Virion electron microscopy

Ear less is known about the process of flavivirus assembly. Electron microscopy has shown that immature virions can be found in the lumen of the endoplasmic reticulum (Murphy, 1980). The nucleocapsid core is not assembled free in the cytoplasm rather, its assembly appears to take place on the cytoplasmic face of membranes with which prM and E proteins are associated (Khromykh et al., 2001). The carboxy-terminal signal sequence of the precursor to the capsid protein is thought to anchor that protein to the membrane (Amberg et al., 1994). This should allow interactions to occur between the capsid protein and the envelope proteins, which are also anchored to the membrane but reside in the lumen of the endoplasmic reticulum or vesicles. The capsid protein also contains a conserved stretch of hydrophobic residues located roughly in the middle of the protein that has been suggested to serve as an additional or alternative membrane anchor (Markoff et al., 1997). 

A quantitative Amp-RT assay is a useful laboratory tool for several applications, including the determination of HIV-1 levels in culture supernatant or biological fluids (e.g., plasma and serum), and the study of HIV-1 RT susceptibility to RT inhibitors. The authors describe a quantitative Amp-RT assay for measuring RT activity of HIV-1. Quantitation is based on a calibration curve made by external standards. The standards used can be known numbers of HI V-1 particles (from 10,000 to 1) or known numbers of HIV-1 RT molecules. Virion numbers of an HIV-1 stock can be easily determined from the concentration of p24 antigen on the basis of 10 4 pg of p24 antigen per virion as previously estimated (5). Alternatively, the virion numbers can be determined by RT-PCR analysis or by electron microscopy. HIV-1 RT with a well-characterized specific activity and known p66/p51 protein content can also be used to generate standard curves with known numbers of RT molecules. Commercially... 

Nevertheless, the chances of successfully doing that seem to be slim, for now. Notably, Ramses V, who lived some 3000 years ago, is the earliest known victim of smallpox, based on an analysis of his mummy [261]. Many virus-like particles were revealed by electron microscopy and identified serologically as smallpox virus in a 400-year-old mummy from Italy. The antigenic structure of the particles was well preserved [262]. The virions in the mummy s skin had lost their viability. Viral antigen was not be detected by EIA or RPHA, and its DNA was not detected by molecular hybridization [263]. Attempts to recover the virus from the frozen bodies of persons who died of smallpox continue in Arctic sites in Siberia [264], and in Canada [265]. 

Two orthopoxviruses may, very occasionally, be involved as human offenders in occupational life the vaccinia virus and the cowpox virus. Orthopoxviruses are DNA viruses the virions have an ovoid structure, 300 X 250 nm, and are readily identified by negative electron microscopy. 

Viral particles of HCMV and Kaposi s sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) have recently been examined. During the her-pesviral replicative cycle, different viral particles are formed. For HCMV, this includes mature, infectious virions, noninfectious enveloped particles, and dense bodies [6]. Similarly for KSHV, only a portion of the produced virus particles is infectious [40]. Therefore, analysis of infectious virions requires their separation from the noninfectious and immature forms. Density ultacentrifugation gradients are typically used to separate the various forms. Each fraction can be analyzed by electron microscopy to determine the level of purity [6,41] or by assaying for viral DNA and an envelope glycoprotein [40]. 

Figure 1 Electron micrograph of a human adenoviral virion. A negative stained preparation of human adenovims was visualized under electron microscopy at 400,000xmagnification.

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