Large replication compartments are formed in HSV-infected cells by 6 h, compared to between 9 and 12 h in VZV-infected cells 8 , IE62, the predominant VZ virion tegument protein, is predicted to be an important immediate-early protein because it functions as the most potent and promiscuous transactivator of VZV gene promoters, including its own, and is essential for VZV replication 47 , The functions of ORF61 are much less well defined, although it also has regulatory activity in transient expression experiments Using drug inhibition protocols and spatiotemporal analyses of individual newly infected cells, we demonstrated synthesis and nuclear translocation of both ORF61 and IE62 within 1 h after VZV infection.
ICP0 localization to ND10 bodies and its ubiquitin ligase function is essential for their molecular rearrangement and degradation in HSVinfected cells 6 , Whether ORF61 shares the other functions of ICP0 that are necessary to inhibit the host cell antiviral response during initial replication is not known. At the earliest time points, IE62 tended to be enriched at the inner nuclear rim in the newly infected cell that was nearest to an infected inoculum cell.
Since ICP4 has been demonstrated to mark the location of HSV-1 prereplication compartments 19 , 20 , 32 , it is likely that the first detection of IE62 punctae at the inner nuclear rim adjacent to an attached VZV-infected input cell represented its localization with incoming VZV genomes.
In contrast, although earlier reports differ, recent studies indicate that ICP4 associated with these structures within 30 min after HSV-1 entry However, ND10 components, represented by PML, did not seem to be deposited in these locations within infected cell nuclei at these early time points data not shown. Although delayed compared to that for HSVinfected cells, a similar sequence of events was observed in VZV-infected cells in that IE62 was first observed at less than 4 h in nuclear punctae that did not yet contain ORF29, the VZV single-stranded DNA binding protein, and probably represented prereplicative sites.
ORF29 was first detectable at low levels in a diffuse nuclear distribution at 4 h. IEexpressing domains grew into larger globular nuclear structures and ORF29 began to appear at these sites from 4 to 6 h after infection. When evaluated at 4 h and 8 h, small ORF29 punctae were localized specifically and exclusively at the margins of IE62 punctae, and small, although somewhat more globular IE62 sites, followed at later times by a distribution together with IE62 in larger globular compartments.
Similarly, ORF29 seems to be recruited to sites where IE62 has accumulated and that may serve as platforms for the assembly of proteins required for VZV replication. IE63 has been designated an immediate-early protein based on its presumed kinetics However, IE63 nuclear expression was detectable only as a weak, diffuse signal at 4 to 6 h, which was substantially later than that of IE62 and ORF A difference in the sensitivity of IE63 detection is unlikely, because the anti-IE63 antibody is a high-potency reagent 34 and the intensity of the IE63 signal showed a rapid increase when evaluated between 6 to 8 h after infection.
However, because IE63 was diffusely expressed and was not enriched in discrete compartments like the IE62 signal, earlier expression of IE63 might escape detection. IE63 expression remained diffuse throughout the nuclei, suggesting that this protein may be present within IEpositive replication compartments but is not specifically enriched at these sites even though IE63 binds to IE62 by residues in the IE63 N terminus 3.
IE63 was predominantly nuclear throughout the 9- to h VZV replication cycle and remained so up to 24 h, but IE63 expression in the cytoplasm increased beginning at 9 h. This extensive cytoplasmic expression of IE63 at later times may be important for its role as an antiapoptotic factor, as has been demonstrated in cultured neurons VZV gE is the most abundant glycoprotein in infected cells and is a prominent envelope glycoprotein.
In contrast to other alphaherpesviruses which replicate in the absence of gE, VZV requires gE for viral replication 11 , While it has been considered to be a late gene product, our time course experiments revealed that gE synthesis occurred quite early in newly infected cells in a pattern similar to that described for HSV gB 4 , 5.
This early gE expression may serve to rearrange the Golgi compartment and prepare Golgi-compartment-derived membranes for VZ virions exiting the nucleus by accumulating tegument proteins such as IE62 on membrane-bound gE 11 , 23 , Because gE was absent from the plasma membrane and did not show a punctate endosome-like cytoplasmic pattern at early time points, it is unlikely that we detected endocytosed gE. ORF23, which is a VZV capsid protein related to HSV-1 VP26, became enriched within nuclei at 9 h, suggesting that viral nucleocapsids could become assembled for release from the nucleus and envelopment in the premodified Golgi-compartment area by 6 to 9 h after infection.
