This CD-ROM version of Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology is an excellent and concise tool for easy reference during sign out of cytology and surgical cases. It is also invaluable as a teaching tool for residents and fellows.Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology CD-ROM (ISBN 0-9736518-0-7), Published by Pathology images Inc Ottawa, Ontario K2B 7L4, Canada The Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology as a CD-ROM version is a very welcome edition to the library of any pathologist for quick reference . The patThe authors have organized this CD-ROM into convenient chapters. In the sections on specific tumors, the authors have nicely provided tables, brief cytologic and surgical text and excellent colored photographs with key features highlighted. Some photographs use arrows to illustrate the salient features. The majority of the photographs are of high quality, although an occasional one has been taken at too low power to fully discern the specific features.I would recommend this CD-ROM in the cytology sign out room as a very useful quick reference for differential diagnosis of salivary gland tumors. The CD-ROM is easy to use, very concise in the information provided with high quality colored illustrations.The author(s) declare that they have no competing interests.The author critically reviewed the CD-ROM of Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology. Thereafter drafted and revised the manuscript of this review article.


The VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown.In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells.The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells. Culicoides sp) transmitted double-layered non-enveloped virus, is the type species virus of the genus Orbivirus, in the family Reoviridae. BTV is an economically important virus and causes hemorrhagic disease in sheep and other ruminants , 1% [v/v] Triton X and protease inhibitors (Sigma-Aldrich) at 4°C for 10 minutes. The lysate was clarified by centrifugation for 10 min at 10,000 rpm  and sample buffer added to a final concentration of  without 1% β-mercaptoethanol, without heating, and resolved by SDS-7% PAGE followed by western blotting.SDS-PAGE separated proteins were transferred onto a Hybond enhanced chemiluminescence nitrocellulose membrane  as described previously  and wereCells were washed 48 hours post transfection and incubated in medium with 50 μM of acrylamide (24 hours) or 5 μg/ml of colchicine (2 hours). They were then processed for immunofluorescence staining as described above. To examine the effect of the inhibitors on viral yield at different time points, the cells were first infected as described previously and then pharmacologically treated. Cell supernatant and cell pellets were collected and were subjected to three freeze/thaw cycles to disrupt cells and to release virus. All samples were collected, the viral titer of each sample was determined in triplicate by virus plaque assay in BSR cell monolayer cultures. Infectivity titers were calculated as log10 pfu/ml, and the mean and standard error of the reduction mediated by each inhibitor was calculated . All the pharmacological treatments were performed in duplicate and the plaque assay in triplicate.The author(s) declare that they have no competing interests.BB carried out the confocal microscopy, mutagenesis and fractionation studies. RN conceived the study, made the initial observations on the localisation of GFP-tagged VP2 and wrote a draft of the manuscript. PR contributed ideas to the design and co-ordination of the study and secured funding for the project. All authors read and approved the final manuscript.


In stalk-eyed flies (Diopsidae) the eyes and antennae are laterally displaced at the ends of elongated eyestalks. Eyespan and the degree of sexual dimorphism in eyespan vary considerably between species and several sexually dimorphic species show sexual selection through female mate preference for males with exaggerated eyespan. The genes on which selection acts to regulate eyespan remain to be identified. This could be achieved by comparing gene expression during eyestalk development in males and females if the sex of pre-adult flies could be reliably assigned. Here we describe two techniques, one morphological and one microsatellite-based, that identify the sex of stalk-eyed fly larvae and pupae.Teleopsis dalmanni have two highly distinct morphologies, compact ("C") and lobed ("L"). Segment composition  was consistent with C morphology being typical of males and L morphology of females. We confirmed the proposed association between disc morphology and sex by evaluating the combined heterozygosity of four X-linked microsatellite markers. We demonstrated that individuals with C genital discs had hemizygous  genotypes while those with L discs were heterozygous  genotypes. Similar dimorphism in genital disc morphology was observed in eight other species spanning three representative Diopsid genera. In every case the segment composition supported C morphology being male and L morphology female. We assigned larval sex by C or L morphology and compared cell division frequencies in male and female eye-antennal discs in two species  sexually dimorphic for eyespan. The number of mitotic (anti-H3-labelled) cells did not differ between the sexes in either species.We showed that genital discs of the stalk-eyed fly We have made novel use of two complementary techniques for identifying the sex of pre-adult stalk-eyed flies. These procedures will facilitate studies of the evolution of sexually dimorphic development in a variety of other species. Morphology and En expression in male and female genital discs are highly conserved within each genus of Diopsidae. Finally, sexual dimorphism for eyespan in two Diopsid species is unlikely to be due to an increased rate of cell division at the third larval instar in males. Understanding the evolution of sexually dimorphic development is a key goal in many biological contexts -3. HowevHypercephaly, in the form of lateral extensions of the head capsule, is observed in several families of Diptera . A partiDrosophila melanogaster it is possible to do this either on the basis of gonad size , or by using X-linked genetic markers such as the cuticle pigmentation gene yellow. Neither method is applicable in stalk-eyed flies in which the larval gonads of both males and females are equally small and undifferentiated and visible genetic markers are lacking. Here we present an alternative, novel method based on genital disc morphology and X-linked DNA markers, which can be applied in principle to many different Diopsid species, and potentially to other taxa.From an evolutionary standpoint there is considerable interest in identifying the timing of expression of the hitherto unknown genes on which selection acts to regulate eyespan in sexually dimorphic Diopsid species. One potentially powerful approach is to compare gene expression during the development of eyestalks in between the sexes, since sexual dimorphism likely results from differential gene expression with respect to sex. However, this requires a method for determining the sex of pre-adult flies. In the model Dipteran D. melanogaster it has been shown that with the exception of the eye-antennal and genital discs all of the imaginal discs are composed of cells derived from a single embryonic segment [Drosophila the genital disc is a single disc in each sex [The adult structures of Dipteran flies, including stalk-eyed flies, develop from the larval imaginal discs, which originate as invaginations of the embryonic ectoderm. In  segment . Imaginaeach sex .Drosophila the genital disc develops into the internal and external genitalia and the analia, collectively known as the terminalia. It is the only imaginal disc with an unambiguous sexually dimorphic morphology. The genital discs of both sexes comprise cells from the eighth, ninth and tenth/eleventh abdominal embryonic segments and the fate of these cells depends on the fly's sex [In ly's sex ,18. In mly's sex .Teleopsis dalmanni  have very different expression patterns in the male and female discs, which reflect later differences in eventual segment fate [en protein expression in T. dalmanni genital discs showing the compact morphology resembled that observed in the male genital discs of D. melanogaster. As in D. melanogaster, male Diopsids have a single X chromosome whilst females have a pair [X-linked microsatellite markers; individuals with a compact form of genital disc had hemizygous  genotypes, whereas individuals with lobed discs tended to exhibit heterozygous  genotypes.In the model stalk-eyed fly species, almanni; ), we fouent fate . The pate a pair . We confT. dalmanni was observed and in all species studied EN expression patterns were consistent with the compact morphology being typical of males across genera.To determine the extent to which male and female genital disc structure and segmental composition is conserved across the Diopsid clade, we characterised morphology and EN expression in a panel of Diopsid species spanning three genera. A compact/lobed dimorphism similar to that of T. dalmanni the larger eyespan of males is due, at least in part, to the eyestalks being composed of more cells. This could reflect a higher rate of cell division or a prolonged period of proliferation in males. We used genital disc morphology to determine whether the frequency of cell division differs between male and female H3 antibody-labelled eye-antennal discs in two sexually dimorphic species, T. dalmanni and Diasemopsis meigenni. We found no significant differences in dividing cell counts between the sexes in either species.We exploited our ability to assign sex by genital disc morphology to compare cell division rates in male and female eye-antennal discs. In D. melanogaster are essentially two-dimensional with folds in the plane of the disc epithelium. In contrast, the genital disc epithelium is folded into a fully three-dimensional structure with distinct ventral and dorsal sides [Most of the imaginal discs in al sides . By the en protein product, the expression of which is restricted to the posterior compartments of all segments. In the male disc, EN expression in the male primordium forms a broad anterior band on both ventral and dorsal sides of the discs. On the dorsal side two lateral stripes representing the anal primordium are visible, while a small central patch corresponding to the posterior compartment of the repressed female primordium is visible ventrally  are contiguous. As each is derived from a different segment, their boundaries can be revealed by staining the discs with an antibody raised against a highly conserved epitope of the y Figure . In the y Figure .T. dalmanni, two distinct genital disc morphologies can be observed. One type, which we have termed "compact", resembles the morphology of the male genital disc in Drosophila. The posterior half of the disc has a relatively simple flattened structure while the anterior forms