BGC, SW conceived and designed the experiments; FW, NC performed the experiments; KWS, TS, DS analyzed the data; FW, SW, TS, DS, NC contributed reagents, materials, analysis tools; KWS, BGC wrote the paper. All authors have contributed to the editing or revision of the manuscript and approve its publication.


The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase.R) vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3–7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated.We have isolated several CDV resistant (CDVResistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents. Furthermore, outbreaks of monkeypox, a virus indigenous to equatorial Africa, have occurred recently in both the US and Western Africa in human populations and demonstrate the potential of viruses to be rapidly transmitted throughout the world [ studies -4.R) orthopoxviruses were isolated previously via serial passage [R VV the mutations responsible for resistance were mapped to the viral DNA polymerase [CDV is a nucleotide analog and thus the proposed target of its interaction is the viral DNA polymerase. CDV resistant  negative thus attenuating the virus and rendering it an unsuitable backbone to later assess the CDVR virus phenotype in animals [We first established the concentration of CDV that would effectively eliminate wt VV plaques without having significant cytotoxic effects on the BSC40 cells Figure . The con animals . Indeed, animals . To insuR 1 and 2 are shown in Figure R 1 and 2 we used PCR fragment 14 containing the A314V mutation. For CDVR15 a fragment containing the M671I mutation was used and for CDVR16, a fragment with the ΔK174 mutation was transfected. The reconstructed viruses are designated with an "A" following the original virus name to distinguish them from the original isolates. Reconstructed viruses containing only the identified mutation in E9L in a wild type VV background were sufficient to confer CDV resistance except for CDVR 15. We were unable to reconstruct the CDVR 15A virus (Table R 16 (ΔK174). This was observed multiple times and indicates quite clearly, that the resistance that allowed the isolation of CDVR15 containing the M671I mutation depends on a second contributing mutation in the original CDVR 15 isolate outside of the DNA polymerase for resistance. Furthermore, this second site mutation in CDVR15, outside the DNA polymerase can be compensated for by a specific second mutation in the DNA polymerase, ΔK174. In contrast, CDVR 16A was successfully reconstructed to contain only the original ΔK174 mutation.In order to confirm that the mutation detected in the E9L gene was responsible for the CDV resistance, we performed a series of marker rescue experiments and the results of the marker rescue experiments for isolates CDVR 1A, 15A and 16A were analyzed for their growth properties compared to wt VV. The growth curves in Figure R 16A did produce titers reaching wild type levels after an initial lag in growth. All three resistant strains produced very small plaques compared to wild type virus, with CDVR 1A producing "pinpoint" plaques after three days. CDVR 1A and 15A ultimately produced much less total virus than wild type or CDVR16A.Each of the three reconstructed viruses, CDV50 for CDV was obtained in HFF and Vero cells and compared to two independently obtained strains of parental VV WR. The resistant viruses had EC50 values that were 3 to 7 fold higher than the parental virus strains. The greatest resistance was with CDVR1A containing the mutation at A314V which produced the smallest plaques and lowest titers.We confirmed that the viruses were resistant to CDV in two additional cell lines at another laboratory Table . The EC5R VV is attenuated in mice. We assessed the virulence of our reconstructed viruses in mice inoculated intranasally . This region represents the catalytic core of the DNA polymerase. By modeling E9 on the crystal structure of Thermococcus gorgonariu DNA polymerase (PDB code 1TGO) it appears that the location of the M671I mutation is not far from the active site in the putative polymerase domain  had demonstrated that a mutation of alanine 314 to threonine conferred resistance to CDV; however, higher levels of resistance were obtained when this mutation was in combination with a second mutation, A684V, found in the original isolate . This stR 15 mutant provided some of the most interesting results. In our original isolation of CDVR15 we found only a single mutation within the E9 gene, however, upon