Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative  Lyssavirus species have been classified: Rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus types -1 and -2, Australian bat lyssavirus, Aravan virus, Khujand virus, Irkut virus, and West Caucasian bat virus (WCBV) . LBV, MOKV, DUVV, and SHIBV are detected exclusively in Africa, whereas RABV is detected worldwide. The predominant RABV variants circulating in Africa are the mongoose and canine biotypes. In South Africa, canine RABV is considered to have been introduced in the eastern Cape Province after importation of an infected dog from England in 1892 and subsequently spread, infecting domestic and wild carnivores  displaying clinical signs consistent with rabies was killed by rangers in Ikoma Ward within Serengeti National Park , including 12 canine RABV sequences from domestic and wild animals in the Serengeti ecosystem (64.1%–65.1% identity). The posterior probabilities indicated that the IKOV and WCBV grouping was strongly supported, despite low sequence identity. Further phylogenetic analysis of representatives from other rhabdoviruses demonstrated that IKOV is a member of the Lyssavirus genus (41.6%–50.9% identity to representative rhabdovirus sequences that are available for this region of the genome) (data not shown).RNA was extracted by using TRIzol Reagent , and a pan-lyssavirus reverse transcription PCR yielded a specific 606-bp amplicon . A caninWe describe evidence, based on genomic sequences obtained from the brain sample of an African civet with clinical signs consistent with rabies, for the existence of a novel lyssavirus designated IKOV (Ikoma lyssavirus). The sample was frozen and thawed several times before being sent to AHVLA, which had a detrimental effect on the sample quality and resulted in viral RNA degradation and loss in viral viability. The results of confirmatory FATs performed at AHVLA were inconclusive, and attempts to isolate virus by using the rabies tissue culture inoculation test and the mouse inoculation test were unsuccessful. Despite the lack of isolated virus, the pan-lyssavirus sensitivity and specificity of the fluorescent antibody test  and direct rapid immunohistochemistry test support the assertion that a novel lyssavirus exists in the region. In addition, despite the poor quality of the sample, molecular techniques identified a lyssavirus-specific amplicon that was confirmed to be unique by phylogenetic analysis and to be highly divergent from known circulating RABV strains. A real-time PCR also detected this unique lyssavirus sequence, confirming that both molecular tests are pan-lyssavirus specific and are sufficient for the detection of highly divergent novel lyssaviruses  was restricted to the RABV mongoose lineage . Ad


The complete genome sequence of the genotype 3 border disease virus strain Gifhorn has been determined; this strain was originally isolated from pigs. This represents the consensus sequence for the virus used to produce the bacterial artificial chromosome (BAC) cDNA clone pBeloGif3, which yields a virus that is severely attenuated in cell culture. Pestivirus, which includes other important animal pathogens, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). BDV causes disease mainly in small ruminants, but cattle, pigs, and other wildlife may also be infected; indeed, the BDV genotype 3 (BDV-3) strain Gifhorn was originally isolated from both infected pigs and sheep kept on the same farm (Border disease virus (BDV) belongs to the genus ame farm , 2. The ame farm . A complame farm . The comde novo assembly and for mapping of the reads using pBeloGif3 (GenBank accession no. GQ902940) as a reference sequence. Finally, the consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).Viral RNA was extracted from BDV-3 Gifhorn-infected sheep fetal thymoid (SFT-R) cells, and full-length viral cDNAs were amplified by long reverse transcription-PCR (RT-PCR) as previously described . cDNA waThe consensus sequence for the BDV-3 Gifhorn genome was obtained by replicate sequencing of RT-PCR products and RNA. The replicate samples generated the same consensus sequence, which was 12,325 nucleotides (nt) long. The polyprotein-coding sequence is 11,694 nt long and contains 3,898 codons. The 5′ and 3′ untranslated regions (UTRs) are 375 and 256 nt long, respectively. A comparison with the cloned Gifhorn genome sequence  revealed a 1-nt deletion in the 5′ UTR and 11 nt differences in the coding sequence, including 8 that are nonsynonymous. The complete consensus sequence of this BDV-3 virus and its comparison with the pBelGif3 sequence should enhance our understanding of the factors that are important for viral attenuation. The generation of additional genomic data for BDV will assist further investigations into the properties of this group of viruses.KF925348.The genome sequence of BDV-3 Gifhorn has been deposited in GenBank under the accession no.


Two gH/gL complexes are used for entry into cells. gH/gL/pUL shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.Human cytomegalovirus (HCMV) can infect many different cell types  gH/gL complexes of herpesviruses are supposed to promote fusion of the viral envelope with cellular membranes. The gH/gL core complex associates with additional proteins which define the tropism for certain cell types by promoting binding to specific receptors. Two alternative gH/gL complexes of human cytomegalovirus (HCMV) define the cell tropism, the entry pathway and the spread of virus. Formation of a gH/gL/gO complex during infection determines release of infectious virus into the supernatant. The gH/gL/pUL complex determines the tropism for endothelial cells (EC) and promotes focal spread. Here, we could show that HCMV-infected cells produce EC-tropic and non EC-tropic virus populations. While fibroblasts release both populations into the supernatant, EC predominantly release the non EC-tropic population. Different host cells of HCMV thus may direct the distribution of virus progeny. In immunocompetent adults infections are mainly asymptomatic, but in immunocompromised patients like transplant recipients or AIDS patients life threatening infections occur at a high rate. HCMV is also the leading cause of birth defects among congenitally transmitted viral infections. HCMV replicates The best candidates for binding to entry receptors are the HCMV gH/gL complexes. The gH/gL complex has been shown to promote fusion of cellular membranes It is currently not clear whether gH/gL/gO complexes exert their function by directly initiating entry In contrast to the gH/gL/gO complex, the gH/gL/pUL complex has been found to be consistently incorporated into virions Viruses lacking both, gO and pUL, are not viable, indicating that at least one of the two gH/gL complexes is needed for infection vß6 and αvß8 and promotes entry into epithelial cells by fusion at the plasma membrane The formation of distinct gH/gL complexes is not restricted to HCMV and has also been described for EBV and HHV-6 Here, we show that, similar to EBV, also HCMV progenies derived from different cell types differ in their cell tropism. Fibroblast-derived virus progeny could readily infect fibroblasts and EC, whereas EC-derived virus progeny was barely able to infect EC, and this difference in tropism was reflected by a respectively high or low content of the gH/gL/pUL complex in virus particles. EC-tropism could be depleted from fibroblast-derived virus progeny, indicating that this progeny is composed of distinct populations of virus particles with different EC infection capacities. Spread patterns in culture and cell disruption experiments indicated that fibroblasts readily released EC-tropic and non EC-tropic virus particles, whereas EC selectively retained the EC-tropic population.in vitro, virus homogeneously spreads in fibroblast cultures whereas spread in endothelial cell cultures stays focal When fibroblasts and EC are infected with HCMV To test whether the focal spread could be attributed to differences in release of infectious virus, we performed growth curves of vTB40-BAC4 on HFF, TIME cells and HUVEC, and measured virus release into the supernatants by titration on fibroblasts. HFF and EC equally released high amounts of virus into their supernatants . As spreInfection capacities on different cell types are often compared by methods, which depend on counting infected cells which are either stained for viral antigen or GFP- expression. These methods reach their technical limits, when the infection capacities strongly differ on the cell types to be tested. It is very difficult to obtain reliable cell counts on the less permissive cell type, without at the same time saturating infection on the more permissive cells. Saturation yet, would lead to an overestimation of the infection capacity on the less permissive cells, when related to the more permissive cells.To circumvent these problems and to simplify the analysis, we used a luciferase reporter virus to monitor infection. An SV40 promoter-driven luciferase expression cassette was inserted into BAC4-FRT5-9, a TB40-BAC4-derived BACmid lacking the genes UL5 to UL9 and carrying an FRT site at the position of the deleted locus . Virus wWe used vBAC4-luc to evaluate EC and fibroblast infection capacities of virus preparations on HFF and TIME cells. The luciferase signals