a series of concentric folds. EN is expressed in two posterior strips similar to the staining of the anal primordium in Drosophila and in two large anterior patches, which could correspond to male primordium expression  were hemizygous at all four loci and 90% of females (n = 20) were found to carry two different alleles for at least one marker locus  and lobed  genital discs of les n = 1 were hemrd instar larvae, stained with methylene blue and typed for morphology (compact or lobed). DNA for microsatellite analysis was extracted from the remaining larval tissue of 16 with the compact type of genital disc and 16 individuals with the lobed type . The expected values for this test have been adjusted using the observed error levels from the genotyping trials of adults. Note that even if this error level is omitted from the contingency test, the significant association between disc morphology and level of X chromosome zygosity remains . Thus the microsatellite genotyping confirms the inferences from the comparative morphology and en expression analyses that individuals with compact genital disc morphology are male and those with lobed morphology are female.A further assay based on the same 4 loci was used to test for an association between the sex of a larva  and the morphological type of genital disc. Genital discs were dissected from 3en protein expression patterns of the male and female discs in a further eight species sampled from three representative genera of Diopsids in order to characterise morphological variation among the clade. Three further Teleopsis species were chosen  to supplement our findings described above for T. dalmanni. Also we selected three more distantly related Diasemopsis species . The Sphyracephalae have been shown to form a basal genus within extant Diopsidae [S. beccarii and S. europaea). Examples of representative species from each genus are presented in Figure We investigated the morphologies and iopsidae  and so fTeleopsis species, we can assign sex according to genital disc morphology with some confidence given the consistent results of our microsatellite-based assay to identify the male and female disc types in one genus member, T. dalmanni  showed an almost identical pattern of EN expression to that seen in male Teleopsis discs . Cell counts were non-normally distributed and therefore analysed by non-parametric methods (Wilcoxon test). In both species, cell counts did not differ significantly between the sexes .Counts of mitotic cells in the eye-antennal discs at the prepupal stage were obtained for males and females of two sexually dimorphic Diopsid species , present in all Diopsid species examined. However, it is not intrinsically obvious whether these forms reflect sexual identity. A comparative argument can be made based on the similarities between stalk-eyed fly disc morphologies and those of the male and female discs in notyping , correctD. melanogaster, sexual dimorphism in genital disc morphology is not apparent until the third larval instar and it becomes progressively more difficult to observe the genital disc as metamorphosis proceeds. In contrast, the use of microsatellites has the advantage over simple observation of genital disc morphology as they can be used to identify the sex of individuals at any growth stage. However, there are constraints to the adoption of a microsatellite-based approach, as it is also more costly, more time-consuming and currently less reliable than morphological observation; in Diopsids, X-linked microsatellites are currently limited to only a few species within the Teleopsis genus [Each method, morphological or genetic, has advantages and disadvantages. Assays of genital discs in order to assign sex are only possible for a limited period during development. For example, in is genus .T. dalmanni. We are therefore confident that the morphological assay can be used to identify the sex of larvae from these species. In the absence of X-linked microsatellite markers it may be possible to verify this by in vivo culture of male and female discs, as has been achieved for analysis of eye-antennal discs in T. dalmanni [In the three genera we have studied at least one of the discs has proved almost identical in terms of morphology and EN expression to either the male or the female disc in dalmanni .D. melanogaster. EN expression patterns are consistent with the hypothesis that the genital discs are derived from more than one embryonic segment and suggest that as in D. melanogaster, the posterior part of each disc gives rise to the analia and the anterior to the genitalia. It should be possible to verify these assumptions by in vivo culture of male and female discs.We have described differences between the genital discs of males and females in three stalk-eyed fly genera. EN expression patterns and morphology are very highly conserved within Diopsid genera and, with one notable exception, quite similar to that described in Teleopsis and Diasemopsis are similar in appearance, suggesting a conserved morphology since the two lineages diverged. Female discs in the phylogenetically basal Sphyracephala exhibit a more complex morphology quite unlike their counterparts in the other Diopsid genera or in D. melanogaster so that it is unclear which regions of the disc represent the genital and anal primordia. Although we have not tried to assign functionality to these differences, our data provide a firm basis for future studies of the evolution of genital disc morphology and sexual dimorphism in stalk-eyed flies.Male discs are highly conserved within and between Diopsid genera. The female discs of Calliphora erytrocephala [Musca domestica [D. melanogaster possesses a single genital disc [T. dalmanni shows that for a second acalypterate species, individuals of each sex possess one genital disc and lateral genital discs are absent. Two recent studies have established that the acalypterate Diopsidae are the basal family within Schizophorans based on morphological [Phylogenetically the Schizophoran dipterans are traditionally viewed as comprising two sister groups, the acalypterates and calypterates ,26. Geniocephala ; Musca domestica ,29). In tal disc . Our stuological  and moleological . Consequological ,33, or oological  are needT. dalmanni may be due, at least in part, to differences in cell number. We have exploited our ability to identify larval sex in this species to compare rates of cell division in male and female eye-antennal discs. The difference between the number of mitotic H3-labelled cells in males and females at the prepupal stage was not significant, suggesting that the rate of cell division is similar in both sexes at this timepoint. We are currently developing techniques for assessing cell division at subsequent stages of metamorphosis, when the developing eyestalks are less accessible to experimental manipulation.The cellular basis of the sexual dimorphism for eyespan in We describe the novel application of two complementary techniques, one morphological and one microsatellite based, for identifying the sex of pre-adult stalk-eyed flies. Both procedures will aid studies of the evolution of sexually dimorphic development in Diopsid and other flies and the microsatellite-based approach may be applied to a wide range of species. To demonstrate the potential value of our methods, we used them to assign larval sex in a study comparing cell division frequencies in male and female eye-antennal discs of two sexually dimorphic species. Our findings indicate that sexual dimorphism for eyespan in Diopsids is unlikely to be due to an increased rate of cell division in males at the end of the third larval instar.T. dalmanni for this study were obtained from a pair of laboratory populations originally derived from field collections in 1989 and 1993 near the Gombak River in Malaysia and maintained subsequently as independent stocks. To minimise the potential effects of inbreeding, both stocks have been maintained in population cages containing at least 200 individuals, kept at at 25°C on a 12 hour: 12 hour light: dark cycle, and fed ground sweetcorn. In order to maximise X-chromosomal heterozygosity in females when genotyping microsatellites, we analysed the hybrid offspring of males from the 1989 stock crossed to females of the 1993 stock.Experimental samples of T. quinqueguttata , T. species n , T. breviscopium , Sphyracephala beccarii , S. europaea , Diasemopsis dubia , Diasemopsis meigenii  and D. comoroensis . All species were maintained under similar conditions to those of T. dalmanni.A further six stalk-eyed fly species were used during this study. They were D. melanogaster came from a stock established in 1972 from a collection in Dahomey . Subsequently this has been maintained on standard sugar-yeast food medium at large population size in cage culture at 25°C and exhibits high levels of genetic variation [The sample of wild-type ariation .T. dalmanni larvae were dissected in 1 × PBS, with the larval cuticle being inverted to expose the genital disc. Discs were stained in 0.1% methylene blue (Fisher Scientific) for 5 minutes and fixed in 1% gluteraldehyde (Sigma) in 1 × PBS, before being removed from the cuticle wall and scored for their morphology.Third instar D. melanogaster and the suite of stalk-eyed fly species, third instar genital discs were labelled with a 1:20 dilution of mouse monoclonal anti-EN mAb 4F11 antiserum (gift from N. Patel). T. dalmanni eye-antennal discs from the early pupal stage were labelled with a 1:1000 dilution of rabbit polyclonal anti-phospho-Histone H3 (Ser10) antiserum (Upstate). Labelled discs were mounted in 90% glycerol and photographed using a Nikon Coolpix 4500 digital camera attached to a Leica DMLB microscope. Labelled cells from each eye disc  were counted from the photographs.Antibody staining was performed using the protocol of . For D. Tissue from individual adults or larvae was ground in TNES , to which Proteinase K (1 mg/ml) was added. The mixture was incubated overnight at 37°C. 5 M NaCl was then added and the DNA was precipitated with absolute EtOH.X chromosome zygosity levels were determined by genotyping individuals for the X-linked microsatellite loci ms-054, ms-125, ms-167 and ms-395 . PCR was initiated with a two minute denaturing step at 94°C, followed by thirty cycles of denaturing at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 45 seconds. The final step was a ten minute extension at 72°C. Microsatellite genotyping was carried out using a 3100 DNA Analyzer (Applied Biosystems) and analysed with Genescan 3.1.2 software (Applied Biosystems).MC conceived the study, contributed to the design and execution of the experiment, and helped to draft the manuscript. SC participated in the execution of the experiment and helped to draft the manuscript. DR contributed to the design of the experiment. KF conducted the statistical analysis. HS, AP and KF jointly conceived the study with MC, contributed to design and coordination, and drafted the manuscript. All authors have read and approved the final manuscript.