In contrast to its Golgi-compartment localization, substantial gE expression on plasma membranes was evident only at 9 h. While syncytium formation is a hallmark of VZV replication in cultured cells, our analyses of the VZV replication cycle were done by tracking events during the infection of individual cells that were located next to an infected inoculum cell but had no evidence of green-fluorescent-dye transfer into the cytoplasm.
These experiments suggested that VZV infection was initiated without cell-cell fusion and could be attributed to the transfer of extracellular virions on surfaces of inoculum cells, as visualized by EM.
This explanation was supported by the observation that newly infected cells were consistently found adjacent to inoculum cells. The inoculum cells were also washed extensively to eliminate any unabsorbed fluorescent dye, which also removed the original culture medium and any cell-free VZ virions it may have contained.
Furthermore, it is known that secondary plaques attributable to virus release do not appear in VZV-infected monolayers; in addition, extracellular VZ virions are quite labile, whereas enveloped VZV particles are abundant on the surfaces of heavily infected fibroblasts and are also present in intracellular vacuoles near the plasma membranes When the inoculum cell was attached in proximity to the uninfected cell, the space between the plasma membranes appeared to be minimal, therefore placing surface virions next to the uninfected cell membrane and potential entry receptors of the uninfected cell and limiting the dispersal of virions.
These conditions would favor entry of cell-associated extracellular virus particles without requiring cell-cell fusion Fig. Of interest, VZV gE may promote these conditions, since gE enhances the formation of tight junctions between plasma membranes Model of the cell-to-cell spread of VZV before cell fusion in vitro. Mature VZV particles are transported in an export vacuole to the plasma membrane of an infected inoculum cell gray cell on left.
A VZV particles released by exocytosis adhere to the plasma membrane at the interface with culture media. Infectious VZV particles are not released into the media or rapidly lose infectivity, since secondary plaques do not form in VZV-infected monolayers. B Some VZV particles are released from the inoculum cell in close proximity to the plasma membrane of the adjacent uninfected cell white cell on right.
C Envelope glycoproteins of extracellular VZV particles at these sites have an increased probability of binding to cell surface receptors on the plasma membrane of the uninfected cell. Entry into the adjacent cell occurs by endocytosis D or direct entry E , which is followed by the release of partially tegumented capsids into the cytoplasm of the newly infected cell.
Our model predicts that the spread of cell-associated extracelluar VZ virions in vitro is determined by the proximity of infected and uninfected cells and does not require cell fusion. Although VZV infection occurred immediately after the exposure of uninfected cells to the infected cell inoculum without the formation of polykaryocytes, the kinetics experiments done using two cell dyes with different emission wavelengths red and green fluorescence to label inoculum and uninfected cells revealed that abundant cell-cell fusion and syncytium formation occurred by 9 h after infection.
Some earlier fusion may have been undetected if a syncytium consisting of several green-labeled inoculum cells had fused with one red-labeled output cell, diluting the red fluorescence signal. In any case, while viral entry into uninfected cells occurs without it, cell-cell fusion at 9 h should amplify VZV replication substantially, since this time point coincides with the detection of enveloped cytoplasmic virions and precedes their appearance in large numbers on the surfaces of plasma membranes, which became prominent at 12 h.
In summary, this kinetic analysis of the VZV replication cycle in individual fibroblasts demonstrated the spatiotemporal expression of six VZV proteins, newly synthesized viral DNA, and virion morphogenesis. Our results reveal that one complete VZV replication cycle leading to a new generation of infectious VZV particles takes between 9 and 12 h. National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Published online Feb 4.
Ann M. Author information Article notes Copyright and License information Disclaimer. Phone: Fax: E-mail: ude. Received Oct 9; Accepted Jan This article has been cited by other articles in PMC. Abstract Varicella-zoster virus VZV is a human alphaherpesvirus that is highly cell associated in cell culture. Fluorescent cell tracker labeling. Cell labeling by endocytosis of PA-gold particles.
