Unenveloped Particles Research Articles

Analysis of Rab GTPase-Activating Proteins Indicates that Rab1a/b and Rab43 Are Important for Herpes Simplex Virus 1 Secondary Envelopment

Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment.

Evidence of a role for nonmuscle myosin II in herpes simplex virus type 1 egress.

After cell entry, herpes simplex virus (HSV) particles are transported through the host cell cytoplasm to nuclear pores. Following replication, newly synthesized virus particles are transported back to the cell periphery via a complex pathway including a cytoplasmic phase involving some form of unenveloped particle. These various transport processes are likely to make use of one or more components of the cellular cytoskeletal systems and associated motor proteins. Here we report that the HSV type 1 (HSV-1) major tegument protein, VP22, interacts with the actin-associated motor protein nonmuscle myosin IIA (NMIIA). HSV-1 infection resulted in reorganization of NMIIA, inducing retraction of NMIIA from the cell periphery and condensation into a spoke-like distribution around the nucleus along with a second effect of accumulation in a perinuclear cluster. VP22 did not appear to colocalize with the reorganized cagelike distribution of NMIIA. However, VP22 has been previously reported to localize in a perinuclear vesicular pattern, and significant overlap was observed between this pattern and the perinuclear clusters of NMIIA. Inhibition of the ATPase activity of NMIIA with the myosin-specific inhibitor butanedione monoxime impaired the formation of the perinuclear vesicular VP22 accumulations and also the release of virus into the extracellular medium while having much less effect on the yield of cell-associated virus. Virus infection frequently results in the induction of highly extended processes emanating from the infected cell, and we observed that VP22-containing particles line up along NMIIA-containing filaments which run through these protrusions.

Squid axoplasm supports the retrograde axonal transport of herpes simplex virus.

Squid axoplasm supports the retrograde axonal transport of herpes simplex virus.

Partial cloning of the genome of infectious hypodermal and haematopoietic necrosis virus, an unusual parvovirus pathogenic for penaeid shrimps; diagnosis of the disease using a specific probe

The infectious hypodermal and haematopoietic necrosis virus (IHHNV), pathogenic for penaeid shrimp, is an icosahedral unenveloped particle, 22 nm in diameter, with an ssDNA linear genome, and proposed to be a member of the Parvoviridae. A large majority of minus-strand DNA is incorporated into the capsids compared to the plus-strand. A small amount of reannealed plus- and minus-strands (dsDNA) obtained after nucleic acid extraction was blunt-ended and cloned into the system pUC18/Escherichia coli strain DH5 alpha. Selected clones were studied and characterized using restriction enzymes. One of them, BQ31, was used to construct different sized probes labelled with digoxigenin-11-dUTP. These probes failed to hybridize with DNA of some insect parvoviruses and with DNA of a parvo-like virus of shrimp. They reacted strongly with dilutions of homogenized IHHNV-infected shrimp tissues and, conversely, did not react with uninfected shrimp tissues. They hybridized in situ, in sections of infected animals, labelling strongly the target cells and particularly the nuclear Cowdry type A inclusion body, which is the most diagnostic characteristics of this disease.

HT-29 cells: a new substrate for rotavirus growth

Susceptibilities of a continuous rhesus monkey kidney cell line (MA-104) and that of a human colon carcinoma cell line (HT-29) to infection by different human and animal rotavirus strains were compared. HT-29 cells appeared to be more sensitive to human rotavirus infection than MA-104 cells, whereas the latter cell line was more susceptible to animal rotavirus replication. The greater sensitivity to human rotavirus infection of HT-29 cells was confirmed by the successful, direct isolation of these viruses from faecal specimens. Human rotavirus infection of HT-29 cells was also followed by transmission electron microscopy. In ultra-thin sections, unenveloped particles of rotaviruses, representing infectious mature virions, were observed in large number. Moreover, many "double-shelled" particles were detected in negative-stained supernatants from infected cultures. Scanning electron microscopy of uninfected HT-29 cells showed that in the presence of Ca++, required for rotavirus growth, they are able to express some of the features of mature intestinal cells. In view of these results, HT-29 cells appear to be a useful in vitro model for the study of rotavirus infection.