To the Editor: Free-range domestic ducks can be a key factor in regional spreading of Asian subtype H5N1 avian influenza (AI) virus (Anas platyrhynchos var. domestica) and influenza A virus (H5N1) A/chicken/Miyazaki/K11/2007 as previously described (7 50% egg infectious doses (EID50) of 0.1 mL. All experimental procedures were approved by the Ethics Committee of the National Institute of Animal Health in Japan.An experimental infection study was conducted with Japanese domestic ducks . Samples were examined by rapid tests, virus isolation, and reverse transcription–PCR (RT-PCR). Feathers were also examined by immunohistochemical testing.A). Briefly, we put the calamuses into the test tube containing attached reagent solution (340 μL) and chopped them into small pieces with an iris scissor and then placed the test strip in the tube. We obtained the following results: feathers tested positive for influenza A virus from days 3 through 6 pi in 1 duck (a), and on days 3 and 4 pi in 2 ducks (b and c), whereas all oropharyngeal and cloacal swabs were negative (B).On-site rapid tests were performed with a commercial kit, QuickVue Influenza A+B , which can detect influenza virus nucleoprotein. The first set of swabs was used for rapid tests according to the manufacturer’s instructions. We also tested 1–2 sticks of the feather calamus (≈15–30 mg per stick) for rapid tests , panel Anegative , panel B50/mL. Viruses were isolated from the oropharyngeal swabs, cloacal swabs, and feathers of all birds, and feathers tested positive for the virus for a longer period than did the swabs  feather homogenate supernatants were calculated with 10-day-old embryonated chicken eggs and expressed as EIDhe swabs . AlthougOne-step RT-PCR was performed on the total RNA extracted from the same samples as in virus isolation to detect the H5 AI virus gene . The 1:10 dilution of RNA templates was used for feathers. The primers used were H5–248–270F and H5–671–647R; the expected product was 424 bp .C).Immunohistochemical testing was performed to detect influenza virus nucleoprotein in the feather tissue by using a rabbit polyclonal antibody . Virus antigens were detected in feather epidermal cells from days 3 through 6 pi, and in a few stromal cells in the feather pulp on days 3 and 4 pi , panel COur results indicate that larger amounts of viruses can be isolated for a longer time from feathers than from swabs. Therefore, feathers can be considered useful samples for surveillance or diagnostic examination of AI virus (H5N1) in domestic ducks. The epidermis, the outer layer of the feather, is a tissue that has poor host immune response against viral replication  Developing contour feather. The calamus was used for examination (bar = 1 cm). B) Result of the rapid test with feathers. A pink line (arrowhead) indicates a positive result for influenza A virus. C) Immunohistochemical stain of a biopsied feather composed of feather epidermis (fe) and feather pulp (fp). Influenza virus nucleoprotein was detected in the fe epidermal cells (arrowheads) (bar = 200 m).


West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter . Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus  and the highest number of human cases (n=10).


Umbre (UMB) virus was first isolated from India in 1955 and classified as Orthobunyavirus (Turlock serogroup). Eight isolates of this virus, isolated from Culex mosquitoes were characterized on the basis of partial glycoprotein (G2) gene. Twenty-six percent differences at nucleotide level while 17% differences at amino acid level were noted within different isolates. Phylogentic data shows that this virus represents a distinct group within the genus Orthobunyavirus. Bunyaviridae family are spherical particles, range 80 to 120 nm in diameter and share a common genetic organization of three predominantly negative stranded RNA segments . Based on antigenic, genetic and ecological relatedness, the Bunyaviruses are divided into five genera. The genus Orthobunyavirus includes approximately 60 viruses, which are known to cause disease in humans . Virological surveillance of these viruses depends primarily on detecting the viruses in arthropod vector populations in nature. Although, serological test like immunoassays are available for antigen detection for a few viruses, cross-reaction in closely related viruses cannot be ignored .The viruses of Culex bitaeniorhynchus mosquitoes, collected in 1955 at Umbre, Kolaba district, Maharashtra State, India. The virus has been registered in the International Arbovirus Catalogue No. 43 . During further field investigations, seven more strains of virus were isolated from Cx. vishnui mosquitoes. Recent reports on Bunyaviruses via. Ganjam virus isolation from Maharashtra  and antibody detection of Hantan virus in India , has provided evidences that Bunyaviruses are circulating in this country but their involvement in causing human and animal disease are not known yet. In the Gene Bank, only one sequence of Turlock serogroup i.e. N gene of M'poke virus is available.Umbre virus (UMB) [strain G-1424] was first isolated from VeroE-6 cells. Cytopathic effect (CPE) was observed during 4th – 6th post infection day. Infected cells were harvested, centrifuged and supernatant was used for molecular characterization of the virus.UMB viruses used in this study are listed in Table  along wiet. al., (2001), represents the nucleotide sequences of the N-terminal half of the G2 glycoprotein. Superscript III single step RT-PCR with Platinum Taq DNA polymerase kit (Invitrogen) was used for amplification of partial M gene according to the manufacturer's instructions.RNAs were isolated using chloroform, isoamylalcohol and further purified using RNAaid kit (Biogene), according to the manufacturer's instructions. RNAs were dissolved in 50 μl nuclease free water. Different sets of primers were used to amplify partial N, L and M gene. Partial M gene of 570 bp could be amplified using primer pair M14C and M619R, as described by Bowen Amplified products were detected in 2% agarose gel after staining with ethidium bromide in Tris/acetate/EDTA buffer (TAE). A desired size of 575 bp product was purified using QIAquick gel extraction kit (Qiagen), as per manufacturer's instructions. The sequences of amplified products were determined by using ABI PRISM BigDye Terminator V3.1 cycle sequencing ready reaction kit (Applied Biosystems). Amplification primers were used to sequence the amplified products. Cycle sequencing PCR program was used for 96°C-1 min, 96°C-10 sec, 50°C-5 sec and extension of 2 min at 60°C for 30 cycles.W program. Phylogenetic analysis was performed using Mega 3.0 by using neighbor-joining algorithm with thousand bootstrap values.The partial M gene sequence was curetted with the help of KODON Software and aligned with known Gene Bank sequences of Bunyamwera serogroup, California serogroup, and Kaeng Khoi viruses using clastal Partial M gene sequences showed maximum homology with Bunyamwera serogroup virus. Nucleotide and amino acid similarity within eight isolates varied from 74–100% and 83–99% respectively. Isolate G1424 and 809365 come together with 6% and 1% difference of nucleotide and amino acid respectively, while other six isolates club together with 5% nucleotide and 4% amino acid differences  are divides into six distinct lineage viz. Bunyamwera, Simbu, California encephalitis, Group C, Kaeng Khoi (KK) and UMB virus. Comparison of 550 bp of partial M gene showed four lineages, excluding Group C viruses and Simbu group of virus . Availability of more sequences of Turlock group may answer about placement of this group of viruses. Bunyaviruses being three segmented RNA viruses have the capacity to reassort their segments into new genetically distinct viruses, if the target cells are subject to dual infection. The possibility of drift, shift and UMB virus evolution towards an emerging disease pathogen cannot be predicted based on partial sequences. Complete genome sequencing of UMB virus can possibility suggest whether there is any reassortment between three genes of this virus as known for Ngeri, Batai and Jatobal virus .Turlock and Umbre virus are distinct from each other based on neutralization test (Calisher The authors declare that they have no competing interests.PDY performed the PCR and sequencing. ACM helped in preparation of manuscript. DTM and PDY designed, coordinated the study and prepared the manuscript.


Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in  Hantavirus genus (family: Bunyaviridae) have been identified worldwide. Rodents are the natural reservoir for hantaviruses, although one virus strain has been isolated from the house shrew (Suncus murinus), an insectivore Currently, at least 20 serotypes and genotypes of the Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), and Dobrava-Belgrade virus (DOBV) Apodemus agrarius), Norway rat (Rattus norvegicus) and black rat (R. rattus), bank vole (Clethrionomys glareolus), and yellow-necked field mouse (A. flavicollis), respectively. DOBV was also found in A. agrarius in Europe (Sin Nombre virus (SNV), New York virus (NYV), Black Creek Canal virus (BCCV), Bayou virus (BAYV), Andes virus (ANDV), and other related viruses cause HPS in the New World and are carried by the deer mouse (Peromyscus maniculatus), white-footed mouse (P. leucopus), cotton rat (Sigmodon hispidus), marsh rice rat , and Oligoryzomys longicaudatus, respectively (Microtus fortis) in Far East Russia were found to harbor two novel hantaviruses, Khabarovsk virus (KHAB) and Vladivostok virus (Topografov virus (TOPV), was isolated from brown lemmings (Lemmus sibiricus). The correlation between these three viruses and their pathogenicity for humans are not yet known Hantaviruses cause two forms of human disease, hemorrhagic fever with renal syndrome (HFRS), and hantavirus pulmonary syndrome (HPS); human infection occurs after the inhalation of aerosolized rodent excreta. HFRS is manifested as high fever, renal dysfunction, and hemorrhage; HPS is characterized by an acute progressive pulmonary edema and a fatality rate of about 40%. Among the hantaviruses that cause HFRS in Eurasia are n Europe . Sin Nomectively ,6. Althook virus ,8. AnothA. peninsulae, according to a recent study on nucleotide sequence comparisons by Yashina et al. A recent study reported two novel hantaviruses, designated as Amur (AMR) and Far East (FE), that were identified from HFRS patients in Far East Russia A. peninsulae is a carrier of pathogenic hantaviruses. We detected antibodies to hantaviruses in A. peninsulae, and the viral genome characteristics were extremely similar to the newly identified genotype, AMR A. peninsulae, we were able to corroborate the assumption of Yashina et al. A. peninsulae-related viruses are pathogenic for humans and are distributed over a large area of east Asia that includes Far East Russia.In 1999, we carried out an epizootiologic survey in a suburb of Vladivostok, Russia, to determine the characteristics of hantaviruses circulating in Far East Russia and to examine the possibility that We collected sera and organs from wild rodents captured during 1999. We also collected sera and autopsy materials from HFRS patients in two rural villages in the Primorye region of Russia, located 400 km and 600 km from Vladivostok.Rodent sera were screened for antibodies to HTNV and PUUV or both by indirect immunofluorescent antibody assay (IFA). Vero E6 cells infected with the Hantaan 76-118 strain of HTNV or the Sotkamo strain of PUUV were used as antigen slides. Diluted sera (1:16 and 1:64) were spotted onto the antigen slides and incubated at 37°C for 1 h. After three washes with phosphate-buffered saline (PBS), protein G-conjugated fluorescein isothiocyanate (FITC)  was spotted onto the slides. After incubation at 37°C for 1 h, the slides were washed and observed by fluorescence microscopy. Scattered, granular fluorescence in the cytoplasm of infected Vero E6 cells was considered a positive reaction. Antibodies in HFRS patient sera were detected by the same protocol, except for the substitution of FITC-conjugated antihuman immunoglobulin (Ig) G .A. peninsulae with Isogen , which is based on the acid guanidium-phenol-chloroform technique, according to manufacturer’s instructions. Similarly, total RNA was extracted from lung, liver, kidney, spleen, and brain tissues of HFRS patients. Reverse transcription (RT) was carried out at 42°C for 30 min by using Superscript II and random primer . Full-length S segments were amplified with Platinum Taq (Gibco-BRL) and HTNV–full S primer for 30 polymerase chain reaction (PCR) cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 2 min. Amplification of M segments was identical to that of S segments, except for the use of M genome-specific primers  to generate the phylogenetic trees by using the neighbor-joining method with 1000 bootstrap replicates. Hantavirus sequences used in the comparisons were obtained from GenBank. The S and M genome sequences used in this study are listed in We used the ClustalX program package  A. agrarius, (8) C. rufocanus, (3) M. fortis, and (2) Tamias sibiricus. Screening by IFA showed that one A. agrarius (2.5%), four A. peninsulae (5.7%), and one C. rufocanus (12.5%) had antibodies to HTNV or PUUV or both. HTNV-antibody titers ranged from 1:32 to 1:512. All the seropositive rodents, except for C. rufocanus, lacked antibody against PUUV (A. peninsulae were subjected to RT-PCR to amplify the virus genomes. Two of the four rodents with high IFA titers to HTNV (1:256 and 1:512) were positive by PCR for both the S and M segments of hantavirus.We carried out the epizootiologic survey on 122 rodents captured in a suburb of Vladivostok; results of serologic screening of rodent sera by IFA are shown in nst PUUV . Lung tiWe obtained the clinical histories of two fatal cases of HFRS in the Primorye region. The patients, who lived in villages 400 km and 600 km from Vladivostok, died 8–13 days after the onset of illness; gastrointestinal bleeding and acute renal failure were the causes of death. Serologic screening showed that both patients were positive for hantaviral antibodies. Antibody titers to HTNV and SEOV were apparently higher than to PUUV. We used lung, liver, kidney, spleen, and brain tissues of these HFRS patients for RT-PCR analysis; the lung and kidney tissues of patient no. 1 and the spleen tissue of patient no. 2 were positive for hantaviral M segment.To examine the histopathologic changes in HFRS patients, we used light microscopy to examine sections of formalin-fixed lung, liver, kidney, spleen, and brain tissues from patient no. 2, who had died of acute renal failure . The kidA. peninsulae were amplified and sequenced. We designated these segments as Solovey/AP61/1999 and Solovey/AP63/1999 based on the name of the village closest to the survey point, the rodent species from which the sample was taken, and the year in which the epizootiologic survey was done. We compared the coding regions of these sequences with those of other hantaviruses . We also sequenced the partial M segments of genetic lineages identified in the two HFRS patients from the Primorye region, designated as Primorye/H1/2000 and Primorye/H2/2000. The M segment of Solovey and Primorye sequences were compared with those of other hantaviruses (A. peninsulae in Far East Russia (A. peninsulae) in Far East Russia and China. Our results also suggest that this genetic lineage is widely distributed throughout east Asia.To explore the genetic diversity of hantaviruses identified in aviruses . Nucleott Russia ,11. The The M segments of Solovey, Primorye, and AMR sequences formed a common phylogenetic lineage with high bootstrap support values, regardless of viral origin . FurtherTo identify signature amino acids for each virus type, we compared the deduced partial amino acid sequences of their G2 regions using ClustalX multiple-sequence alignment . The preP. maniculatus could transmit highly virulent hantavirus to humans until SNV was identified . Recently, AMR sequences were found in both HFRS patients and A. peninsulaeA. peninsulae. Comparison of the deduced hantaviral amino acid sequences showed that aspartic acid and methionine represented signature amino acids for AMR genetic lineage, regardless of the region in which the virus was identified or its origin  from Primorye region, we detected pathologic changes typical of severe HFRS caused by hantavirus infection –35. We aA. peninsulae as one of the causative agents of HFRS. We think that this information may be helpful in preventing human infections in East Asia. Controversy persists over whether A. peninsulae carries a distinct virus type or a subtype of HTNV. A similar question arises with Dobrava/Slovenia and Dobrava/Saaremaa, which are carried by A. flavicollis and A. agrarius, respectively. The S segment identities between Dobrava/Slovenia and Dobrava/Saaremaa (both obtained from GenBank for comparison purposes) were 87.8% (nucleotide) and 92.7% (amino acid). Similarly, the nucleotide and amino acid sequence identities of the S segments of Solovey sequences and HTN 76-118 were 82.7% and 92.2%, respectively. We suggest that Solovey sequences belong to a sublineage within the HTNV clade.Through epizootiologic, clinical, pathologic, and sequencing studies, we identified a hantavirus carried by


