A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.

Intestinal microsporidiosis in patients diagnosed with acquired immunodeficiency syndrome (AIDS) and having chronic diarrhea was first reported in 1985 and the associated microsporidian was named Enterocytozoon bieneusi. The intracellular developmental cycle of E. bieneusi in enterocytes has been demonstrated and many cases have been reported worldwide. This report presents the life cycle of a second intestinal microsporidian, associated with the same symptoms, in five AIDS patients. This new microsporidian also infects enterocytes but its pathology and morphology differ from that of E. bieneusi. It involves lamina propria macrophages, fibroblasts, and endothelial cells and can disseminate to infect other parts of the body, e.g. the kidney and gall bladder. The parasite cycle includes development of rounded uninucleate and elongated bi- or tetranucleate cells without the formation of plasmodial stages. Sporogony is similar to the more typical development of microsporidia with sporoblast morphogenesis occurring after the last cell division. The development of cells within chambers of a septate, honeycomb-like, parasite-secreted fibrillar network and surrounded by a parasitophorous vacuole, however, is unique to this microsporidian, justifying the establishment of a new genus and species, Septata intestinalis n. g., n. sp.

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

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Brachiola vesicularum, n. g., n. sp., is a new microsporidum associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 x 2 microns diplokaryotic spores containing 8-10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron-dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae, its development occurred at warm blooded host temperature (37-38 degrees C) and unlike Nosema connori, which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema, this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.

The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the "thickened" plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1-h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane invaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin "unit limiting membrane", present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross-connections forming the "thick blistered" surface by 30 min-postextrusion.

In the past several years, microsporidia have become recognized as another important group of opportunistic infections of immunocompromised patients, especially those with AIDS. Enteric infections with the noncultivatable microsporidian parasite Enterocytozoon bieneusi have been diagnosed from AIDS patients with chronic diarrhea, malabsorption, and wasting. The incidence of infection and mechanism of transmission of these organisms in humans is unknown. Several recent tests for human pathogens have been developed using rRNA genes as diagnostic probes. Using the polymerase chain reaction and conserved regions of the small subunit rRNA (SSU-rRNA) gene, the SSU-rRNA gene of E. bieneusi was successfully cloned and subsequently sequenced. Amplification of E. bieneusi rRNA could be demonstrated from intestinal biopsies from HIV-1-infected patients infected with E. bieneusi but not from intestinal biopsies from noninfected patients. This cloned SSU-rRNA gene was used to develop improved probes for detection of E. bieneusi in tissue of infected patients.