reconstruction this mutation alone cannot confer resistance and attempts to reconstruct this virus resistant to CDV always contained the ΔK174 mutation as well. This implied that the original CDVR15 virus we isolated must contain a second mutation elsewhere in the genome other than in the DNA polymerase. Further analysis of CDVR15 by marker rescue experiments should allow identification of this second target gene. The lower titers of the CDVR stocks that were produced severely limited the amount of virus that could be used in this model. However, as expected from previous work, all three of our reconstructed virus strains grew somewhat less well than wild type virus and were attenuated in the mice by more than one log [Two other mutations conferring resistance were also isolated. One is a novel mutation of the deletion of amino acid K174 within the putative exonuclease domain of the DNA polymerase. Again, this mutation alone conferred resistance; however, the level of resistance is not as great as that for A314V. Examination of the CDVThe results obtained from these studies provides further evidence that the primary but not sole target of CDV is the viral DNA polymerase and that drug resistance can be a significant problem even in the presence of relatively low doses of drug. It is important to note that these drug resistant mutants all had reduced virulence in mice and suggest that the development of these mutants may not contribute to enhanced disease.2.Monolayer cultures of BSC40 cells (Dr. Richard Condit) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), 50 IU of penicillin, and 50 μg of streptomycin per ml  . CV1 celR mutants were VV WR and VV TK::GFP. VV TK::GFP contains the GFP gene driven by the synthetic VV early-late promoter inserted into the thymidine kinase (tk) gene. This virus was generated via standard methods using a pSC65GFP clone in order to recombine the GFP gene into the TK locus of wild type VV [R mutants, 20 independent virus stocks were generated from single plaques from the original VV TK::GFP (lines 1–10) and VV WR (lines 11–20) virus stocks.The parental viruses used for isolation of CDV type VV . Virus tCDV was provided by Gilead Sciences, Foster City, CA. Stock solutions of CDV (5 mM) in DMEM without serum was stored at 4°C and protected from light.4 PFU/well of either VV WR or VV TK::GFP in DMEM with no supplements except for 150 μM CDV. After 60 min of adsorption an additional 1.5 ml of DMEM with 10% FBS, antibiotics and 150 μM CDV was added to each well. Plates were incubated at 37°C for 48 h and examined for plaques under the light microscope, or with fluorescence for GFP containing plaques. To isolate identified plaques, the liquid medium was carefully removed and the plaque was scraped with a 1 ml large bore pipette tip and transferred into 1 ml of DMEM without serum and stored at -80°C. Routinely, 2 plaques from each individual virus stock were isolated. The virus from the original plaque was plaque purified one additional time under agarose and in the presence of 150 μM CDV to ensure that it was a single isolate. From these dishes, plaques were picked and subsequently amplified.Confluent monolayers of BSC40 cells in 6-well plates were infected with 2 × 10DNA sequences of the DNA polymerase (E9L) gene from the viruses were obtained by direct sequencing of the PCR products amplified from the total DNA of infected cells. The DNA was prepared from virus infected cells with the DNeasy Tissue Kit , according to the manufacturer's protocol. The entire E9L gene was PCR amplified using two primers IDT327, 5'-ATGGATGTTCGGTGCATTAATTGGT-3' and IDT328, 5'-TTATGCTTCGTAAAATGTAGGTTTTGAACC-3' and then sequenced with primers that hybridize within E9L to give overlapping sequence data. Sequencing was performed by the University of Florida ICBR DNA Sequencing Core Laboratory.3 PFU of wt VV WR in a volume of 0.5 ml and 30 min later transfected with 1.5 – 2 μg DNA complexed with 12 μl Lipofectamine 2000 per manufacturer's instructions (Invitrogen). Different PCR products from the mutant CDVR viruses were used for transfection including products containing the entire E9L gene or products containing only portions of E9L gene. A map of the fragments used is found in Figure R plaques were picked from the plaques obtained from the infection/transfection mixture and later amplified on BSC40 cells for stocks. The E9L gene of the reconstructed viruses was sequenced as described above and compared to the original mutation from