猴痘、天花病毒感染快速分子诊断荧光定量实时PCR方法的建立

目的:天花、猴痘可感染人并引起严重皮疹、发热等临床症状,均为烈性传染病,是潜在的生物恐怖因子,因此需要建立针对其感染的快速特异的诊断方法。方法:分别设计正痘病毒属通用型、天花病毒特异、猴痘病毒特异的引物与荧光标记探针,建立荧光定量实时PCR方法,对人工合成或模拟样本进行检测。结果:可在4h内对天花或猴痘病毒感染进行特异性鉴别诊断,检测灵敏度可达100拷贝/25μL反应体积。结论:本方法可作为一种检疫与反恐应急储备技术。     

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.     

Book Reviews

M icrobial I nteractions and C ommunities Vol. 1. (1982). Edited by A.T. Bull & J.H. Slater. 567 pp. London, New York: Academic Press. £33.80, US $69.50.
B ioactive M icrobial P toducts : S earch and D iscovery (Special Publications of the Society for General Microbiology No. 6) (1982). Edited by J.D. Bu'Lock, L.J. Nisbet & O.J. Winstanley. 148 pp. London and New York: Academic Press. £10.00, US $20.50.
I solation and I dentification M ethods for F ood P oisoning O rganisms (Society for Applied Bacteriology Technical Series No. 17). (1982). Edited by Janet E.L. Corry, Diane Roberts & F.A. Skinner. 406 pp. London and New York: Acadmic Press. £30.00, US $55.50.
P lasmids (Study in Biology No. 142) (1982). M.J. Day. 52 pp. London: Edward Arnold. £2.50.
S mith's I ntroduction T o I ndustrial M ycology 7th Edition (1981). A.H.S. Onions, D. Allsopp & H.O.W. Eggins. 398 pp. London: Edward Arnold. £37.50.     

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.     

Human monkey pox: its clinico-epidemiological characteristics

During the course of the smallpox eradication programme, a new eruptive disease clinically resembling smallpox was discovered in Zaire. The disease, which was named monkeypox after the virus, is a zoonosis occurring sporadically in countries of western and central Africa with tropical rain forest. The studies carried out in Zaire from 1980 through 1985 showed that monkeypox affects mainly children in relatively small remote villages whose population has traditionally frequent contacts with wild animals. Apart from the wildlife, the virus can be transmitted from man to man, but among other sources of infection sick persons did not exceed 20%. Presumed human transmission has occurred in 38 out of 61 outbreaks of human monkeypox and only once reached the third and once the fourth generation; the transmission in all affected villages under observation has extinguished itself. Considering the sporadic and relatively rare occurrence of the disease and expected complications following the immunization with vaccinia which protects from monkeypox, introduction of mass vaccination in the areas at risk is hardly justified at present.     

Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.

When influenza (H3N2) viruses from infected individuals are grown in embryonated chicken eggs, viruses are isolated which differ antigenically and structurally from viruses grown in mammalian Madin-Darby canine kidney (MDCK) cell culture [G.C. Schild, J.S. Oxford, J.C. de Jong, and R.G. Webster, Nature (London) 303:706-709, 1983]. To determine which of these viruses is most representative of virus replicating in the infected individual, a region of the HA gene of virus present in original clinical samples was amplified by using the polymerase chain reaction and sequenced directly. Comparison of 170 amino acid residues of HA1 flanking and containing the receptor-binding site and antigenic sites indicated that over this region, the HA of virus replicating in the infected individual was identical to that of virus after growth in MDCK cells and was distinct from the HA of viruses grown in eggs. Therefore, cultivation of human influenza H3N2 virus in mammalian MDCK cells results in a virus similar to the predominant population of virus found in the infected individual.     

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.     

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.     

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.     

Some Aspects of the Ecology and Biology of Some Small Mammal Fleas from Yorkshire

The Invertebrates R. MCNEILL ALEXANDER 561 pp. London: Cambridge University Press, 1979. £28.00/£7.95. ISBNs 0 521 22120 X/29361 8 Reviewed by Andrew C. Campbell

Lecture Notes on Invertebrate Zoology Second edition M. S. LAVERACK and J. DANDO 194 pp. Oxford: Blackwell Scientific, 1979. £5.50. ISBN 0 632 00325 1 Reviewed by Andrew C. Campbell

An Illustrated Guide to River Phytoplankton H. BELCHER and E. SWALE 64 pp. London: Institute of Terrestrial Ecology/HMSO, 1979. £1.50. ISBN 0 11 886602 8 Reviewed by J. W. G. Lund

The Cell as a Habitat 286 pp. London: The Royal Society, 1979. £8.50. ISBN 0 85403 113 8 H. V. Wyatt

Cell Motility Integrated Themes in Biology H. STEBBINGS and J. S. HYAMS 192 pp. Harlow, Essex: Longman, 1979. £4.95. ISBN 0 582 44380 6 Reviewed by K. R. Tyler

Concepts in Cell Biochemistry W. K. STEPHENSON 221 pp. Chichester, Sussex: John Wiley, 1978. £5.00. ISBN 0 471 03390 1 Reviewed by D. A. Kennedy

The Kindly Fruits of the Earth G. E. HUTCHINSON 264 pp. London: Yale University Press, 1979. £13.35. ISBN 0 300 02272 7 Reviewed by W. H. Dowdeswell

Brain, Behaviour and Evolution D. A. OAKLEY and H. C. PLOTKIN (Ed.) 237 pp. Andover, Hants: Methuen, 1979. £9.00/£4.95. ISBNs 0 416 71260 6/71270 3 Reviewed by W. H. Dowdeswell