Identification of HPeV-6 will advance HPeV diagnosis and epidemiology. Picornaviridae. Neutralization test showed that the NII561-2000 virus had distinct antigenicity to HPeV-1, HPeV-2, and HPeV-3, and that the sequence was distinct from these types as well as from HPeV-4 and HPeV-5. Thus, we propose the virus (NII561-2000) as the prototype of HPeV-6. We isolated 10 NII561-2000–related viruses, 14 HPeV-1, 16 HPeV-3, and 1 HPeV-4 of 41 HPeVs from various clinical samples collected in Niigata, Japan. Clinical symptoms of the persons infected with the NII561-2000–related viruses were infectious gastroenteritis, rash, upper respiratory tract infection, and paralysis, in addition to Reye syndrome in the 1-year-old girl.Using Vero cells, we isolated a virus (NII561-2000) from a cerebrospinal fluid specimen of a 1-year-old girl with Reye syndrome. The determined amino acid sequence of the virus indicated that the isolate was a human parechovirus (HPeV), a member of  Picornaviridae family (Human parechovirus (HPeV) is a small, nonenveloped RNA virus with a single-stranded genome of positive polarity, ≈7.3 kb in length; it is a member of the We report a 1-year-old girl who died with Reye syndrome, which is characterized as an acute, noninflammatory encephalopathy with hepatic dysfunction and fatty infiltration of the viscera; the syndrome is frequently associated with an antecedent viral infection, such as influenza or varicella , and 8% fetal bovine serum at 37°C under 5% carbon dioxide.g for 24 h in cesium chloride (CsCl) solution at an initial density of 1.34 g/mL, and the virion-containing fraction was collected and used for viral RNA isolation.MDCK, Caco-2, RD-18S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1 cells were cultured on 24-well plates for 3 days. Then, these cells were inoculated with a CSF specimen (100 µL per well) from a patient with a diagnosis of Reye syndrome and cultured at 33°C for 2 weeks. To check for cytopathic effect (CPE), we examined the cells under a light microscope. To purify the virus particles, we mixed 900 mL culture fluid of Vero cells inoculated with the NII561-2000 virus with 20 g NaCl and 76 g polyethylene glycol #6,000 at 4°C overnight. Next, the sample was centrifuged at 5,000 rpm for 30 min, and the pellets were suspended in 4.5 mL of phosphate buffer (pH 7.2). After treatment with 4.5 mL of chloroform at 4°C for 5 min, the sample was centrifuged at 2,500 rpm for 20 min. The supernatant was centrifuged at 120,000 × Taq polymerase  was used as a polymerase. Because 5′ RACE did not reach to the 5′ end of the NII561-2000 virus cDNA, the 5′ end cDNA fragment of the NII561-2000 virus was amplified from the NII561-2000 viral RNA by reverse transcription–PCR (RT-PCR). The primers used for RT-PCR were HPeV-head  and HPeV-mid . Two cDNAs isolated contained part of the NII561-2000 gene. To isolate the 3′ cDNA fragment of the isolated NII561-2000 cDNAs, the 3′ rapid amplification of cDNA ends (RACE) was performed by using the RNA PCR Kit (AMV) according to the instructions provided by the supplier . The primers used for 3′ RACE were 35F-out , 35F-in , and M13M4 . Herculase Hotstart DNA Polymerase  was used. To isolate the 5′ cDNA fragment of the isolated NII561-2000, the cDNA fragment containing part of the 5′ untranslated region (UTR) and capsid precursor protein VP0 was amplified by PCR from the cDNA prepared from the NII561-2000 virus with degenerate primers corresponding to this region. The degenerate primers used were E23P1  (50]) of the indicated viruses and 25 µL of the serially diluted antisera  were mixed in a 96-well plate and incubated at 37°C for 2 h. Then suspended Vero cells (100 µL/well) were added into these wells. Three to 10 days later the CPE of Vero cells was checked by light microscopy. To prepare the antiserum against the NII561-2000 virus, the virus particles grown in Vero cells were purified by CsCl centrifugation as described above. By using this purified virus, we prepared antiserum by Nippon Biotest Laboratory . In brief, the purified viruses were subcutaneously injected into rabbits 3× every 2 weeks. After we checked the antiviral titer by the Ouchterlony method, blood was collected from the vaccinated rabbits.Twenty-five microliters . The amplification reaction consisted of 30 cycles at 95°C for 30 s, at 59°C for 30 s, and at 72°C for 1 min. The primer set used was E23P1 as a forward primer and HPV-N1 . cDNA was synthesized from 8 µL of the viral RNA by using 1 U Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 20 U of recombinant RNAs in ribonuclease inhibitor . The 810-bp fragment containing part of 5′ UTR and VP0 was amplified by PCR, using 5 µL of cDNA in a 50-µL reaction mixture containing 50 mmol/L KCl, 10 mmol/L Tris/HCl (pH 8.5), 2.5 mmol/L MgClThe bootstrap values were calculated from P1 amino acid sequences of parechoviruses with the CLUSTAL X software program . Taken together, these results suggested that the CSF from the patient contained a virus, which we refer to here as NII561-2000.In January 2000, a 1-year-old girl with croup and high fever (39.6°C) was hospitalized in a regional general hospital in Niigata Prefecture, Japan. The infant died, and her condition was diagnosed as Reye syndrome after postmortem pathologic examination. To identify the pathogenic agent, we added a CSF specimen collected before death to 8 cell lines: MDCK, Caco-2, RD-18S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1 cells. Only Vero cells inoculated with the specimen exhibited a CPE. The CPE titer of the culture fluid was 10Neutralization tests were performed to examine whether the NII561-2000 agent is related to known viruses. The NII561-2000 virus infection of Vero cells was not neutralized by a pool of enterovirus typing antisera or 3 HPeV typing antisera [ (A308/99)] . Convers2 did not reduce the infectivity. These results indicate that the NII561-2000 virus has similar properties to human enteroviruses and HPeVs.We next examined the sensitivity of the virus to 5-iodo-2′-deoxyuridine (IUDR) (www.ncbi.nlm.nih.gov/blast) search identified that the nucleotide sequences of 2 isolated cDNAs showed high similarity with that of HPeV-2. By using these cDNAs as a starting material, the cDNAs containing the 5′ portion and 3′ portion of the NII561-2000 gene were isolated by RT-PCR with degenerated primers, 5′ RACE and 3′ RACE. The determined nucleotide sequence of NII561-2000 was 7,347 nt in length, excluding a poly (A) tract. Following a 709-nt 5′ UTR, a long open reading frame encoded a putative polyprotein precursor of 2,182 aa, which was followed by an 89-nt 3′ UTR. To verify the nucleotide sequence of the determined NII561-2000 genome, RT-PCR with a set of primers and RNA sample extracted from the virus-infected Vero cells was carried out. The nucleotide sequences of the NII561-2000 genome obtained by using this RT-PCR method were perfectly matched with those of the originally determined sequences (data not shown).To determine the nucleotide sequence of the NII561-2000 virus, the viral RNA was extracted from the purified virus. The partial nucleotide sequences of cDNA clones derived from this viral RNA were determined. BLAST  amino acid sequences showed that the NII561-2000 virus was most similar to HPeV-1/HPeV-2 , panel AHPeVs have 9 polyprotein cleavage sites: VP0/VP3, VP3/VP1, VP1/2A, 2A/2B, 2B/2C, 2C/3A, 3A/3B, 3B/3C, and 3C/3D . CompariThe NII561-2000 virus, HPeV-1, HPeV-2, HPeV-4, and HPeV-5, but not HPeV-3, had an RGD (arginine-glycine-aspartic acid) motif at the C terminus of VP1 , we have isolated 8,195 CPE-inducing agents from 13,656 clinical samples  collected between 1991 and 2005, at Niigata, Japan , Table 5Phylogenetic tree analysis showed 14 HPeV-1, 16 HPeV-3, and 1 HPeV-4, but no HPeV-2 and HPeV-5 among the examined 41 HPeVs. In addition, 10 viruses, including NII561-2000, formed a distinctive tree from those of the other HPeVs , panel BIn this study, we isolated a novel HPeV (NII561-2000) from a 1-year-old girl with Reye syndrome and determined the nucleotide sequence. Nucleotide sequence analysis and mutual neutralization test indicated that the NII561-2000 virus was distinct from 5 known HPeVs , panel AThe NII561-2000 virus was originally isolated from a patient with Reye syndrome, an acute noninflammatory encephalopathy characterized by an antecedent viral infection, such as influenza or varicella , HPeV-1 were isolated in 5 years, and HPeV-3 were isolated in 6 years. The genetic variations of these NII561-2000–related viruses were small , panel BThirteen of 16 HPeV-3 isolated in Niigata were from the patients <3 years of age, consistent with the previous report (HPeV-4 was isolated from a 5-year-old patient with lymphadenitis in Niigata . Thus, tThe degenerate primer set we used here was originally developed by Ito et al. (


Françoise Barré-Sinoussi and Luc Montagnier, codiscoverers of HIV, the causative agent of AIDS, have been awarded the 2008 Nobel Prize in Physiology or Medicine. They share this prize with Harald zur Hausen who was responsible for establishing the link between human papilloma virus infection and cervical carcinoma. Nobel prizes have often attracted controversy and controversial figures. William Ramsay was awarded the Nobel prize in Chemistry for the discovery of inert gases and subsequently went on to claim chemical transmutation of elements. Individual, institutional and national pride are all at stake when they are announced. One small Cambridge college boasts unashamedly of having been the workplace of no less than four Nobel Laureates including Perutz, Kendrew and Klug. Their value is more than financial and the method of their choosing somewhat mysterious and, to some peoples' minds flawed. Arrhenius having won in 1903, reputedly then manipulated future choices in favour of his friends and attempted (unsuccessfully) to exclude some very significant scientific figures such as Paul Ehrlich.This year virology has been honoured and three scientists chosen who worked with two very different viruses. Harald zur Hausen, professor emeritus of the German Cancer Research Center in Heidelberg (Germany) identified the link between human papilloma virus (HPV) infection and cervical cancer. It is timely that with the recent development of a safe and effective vaccine he should be recognised for first identification of this human pathogenic virus. He made several particularly noteworthy findings in papilloma virus research. The earliest was the recognition that there are multiple HPV genotypes, particularly that HPVs that cause non-genital warts are distinct from those that cause genital warts. His most important finding was the identification and molecular cloning of the HPV types 16 and 18 genomes, and that a majority of cervical cancers contained DNA from these two HPV types. A third critical finding was that the HPV DNA becomes integrated into the host genome in cervical cancer cell lines and that the viral E6 and E7 (onco)genes are preferentially retained and expressed in the tumors. His work is even more noteworthy because it was carried out in the 1980's when there was considerable scepticism whether viruses in general, and HPV in particular, caused any common cancers. Professor zur Hausen has continued to run a highly productive laboratory since these seminal early findings. No controversy thus far.Retroviruses have generated more than their share of Nobel prizes. Peyton Rous for retroviral oncogenesis (1966), Temin and Baltimore for reverse transcriptase (1975) and Varmus and Bishop again for oncogenes that were originally described for retroviruses (1989). The human immunodeficiency virus (HIV) as the largest public health problem in the world was destined to follow sooner or later. The difference is that, unlike in papilloma viruses, and whilst antiviral treatment has made astonishing progress in 20 years, we are no nearer a cure and certainly years from a vaccine (should one ever be possible) against HIV. The Nobel