which it was derived.Mapping of the individual mutations conferring resistance and the reconstruction of the mutation(s) in a wild type VV background were performed as described previously . ConflueR mutant virus suspension in 0.5 ml DMEM without serum and with CDV concentrations of 0, 50, 150, 250 or 500 μM. After adsorption for 60 min at 37°C, the medium was carefully aspirated and the wells were overlaid with 1% agarose mixed with an equal volume of 2 × DMEM, 10% FBS and the same concentration of CDV used during the initial infection. The plates were incubated for 4 days at 37°C and then stained with 0.26% crystal violet in 10% ethanol, 22% formaldehyde and plaques were counted.BSC40 cell monolayers in 60 mm dishes were infected with 200 PFU of VV WR, VV TK::GFP or CDVHFF cells were added to 6-well plates two days prior to the assay. On the day of assay, drug at two times the final desired concentration was diluted serially 1:5 in 2× MEM with 10% FBS to provide six concentrations. Culture medium was aspirated from triplicate wells for each drug concentration and 0.2 ml per well of diluted virus was added which yielded 20–30 plaques per well. The plates were incubated for one h with shaking every 15 minutes. Equal volumes of 1% agarose and drug solutions were mixed and added to each well in 2 ml volumes and the plates incubated for three days. Cell monolayers were stained with neutral red and plaques were enumerated using a stereomicroscope at 10× magnification. 50% effective concentration (EC50) values were calculated by standard methods.R viruses were compared to growth of wild type VV. BSC40 cells (1 × 105) in twelve well dishes were infected individually with each virus, wt VV, CDVR 1A, CDVR 15A, CDVR 16A, at an MOI = 0.01 PFU/cell, in duplicate. After 1 h adsorption, the virus was removed and the cells washed with PBS. One ml of DMEM containing 10% FBS was added to cells. Infected cells were incubated at 37°C and harvested by scraping at the following time points: 1, 3, 6, 9, 12, 18, 24, 48, 72 h post infection. The virus was released from the cells by 3 freeze thaw cycles and titered on CV1 cells.Growth properties of the reconstructed CDV5 to approximately 1 PFU/animal, depending on the titer of each virus stock. Virus suspension was instilled into both nostrils using a micropipetor and a total volume of 40 μl per animal. For these experiments mice were checked for mortality at least once daily for 21 days, but twice daily during the period when peak mortality was expected to occur. The mortality observed for the wild type virus, such as VV WR, was compared with that observed with the CDVR VV.Female BALB/c mice, 3 weeks of age, were obtained from Charles River Laboratories, Raleigh, North Carolina. Mice were group housed in microisolator cages and utilized at a quantity of 10–15 mice per group. Mice were obtained, housed, utilized and euthanized according to USDA and AAALAC regulatory policies. All animal procedures were approved by University of Alabama at Birmingham, Institutional Animal Care and Use Committee prior to initiation of studies. BALB/c mice were anesthetized with ketamine-xylazine prior to virus inoculation. VV infections were initiated by intranasal inoculation of media containing varying concentrations of wild type and drug resistant mutants of VV ranging from 8 × 10Thermococcus gorgonariu (derived from PDB file 1TGO) by Blast. A homologous model was calculated based on the amino acid sequence alignment and the known structure 1TGO using Modeller (version 9.2) [The amino acid sequence of E9 was aligned with that of Thermostable B Type DNA Polymerase from ion 9.2) . The mod50; vaccinia virus Western Reserve strain: VV WR; lethal dose 90%: LD90; Dulbecco's modified Eagle's medium: DMEM; hours post infection: hpi; plaque forming unit: PFU.Wild type: wt; effective concentration: ECThe authors declare that they have no competing interests.MNB contributed to the experimental design, sequence alignments, data analysis, and drafted the manuscript. MO isolated the resistant viruses and mapped the mutations. ERK contributed to the experimental design and provided a critical review of the manuscript. DCQ directed all mouse experiments and analyzed the resulting data. KAK contributed to the acquisition and interpretation of data. MNP contributed to the interpretation of data and the critical review of the manuscript. ML modelled the DNA polymerase. RWM contributed to the experimental design and assisted in writing the manuscript.