obtained from HFF and TIME cell infections were related to each other and expressed as TIME/HFF infection ratios, and thus, represent relative EC infection capacities. After infection, phosphono acetic acid (PAA) was added to block the viral DNA replication and the further amplification of the luciferase signal. Thus, the luciferase activity evaluates infection of cells in a fashion analogous to staining cells for HCMV ie1 protein expression. Indeed, when infection with one and the same virus preparation was evaluated either by counting ie1-positive cells or by measuring the luciferase activity in cell lysates, both methods always gave comparable results .It is a standard observation in the field that one and the same HCMV preparation yields variable results, when repeatedly titrated on different target cell batches. When we tested HFF- and TIME cell-derived supernatants in independent luciferase assays, the results strongly depended on the quality of the cells used and varied with e.g. passage number and time after passage . Virus pEC infection strictly depends on the gH/gL/pUL complex With the luciferase assay described above, we could compare the properties of virus progenies from HFF and EC. Supernatants of infected HFF and EC were harvested 6 days after infection, titrated on HFF, and the viral DNA content determined by real-time PCR. The ratios of infectious virus to viral DNA copy numbers were comparable for HFF- and EC-derived supernatants (data not shown). These supernatants were then used to infect HFF and TIME cells. Forty-eight hours later, infection was monitored by the luciferase assay. Although virus derived from all three cell types showed a comparable infection of HFF (data not shown), EC-derived virus was significantly less capable in infecting EC than fibroblast-derived virus . On averIncorporation of gH/gL/pUL glycoprotein complexes into virions Whereas the virus released by EC is low in gH/gL/pUL complexes, focal spread in EC cultures was highly efficient. Like EC infection by supernatant virus, it can be blocked by anti-pUL128, anti-pUL130 and anti-pUL131A antibodies Virus released from EC is only poorly tropic for EC, whereas virus associated with EC shows a much higher tropism for EC. Virus released from HFF is highly EC-tropic and virus found in the particulate fraction of disrupted HFF rather shows a lower tropism for EC. One explanation would be that HCMV progeny is heterogeneous and consists of distinct virus populations with regard to their EC-tropism. EC show a propensity to retain EC-tropic virus and release non EC-tropic virus, whereas HFF readily release both, EC-tropic and non EC-tropic virus. The hypothesis, that HFF progeny is a mixture of EC-tropic and non EC-tropic virus is testable by separation of EC-tropic and non EC-tropic virus. As HUVEC strongly retain EC-tropic virus, they might serve to specifically bind EC-tropic virus and deplete HFF virus progeny of its EC-tropic fraction. We preincubated HFF-derived supernatant virus with HUVEC or with HFF, pelleted the cells, and analysed the HFF and TIME cell infectivity of virus remaining in the supernatants . Preincuin vivo. For EBV, the group of L. Hutt-Fletcher has pioneered the paradigm that epithelial cells produce a virus progeny high in gH/gL/gp42 complexes, which promotes B-cell infection. B-cells in turn, produce virus progeny low in gH/gL/gp42 complexes which efficiently infect epithelial cells, but not B-cells. Although not absolute, this relative switch of cell tropism after alternate replication in epithelial and B-cells directs infection from one cell type to the other.The use of different receptor binding proteins to mediate entry into different cell types, and the use of different entry pathways even into one cell type is a common feature of herpesvirus entry. Herpesviruses have additionally developed strategies, which may route infection Here, we propose that also different producer cells of HCMV may direct the infection. gH/gL/gO complex formation is needed for release of infectious virus from any infected cell type tested so far Our initial observation was that virus spread in fibroblast cultures differed from virus spread in EC cultures Similar to the EBV model, supernatants from infected HFF showed a higher capacity to infect EC than EC-derived supernatants, and we could show that the biochemical basis for that is a respectively high and low content of gH/gL/pUL complex in virions.The question, which arose then, was, what causes the observed difference in gH/gL/pUL content. For EBV it has been described that in infected B-cells HLA-DR ß binds the gp42 protein of the gH/gL/gp42 complex, which promotes B-cell infection, holds it back intracellularly, and thus, makes it vulnerable for degradation. As a consequence, B-cells release mainly virions containing a two-part gH/gL complex, which cannot infect B-cells. Epithelial cells, which do not express HLA-DR ß, do not retain gp42 and thus, release virus, which contains more of the three-part gH/gL/gp42 complex. The mechanisms, by which the differences in the released populations of virions in HCMV are achieved, appear to be different. For HCMV, we found that EC and fibroblasts produce heterogeneous virus progenies. EC release a virus progeny, which is not EC-tropic, and retain a progeny, which is highly EC-tropic. HFF release an EC-tropic progeny. which can be depleted of its EC-tropism by using HUVEC or protein G sepharose beads coated with antibodies directed against pUL131A. This strongly suggested that HFF progeny is composed of distinct EC-tropic and non EC-tropic virus populations, and that the EC-tropic population most likely is a population with a high gH/gL/pUL content. If HFF-derived virus progeny was homogeneous, a specific depletion only of EC-tropic virus would not be possible. Interestingly, HUVEC, which retain EC-tropic virus in infection experiments, were a good matrix to retain EC-tropic virus in the test tube, whereas HFF, which readily release EC-tropic virus in infections, were a bad matrix.Thus, we propose that the difference in cell tropism of virus released from EC and fibroblasts is the result of a sorting process. EC strongly and specifically retain EC-tropic virus through the gH/gL/pUL complex. HFF release EC-tropic and non EC-tropic virus. Thus, for HCMV, not protein components of gH/gL complexes are retained in a cell-type specific manner, but rather mature virions carrying the gH/gL/pUL complex in their envelopes. Future experiments will have to show where and how EC-tropic virus is held back. It has recently been shown that overexpression of gH/gL/pUL in epithelial cells interferes with HCMV infection. It has been postulated that this reflects binding of the gH/gL/pUL complex to the respective entry receptor Assuming that gH/gL/pUL complexes are incorporated into virus progeny at random, EC-tropism of a virus particle might be defined by a threshold level of gH/gL/pUL complexes. Accordingly, high levels of gH/gL/pUL complexes in turn could also block release from EC. Thus, the levels of gH/gL/pUL complexes could define whether a particle is EC-tropic or not, whether it is retained by EC during infection, and whether it can be depleted from supernatants by EC-preincubation. This could also explain, why progeny of a ΔgO virus, which expresses only the gH/gL/pUL complex, readily spreads cell-associated and can barely be released from EC 3-104 times more infectious for epithelial cells than cell-free virus For EBV, it has been observed that virus bound to the surface of resting B cells is 10We restricted our experiments to endothelial cells and fibroblasts. Macrophages, dendritic cells, and epithelial cells also strictly depend on the gH/gL/pUL complex for their infection. Whether their infection also follows the pattern of the EC infection shown here, will have to be investigated in the future. Recently, it has been published by Wang et al. in vivo, will be the subject of future research. It will be of particular interest to find out whether the relative propensity of different cell types to release virus plays a crucial role in establishment of infection and transfer of virus to new hosts or the fetus. For HCMV, it has been shown that primary isolation of EC-tropic virus depends on infected cells as a source of virus, whereas fibroblast infection can also be achieved with cell-free virus sources like throat washes and amniotic fluid Whether our observations made in cell culture, reflect features valid for all HCMV strains, and what role a switch in tropism and spread patterns may play Primary human foreskin fibroblasts (HFF)  were used from passage 12 to 22 and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. Primary human umbilical vein endothelial cells (HUVEC)  were used from passage 1 to 6. HUVEC and TIME  cells The HCMV strains used were VR1814 5′-ATGTTTCTAGGCTACTCTGACTGTGTAGATCCCGGCTTTGCTGTATATCGTGTATCTAGACGGGGGTGTCCAGGGTTTTCCC-3′) and UL9pcp15rev (5′-ATTGTTGTAACGATAACTAAGGGTATGATCCACATTGTATGTGGGGTGGCAGTATCGTGTCTTCCGGCTCGTATGTTGTGTGG-3) and pCP15 as template E. coli, thereby deleting 3,066 kb. The kanamycin-resistance gene was subsequently excised by FLP-mediated site-directed recombination The HCMV strain TB40/E cloned as a bacterial artificial chromosome (BAC) (TB40-BAC4) Sal I and Bgl II, filled in by Klenow polymerase and inserted into