A mixed virus infection in midgut cells of Autographa californica and Trichoplusia ni larvae

Ultrastructural examination of the midguts of Autographa californica and Trichoplusia ni infected with a viral preparation obtained from Autographa nuclear polyhedrosis virus (AcMNPV)-infected insects revealed the presence of five morphologically distinct virus particles. Two virus particles were observed simultaneously in the nuclei, AcMNPV and an approximately 40-nm icosahedral unenveloped particle. Within the same cell two other viruses were observed in the cytoplasm, cytoplasmic polyhedrosis virus and a dense icosahedral unenveloped particle around 28 nm. A fifth particle resembling an icosahedral cytoplasmic deoxyribovirus was occasionally observed. Both the 40- and the 28-nm particles were observed to be occluded within polyhedra.

Ultrastructure of white-streaked Dendrobium phalaenopsis orchid flowers infected with a rhabdovirus

This report describes the first true rhabdovirus in orchids. Virions occurred as long bacilliform particles with a mean length of 320 ± 10 nm and as enveloped bullet-shaped particles and short bacilliform particles with a mean length of 180 ± 10 nm. Semienveloped bullet-shaped particles also were present. Both enveloped and unenveloped particles were in the perinuclear space. Inclusion bodies of similar appearance and density were in the nucleoplasm and the perinuclear space. No virus particles were observed in association with the inclusion bodies in the nucleoplasm but unenveloped virions were found on the margin of the inclusion bodies in the perinuclear space. Perinuclear pockets containing virions were often invaginated into the nucleoplasm or evaginated into the cytoplasm. In contrast to other bacilliform viruses studied in orchids, this virus did not induce the formation of membrane-bounded vesicles lined with unenveloped particles (“spokewheels”) in the cytoplasm. The morphological differences between this virus and other bacilliform viruses found in orchids may represent distinct modes of development of different viruses.

Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus--a new taxonomic group. Electron microscopic studies.

SUMMARY The morphology and mode of maturation of a number of relatively solvent resistant arboviruses were examined by thin-section and negative-stain electron microscopy of infected mouse brain and cell culture specimens. These viruses, which have physicochemical properties distinct from other arboviruses, included Colorado tick fever, Tribec, Wad Medani, Chenuda, Irituia, Palyam, Lebombo, epizootic haemorrhagic disease of deer and bluetongue. They were 65 to 80 nm. in diameter and matured in the cytoplasm as unenveloped particles with an electron-dense core. Virus development occurred in association with a cytoplasmic granular matrix and was accompanied by formation of regularly substructured filaments and tubules. Surface architecture was compatible with icosahedral symmetry with T = 3 (32 capsomeres). The combination of taxonomic parameters, morphologic and morphogenetic as well as physicochemical, was distinct from that of any presently recognized virus group. The independent classification of these viruses with bluetongue as the type virus is thus proposed.

The Ultrastructural Changes Produced by Infectious Laryngotracheitis Virus in Tracheal Epithelium of the Fowl

SUMMARY The ultrastructural changes produced by infectious laryngotracheitis virus in the tracheal epithelium of minimal diseased fowls are described. The giant cell structures seen using the light microscope were identified as syncytia. Virus particles were first seen in the nuclei and in cytoplasmic vacuoles of the syncytia on the third day after exposure. The majority of the particles within the nucleus consisted of a core and capsid only. By the fourth day after infection most of the virus particles within the nucleus had acquired an outer envelope and were most often seen close to or between the layers of the nuclear membranes. Structures morphologically consistent with inclusion bodies were present on the third and fourth days after exposure and consisted of fine granular electron dense material through which a few unenveloped particles were scattered. Regeneration commenced with the spread of a single layer of cells over the surface of the lamina propria. Mitotic figures were numerous in the regenerating epithelium on the sixth and seventh days after exposure when several layers of these cells had formed. Numerous microvillus-like processes were seen extending from the regenerating epithelial cells on the ninth day after exposure and cilia were first seen on the tenth day. By the twelfth day the epithelium appeared normal.

A study of Australian arboviruses resembling bluetongue virus.

Five Australian arboviruses, D'Aguilar, Eubenangee, Corriparta, and two as yet unnamed isolates Ch9935 and MRMI0434 were studied by thin-section and negative-contrast electron microscopy and were found to be very similar in both morphology and morphogenesis. In thin section they had an extremely electrondense core surrounded by a less dense capsid. All developed in association with a fibrillogranular intracytoplasmic matrix and to varying degrees were released as either enveloped or unenveloped particles from the infected cell. The diameters of enveloped and non-enveloped particles were 100-104 and 66-67 nm respectively. Negatively stained particles exhibited a surface capsomer structure.