committee have chosen to honour the scientists who most people agree were the first to actually isolate the virus at the Pasteur Institute in Paris (France) in 1983: Françoise Barré-Sinoussi and Luc Montagnier. Following medical reports of a novel immunodeficiency syndrome in 1981, the search for a causative agent was on. The French researchers isolated and cultured cells from lymph nodes of patients with the lymphadenopathy characteristic of the early stage of acquired immune deficiency. They detected activity of the retroviral enzyme reverse transcriptase, a direct sign of retrovirus replication. They also observed retroviral particles budding from the infected cells. Isolated virus infected and killed lymphocytes from healthy donors and reacted with antibodies from infected patients. By 1984, Barré-Sinoussi and Montagnier had obtained several isolates of the novel human retrovirus from sexually infected individuals, haemophiliacs, mother to infant transmissions and transfused patients.Nobel prizes can be split between a maximum of three individuals and sometimes a deserving fourth person has been omitted on what appear to be relatively tough pragmatic grounds. Had the Nobel committee decided this year that the prize was for the discovery of HIV alone there would have been only one possible third candidate. That they didn't may be perceived in some quarters as a more pointed statement. However the subjects into which the prize is divided may be very diverse. Peyton Rous shared his with Charles Huggins, honoured for his contribution to hormonal treatment of cancer. Luc Montagnier (perhaps with a winner's magnanimity) has said that the obvious third recipient should have been Robert Gallo. Others will quote Gallo's identification of HTLV-1 with Bernie Poiesz and the fact that many of the techniques used to grow viruses like HIV were dependent on discoveries from the Gallo lab as were some of the earliest blood tests for the virus. Had the Nobel committee honoured only those who contributed to the discovery of HIV rather than HPV as well the picture may have been different. Instead they chose to reward the two scientists most responsible for the first isolation of the virus. These two indeed deserve our congratulations. It would be churlish however not to acknowledge that the work of others, notably the Gallo lab – but also other scientists working at the time, and since – made very significant contributions without which it is likely that the unprecedented advances we have seen in our understanding and successful therapy of HIV might have been much slower in coming.The authors declare that they have no competing interests.Both authors contributed equally to the work.


Background and purpose Late infections after total hip arthroplasty are still a problem. Treatment procedures include resection arthroplasty with implantation of antibiotic-loaded beads or implantation of an antibiotic-impreganted spacer. However, little is known about antibiotic elution from bone cement beyond the first 2–3 postoperative days in humans.Methods 17 hip spacers  and 11 chains  in 28 patients were studied. The release of both agents was measured in the drainage fluid on a daily basis. The drains were left in situ until less than 50 mL was produced per day. The elution of both antibiotics was determined by fluorescence polarization immunoassay. Systemic antibiotics were given postoperatively according to antibiogram. If possible, no gentamicin or vancomycin was given.Results Peak mean concentrations from beads and spacers were reached for gentamicin  µg/mL and 21 (0.7–39) µg/mL, respectively) and for vancomycin (80 (21–198) µg/mL and 37 (3.3–72) µg/mL) on day 1. The last concentrations to be determined were 3.7 µg/mL gentamicin and 23 µg/mL vancomycin in the beads group after 13 days, and 1.9 µg/mL gentamicin and 6.6 µg/mL vancomycin in the spacer group after 7 days. Between the fifth and seventh day, an intermittent increase in elution of vancomycin from both beads and spacers and of gentamicin from spacers was noticed. No renal or hepatic dysfunction was observed.Interpretation Beads showed higher elution characteristics in vivo than the spacers due to their larger surface area; however, a great amount of inter-subject variability was seen for both beads and spacers. The inferior elution properties of spacers emphasize the importance of additional systemic antibiotics for this treatment procedure during the postoperative period. Future studies should clarify whether the dose of antibiotics or length of antibiotic therapy may be reduced in the case of bead implantation, without jeopardizing the control of infection. Late infections after total hip arthroplasty (THA) still remain a problem. Usually, a two-stage procedure is chosen for treatment of infection, whereby either a resection arthroplasty (Girdlestone procedure) is performed or an antibiotic-loaded spacer is inserted .Recent reports have emphasized the importance of studying and understanding the elution of antibiotics from beads and spacers in such treatment procedures, in order to define the ideal moment for explantation of beads or spacer and to plan for reimplantation of the prosthesis . The maiWe conducted a prospective study to evaluate the local antibiotic concentrations of gentamicin and vancomycin in patients undergoing two-stage exchange for infected THA: either by inserting antibiotic-loaded beads in the case of a Girdlestone procedure, or by implantation of an antibiotic-loaded spacer.S. epidermidis was isolated 6 times, S. aureus 5 times, a methicillin-resistant S. aureus (MRSA) 3 times, and there were single isolates of P. mirabilis, E. faecalis, and E. faecium. In the beads group, S. epidermidis, S. aureus and MRSA were isolated 3 times each and there were single isolates of S. haemolyticus and E. faecalis. All organisms were sensitive to both gentamicin and vancomycin, except for the MRSA strains, which were gentamicin-resistant. Both spacers  and chains  were produced intraoperatively using molds, as reported previously   and 11 chains   in 28 patients  years; beads group: 6 males and 5 females, mean age 67 (41–76) years). In all cases, a causative pathogenic organism could be identified in the wound. In the spacer group, eviously . In shorIn all cases, the primary indication for implantation of spacers and beads was late infection after total hip arthroplasty. The choice of bead or spacer implantation was made according to the general condition of each patient. If a patient was compromised by his general medical condition and thus could not afford a later prosthesis reimplantation, or was not willing to undergo a two-stage procedure, a Girdlestone arthroplasty with insertion of beads was performed. In each of these cases, half of the chain produced was inserted into the femur and the other half was inserted into the acetabular cavity. In the other cases, an articulated antibiotic-loaded spacer was implanted. All spacers were fixed to the femur by cementation to its proximal part. In all cases, the bone cement was loaded with gentamicin and vancomycin due to the broad antimicrobial spectrum and the known synergistic effect of these 2 agents regarding elution .Over the first postoperative days, a wound secretion specimen was taken from the redon drainage bottle every 24 h via the drainages placed directly at the head of the spacers or near the beads . The redA fluorescence polarization immunoassay (FPIA)  was used to determine the concentrations of gentamicin and vancomycin released. The lowest measurable concentration of drug was 0.27 µg/mL for gentamicin and 2.0 µg/mL for vancomycin.Both agents showed similar release characteristics over the first postoperative days  and 3. IAfter reaching their highest concentrations on day 1, the elution of both antibiotics decayed constantly at a similar rate over the following days. . The last concentrations to be determined were 3.7 µg/mL gentamicin and 23 µg/mL vancomycin in the beads group after 13 days, and 1.9 µg/mL gentamicin and 6.6 µg/mL vancomycin in the spacer group after 7 days. Between the fifth and the seventh day, we found an intermittent increase in the elution of vancomycin from both beads and spacers and of gentamicin from spacers. There was a substantial degree of inter-subject variability with regard to the peak amounts released  and 5.No renal or hepatic dysfunction was observed in any of the cases.The total amount of gentamicin and vancomycin released from beads and spacers was not determined.Since the introduction of antibiotic-loaded bone cement by Buchholz and Engelbrecht in the early 1970s , antibioDue to the increasing emergence of multiresistant pathogenic organisms over the past few years, cement media loaded with two different antibiotics are being used more frequently in the treatment of orthopedic infections . It is kWe have not found any previous studies describing measurement of antibiotic elution from PMMA on a daily basis beyond the first 2–3 postoperative days. The greater amount of vancomycin incorporated into PMMA should explain our findings of higher concentrations released over the early postoperative period, except for the first day. Despite the fact that the beads were impregnated with half of the amount of antibiotic that the spacers were, in the first days the beads released greater amounts of both agents due to their larger surface area . AlthougOur results also show high inter-subject variability with regard to the peak concentrations of antibiotic measured. For example, in the beads group the mean gentamicin concentration was 116 µg/mL on day 1, while the maximum concentration measured was at 370 µg/mL. Moreover, peaks were measured between the fifth and seventh day. This discrepancy may be attributable to several factors such as local blood perfusion or pH value of the tissues; however, we believe that the manual incorporation of the vancomycin into the cement powder is the main parameter responsible for the variability. This inhomogenity of antibiotic elution has been seen by the authors in in vitro studies  and can The fact that a sufficient amount of antibiotic release from beads lasts longer than from spacers emphasizes the importance of systemic antibiotics, given as a complement to the spacer implantation, over the first 4–6 postoperative weeks in order to avoid not only spread of the infection, but also persistence of the infection locally. Perhaps in the case of a bead