After the 1999 West Nile (WN) encephalitis outbreak in New York, 2,300 overwintering adult mosquitoes were tested for WN virus by cell culture and reverse transcriptase-polymerase chain reaction. WN viral RNA and live virus were found in pools of Culex mosquitoes. Persistence in overwintering Cx. pipiens may be important in the maintenance of WN virus in the northeastern United States.


We are reporting a case of bilateral Fuchs’ heterochromic iridocyclitis with chikungunya virus infection in the left eye. A 20-year-old female was presented with a past history of fever suggestive of chikungunya with bilateral Fuchs’ heterochromic iridocyclitis and complicated cataract. She had a tripod dendritic pattern of keratic precipitates by confocal microscopy in the left eye with a stippled pattern of keratic precipitates in both eyes. The real-time polymerase chain reaction (RT-PCR) assay in the aqueous humor detected 98 copies/ml of chikungunya virus RNA. The patient underwent clear corneal phacoemulsification with in-the-bag intraocular lens implantation in the left eye with a good visual outcome. This is the first report where the presence of chikungunya virus RNA has been associated with a case of bilateral Fuchs’ heterochromic iridocyclitis. Fuchs’ heterochromic iridocyclitis is a specific form of uveitis. Earlier studies suggest its association with infectious agents like toxoplasmosis, toxocariA 20-year-old female of Indian origin presented with the complaint of decreased vision in the left eye since 2 weeks. One week prior to the onset of ocular symptoms, the patient had fever lasting for 3 days suggestive of chikungunya fever. Her ocular history was negative for trauma and previous inflammation. Her old investigational reports ordered by the physician revealed positive serum anti-rubella virus IgG (153 IU/ml), and serum toxoplasma, cytomegalovirus, and herpes simplex virus antibodies were negative. Her best corrected visual acuity was 20/30 in the right eye and counting finger close to face in the left eye. Intraocular pressure was normal in both the eyes. Slit-lamp biomicroscopy in the right eye demonstrated fine-to-medium-sized keratic precipitates, flare+, cells+, stromal atrophy of the iris , nodulesDiagnosis of bilateral Fuchs’ heterochromic iridocyclitis with a complicated cataract was made probably of viral origin. A sample of aqueous humor (AH) was drawn through a paracentesis at the beginning of the cataract surgery for RT-PCR and the anterior capsule was removed during the capsulorrhexis in the left eye for histopathological examination.Reverse transcriptase PCR for rubella virus on the AH was carried out using primers and protocols as described earlier found toChikungunya is a single-stranded RNA virus. It can cause conjunctivitis, iridocyclitis, episcler6et al.[Confocal microscopy of keratic precipitates in Fuchs’ heterochromic iridocyclitis are globular keratic precipitates, infiltrating and dendritiform, stippled, and cruciform as described by Kanavi et al.et al. whereas the tripod dendritic pattern of keratic precipitates was noted in the left eye by confocal microscopy. We had an epidemic of chikungunya. The patient had fever suggestive of chikungunya which made us do the RT-PCR for chikungunya in the aqueous humor at the time of cataract surgery. The presence of chikungunya RNA virus in the AH of Fuchs’ heterochromic iridocyclitis suggests that chikungunya virus infection is present in a case of Fuchs’ heterochromic iridocyclitis, although it is not clear if the chikungunya virus was the initiator of the disease or a coincidental finding. This association reinforces the theory based on the putative role of an infectious agent associated with different forms of Fuchs’ heterochromic iridocyclitis. To the best of our knowledge, this is the first report where the presence of chikungunya virus RNA has been demonstrated in the case of Fuchs’ heterochromic iridocyclitis.The stippled pattern of keratic precipitates in both eyes was present which is similar to that reported by Kanavi