the pOriR6K-zeo plasmid linearized by EcoR V. The resulting plasmid pO6-Luc was inserted into BAC4-FRT5-9 via FLP-mediated FRT recombination mutagenesis using the temperature-sensitive expression plasmid pCP20 To generate a luciferase reporter HCMV, the SV40-driven firefly luciferase expression cassette was excised from pGL3-promoter (Promega) with The BAC mutants BAC4-Luc/ΔgO and BAC4-Luc/UL131Astop were cloned into the BAC4-Luc background as described previously Deletions and insertions were controlled by restriction pattern analysis and subsequent sequencing.BACmids were reconstituted to virus by transfection of BAC DNA into HFF using FugeneHD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions. Transfected cells were propagated until viral plaques appeared and the supernatants from these cultures used for further propagation of virus.Virus stocks were prepared from supernatants of infected HFF, HUVEC or TIME cells. Supernatants were cleared of cellular debris by centrifugation for 15 min at 3,500×g and stored at −80°C.For Western Blot analysis of HCMV particles, virus was concentrated from cell culture supernatants. Briefly, 200 ml supernatant from infected cells showing about 90% CPE was cleared of cellular debris by centrifugation at 3,500×g for 15 min. Then, virus was pelleted from cleared supernatant by ultracentrifugation at 80,000×g for 70 min. Virus pellets were resuspended in 1.5 ml 0.04 mol/l sodium phosphate pH 7.4.50 assay performed on 96 well plates on HFF.Virus titers of cleared supernatants were determined by a TCIDTo infect cells, medium was removed from 90% confluent cell monolayers and replaced by virus diluted in DMEM containing 5% FCS. For some experiments, virus infection was enhanced by a centrifugation step , followed by incubation at 37°C for 90 min. To compare infectivity of virus derived from fibroblasts and endothelial cells, subsequent infections were performed in DMEM 5% FCS/EGM-2 mixed at a ratio of 1∶1, to exclude medium effects. During infections, medium was exchanged every second day in a way that supernatants harvested contained virus released during the preceding 48 hours.As HCMV in general more readily infects fibroblasts than EC, in all experiments, where infections of EC and fibroblasts (spread patterns and growth curves) were compared, the infections were adapted in a way that EC were infected with more virus than fibroblasts to achieve comparable numbers of ie1-positive cells after 48 hours.For gradient purification of virions, supernatants from infected cell cultures showing approximately 100% late-stage CPE were cleared of cell debris by centrifugation for 10 min at 2,800×g. Supernatants were then ultracentrifuged for 70 min at 80,000×g. Pellets containing virions were resuspended in 1 ml PBS and transferred onto a preformed, linear glycerol/tartrate gradient (15–35% sodium tartrate and 30–40% glycerol in 0.04 mol/l sodium phosphate pH 7.4), which was ultracentrifuged for 45 min at 80,000×g. The virion-containing band was harvested with a syringe and the virions were washed and pelleted by an additional ultracentrifugation for 70 min at 80,000×g. The pellet was resuspended in 0.04 mol/l sodium phosphate.HCMV-infected cells were fixed in 50% acetone/50% methanol, stained using a mouse anti-ie1 antibody  and detected with a Cy3-coupled goat anti-mouse antibody (Dianova). For counterstaining of cell nuclei, cells were incubated in PBS containing 5 µg/ml Hoechst 333258 (Invitrogen) for 1 min.3PO4, 2 mM CDTA, 2 mM DTT, 10% glycerol, 5% Triton-X 100) and luciferase activity was determined for 20 µl of lysate with a luciferase assay system (Promega) according to the manufacturer's instructions.HFF and TIME cells were grown in 96 well plates  and infected in triplicates at an m.o.i. between 0.02 and 0.5 for 90 min. Inoculi were then replaced by medium supplemented with 300 µg/ml phosphono acetic acid (PAA). 48 h after infection cells were lysed in 50 µl lysis buffer (25 mM Tris/HVirus particles or infected cells were lysed in 6× sample buffer (300 mM Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, 5% ß-mercaptoethanol, 0.01% (w/v) bromphenolblue, 0.01% (w/v) phenolred), separated on 15% polyacrylamide gels and transferred onto nitrocellulose (Amersham Biosciences). Membranes were blocked with 5% low-fat milk in TBS and stained for gB or pUL128 using mouse anti-gB antibody  or mouse anti-pUL128 antibody , respectively. The specific protein bands were detected by using an peroxidase-coupled anti-mouse antibody (Dianova) and the SuperSignal West Dura Extended Duration Kit (Perbio).The intensities of protein bands were quantified using the Fujifilm Intelligent Light Box LAS-300 and the Image reader LAS-300. Non-saturated light signals were analysed to determine the protein amounts using ImageQuant 5.0 software. The pUL128 levels were related to gB levels of the respective samples.Cells were infected in 6 cm dishes, and 6 days after infection the supernatants (4 ml) harvested and cleared of cellular debris . Cell monolayers were washed with cold PBS, scraped and cells dounced in 4 ml DMEM medium supplemented with 5% FCS using tight fit hand homogenizers (Sartorius-Stedium). 3.5 ml of the total homogenized cells were pelleted , the supernatant removed (supernatant of homogenized cells) and the pellets resuspended in fresh 3.5 ml DMEM medium supplemented with 5% FCS.GeneBank/EMBL/DDBJ accession number for TB40-BAC4 is EF999921.Figure S1Neutralizing antibodies block focal spread of HCMV in EC cultures. HFF and HUVEC were infected with vBAC4-luc as described in (0.56 MB TIF)Click here for additional data file.Figure S2Growth properties of vBAC4-FRT5-9 and vBAC4-luc. (A) Schematic presentation of TB40-BAC4 derived mutants BAC4-FRT5-9 and BAC4-luc. The UL and US regions, the positions of internal and terminal repeats (dark grey), the open reading frames UL5 and UL9, the BAC cassette (light grey), the position of the FRT-site and the insertion of a luciferase expression cassette are indicated. (B) Growth curves of vTB40-BAC4, vBAC4-FRT5-9 and vBAC4-luc on HFF, TIME cells and HUVEC. Cells were infected as described in (0.63 MB TIF)Click here for additional data file.


Molecular Pathogenesis of Virus Infections describes our current understanding of the pathogenesis of selected virus and prion infections. The innate response is an early barrier to virus spread. In this context, O. Haller et al. describe the antiviral activity of type I interferons and the various virus-encoded countermeasures. R.P. van Rij and R. Andino review the role of RNAi as a therapeutic antiviral agent and its use by the host and virus during viral infections. J.L. Whitton gives an overview of the adaptive CD8+ T-cell immune response in the context of virus infections. G. Screaton and J. Mongkolsapaya explain potential roles of T-cell responses in dengue hemorrhagic fever. E. Turnbull and P. Borrow provide a detailed description of the ineffectual roles of the innate and immune responses in the control of HIV and the long road ahead for development of either a prophylactic or therapeutic vaccine.Transmissible spongiform encephalopathies have perhaps the most unconventional natural history of any infectious agent. J.C. Manson and R.M. Barron describe the diagnosis of transmissible spongiform encephalopathies, the appearance of new strains, and the nature of host susceptibility to disease. C.M. Dixon et al. depict the special problems presented to the host by certain RNA viruses that are maintained and persist in human populations through avoidance or inhibition of apoptosis, innate immune response, and adaptive immune response.Other viruses infect humans only as incidental hosts and cause epizootics of varying degrees. A.L. Hartman et al. review our current understanding of the pathogenesis of Ebola and Marburg filoviruses, paying particular attention to the factors that contribute to lethal disease. C. Dye and S. Siddell discuss the pathogenesis of feline coronavirus, an animal disease model that has provided insights into the study of the newly recognized disease, severe acute respiratory syndrome. R.G. Webster et al. enumerate the key influenza genes responsible for human pathogenicity, their roles in past pandemics, and the potential of avian influenza virus strains to evolve into highly pathogenic and transmissible viruses for human populations.Many viruses modify host metabolism and innate/immune responses to their own ends. L. Gray et al. describe the impact of human papillomaviruses on cell cycle and apoptosis. S.M. Lemon and K. Li review the data documenting hepatitis C virus disruption of innate intracellular antiviral defenses, including interferons and toll-like receptors. M.B. Ruiz-Arguello et al. enumerate the multiple, distinct receptor homologs and binding proteins encoded by poxviruses that target tumor necrosis factor. L.K. Dixon summarizes the multiple host pathways that are targeted at multiple levels by African swine fever virus. J.P. Stewart et al. describe the pathogenesis of murid herpesvirus 4 that supports its use as model for gammaherpesviruses. M.L. Freeman et al. provide an overview of the potential role of the immune system in the latency of the alphaherpesvirus, herpes simplex virus 1. This book is suitable for the serious student and professional and is well referenced for further reading.


Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding  molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.A recent emergence of  Cryptococcus gattii is an emerging cause of community-acquired fungal pneumonia, meningitis, and disseminated disease. In the past decade, infections caused by C. gattii have been increasingly identified outside of the previously recognized tropical and subtropical endemic regions and have emerged in temperate regions, including British Columbia (BC), the US Pacific Northwest (PNW), and Japan C. gattii in BC and the PNW is remarkable for a number of reasons, including the adaptation of the pathogen to a climate previously unrecognized as potentially hospitable, an apparent increased virulence of certain molecular subtypes in mouse models Emergence of C. gattii have been differentiated through the use of restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and multi-locus sequence typing (MLST) into four main molecular types: VGI, VGII, VGIII and VGIV. Further work using MLST has determined that these groups are monophyletic and genotypically distinct enough to deserve varietal status C. gattii molecular types, a unique feature of the C. gattii emergence in BC and the PNW is the distinct clonality of these isolates. The predominant C. gattii molecular type VGII sorts into three closely related clonal subtypes designated VGIIa, VGIIb, and VGIIc Strains of Coccidioides immitisC. gattii using isolates from the PNW emergence. This next generation typing methodology allowed us to generate a unique genetic fingerprint for each study isolate, effectively subtyping a “clonal” population.While MLST has been able to distinguish adequately between the VGIIa, VGIIb and VGIIc subtypes within the BC and PNW emergence, neither MLST nor variable number tandem repeat analysis (VNTR) have been successful at distinguishing among isolates within each clonal group. To date, only one single nucleotide polymorphism (SNP) has been identified within the VGIIc clonal group, and one VNTR difference has been identified within the VGIIa clonal group using these methods C. gattii isolates were received at the Centers for Disease Control and Prevention from state and local health departments, clinicians, veterinarians and ongoing environmental studies  and two published C. gattii genomes  studies  in the PMLST gene fragments were amplified using the seven consensus primers of Meyer et al. Cryptococcus libraries were prepared at a concentration of at least 1 nM using 10 mM Tris-HCl, pH 8.0+0.05% Tween 20 as the diluent. To ensure accurate loading onto the flowcell, the same quantification method was used to quantify the final pools. The pooled libraries were sequenced on the Illumina GAIIx. A 75 bp read paired end run was used for the first 2 isolates sequenced; the remaining isolates were done with a 101 bp read paired end run.The DNA samples were prepared for multiplexed, paired-end sequencing on the Illumina GAIIx Genome Analyzer . For each isolate, 1–5 µg dsDNA in 200 µl was sheared in a 96-well plate with the SonicMAN™  to a size range of 200–1000 base pairs with the majority of material at ca. 600 base pairs using the following parameters: Pre Chill - 0°C for 75 sec; Cycles - 20; Sonication - 10 sec; Power - 100%; Lid Chill - 0°C for 75 sec; Plate Chill - 0°C for 10 sec; Post Chill - 0°C for 75 sec. The sheared DNA was purified using the QIAquick PCR Purification kit . The enzymatic processing  of the DNA followed the guidelines as described in the Illumina protocol . The enzymes for processing were obtained from New England Biolabs  and the oligonucleotides and adaptors were obtained from Illumina (Cat #PE-400-1001). After ligation of the adaptors, the DNA was run on a 2% agarose gel for 2 hours, after which a gel slice containing 500–600 bp fragments of each DNA sample was isolated and purified using the QIAquick Gel Extraction kit . Individual libraries were quantified with qPCR on the ABI 7900HT  in triplicate at two concentrations, 1∶1000 and 1∶2000, using the Kapa Library Quantification Kit . Based on the individual library concentrations, equimolar pools of no more than 5 indexed The seven MLST gene fragment sequences were concatenated, aligned, and subjected to maximum parsimony analysis using MEGA4 Cryptococcus neoformans var gattii R265 genome Illumina WGS data sets were aligned against the http://sourceforge.net/projects/solsnp/). SNPs were excluded if they did not meet a minimum coverage of 10× and if the variant was present in less than 90% of the base calls for that position. In parallel, publically available genomes were aligned against R265 using MUMmer 3.22. SNPs were extracted from the alignments using a custom script. Subsequently, regions found to be duplicated with the R265 reference genome were identified using MUMmer version 3.22 http://www.ncbi.nlm.nih.gov/sra, accession # SRP006436).Reads containing insertions or deletions, and those mapping to multiple locations in the reference were removed from the final alignments. Each alignment was analyzed for SNPs using SolSNP  were subjected to MLST analysis using the consensus seven loci C. gattii genomes, 211 of which were parsimonious, with the majority being among the different subtypes  demonstrate that WGST has superior resolving power as compared to available sub-typing tools; (2) reveals the presence of unique genotypes within VGII “clonal” subtypes; and (3) yields evidence that while the VGIIb sublineage may have radiated earlier than the other two sublineages, it may not have given rise to them.Employing next generation sequencing to analyze a large number of C. gattii WGST analysis identified a substantial number of genomic differences that were undetectable by the currently available typing methodologies that include MLST and VNTR analysis In the present study, Of note is the observation that most of the SNPs in VGIIa are found on the branch leading to the single R265 genome; when this genome is removed from the analysis, only 107 SNPs are identified among the remaining VGIIa isolates, seven of which are parsimony-informative . One reaWhile VGII subtypes had been identified in several collections globally distributed in Australia, Europe and South America C. gattii clonal subtypes causing disease in the PNW. WGST revealed definitively that the VGIIc subtype of C. gattii, the predominant causative agent of recent cryptococcal illness in Oregon Our study provides further evidence of the distinct nature of the C. gattii molecular types and subtypes as well as differences in pathogenesis The VGI and VGIII isolates were included in this study as outgroups to anchor the analysis, but the genome data provide important information. The diversity  seen among the VGI, VGII and VGIII molecular types is consistent with previous reports that assigned varietal status to these types C. gattii isolate collected. Large numbers of SNPs enable robust phylogenetic analyses, leading to more accurate phylogenetic reconstructions, especially in recombining organisms The comprehensive SNP data sets provided by WGS analysis will help facilitate the development of an accurate molecular clock, which will facilitate a better understanding of the evolutionary history of this organism. One of the most important aspects of this analysis is that we will now be able to individually delineate every C. gattii and to provide robust tools for rapidly detecting emerging strains and efficiently responding to outbreaks.Data generated from this study have established a framework from which computational analyses can be designed and conducted to better characterize the Figure S1Phylogenetic analysis of WGST data from C. gattii, C. grubii and C neoformans isolates. Maximum parsimony phylogenetic analysis was performed in MEGA4 on C. gattii, C. neoformans and C. grubii whole genome sequence data C. grubii/C. neoformans branch; one of 190 most parsimonious trees is shown. Bootstrap values less than 50% and for intra-VGIIa, VGIIb and VGIIc nodes are not shown. The numbers of SNPs included in the analysis is indicated (number of parsimony-informative SNPs in parentheses), as is the consistency index (CI) as calculated by MEGA4. While WGST analysis found unique genotypes for all isolates, they are not visible on this tree due to the large numbers of SNPs separating the VGII isolates from the other isolates.(TIF)Click here for additional data file.


Culicoides midges. The equids most severely affected by the virus are horses, ponies, and European donkeys; mules are somewhat less susceptible, and African donkeys and zebra are refractory to the devastating consequences of infection. In recent years, Bluetongue virus, an Orbivirus similar to African horse sickness, which also utilises Culicoides spp. as its vector, has drastically increased its range into previously unaffected regions in northern Europe, utilising indigenous vector species, and causing widespread economic damage to the agricultural sector. Considering these events, the current review outlines the history of African horse sickness, including information concerning virus structure, transmission, viraemia, overwintering ability, and the potential implications that an outbreak would have for Ireland. While the current risk for the introduction of African horse sickness to Ireland is considered at worst ‘very low’, it is important to note that prior to the 2006 outbreak of Bluetongue in northern Europe, both diseases were considered to be of equal risk to the United Kingdom (‘medium-risk’). It is therefore likely that any outbreak of this disease would have serious socio-economic consequences for Ireland due to the high density of vulnerable equids and the prevalence of Culicoides species, potentially capable of vectoring the virus.African horse sickness is an economically highly important non-contagious but infectious Orbivirus disease that is transmitted by various species of  Culicoides midges during a blood meal, which they require for reproduction . Of the Culicoides identified, approximately 73% were considered to be potential AHSV vectors  [In response to the outbreak of BTV in northern Europe, the BTV vector surveillance programme was initiated in Ireland . This pre R.O.I. . A similoup 22%) . Initialoup 22%) . TherefoIt is evident from previous outbreaks of AHSV worldwide that this disease can have serious consequences for animal health and mortality. As such, it is likely that any introduction to Ireland would be catastrophic, considering the high population density of vulnerable animals, and the importance of the equine industry to the rural economy. Despite the current risk of importation of AHSV being considered at worst ‘very low’, a stockpile of live, attenuated vaccines, which are currently used in sub-Saharan Africa, has been created for the potential use in the European Union should an outbreak occur . These vAHS = African horse sickness; AHSV = African horse sickness virus; AHSV-1 = African horse sickness virus serotype 1; AHSV-4 = African horse sickness virus serotype 4; AHSV-9 = African horse sickness virus serotype 9; BT = Bluetongue; BTV = Bluetongue virus; BTV-8 = Bluetongue virus serotype 8; DARD = Department of Agriculture and Rural Development Northern Ireland; DEFRA = Department for Environment, Food and Rural Affairs; EFSA = European Food Safety Authority; N.I. = Northern Ireland; OIE = The World Organisation for Animal Health; RNA = Ribonucleic acid; R.O.I. = Republic of Ireland.None of the authors of this manuscript has any financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper.GMT, SJ, and AKM drafted the manuscript and compiled the literature. All authors made substantial inputs to the review, critically discussed, and approved the final manuscript.


Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3), was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.O'nyong nyong virus (ONNV) and Chikungunya virus (CHIKV) are two closely related alphaviruses with very different infection patterns in the mosquito,  Anopheles gambiae mosquitoes. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from chikungunya virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. That ONNV nsP3 is largely responsible for ONNV's unique ability to infect An. gambiae is especially interesting since the exact mechanisms and functions of this highly-variable protein remain poorly understood.O'nyong nyong virus (ONNV) is unique in that it is the only alphavirus, and one of few viruses in general, to be transmitted to humans by the bite of an anopheline mosquito. The genetics responsible for this unique vector specificity would be useful information in helping to develop antivirals, vaccines, and other methods for interrupting virus transmission. Previous research using other arboviruses has shown that specific viral genomic regions, amino acid sequences, or even single nucleotide mutations can have a profound effect on virus growth, infection, and virulence characteristics. Using chimeric viruses that substitute a gene from one virus with a gene from a closely related virus is a proven method of evaluating the relative contribution of each gene to a given phenotype. Our study analyzed both structural and non-structural regions of the ONNV genome using chimeric viruses and artificially infected  Anopheles gambiae and Anopheles funestusO'nyong nyong virus (ONNV) is an arthropod-borne virus (arbovirus) associated with a small number of large- scale epidemics. One such epidemic began in 1959 in Uganda, lasted three years and affected over 2 million people Aedes aegypti or, during epidemics, Aedes albopictus mosquitoes. Patients infected with CHIKV suffer from clinical symptoms similar to those infected with ONNV except that the fever is a higher, there is typically no lymphadenopathy, and the arthralgia is both incapacitating and chronic.Chikungunya (CHIKV) virus is a closely related alphavirus which has caused millions of cases of disease throughout countries in and surrounding the Indian Ocean since its re-emergence in 2004 An. gambiae mosquitoes have been shown to be highly susceptible to ONNV infection and refractory to CHIKV infection CHIKV and ONNV diverged from a common ancestor thousands of years ago Alphavirus contain a single-stranded, positive-sense RNA genome (∼11.7 kb) that contains two regions, a non-structural domain making up the 5′ two-thirds of the RNA and a structural domain at the 3′ end making up the remaining one-third of the genome An. gambiae mosquitoes Members of the genus All plasmid clones used in this study were designed and constructed in house. The full length clone pONN.AP3 was constructed from ONNV strain SG650 All plasmid clones designed to evaluate structural regions of the genome were constructed in a similar fashion, with the substituted region produced from a PCR product and the backbone region produced from the parental plasmid clones described previously. For example, to construct pCHIK/ONN E2, the E2 region of ONNV was amplified from parental pONN.AP3 by PCR with PFU turbo polymerase . The ONNV amplicon and pCHIK.B were digested with the same restriction enzymes . When nePlasmid clones designed to evaluate non-structural regions of the genome were engineered as exact gene substitutions by using a series of subclones, as described in the in vitro transcription were generated by linearizing each full-length clone with a unique Not I restriction site present downstream of the poly (A) tail. Linearized plasmids were treated with Proteinase K  to digest any endogenous RNases or DNases. DNA was purified using a phenol-chloroform extraction followed by an ethanol precipitation. A 20 µl aliquot of linearized and treated DNA was then transcribed in vitro by incubating the DNA with 0.4 µl (100 mM) each rNTP , 0.4 µl BSA (10 mg/ml) , 2 µl DTT (100 mM) , 8 µl (5x) transcription buffer , 1.3 µl (15 U/µl) T7 RNA polymerase , and 4 µl (10 mM) A-cap-structure analog  for one hour at 39°C. Transcribed RNA (10 µl) was mixed with 400 µl BHK-21 cells (1×107cells/ml) in a 2 mm gap cuvette  and electroporated twice using a BTX ElectroCell Manipulator with the following settings: 460volts, 725ohms, 75 µF Templates for Each time a virus was generated, the entire virus was sequenced to verify fidelity to the original sequence. Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Extracted RNA was then added to a reverse-transcriptase PCR reaction using the Titan One Tube RT-PCR System . Complementary DNA for sequencing the 5′ end of each viral genome was generated using a FirstChoice RLM-RACE Kit . This complementary DNA was then sequenced using virus-specific primers with the Big Dyev3.1 kit on an ABI 3130xl genetic analyzer . Sequence files were aligned and analyzed for sequence quality and genome coverage using Lasergene suite software .−1 to 10−7 were added to individual well of 6-well plates covered in monolayers of VERO cells. Plates were incubated at 37°C, with 5% CO2. Cells were fixed 48–72 hours later using a solution of 40% methanol and 0.25% crystal violet in water. Plaques were then counted and titers were calculated as plaque forming units per milliliter (PFU/ml).Virus rescued from clones was titered by plaque assay. Ten-fold virus dilutions from 10An. gambiae, originally obtained from the National Institute of Health. This strain has been maintained as a colony in our lab with rearing conditions that include a 12∶12 hour light∶dark cycle in chambers maintained at 28°C with approximately 95% humidity The ability of each chimeric virus to infect mosquitoes was evaluated using the G3 strain of log 10 PFU/ml of virus. Mosquitoes were allowed to feed on the warmed infectious blood meal for one hour through an artificial membrane feeder . Fully engorged females were separated and maintained for an incubation period of up to 12 days. Mosquitoes were sacrificed at days 4, 8, and 11 or 12 post-infectious-blood meal. Heads and bodies were separated into individual tubes and stored at −80°C until subsequent processing. Infection rates were determined using individual bodies and dissemination rates were calculated as the number of positive heads among the positive bodies. At least two replicate infectious feeds were done for each chimeric virus, with replicate feeds performed entirely independent of one another. No less than 140 mosquitoes were tested for any one chimeric virus.Infectious blood meals were prepared from equal volumes of packed, calf erythrocytes, 10% sucrose in fetal bovine serum, and 4.4–610 cells/well). Inoculated tissue-culture plates were incubated at 37°C for 5 days. Cells were observed daily for CPE due to virus replication. Virus replication in mosquito body samples indicated that virus had infected the mosquito's midgut, while replication in the mosquito heads showed a disseminated infection.Individual frozen mosquito bodies and heads were triturated in 300 µl of DMEM supplemented with (by volume): 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 0.1% gentamicin, and 0.1% Fungizone. The mosquito homogenates were passed through a 0.2 µm Gelman Acrodisc filter  to remove potential bacterial or fungal contaminates. Filtrate from each body or head was added to a single well of a 96-well flat-bottom tissue-culture plate, along with 50 µl of prepared BHK-21 cell suspension  were seeded with Vero (African green monkey) cells. Monolayers at 90% confluency were infected with virus at a multiplicity of infection (MOI) of 0.1. At specified times post infection, supernatant was removed from two wells for each virus and placed in a screw-cap cryovial at −70°C until titration by plaque assay. Titration results for each virus were compared at all time points by the student t-test.5′ GCA GGG AGG CCA GGA CAG T, 10840 REV- 5′ GCC CCT TTT TCY TTG AGC CAG TA). The real-time probe was labeled with a 5′ end HEX reporter dye and a 3′ end BHQ1 quencher dye (10759 FWD- 5′ AAA GAC CAG CGG CAG GAG CAA TAC AC) and PCR results are reported here as PFU-equivalents/mosquito by comparison with known concentration standards.To confirm virus replication (and not just persistence of the input virus) within the mosquito, five females that had fed on the ONNV infectious blood meal were sacrificed every other day post-infectious feed. Each body and head was processed separately, as described earlier. RNA was extracted from homogenized mosquitoes using QIAamp Viral RNA Mini Kit (Qiagen). The amount of RNA in each head and body was determined using the Quanti Tect Probe RT-PCR kit (Qiagen) and a TaqMan Real-Time PCR assay as previously described Fisher's exact probability test was employed to evaluate whether infection rates with chimeric viruses were statistically different from those with parental viruses. The infection rate was defined as significantly different from parental CHIKV if the two-tailed p-value was <0.007. The two-tailed p-value had to be <0.01 to be statistically different from parental ONNV infection rates. Both adjusted alphas were obtained using the Bonferroni correction for multiple comparisons to ensure an overall Type I error of 0.05. Computations were made using freely-available software An. gambiaeAn. gambiae mosquitoes were infected with ONNV, whereas a maximum of only 6% were infected with CHIKV. These values are similar to previously published work An. gambiae, viral replication (and not just persistence of input virus) within both cell culture and in the mosquito was confirmed. Cell culture growth curves of all of the chimeras were performed in Vero cells to confirm that all viruses were indeed replication competent and replicated in a manner similar to their parental viruses  of mosquitoes had replicating virus when harvested on day 8 post infection  of mosquitoes feeding on ONN/CHIK 3′STR and 53.2% (n = 77) of ONN/CHIK 5′STR