Time course experiments. Antibodies and IF staining. Drug inhibition experiments. Confocal microscopy. Electron microscopy. Open in a separate window. ORF61 and IE62 proteins are targeted to different subnuclear compartments during very early infection. VZV nucleocapsid formation and plasma membrane expression of glycoprotein gE. The kinetics of cell-cell fusion after VZV infection. Detection of VZV nucleocapsids, enveloped virions in cytoplasmic vacuoles, and extracellular enveloped virions.
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Varicella-zoster virus gene 63 encodes an immediate-early protein that is abundantly expressed during latency.
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Breakthrough varicella occurs less frequently among those who have received two doses of vaccine compared with those who have received only one dose; disease may be even milder among two-dose vaccine recipients, although the information about this is limited.
Varicella is highly contagious. The virus can be spread from person to person by direct contact, inhalation of aerosols from vesicular fluid of skin lesions of acute varicella or zoster, and possibly through infected respiratory secretions that also may be aerosolized. A person with varicella is considered contagious beginning one to two days before rash onset until all the chickenpox lesions have crusted.
Vaccinated people may develop lesions that do not crust. These people are considered contagious until no new lesions have appeared for 24 hours. It takes from 10 to 21 days after exposure to the virus for someone to develop varicella.
People with breakthrough varicella are also contagious. However, people with breakthrough varicella with 50 or more lesions were just as contagious as unvaccinated people with the disease. Severe complications caused by the virus include cerebellar ataxia, encephalitis, viral pneumonia, and hemorrhagic conditions.
Other severe complications are due to bacterial infections and include:. Immunocompromised people who get varicella are at risk of developing visceral dissemination VZV infection of internal organs leading to pneumonia, hepatitis, encephalitis, and disseminated intravascular coagulopathy.
They can have an atypical varicella rash with more lesions, and they can be sick longer than immunocompetent people who get varicella. New lesions may continue to develop for more than 7 days, may appear on the palms and soles, and may be hemorrhagic. Children with HIV infection tend to have atypical rash with new crops of lesions presenting for weeks or months. The lesions may initially be typical maculopapular vesicular but can later develop into non-healing ulcers that become necrotic, crusted, and hyperkeratotic.
The rate of complications may also be lower in HIV-infected children on antiretroviral therapy or HIV-infected people with higher CD4 counts at the time of varicella infection. Retinitis can occur among HIV-infected children and adolescents. As a result, varicella is relatively uncommon among HIV-infected adults. Pregnant women who get varicella are at risk for serious complications, primarily pneumonia, and in some cases, may die as a result of varicella.
Some studies have suggested that both the frequency and severity of VZV pneumonia are higher when varicella is acquired during the third trimester, although other studies have not supported this observation. If a pregnant woman gets varicella in her first or early second trimester, her baby has a small risk 0. The baby may have scarring on the skin; abnormalities in limbs, brain, and eyes, and low birth weight. If a woman develops varicella rash from 5 days before to 2 days after delivery, the newborn will be at risk for neonatal varicella.
The vaccine is contraindicated for pregnant women. Top of Page. Varicella-Zoster Immune Globulin For people exposed to varicella or herpes zoster who cannot receive varicella vaccine, varicella-zoster immune globulin can prevent varicella from developing or lessen the severity of the disease.
Varicella-zoster immune globulin is recommended for people who cannot receive the vaccine and 1 who lack evidence of immunity to varicella, 2 whose exposure is likely to result in infection, and 3 are at high risk for severe varicella. VariZIG is commercially available from a broad network of specialty distributors in the United States list available at www.
Acyclovir Treatment The American Academy of Pediatrics AAP recommends that certain groups at increased risk for moderate to severe varicella be considered for oral acyclovir or valacyclovir treatment. These high risk groups include:. Some healthcare providers may elect to use oral acyclovir or valacyclovir for secondary cases within a household.
For maximum benefit, oral acyclovir or valacyclovir therapy should be given within the first 24 hours after the varicella rash starts.
VZV is found in a worldwide geographic distribution but is more prevalent in temperate climates. Virus-specific cellular immunity is critical for controlling viral replication in healthy and immunocompromised patients with primary or recurrent VZV infections. Rapid laboratory confirmation of the diagnosis of varicella or herpes zoster, which can be accomplished by detecting viral proteins or DNA, is important to determine the need for antiviral therapy.
Acyclovir is licensed for treatment of varicella and herpes zoster, and acyclovir, valacyclovir, and famciclovir are approved for herpes zoster.
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