The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease

Since time immemorial animals have been a major source of human infectious disease. Certain infections like rabies are recognized as zoonoses caused in each case by direct animal-to-human transmission. Others like measles became independently sustained with the human population so that the causative virus has diverged from its animal progenitor. Recent examples of direct zoonoses are variant Creutzfeldt-Jakob disease arising from bovine spongiform encephalopathy, and the H5N1 avian influenza outbreak in Hong Kong. Epidemics of recent animal origin are the 1918-1919 influenza pandemic, and acquired immune deficiency syndrome caused by human immunodeficiency virus (HIV). Some retroviruses jump into and out of the chromosomal DNA of the host germline, so that they oscillate between being inherited Mendelian traits or infectious agents in different species. Will new procedures like animal-to-human transplants unleash further infections? Do microbes become more virulent upon cross-species transfer? Are animal microbes a threat as biological weapons? Will the vast reservoir of immunodeficient hosts due to the HIV pandemic provide conditions permissive for sporadic zoonoses to take off as human-to-human transmissible diseases? Do human infections now pose a threat to endangered primates? These questions are addressed in this lecture.     

The Leeuwenhoek Lecture, 1991. The influence of the host on microbes that cause disease.

Microbial pathogenicity or virulence, the capacity to cause disease, depends on microbial gene products that promote infection and penetration of mucous membranes, multiplication in the tissues, interference with host defence and sickness. Formation of these virulence determinants by microbes is influenced by the environment of the host, which differs from that in laboratory cultures. Studies of microorganisms grown in vivo, and of the host's influence on the production of virulence determinants, are increasing. In most studies, however, the complex conditions in vivo are not dissected to show the influence of particular factors. In future we should define specific host factors that are responsible for producing identified virulence determinants. There are three studies which point the way. Iron limitation in vivo causes production of bacterial siderophores, outer membrane receptors and some toxins. Erythritol, a growth stimulant for brucellae, causes intense placentitis and hence abortion in cattle, sheep and pigs. Cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) sialylates a conserved component of gonococcal lipopolysaccharide (LPS), thereby rendering gonococci in patients resistant to complement-mediated killing by serum. Although the lecture uses bacteria for examples, the principle applies equally to studies of viral and fungal pathogenicity.     

Molecular characterization of Marek's disease herpesvirus B antigen.

The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B-antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O-linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed R alpha PM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement and interference in chickens inoculated with Marek's disease herpesvirus and oncornaviruses.

Enhancement of mortality rates and symptomatology was observed in isolator-held LSI-SPF chickens concurrently inoculated with MDHV and avian oncornaviruses (RAV-1, RAV-2, RAV-7, RAV-50, or REV). Interference with MD antigen production also was demonstrated in extracts of the feather follicle epithelium from chickens inoculated with both MDHV and RAV-1.     

The Leeuwenhoek Lecture 2000 the natural and unnatural history of methane-oxidizing bacteria

Methane gas is produced from many natural and anthropogenic sources. As such, methane gas plays a significant role in the Earth's climate, being 25 times more effective as a greenhouse gas than carbon dioxide. As with nearly all other naturally produced organic molecules on Earth, there are also micro-organisms capable of using methane as their sole source of carbon and energy. The microbes responsible (methanotrophs) are ubiquitous and, for the most part, aerobic. Although anaerobic methanotrophs are believed to exist, so far, none have been isolated in pure culture. Methanotrophs have been known to exist for over 100 years; however, it is only in the last 30 years that we have begun to understand their physiology and biochemistry. Their unique ability to use methane for growth is attributed to the presence of a multicomponent enzyme system-methane monooxygenase (MMO)-which has two distinct forms: soluble (sMMO) and membrane-associated (pMMO); however, both convert methane into the readily assimilable product, methanol. Our understanding of how bacteria are capable of effecting one of the most difficult reactions in chemistry-namely, the controlled oxidation of methane to methanol-has been made possible by the isolation, in pure form, of the enzyme components.The mechanism by which methane is activated by sMMO involves abstraction of a hydrogen atom from methane by a high-valence iron species (FeIV or possibly FeV) in the hydroxylase component of the MMO complex to form a methyl radical. The radical combines with a captive oxygen atom from dioxygen to form the reaction product, methanol, which is further metabolized by the cell to produce multicarbon intermediates. Regulation of the sMMO system relies on the remarkable properties of an effector protein, protein B. This protein is capable of facilitating component interactions in the presence of substrate, modifying the redox potential of the diiron species at the active site. These interactions permit access of substrates to the hydroxylase, coupling electron transfer by the reductase with substrate oxidation and affecting the rate and regioselectivity of the overall reaction. The membrane-associated form is less well researched than the soluble enzyme, but is known to contain copper at the active site and probably iron.From an applied perspective, methanotrophs have enjoyed variable successes. Whole cells have been used as a source of single-cell protein (SCP) since the 1970s, and although most plants have been mothballed, there is still one currently in production. Our earlier observations that sMMO was capable of inserting an oxygen atom from dioxygen into a wide variety of hydrocarbon (and some non-hydrocarbon) substrates has been exploited to either produce value added products (e.g. epoxypropane from propene), or in the bioremediation of pollutants such as chlorinated hydrocarbons. Because we have shown that it is now possible to drive the reaction using electricity instead of expensive chemicals, there is promise that the system could be exploited as a sensor for any of the substrates of the enzyme.     