were shown to be infected at day 8. Infection rates for mosquitoes sacrificed at days 4, 11, or 12 corroborate day 8 results (data not shown).Each chimeric virus constructed from the parental CHIKV genome maintained a CHIKV-like infection profile (<10% infection rate), with one exception. When allowed to feed on a blood meal containing approximately 5.5 lognfection . None ofnfection . Three oAn. gambiae was very low and all were comparable for both day 8 and day 11 samples. Only 5 viruses showed any dissemination  was not able to be rescued from cDNA in either mammalian or insect cells. This would further suggest that nsP3 plays a critical role in viral replication that is distinct in these two closely related viruses that exhibit 81% and 72% amino acid and nucleotide identity respectively in nsP3. That nsP3 should be found to be essential to infection is especially interesting given the fact that the precise functions of this protein are not fully defined. It is required for the correct formation and localization of replication complexes and does provide essential functions in both minus strand and subgenomic RNA synthesis, but specific mechanisms are not yet resolved A panel of 15 chimeric viruses were developed here to study specific elements of the ONNV genome and to determine which of these regions are necessary for ONNV to infect Ae. aegypti, the main vertebrate vector for that virus To further add to the intrigue of this protein, it has been shown that some members of the alphavirus family actually contain inserts of foreign genetic material within nsP3. An eight amino acid sequence from the carboxyl-terminus of CHIKV nsP3 maps to a putative zinc finger protein in An. gambiae could be attributed solely to either of the distinct domains within nsP3. Interestingly, the addition of just the carboxyl-terminus of ONNV nsP3 did produce a small, although not a statistically significant, increase in infection rates as compared with parental CHIKV in An. gambiae.Alphavirus nsP3 can be clearly divided into two distinct domains. The macro domain, or amino-terminal region, is highly conserved, not just among alphaviruses but also among coronaviruses, hepatitis E virus, rubella virus and even cellular proteins The carboxyl-terminus of nsP3, which has been subject to rapid alteration during alphavirus evolution, may also be involved in the optimization of replication in diverse host cell types An. gambiae infection. The former seems more probable since substituting CHIKV for either half of ONNV nsP3 results in infection rates not significantly different from rates with parental CHIKV.Another interesting characteristic of the carboxyl-terminus of nsP3 is that it is phosphorylated at multiple serine and threonine residues An. gambiae mosquitoes An. gambiae, as shown with the chimera CHIK/ONN eSTR  tail, has a sequence which is highly conserved among all alphaviruses and which is absolutely required for efficient virus replication An. gambiae. There are also numerous interactions within nsP3 itself, within the two halves of the structural region, and possibly the 3′ NTR which, when disrupted, can eliminate mosquito infection.Studies with chimeric viruses must be viewed in the overall context of the virus' life cycle. When substitutions are made to construct chimeric viruses, numerous aspects of the virus-host interactions and virus-virus regulatory functions can be disrupted resulting in reduced infection rates. Reduced infection rates may be a direct consequence of missing the essential genomic region or may be an indirect result of disrupting an essential regulatory interaction. Conversely, when the addition of a specific region increases mosquito infection rates, we must conclude the region itself to be essential for infection. Interestingly, because there was such a low dissemination rate of all viruses within this study, elements involved in dissemination throughout the mosquito may be distinct from those important in initial infection. However, this study has shown that ONNV nsP3 is directly responsible for ONNV infection of Figure S1Illustration of exact nsP3 substitution made to create CHIK/ONN nsP3.(TIFF)Click here for additional data file.Figure S2Construction of CHIKV nsP3 receiving plasmid. PCR primers were designed to generate two amplicons flanking the DNA insertion sites and extend outward to include unique restriction enzyme sites and inward to include a unique type II restriction site. Amplification with these primers, subsequent digestion with PciI/SacI or EcoRI/SacI, followed by a 3-part ligation produce a pUC-based vector containing CHIKV sequence flanking the site where ONNV nsP3 will later be inserted.(TIFF)Click here for additional data file.Figure S3Amplifying ONNV nsP3. PCR primers were designed to amplify the desired DNA insert, with the addition of type II restriction enzyme sites to the termini. Type II sites were oriented such that they will be removed upon later digestion.(TIFF)Click here for additional data file.Figure S4Expanded sequence of assembled CHIKV nsP3 receiving plasmid (top). Termini of ONNV nsP3 amplicon (bottom). The lines indicate the cut sites for the type II restriction enzymes.(TIFF)Click here for additional data file.Figure S5Products produced after digestion with appropriate type II restriction enzymes. These products were ligated to build the CHIKV/ONNV nsP3 cassette plasmid.(TIFF)Click here for additional data file.Figure S6Construction of final clone, Chik/Onn nsP3. CHIKV/ONNV nsP3 cassette plasmid and pCHIK.b were digested with SpeI and AvrII. The resulting products were ligated to generate the final clone with the complete ONNV nsP3 gene replacing the like gene in CHIKV.(TIFF)Click here for additional data file.Protocol S1Methods for construction of gene specific clones.(DOCX)Click here for additional data file.


Vitamin B12 deficiency causes a wide range of hematological, gastrointestinal, psychiatric and neurological disorders. Hematological presentation of cobalamin deficiency ranges from the incidental increase of mean corpuscular volume and neutrophil hypersegmentation to symptoms due to severe anemia, such as angor, dyspnea on exertion, fatigue or symptoms related to congestive heart failure, such as ankle edema, orthopnea and nocturia. Neuropsychiatric symptoms may precede hematologic signs and are represented by myelopathy, neuropathy, dementia and, less often, optic nerve atrophy. The spinal cord manifestation, subacute combined degeneration (SCD), is characterized by symmetric dysesthesia, disturbance of position sense and spastic paraparesis or tetraparesis. The most consistent MRI finding is a symmetrical abnormally increased T2 signal intensity confined to posterior or posterior and lateral columns in the cervical and thoracic spinal cord. Isolated peripheral neuropathy is less frequent, but likely overlooked. Vitamin B12 deficiency has been correlated negatively with cognitive functioning in healthy elderly subjects. Symptoms include slow mentation, memory impairment, attention deficits and dementia. Optic neuropathy occurs occasionally in adult patient. It is characterized by symmetric, painless and progressive visual loss. Parenteral replacement therapy should be started soon after the vitamin deficiency has been established. Vitamin B12 or cobalamin  is produced by bacteria in the large bowel of humans and by external bacteria and fungi. However, cobalamin from the former source is not absorbed, and humans need to introduce it solely from the diet .Cobalamin is stable at cooking process temperatures; the likelihood of its transformation into inactive compounds by ascorbic acid  has not Cobalamin from biological sources is provided in the coenzyme form  as a protein-vitamin complex, through non-specific protein-vitamin binding. At the low pH of the stomach, proteolytic digestion by pepsin occurs, which is the prerequisite for cobalamin release. Once released, cobalamin rather than the gastric intrinsic factor (IF) binds preferentially to haptocorrin (R-protein), a high-affinity cobalamin-binding protein in the saliva and gastric juice, resulting in the so-called “holo-R-protein”. In the second part of the duodenum, pancreatic proteases degrade the holo-R-protein complex, and the resulting free cobalamin binds to IF. The cobalamin-IF complex is stable and resistant to proteolysis in pH ranges of 3 to 9. Ileal IF-cobalamin receptors are selective for IF-bound cobalamin and not for R-bound cobalamin; thus, ileal pancreatic proteases are necessary as much as gastric peptic proteases to ensure optimal cobalamin absorption . IF secrThe final step of cobalamin absorption takes place in the ileum through specific membrane-associated IF-cobalamin receptors on the enterocytes. The ileal IF-cobalamin receptor is composed of a protein complex called “cubam” . Dysfunci.e., adenosylcobalamin  and methylcobalamin . In the latter reaction, 5-methyl-tetrahydrofolate donates its methyl group to cobalamin, regenerating methylcobalamin. In its turn, methionine can be adenylated to form adenosyl-methionine, which, in turn, donates its methyl group in a large number of biologic methylations involving proteins, neurotransmitters and nucleic acids.The TC II-cobalamin complex has a very short plasmatic half-life, being rapidly cleared mostly because of its rapid binding to specific TC II surface receptors on several cells . Once inTetrahydrofolate (THF) is required for the activity of thymidylate synthetase (TS) and DNA synthesis. Other than folate precursor deficiency itself, also cobalamin deficiency contributes to functional intracellular deficiency of THF. Cobalamin indeed is required by methione synthetase for the generation of methionine from homocysteine; meanwhile, THF is generated from 5-methyl THF. In fact, only THF can ultimately act as a coenzyme of the TS.As a consequence of the deficient TS activity, the synthesis of deoxythymidine monophosphate (dTMP) and deoxythymidine triphosphate (dTTP) from deoxyuridine monophosphate (dUMP) via