Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.
Understanding of the burden of Chlamydia trachomatis infection and its clinical sequelae is hampered by the absence of accurate, well-characterized tests using serological methods to determine past exposure to infection. An "in-house" immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) based on the C. trachomatis-specific antigen Pgp3 was produced and evaluated against three commercial ELISAs derived from the major outer membrane protein: the Medac pELISA plus, the Savyon SeroCT-IgG ELISA, and the Ani Labsystems IgG enzyme immunoassay. Sensitivities and specificities were determined using sera from both male and female patients (n ؍ 356) for whom C. trachomatis had been detected in the lower genital tract at least 1 month prior to the testing of the sample and from 722 Chlamydia-negative children aged 2 to 13 years. Chlamydia trachomatis is the commonest sexually transmitted bacterial infection in developed countries, with national surveillance programs consistently showing rising rates of diagnosed infections over the past decade. In the United Kingdom, figures based on cases diagnosed in departments of genitourinary medicine (GUM) suggest a population rate of 190 per 100,000 men and 187 per 100,000 women (52). Reported rates are highly dependent on the level of testing at different clinics, with the probability that many Chlamydia cases are not diagnosed. The population prevalence of uncomplicated genital Chlamydia in 16-to 24-year-olds in the United Kingdom is thought to be between 2% and 6% in both men and women (17,33), while the opportunistic National Chlamydia Screening Programme (2008) indicates a higher prevalence of around 10%, likely due to selective testing of higher-risk individuals (15). Nucleic acid amplification tests, commonly used in GUM clinics, identify infection only when the organism is present. Once infection has been resolved, these tests provide no information on past exposure. While detection rates are rising, due in part to increased screening and testing, the overall prevalence of past C. trachomatis exposure is not known.The prevalence of past exposure to genital C. trachomatis and changes over time in age-specific prevalence can be explored serologically. For instance, in Finland, Lyytikäinen et al. (32) studied pregnant women under the age of 29 using a commercial enzyme-linked immunosorbent assay (ELISA) based on C. trachomatis-specific peptides derived from the major outer membrane protein (MOMP). However, for wider application, confidence in the sensitivity and specificity of available antibody tests is critical (24). None of the current ELISAs have ever been rigorously evaluated against large numbers of well-defined serologically positive and negative control sera; hence, their sensitivity and specificity remain open to question.Chlamydia trachomatis is from the same family, Chlamydiaceae, as Chlamydia pneumoniae, a common respiratory pathogen with which it shares genetic homology (26). Sera from patients exposed to C. trachomatis show diverse serological
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