Status of Marek's disease virus in established lymphoma cell lines: herpesvirus integration is common.

Six cell lines derived from Marek's disease lymphomas of chickens and turkeys were investigated for the status of Marek's disease virus (MDV) DNA. In the transformed T- and B-cell lines, viral DNA could be detected by conventional Southern blot hybridization, by Gardella gel electrophoresis, and by in situ hybridization of metaphase and interphase chromosomes. Integration of viral DNA into the host cell chromosome was observed in all cell lines. Two to 12 integration sites of viral DNA could be detected in metaphase chromosome spreads. The integration sites were characteristic for the individual cell lines and were preferentially located at the telomers of large- and mid-sized chromosomes or on minichromosomes. In four of six cell lines, a minor population of latently infected cells supported the lytic cycle of MDV, giving rise to linear virion DNAs. In one of these cell lines, a third species of MDV DNA could be detected with properties reminiscent of covalently closed circular DNA. The finding that MDV integrates regularly into the genomes of latently infected cells is crucial to understanding the molecular biology of herpesvirus-induced tumors in the natural host.     

Marek's disease virus and herpesvirus of turkey noninfective to chickens, obtained by repeated in vitro passages.

The relation between the passage level of Marek's disease virus, C2 strain, and of herpesvirus of turkey (HVT), O1 strain, in cell culture and the level of the serological response of chickens to these viruses was examined. In both cases the immune response of chickens to these viruses decreased with increase in the number of in vitro passages of virus. Virus was not recovered from chickens inoculated with HVT highly passaged in vitro, which had become a high producer of cell-free virus in vitro, and grew equally well at 37 C and 41 C.     

Kaposi's sarcoma-associated herpesvirus and mechanisms of oncogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8), is well known to be responsible for Kaposi's sarcoma, the most common AIDS-related cancer. KSHV is also associated with the B cell malignancies primary effusion lymphoma and multicentric Castleman's disease. Cellular signaling pathways regulate the proliferation and differentiation during normal development and a small number of signaling pathways are involved in tumors. KSHV utilize those pathways, such as pRb-E2F, Wnt and Notch pathways, to promote driving of cell cycle and to regulate their own life-cycles (i.e., latency and lytic cycle). This review focuses on signaling pathways which KSHV gene products manipulate and discusses their contributions to tomorigenesis and regulation of viral life-cycles.     

Roles of avian herpesvirus microRNAs in infection, latency, and oncogenesis

MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation.     

The Leeuwenhoek Lecture, 1981. The biochemical and genetic approach to the study of bioenergetics with the use of Escherichia coli: progress and prospects

How the 'energy currency' of the cell, adenosine triphosphate (ATP), is produced consequent upon the oxidation of foodstuffs (oxidative phosphorylation) is, despite prolonged research, still a matter of debate and the molecular mechanism of the process is unknown. It appears that the problem of oxidative phosphorylation can be approached with the aid of the biochemical genetics of the bacterium Escherichia coli. The ease of manipulation of bacteria and definitive results obtained by this approach have been invaluable in solving other major biochemical problems. Mutants affected in oxidative phosphorylation have been isolated and characterized by genetic and biochemical techniques. These 'unc' mutants are affected in the adenosine triphosphatase (ATPase) multiprotein complex which is part of the cell membrane and responsible for the terminal stages of ATP synthesis. Seven distinct genes concerned with oxidative phosphorylation have been characterized in E. coli and shown to be part of an operon. The relationships between the different classes of unc genes and the various components of the ATPase have been established. Information about the assembly of the ATP synthesizing complex in the cell membrane has also been obtained and the stage set for further studies on the assembly, control and function of the ATP synthesizing system.     

Sequential autoprocessing of Marek's disease herpesvirus protease differs from that of other herpesviruses

Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)(P3)-X(P2)-A(P1)/S(P1') (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M(P3)-N(P2)-A(P1)/S(P1'). Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.