thymidylate synthase is impaired and dUMP ) accumulates. Since DNA-polymerase does not distinguish dUTP from dTTP, increased amounts of dUTP are misincorporated into DNA. Appropriate DNA-repair enzymes (DNA uracil glycosylase) recognize the faulty incorporation and excise dUTP, but, lacking an adequate dTTP supply, effective repair does not occur, leading to DNA strands breaking.Megaloblastic changes due to cobalamin or folate deficiency are clinically indistinguishable. The cause of cobalamin deficiency is not generally revealed until specific laboratory tests are done; on the contrary, the recent patient’s history may give clues to the possible folate deficiency. Likely due to the widespread folate supplementation in Western countries, the hematologic picture of cobalamin deficiency is often attenuated, and neurological presentations may become more common and overlooked. It is currently not generally accepted that folate deficiency may induce neurological manifestations, so that the occurrence of neurological symptoms in the presence of folate deficiency should prompt investigations aimed at ruling out cobalamin deficiency .A strict vegetarian (vegan) diet contains very little cobalamin; less strict vegetarians  may have subclinical deficiency, as shown by increased blood methylmalonic acid (MMA) and homocysteine concentrations . Since tIn developed countries, the diet is rich in meat and cobalamin-rich foods; thus, malabsorption is the most common cause of cobalamin deficiency. Infants born to vegetarian mothers are at risk of cobalamin deficiency and may present with megaloblastic anemia, involuntary movements and skin pigmentation ,10. MostSome of the subjects with subclinical cobalamin deficiency may have normal serum cobalamin concentrations and may be classified as “normal asymptomatic subjects”, although the minimum concentrations of serum vitamin B12 for optimal neuronal health are still unknown, especially in the later stages of life.H. pylori infection, as well as in patients on long-term anti-acid therapy, insufficient pepsin or gastric secretion and inadequate proteolytic digestion there is a failure to dissociate cobalamin from food, thus preventing its absorption cobalamin, is administered orally, and its excretion is measured in the urine within the following 24 h. The test is influenced by a number of factors . Since some of these variables are more easily verified directly with appropriate examinations  and it is far more practical to supplement B12 deficiency, the Schilling test has not gained widespread use in the clinical setting.In the Schilling test, radio-labeled cyanocobalamin, In children, assessment of IF and acid content of gastric juice can help differentiate between Imerslund-Gräsbeck syndrome (where both are present), juvenile pernicious anemia (where both are lacking) and congenital IF deficiency (where IF is absent). In adults, gastric acidity upon maximal stimulation helps exclude the diagnosis of pernicious anemia.i.e., renal impairment, age) can result in false positive elevated serum MMA and homocysteine concentrations, only the reduction in the concentration of these metabolites upon cobalamin supplementation is an unequivocal proof of true cobalamin deficiency.Early clinically markers of cobalamin deficiency, occurring even in the presence of normal serum cobalamin concentrations, are increased serum MMA and homocysteine concentrations. Elevation of both markers distinguishes cobalamin-deficient from folate-deficient patients, most of whom display normal MMA values. However, since a number of variables  can falsely lower serum cobalamin concentrations; on the other hand, some clinical conditions, such as myeloproliferative diseases, some leukemias and lymphomas and active liver diseases, may give falsely normal serum values and obscure true cobalamin deficiency. As a matter of fact, 99% of the patients with hematological or a neurological picture consistent with cobalamin deficiency display concentrations of serum cobalamin lower than the low normal limit (300 pg/mL); among cobalamin-deficient patients, approximately 5% have normal cobalamin concentrations, and 10% of true cobalamin-deficient adults have serum cobalamin values in the low normal range [In clinical practice, normal concentrations of serum B12 do not exclude the diagnosis of B12 deficiency. The analysis of serum metabolite concentrations, such as MMA and homocysteine, can reveal patients with borderline B12 concentrations (<300 pg/mL) . ConcentIf MMA concentrations are elevated, it is recommended to rule out other possible causes of elevated MMA, including renal insufficiency or intravascular volume depletion. Hyperhomocysteinemia can be more sensitive to cobalamin deficiency, but it may also reflect folate deficiency, whereas elevated MMA has a similar sensitivity, but more specificity for vitamin B12 deficiency. Vitamin B12 deficiency is suspected when serum cobalamin concentrations are low (350 pg/mL) and when both MMA and homocysteinemia are elevated, or when MMA is elevated in the absence of renal disease or volume depletion, or when homocysteinemia is elevated in the absence of folate deficiency. Several conditions can falsely elevate or decrease serum cobalamin concentrations, but a normal MMA and homocysteinemia concentration suggest the absence of vitamin B12 deficiency .Thus, in some instances, the diagnosis of cobalamin deficiency requires a high index of suspicion and related biochemical alterations, such as high concentrations of both MMA and homocysteine. Should these two marker be within normal limits, cobalamin deficiency is virtually excluded.Vitamin B12 deficiency causes a wide range of hematological, gastrointestinal, psychiatric and neurological disorders. Megaloblastic anemia is a common early symptom leading to the diagnosis, although neurological symptoms may occur in the absence of hematological abnormalities.The mechanisms whereby cobalamin (and folates) deficiency produces megaloblastic changes are not precisely known. Defective DNA synthesis leads to disparity in nuclear-cytoplasmic asynchrony and cobalamin or folate-deficient cells slowly divide until mature daughter cells die in the marrow or are arrested at various stages of the cell cycle. Although megaloblastic changes are most striking in the bone marrow and peripheral blood, many other proliferating cells  may exhibit megaloblastic features. For instance, megaloblastosis in rapidly proliferating cells of the gastrointestinal tract causes atrophy of the epithelial cells of the luminal lining. This may lead to defects in secretion of IF, which, in turn, may aggravate the vitamin B12 deficiency.The increase of the mean corpuscular volume is the earliest manifestation of megaloblastosis. Red blood cells appear larger than normal, and some of them may lose the central pale area; furthermore, there may be an abnormal degree of variation in the shape of the erythrocytes in the blood (anisocytosis and poikilocytosis). Red blood cells may show traces of DNA and non-hemoglobin iron . In the bone marrow, ineffective hemopoiesis with numerous mitotic figures and trilinear hypercellularity occurs, mainly of the erythroid series, leading to a reduced myeloid-to-erythroid ratio from 3:1 to 1:1). Megaloblastic changes are more evident at basophilic and polychromatophilic erythroblast stages. Instead of the usual dense chromatin, megaloblastic erythroid progenitors exhibit reticular and finely dense chromatin. In particular, orthochromatic megaloblast retains its immature-appearing nucleus instead of the usual clumped chromatin of the orthochromatic normoblast . In addiLeukopoiesis is abnormal, too. Progenitor cells are increased, display fine chromatin and may reach large dimensions, making it difficult to cross traverse marrow sinuses and to reach peripheral blood. Cytoplasmic granulation occurs regularly; hypersegmented polymorphonuclear neutrophils (PMN) are the hallmark of megaloblastic leukopoiesis in the peripheral blood. For nuclear hypersegmentation to occur, more than 5% of PMN with more than five lobes is required.Megakaryopoiesis may be involved, with hypersegmented nucleus and liberation of fragments of cytoplasm, generating “giant platelets” in the peripheral blood. However, platelet production and release is impaired, and as a result, various degrees of thrombocytopenia occur.Clinical  presentation of cobalamin deficiency ranges from incidental increased mean corpuscular volume and neutrophils hypersegmentation in otherwise asymptomatic patients to symptoms due to severe anemia, such as angor, dyspnea on exertion, fatigue or symptoms related to congestive heart failure, such as ankle edema, orthopnea and nocturia. Severe anemia may cause dizziness and orthostatic hypotension, too. Food intake may be painful or may be hampered by severe glossitis. Alteration in bowel motility, such as mild diarrhea or constipation, and loss of bladder or bowel control can occur as a result of the involvement of Meissner’s and Auerbach’s plexus within the gastrointestinal tract and the bladder.The clinical spectrum of cobalamin deficiency has changed significantly over the last few decades. First, it appears that neurological disease is more and more frequent in patients with cobalamin deficiency and mild or absent anemia. Second, only a portion of unequivocally cobalamin-deficient patients display classical abnormal values of hematocrit, mean corpuscular volume and serum lactate dehydrogenase. Furthermore, “standard” morphologic examination of peripheral blood smears fails to recognize the hallmarks of cobalamin deficiency, such as macroovalocytes and hypersegmented neutrophils, since the latter finding is consistently found only in overt cobalamin deficiency. Most of the time, increased red blood cell volume is attributed to other causes, among which are reticulocytosis, liver diseases, chronic alcoholism and myelodysplastic syndromes.In the nervous system, vitamin B12 acts as a coenzyme in the methyl malonyl-CoA mutase