Current Protocols in Microbiology最新文献

Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development of anti-viral strategies. The poliovirus genome structure allows replacement of structural genes with a reporter protein, such as a luciferase or a fluorescent protein, whose signals can be detected and quantified in vivo, thus permitting observation of replication kinetics in live cells. This paper presents protocols for poliovirus replicon RNA production, purification, packaging and transfection, as well as techniques for monitoring Renilla luciferase replication signal in living cells. © 2018 by John Wiley & Sons, Inc.

Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of Paenibacillus larvae endospores into a honeybee colony. Progression to fulminant disease and eventual collapse of the colony requires multiple rounds of endospore germination, vegetative replication, endospore formation, and subsequent spread within the colony. This unit includes protocols for the in vitro sporulation and germination of P. larvae to assist investigators in the study of these processes. © 2018 by John Wiley & Sons, Inc.

Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC-145 cell line is the most often used in a laboratory setting. Infectious cDNA clones of many PRRSV strains have been established, from which these viruses can be recovered. PRRSV titration is generally done in MARC-145 cells. PRRSV RNA copy numbers can be assessed by reverse transcription and real-time PCR. Here, protocols for PRRSV propagation, virus recovery from infectious cDNA clones, and quantification are presented. © 2018 by John Wiley & Sons, Inc.

Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver diseases. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2018 by John Wiley & Sons, Inc.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most economically significant bacterial pathogen infecting honeybees. This unit includes protocols for the in vitro growth and laboratory maintenance of P. larvae. © 2018 by John Wiley & Sons, Inc.

Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the visualization of these responses are detailed in this unit. These assays are simple and can be readily implemented with minimum set ups. © 2018 by John Wiley & Sons, Inc.

Virus-like particles (VLPs) are large particles, the size of viruses, composed of repeating structures that mimic those of infectious virus. Since their structures are similar to that of viruses, they have been used to study the mechanisms of virus assembly. They are also in development for delivery of molecules to cells and in studies of the immunogenicity of particle-associated antigens. However, they have been most widely used for development of vaccines and vaccine candidates. VLPs can form upon the expression of the structural proteins of many different viruses. This chapter describes the generation and purification of VLPs formed with the structural proteins, M, NP, F, and HN proteins, of Newcastle disease virus (NDV). Newcastle disease virus-like particles (ND VLPs) have also been developed as a platform for assembly into VLPs of glycoproteins from other viruses. This chapter describes the methods for this use of ND VLPs. Curr. Protoc. Microbiol. 30:18.2.1-18.2.21. © 2013 by John Wiley & Sons, Inc.

Metronidazole and vancomycin remain the front-line therapies for most Clostridium difficile infections (CDI). However, recurrent CDI occurs in ∼25% of patients, causing significant morbidity and mortality and healthcare costs. For this population, traditional antibiotic therapies fail and new treatment options are greatly needed. The US Food and Drug Administration recently approved fidaxomicin for CDI treatment. This narrow-spectrum antibiotic preserves the normal gut microbiota and shows promise as a treatment for severe and recurrent CDI. Monoclonal antibodies and vaccines directed against toxin are currently in clinical trials and represent alternative, non-antibiotic therapies. Less traditional therapeutic interventions include bacteriotherapy with non-toxigenic C. difficile and fecal transplant. This commentary will provide an overview of current and forthcoming CDI therapies. Curr. Protoc. Microbiol. 30:9A.3.1-9A.3.9. © 2013 by John Wiley & Sons, Inc.

West Nile virus (WNV) is a member of the Flaviviridae family of enveloped, single-stranded, positive-sense RNA viruses. WNV, an emerging viral pathogen, is transmitted by mosquitoes to birds and mammals and is responsible for an increasing incidence of human disease in North America and Europe. Due to its ease of use in the laboratory and the availability of robust mouse models of disease, WNV provides an excellent experimental system for studying molecular virology and pathogenesis of infection by flaviviruses. Here, we describe common laboratory techniques used to propagate, quantify, detect, and store WNV. We also briefly describe appropriate safety precautions required for the laboratory use of WNV, which is classified as a Biosafety Level 3 pathogen by the United States Centers for Disease Control and Prevention. Curr. Protoc. Microbiol. 31:15D.3.1-15D.3.18. ©2013 by John Wiley & Sons, Inc.