implantation, the dose of the intravenous antibiotic could be reduced over the first 2 postoperative weeks due to sufficiency of local antibiotic therapy, but after this time period a re-adjustment of the dose might be necessary in order to prevent the emergence of resistant bacterial strains—since the local antibiotic concentrations decrease over time. Future clinical studies should clarify this issue. Some antibiotics require measurement of peak and trough serum levels during therapy, and the dose is usually adjusted based on those levels. Generally, in the case of late infections where the source of the infection is blood-based, the use of systemic antibiotics is important—not only for treatment of the joint infection, but also for treatment of the primary source of the infection and to avoid any other manifestations of infection. However, in the presence of antibiotic-loaded cement devices, complications might occur despite the fact that such media are considered to be safe . Side efIn summary, gentamicin- and vancomycin-loaded beads and hip spacers appear to be capable of releasing sufficient concentrations over the first 7–13 postoperative days in vivo. Future studies will be required to determine whether this also applies to other antibiotics and other amounts of antibiotic incorporated into PMMA. We recommend the use of systemic antibiotics for at least the first 4 postoperative weeks, to ensure eradication of the infection and to avoid re-infections or persistent infections, especially since both beads and spacers have shown an appreciable amount of inter-individual variability regarding peak concentrations of antibiotic release.


In late summer and fall 1997, Aedes albopictus mosquitoes were found in Peoria, Illinois, a long recognized focus of La Crosse virus transmission. Larvae were found in tires and other artificial containers, biting adults were recovered, and eggs were collected in oviposition traps within a 25-ha area. One chipmunk trapped < 0.25 km from the infested area tested positive for neutralizing antibodies against La Crosse virus.


Millions of children die every year in developing countries, from preventable diseases such as pneumonia and diarrhoea, owing to low levels of investment in child health. Investment efforts are hampered by a general lack of adequate information that is necessary for priority setting in this sector. This paper measures the health system costs of providing inpatient and outpatient services, and also the costs associated with treating pneumonia and diarrhoea in under-five children at a health centre in Zambia.Annual economic and financial cost data were collected in 2005-2006. Data were summarized in a Microsoft excel spreadsheet to obtain total department costs and average disease treatment costs.The total annual cost of operating the health centre was US$1,731,661 of which US$1 284 306 and US$447,355 were patient care and overhead departments costs, respectively. The average cost of providing out-patient services was US$3 per visit, while the cost of in-patient treatment was US$18 per bed day. The cost of providing dental services was highest at US$20 per visit, and the cost of VCT services was lowest, with US$1 per visit. The cost per out-patient visit for under-five pneumonia was US$48, while the cost per bed day was US$215. The cost per outpatient visit attributed to under-five diarrhoea was US$26, and the cost per bed day was US$78.In the face of insufficient data, a cost analysis exercise is a difficult but feasible undertaking. The study findings are useful and applicable in similar settings, and can be used in cost effectiveness analyses of health interventions. The challenge to meet the millennium development goal of reducing under-five mortality with two thirds by 2015 has prompted the need for increased investment in child health in low income countries, where about 4.8 million children die every year from preventable diseases .Morbidity and mortality due to these and other diseases impose a huge economic burden on individuals, families and on society at large. This economic burden is more acute in developing countries, where the opportunity costs of resources are very high. This necessitates the need for priority setting in the allocation of resources.However, priority setting in most developing countries is hampered by lack of sufficient information . Little A literature search was conducted between June 2006 and February 2007, for English language articles on healthcare costs and the costs of pneumonia and diarrhoea in the following databases: MEDLINE (January 1966 to date), Cochrane Controlled Trials Register (CENTRAL), the Cochrane economic evaluation database (NHS EED), EMBASE-Medicine (January 1985 to date) and the ISI Web of Science. The search did not yield any studies undertaken in Zambia to measure health care costs or treatment costs of pneumonia and diarrhoea in children. Limited data are also available on the costs of general healthcare services .In an effort to document information that is potentially beneficial to priority setting in the health sector, we set out to measure the health system costs of providing inpatient and outpatient healthcare services to an urban population in Zambia. We demonstrated how our findings can be used to measure disease specific costs by calculating average treatment costs of pneumonia and diarrhoea in under-five children. This was also done in order to justify the adoption of cost-effective disease prevention measures. Research has shown the efficacy of rotavirus and pneumococcal vaccines in preventing diarrhoea and pneumonia in children ,13, and Lusaka district, in which the capital of Zambia is situated, has a population of about 2 million people. Healthcare for the district is provided by the central Government and private for profit institutions, which are mainly situated in urban areas. The public health institutions are classified into health posts, health centres, district referral centres, general hospitals and central hospitals. Health posts are intended to cater for a population of about 500 to 100 households, while health centres serve a population of about 50,000 people in urban areas and 10,000 people in rural areas. District referral centres, general and central hospitals are meant to cater for populations of 200,000; 800,000 and above 800,000 respectively. Lusaka district has 8 health posts, 22 health centres and 2 central hospitals.We undertook the costing study at Kanyama health centre, in Kanyama Township in the capital, Lusaka. The health centre is a public institution funded by the Ministry of Health (MOH), under the Lusaka District Health Management Team (DHMT). Its catchment area includes all the households in Kanyama Township and some other households in surrounding areas. The centre provides both outpatient and inpatient services, and has about 700 visits daily, and a total of 41 beds.Kanyama Township had an estimated population of over 130,000 in 2006 . ApproxiThe study was undertaken from the health provider perspective, and as such, only costs borne by the health centre were taken into consideration. We did not include the costs to households and social costs such as quality of life reductions caused by disease.Cost Analysis in Primary Health Care: A Manual for Programme Managers [Data collection was guided by the Managers . Cost anManagers . The HMIData on the prices and quantities of drugs and other recurrent medical supplies provided to the health centre were obtained from the DHMT pharmacy, which supplies all such items to government health centres in Lusaka District. It has a well developed system of delivery with updated documentation of all transactions to health centres. We were able to access these records to obtain updated information on supplies to Kanyama health centre, and accurate prices of most of the items provided. The prevailing market price was used as proxy for items whose prices were unavailable in the records of both the health centre and the DHMT. All costs were adjusted to 2006 prices using the 2006 Gross Domestic Products (GDP) deflator, valued at K1,175 . Items wCosts were classified as either economic or financial. Economic costs reflected the opportunity cost of resource use, while financial costs considered only expenditures incurred in the purchase of items. Costs were further classified as capital or recurrent. Capital costs included items such as buildings, equipment and vehicles, who's useful life was more than one year. Recurrent costs included items such as personnel and supplies, which could be replaced within a year. Items such as cutlery, buckets and garden implements, which could last for more than a year but cost less than US$100 were regarded as recurrent costs.A physical count of all capital items such as furniture and equipment was undertaken to ascertain the exact number and condition. Only items that were functional were included for costing. The floor areas and costs of buildings were obtained from the Buildings Department at the Ministry of Works and Supply, which is the custodian of all information on government buildings. Where such costs were not available, the floor space of the buildings was measured physically and an estimated value was attached by a Quantity Surveyor at the Department.Capital costs were annuitized to take into account the fact that such resources are bought in one year, but their useful life spans over several years . The annData were, however, sometimes not readily available and had to be estimated using proxies. For example, we did not have access to the original records of some capital and recurrent outlays, which made it difficult to obtain their original purchase prices. We therefore, made estimates which could have been over or under the true value. This was the case also for utilities, for which we did not find any records on bills paid towards water and electricity. We decided to use fixed monthly charges of US$2.78 for water  and US$6In some cases, the records were available but not in order, and the researcher could not sort them due to inadequate time and resources. It was not possible, for example, to allocate some medical supplies received by the health centre's pharmacy to the various wards or departments, as usage of these items was not specified by ward. It was thus decided to treat all such items as overhead costs, to be allocated to each of the wards according to an estimated allocation