reaction, which is necessary for myelin synthesis. Vitamin B12 deficiency therefore results in defective myelin synthesis, leading to several central and peripheral nervous system dysfunctions. Regarding the pathophysiological mechanism, lack of adenosylcobalamin  leads to accumulation of methylmalonyl-CoA, causing a decrease in normal myelin synthesis and incorporation of abnormal fatty acids into neuronal lipids .Neuropsychiatric symptoms may precede hematologic signs and are often the presenting manifestation of cobalamin deficiency. The neurological syndromes associated with vitamin B12 deficiency include myelopathy, neuropathy, neuropsychiatric abnormalities and, less often, optic nerve atrophy.The spinal cord manifestation, called subacute combined degeneration (SCD) is clinically characterized by symmetric dysesthesia, disturbance of position sense and spastic paraparesis or tetraparesis. The involvement of the posterior and lateral columns of the cervical and upper dorsal parts of the spinal cord is responsible for the impairment of position sense, paraparesis and tetraparesis. The first abnormality is usually sensory impairment, most often presenting as distal and symmetrical paraesthesias at lower limbs frequently associated with ataxia. Almost all patients have loss of vibratory sensation, often associated with diminished proprioception and cutaneous sensation and Romberg sign. Corticospinal tract involvement is common in the more advanced cases, with abnormal reflexes, motor impairment and, ultimately, spastic paraparesis . A minorPeripheral neuropathy can be seen in 25% of patients with vitamin B12 deficiency , and somOptic neuropathy due to vitamin B12 deficiency occurs occasionally in adult patients. Lesions in the optic nerve have been demonstrated in post-mortem examination of adults with pernicious anemia. Abnormal visually evoked responses have been reported in patients with pernicious anemia without visual symptoms, suggesting that there may also be subclinical damage to the visual pathway. Optic nerve disease is characterized by symmetric, painless and progressive visual loss. Central and centrocecal scotomas are the main ophthalmologic findings. Clinical response can be seen during the first three months of treatment .S-adenosylmethionine (SAM) production and abnormal methylation of phospholipids in neuronal membranes, potentially affecting ion channels and second messengers [Cobalamin deficiency may present with neuropsychiatric syndromes also in the absence of hematological signs. Among the psychiatric presentations mood disorders (both depression and mania), chronic fatigue syndrome and psychosis are notable . Cobalamssengers . In deprssengers ,30 may bssengers .Psychosis may be the presenting symptom in vitamin B12 deficiency. Reported symptoms include suspiciousness, persecutory or religious delusions, auditory and visual hallucinations and disorganized thought-processes .An association of vitamin B12 deficiency and depressive symptoms in elderly patients has been documented ,33. ThisSymptoms of mania have been described in the presence of vitamin B12 deficiency .Screening for and supplementing with vitamin B12, when appropriate, in the presence of mania is crucial, especially when there is no psychiatric or family history of bipolar disorder.Vitamin B12 deficiency has been also associated with attention deficits, acute mental-status and acute cognitive changes, with electroencephalography abnormalities . Case reet al. [On a retrospective study on the effects of B12 treatment on neuropsychological function and disease progression in patients with dementia or cognitive impairment, Eastley et al.  found thet al. , it is aet al. . Recentlet al. .Mycobacteriumtuberculosis may occur for poorly understood reasons.Cobalamin deficiency may sometimes result in sterility due to the effects on the gonads; defective bactericidal activity may also be impaired, and increased susceptibility to Vitamin B12 deficiency may affect both the central  and the peripheral  nervous system ,52.in vivo by MRI.Since the early 1990s, MRI has been considered pivotal for detecting B12 deficiency-related central nervous system involvement and for excluding possible mimics ,54. The The spinal cord involvement is associated with the most frequent clinical manifestation of vitamin B12 deficiency, namely SCD. The most consistent MRI finding in SCD is a symmetrical abnormally increased T2 signal intensity, commonly confined to posterior or posterior and lateral columns in the cervical and thoracic spinal cord .In our experience, axial T2 images are more effective in detecting spinal cord lesions, as faint signal abnormalities might be easily overlooked, due to partial voluming on sagittal imaging. In acute and severe cases, the spinal cord might also present as swollen . InvolveDifferential diagnoses of SCD include copper deficiency myelopathy , infectiBrain involvement has been reported in B12 deficiency. Fluid attenuated inversion recovery (FLAIR) and T2-weighted images might demonstrate extensive areas of a high-intensity signal in the periventricular white matter .In children with inherited cobalamin-related diseases, the damage is limited to the brain and characterized by white matter loss with delayed myelination; MRI has also revealed delayed myelination in infants with nutritional cobalamin deficiency . RecentlMarchiafava-Bignami disease is a rare disorder causing progressive demyelination and corpus callosum necrosis, primarily associated with chronic alcohol abuse, likely due to vitamin B complex deficiency. Callosal lesions with selective involvement of the middle layers are highly suggestive of Marchiafava-Bignami disease. Concomitant symmetric signal abnormalities might be observed in the centrum semiovale, internal capsule and middle cerebellar peduncle. In some cases, frontal cortex hyperintensity might be detected by FLAIR and DWI imaging (Morel’s laminar sclerosis) .Peripheral nerve abnormalities in B12 deficiency are usually too subtle to be recognized by current ultrasound, CT and MR imaging techniques, while nerve conduction studies reliably unveil the sensori-motor polyneuropathy, due to both demyelination and axonal degeneration. Similarly, neurophysiologic evaluation  is preferred as a diagnostic tool for optic nerve involvement , while nUsually, 1 mg/day of cobalamin given intramuscularly for a week, followed by 1 mg/week for four weeks and then 1 mg/month for life, is an appropriate therapeutic regimen. Obviously, therapy should be started after the vitamin deficiency has been established and the causes thoroughly looked for. Oral daily doses of 1–2 mg seem also to be suitable for patients refusing parenteral therapy who have cobalamin malabsorption or pernicious anemia, provided that cobalamin stores have been rapidly supplied by parenteral cobalamin.Effective therapy reverses megaloblastosis in 24 h and reestablishes normal marrow hematopoiesis in 48 h. The reticulocyte count increases after 3–4 days and peaks after a week; hypersegmented neutrophils remain in the peripheral blood up to two weeks. Rising red blood cell count and hemoglobin will take one week; normalization of the complete blood count requires about eight weeks.Although most patients respond well to cobalamin treatment, residual neurological abnormalities persist in most. Evidence of improvement or the reversal of neuropsychiatric symptoms varies according to symptom severity, duration and clinical diagnosis. It is commonly accepted that treating deficiencies in the early stages yields better results, as structural and irreversible changes in the brain may occur if left untreated. Vitamin B12 status has been associated with the severity of white-matter lesions, especially periventricular ones, in some , but notThe follow-up of B12 deficient patients should aim at verifying the compliance to cobalamin supplementation and carefully monitor patients with pernicious anemia, since they are likely to develop iron-deficiency as a result of impaired iron absorption, due to achlorhydria, and are at risk of developing stomach cancer. In some instances, cobalamin supplementation is advocated as a prophylactic measure in gastrectomized patients and in patients undergoing high-flux hemodialysis, as well as in vegetarians or infants born to mothers with pernicious anemia.Vitamin B12 is a water soluble substance critical for normal functioning of the nervous system and blood cell formation. Vitamin B12 deficiency may result from pernicious anemia, gastric resection, intestinal malabsorption or a strict vegan diet.It is often overlooked and may cause several hematological, gastrointestinal, psychiatric and neurological manifestations. Megaloblastic anemia is an early hematological sign, but neurological symptoms may occur also in the absence of hematological abnormalities. SCD, peripheral neuropathy, neuropsychiatric disorders and optic nerve atrophy are the most common neurologic manifestations. The typical MRI finding in SCD is a symmetrical abnormally increased T2 signal intensity, commonly confined to posterior or posterior and lateral columns in the cervical and thoracic spinal cord. Brain involvement has also been reported in B12 deficiency patients with extensive areas of a high-intensity signal in the periventricular white matter.Physicians should be alert to identifying signs or symptoms of anemia or suspected vitamin deficiency in populations at risk, even in the absence of hematological symptoms or signs. The concentration of serum vitamin B12 is not sufficient for diagnosis, and the metabolites upstream (homocysteine and MMA) should always be looked for. When B12 deficiency is diagnosed, an extensive search of the cause of cobalamin deficiency should always be carried out.Parenteral replacement therapy should be initiated as soon as possible and associated with a careful, life-long, follow-up of the patients. Early diagnosis is crucial for starting replacement therapy and avoiding irreversible neurological damage.