Introduction to Evolution F. A. RACLE 162 pp. Hemel Hempstead, Herts: Prentice/Hall, 1979. £5.80. ISBN 0 13 382869 0 Reviewed by J. A. Dawes

Tissues and Organs: a text-atlas of scanning electron microscopy R. G. KESSEL and R. H. KARDON 317 pp. San Francisco and Reading: W. H. Freeman, 1979. £18.90/£7.00. ISBNs 0 7167 0091 3/0090 5 Reviewed by A. W. Robards

The Cycling Female A. LEIN 135 pp. Reading: W. H. Freeman, 1979. £6.30/£2.90. ISBNs 0 7167 1039 0/1038 2 D. J. Reid

Ecology and Evolution of Animal Behaviour Second edition R. A. WALLACE 284 pp. Hemel Hempstead, Herts: Prentice/Hall, 1979. £7.10. ISBN 0 87620 272 5 Reviewed by Ursula Bowen

Man Against Disease MUIR GRAY 192 pp. Oxford: Oxford University Press, 1979. £2.50. ISBN 0 19 289127 8 Reviewed by H. V. Wyatt

Pollen and Allergy Studies in Biology, No. 107 R. BRUCE KNOX 60 pp. London: Edward Arnold, 1979. £1.75. ISBN 0 7131 2736 8 Reviewed by D. Brookes

Principles of Animal Physiology Second edition J. A. WILSON 890pp. London: Collier Macmillan, 1979. £16.45. ISBN 0 02 428360 6 Reviewed by K. R. Tyler

Morphogenesis of the Vertebrates Fourth edition T. W. TORREY and A. FEDUCCIA 570 pp. Chichester, Sussex: John Wiley, 1979. £11.60. ISBN 0 471 03232 8 Reviewed by F. E. G. Cox

Lizards—A Study in Thermoregulation Studies in Biology, No. 109 R. A. AVERY 56 pp. London: Edward Arnold, 1979. £1.80. ISBN 0 7131 2745 7 Reviewed by Gillian E. Standring

Life at High Altitude Studies in Biology, No. 112 D. HEATH and D. REID WILLIAMS 60 pp. London: Edward Arnold, 1979. £1.80. ISBN 0 7131 2754 6 Reviewed by C. G. Gayford

Seal Cull J. LISTER-KAYE 174 pp. Harmondsworth, Middx: Penguin Books, 1979. 95p. ISBN 0 14052 336 7 Seal Song B. DAVIES and E. PORTER 94 pp. Harmondsworth, Middx: Penguin Books, 1979. £2.50. ISBN 0 1400 4740 9 Reviewed by Ursula Bowen

Fieldwork Projects in Biology M. HINGLEY 170 pp. Poole, Dorset: Blandford Press, 1979. £4.95. ISBN 0 7137 0964 2 Reviewed by Malcolm Watson

Investigative Mycology R. F. SHARP 136 pp. London: Heinemann Educational, 1978. £5.00/£2.20. ISBNs 0 435 60750 2/60751 0 Reviewed by Avice M. Hall

Sexual Incompatibility in Plants Studies in Biology, No. 110 D. LEWIS 60 pp. London: Edward Arnold, 1979. £1.90. ISBN 0 7131 2747 3 Reviewed by David Skibinski

Introduction to the Principles and Practice of Soil Science R. E. WHITE 198 pp. Oxford: Blackwell Scientific, 1979. £8.50. ISBN 0 632 00052 X Reviewed by D. Payne

Bioscience Education in Developing Countries T. RAMASARMA et al. (Ed.) 230pp. Singapore: University of Singapore, 1979 $7.00 Reviewed by Colin Wood-Robinson

Ecology of African Mammals M. J. DELANY and D. C. D. HAPPOLD 434 pp. Harlow, Essex: Longman, 1979. £25.00. ISBN 0 582 44176 5 Reviewed by Colin Wood-Robinson

Modern Biology Made Simple R. BARRASS 304 pp. London: W. H. Allen, 1979. £3.50/£2.25. ISBNs 0491 02452 5/02462 2 Reviewed by John May

Life Science Physics J. W. KANE and M. M. STERNHELM 664 pp. Chichester, Sussex: John Wiley, 1978. £11.00. ISBN 0 471 03137 2 Reviewed by P. Burdett

Warwick Real Time Science Simulations Teacher's Handbook R. A. BEARE £3.00 Eleven biology booklets J. HEWITSON £1.00-1.75. Hatfield, Herts: Association for Science Education, 1978 Reviewed by M. E. Leveridge     

Functional interaction of nuclear transport-defective simian virus 40 large T antigen with chromatin and nuclear matrix.

We analyzed the subcellular distribution of nuclear transport-defective simian virus 40 Lys-128-mutant (cT-3 [R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984] and d10 [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith, Nature (London) 311:33-38, 1984]) large T antigens in various Lys-128-mutant-transformed rodent cells and in Lys-128-mutant d10-infected TC7 cells. Small but significant amounts of the mutant large T antigens were found in association with nuclear substructures, both in mutant-transformed and in mutant-infected cells. Experiments with TC7 cells made incompetent for cell division by 60Co irradiation supported the assumption that Lys-128-mutant large T antigen did not associate with nuclear components during mitosis but most likely was transported into the nucleus because the Lys-128 mutation was leaky for nuclear transport. Low-level simian virus 40 DNA replication and production of infectious mutant virus progeny in TC7 cells indicated that the association of Lys-128-mutant large T antigen with nuclear substructures is functional.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.