factor. The supplies that were 'ward or department specific', e.g. family planning drugs and some antibiotics were allocated directly to the necessary wards or departments.Further, it was not immediately possible to allocate certain items, such as staff time to certain departments, as their usage between departments was not clearly defined. The same personnel who tended to the paediatric ward also serviced the male and female wards. As a result, it was not easy to distinguish to which ward/department some of the staff time belonged. It was thus decided to allocate time equally to those departments that were serviced by the same personnel, as there was no clearly defined pattern of work. The same was done for equipment, where percent usage was divided equally between wards.Figure The health centre's departments were classified into 9 patient care and 6 overhead departments. The patient care departments included all units or wards where patients directly received treatment, such as the inpatient and outpatient wards and the laboratory. The overhead departments were those that serviced the patient care departments and whose activities were not immediately identified with disease treatment ,20. ThesIn each department, costs were categorised as follows: Personnel cost, including salaries, benefits and allowances. Capital costs, including buildings, vehicles, furniture and equipment such as x-ray machines. Supplies, including cleaning supplies, medical and surgical supplies. Maintenance, for buildings, vehicles and general repairs. For inpatient costs, we included beds, inpatient maintenance and supplies and length of stay for each disease.The costs were further divided into patient care costs, which were costs incurred in the patient care departments, and overhead costs, incurred in the overhead departments. Overhead departments' costs were shared by all the patient care units. These costs were allocated to each patient care department according to the estimated proportion of number of visits, bed days, personnel or floor space.The intermediate outputs were the services provided by each patient care department. The units of analysis used to measure the output were bed days for the inpatient department and visits for the outpatient department. In the laboratory, the units of measurement were the total number of lab tests conducted in the study period. In the labour/maternity ward, the units used were visits instead of bed days. This is because the majority of the women spent a day or less in this ward before being discharged. The total number of visits and admissions included both persons attending the health centre for the first time (new attendees) and re-attendees.Total costs were divided by total outputs to obtain average costs. The average cost of inpatient care, for example, was obtained by dividing the total cost in the paediatric ward by the total number of bed days in the reference period. Average costs of service provision are presented for all the patient care departments except Anti Retroviral Treatment (ART), for which information on usage was not available.To decide on the introduction of pneumococcal and rotavirus vaccines, the economic burden of pneumonia and diarrhoea is needed for inclusion in cost-effectiveness studies. Our result can help estimate these costs. To do this, we measured the average treatment costs of pneumonia and diarrhoea, using total under-five pneumonia and diarrhoea visits and bed days recorded in the health centre's records, and the total outpatient and in-patient costs for treating these diseases. All resources related to the treatment of childhood pneumonia, including personnel, buildings, medical supplies, laboratory equipment and utilities were costed. Unit costs for each disease were calculated by dividing the total outpatient and inpatient costs by the corresponding visits or bed days for the condition. The average healthcare costs provided for both pneumonia and diarrhoea thus include the outpatient (cost per visit) and inpatient (cost per bed day) costs.The annual costs incurred in the patient care departments are presented in table The outpatient ward had the highest total cost at US$426,847 (33%), while the Voluntary Counselling and Testing Ward (VCT) ward had the lowest at US$9,659 (1%). The outpatient ward also had the highest recurrent cost (35%), while the inpatient ward had the highest capital cost (28%).Table There were an estimated 14 outpatient visits per day per medical personnel for the entire study period. The health centre's bed occupancy rate, defined as the bed days per number of beds was 82%; while the bed turnover, defined as the number of admissions per number of beds was 71%. The average length of stay was 4 days.Table The average costs per unit of service in all the departments are given in table Table This paper provides information on the average outpatient and inpatient treatment costs in an urban health centre in Zambia, and also measures the average cost of pneumonia and diarrhoea treatment in children under the age of 5 years. The average costs incurred in the patient care departments ranged from US$1 to US$20, with the highest cost observed in the dental department. Except for dental services, all out-patient services costs were relatively much lower than in-patient costs. This is reflective of the severity of inpatient cases, which require relatively more care and consequently more resources to manage. Additional costs such as lodging facilities and meals tend to increase admission costs [We estimated the total annual cost of operating the health centre at US$1,284,305. It was however not possible to isolate the government budget for Kanyama health centre and compare it with our result. Most of the budget components were financed through various departments, and aggregated together with information from other clinics and health centres. The salaries, for example were paid through MOH and the health centre's supplies and equipment were provided by DHMT.The government per capita expenditure on health was US$17 in 2005 . Taking The annual per capita cost of providing healthcare calculated in this paper is lower than the cost estimated by the World Bank , but is In comparison to a study undertaken in Kenya, which recently reported costs of in-patient pneumonia treatment in children, ranging from about US$46 to US$172 , our estA limitation of this study is that it did not measure the supervisory costs incurred by the Ministry of Health, and the costs of support incurred by non-governmental organisations and the donor community, thereby underestimating the true health systems cost of service provision to Kanyama Township. Furthermore, our exclusion of family costs incurred in accessing healthcare does not give a true reflection of the burden of disease and healthcare. It is necessary for social planning to also have an insight into the cost structures of individuals, households and society at large. Future research should consider both the direct and indirect costs of treatment, and the long term effects of quality of life reductions due to illness. This perspective is more informative to policy makers as it helps them plan for a wider section of the community.The paper, however, fills a large gap in the literature on the cost of healthcare provision in Zambia, particularly on the cost of childhood pneumonia and diarrhoea to the health system. It underscores the challenge of undertaking a cost analysis in the face of unavailability of reliable, consistent and accurate data, which is the case in many developing countries ,20. In ZThe study provides information that is potentially useful for planning in the area of child health. It documents the procedures for undertaking cost analyses of healthcare services which can be used in similar settings. Our experiences in this study could be a guide for others as far as highlighting what is expected in the course of undertaking such work.This study was undertaken in a particular institutional and epidemiological setting. Although generalization must be made with great caution, the study can still be useful in similar Zambian settings since the costs of large items such as staff, equipment and drugs are fairly standard. The costs of medication, treatment and other facility costs may also be generally comparable.The authors declare that they have no competing interests.Both authors participated in the design and planning of the study. Field work was conducted by LC; the analysis and write-up was carried out mainly by LC. Both authors read and approved the final manuscript.


To the Editor:Cantagalo virus (CTGV) is a strain of vaccinia virus  that was isolated from pustular lesions on dairy cattle and dairy workers in Rio de Janeiro State, Brazil, in 1999  cattle on a dairy farm in the municipality of Muricilândia. Small papules progressed to vesicles and pustules , panel BFour scab samples were sent for virus identification by PCR. Parts of the samples were used to infect BSC-40 cells and for DNA isolation by phenol-chloroform extraction, as described (The full-length hemagglutinin gene (≈900 bp) was detected in all clinical isolates and in the control DNA samples from CTGV, VACV strain WR, and cowpox virus strain Brighton-red , panel CFor phylogenetic inference, we used DNA from the isolate MU-07 to sequence the genes A56R (927 bp), C7L (453 bp) that encodes a host-range virulence factor, and K2L (1095 bp); primers aligned externally to the open reading frames. PCR and sequencing were performed as described elsewhere , panel DWe consider the etiologic agent of the infection in Tocantins State to be a CTGV isolate, indicating spread of CTGV infection to northern Brazil. This spread could reflect interstate propagation of the virus due to movement of animals or people, which is particularly intense at the southern border with Goiás State (location of the nearest CTGV outbreak) (www.ibge.gov.br/english). Infected buffalo have not yet been reported in Brazil, but the establishment of VACV strains in buffaloes has long been reported in India; economic losses have been substantial (9). Therefore, a careful survey should be conducted to evaluate dissemination of the virus to other states and species in the Amazon region.Another concern is spread of the virus to water buffalo, which account for a growing farming industry in Brazil, specifically in the northern states (Spread of Cantagalo Virus to Northern Brazil