Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.Bovine herpesvirus 1 (BoHV-1) and BoHV-5 belong to the subfamily Alphaherpesvirinae and are closely related pathogens of cattle (22). The protein repertoire of the two viruses averages 82% amino acid identity (20). Both viruses are neurotropic, but only BoHV-5 can significantly replicate in the central nervous system (CNS) to cause encephalitis of either naturally infected cattle or experimentally inoculated laboratory animals (2,5,6,12,40,41,44). Glycoprotein D (gD) is accepted as the critical and essential receptor-binding protein of many alphaherpesviruses (reviewed in references 8 and 48). The main gD receptors identified to date include members of the tumor necrosis factor (TNF) receptor family (HveA) and the poliovirus receptor family (HveB or nectin 2 and HveC or nectin 1) (28,42,51). Furthermore, a modified form of heparan sulfate, 3-O-sulfated heparan sulfate, can mediate herpesvirus entry (46). J1.1-2 cells (J cells) represent a subpopulation of thymidine kinase-negative baby hamster kidney (BHK) cells selected for their property of being resistant to infection with herpes simplex virus type 1 (HSV-1), HSV-2, and BoHV-1. The expression of nectin 1 in those cells rendered them susceptible to BoHV-1 infection and replication, which suggests that nectin 1 can serve as a receptor for BoHV-1 gD (gD1) (16,18,28). Interestingly, we observed that BoHV-5 was able to productively replicate in J cells without the nectin 1 receptor.According to a previously reported sequence comparison of BoHV-1 and BoHV-5 (20), the highest divergence between the two viruses mapped to the latency-related region and the immediate-early proteins (less than 75% amino acid identity) BICP0, BICP4, and BICP22. Glycoprotein E (gE) was also listed in this category, with 74% amino acid identity between gE of BoHV-1 (gE1) and gE5. This fact also gave ample reason for attempts to map the neurovirulent phenotype of BoHV-5 to the gE5 molecule (3, 4, 13). In contrast, the highest sequence similarities between the two viruses were described for proteins involved in viral DNA replication and proc...

BackgroundBovine herpesviruses type 1 (BoHV1) and type 5 (BoHV5) are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves.FindingsThe viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs). First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types.ConclusionThe two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.

Bangham-type liposomes, undergoing freeze-thawing cycles, were detected electron microscopically in the brain, liver and spleen of experimental animals 24 h after rectal administration. This novel approach has several important advantages over intravenous, intraperitoneal and other usual means of administration of liposome drug-entrapped treatment and diagnostics for human application. The location of the liposomes in the brain after rectal administration shows that the brain-blood barrier can be overcome successfully by this method. Moreover, the risk of embolism and hypersensitivity, strict control of sterility and some other undesirable effects can be avoided. The possible role of rectal administration in the development of liposome drug-entrapped treatment and diagnostics is discussed.

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The effect of specific cell growth inhibitor (chalone) on the mitotic activity of ascites ISM applied entrapped in liposomes compared to the non-entrapped 'free' form was investigated. The 'free' chalone injected intraperitoneally in BALB/c ascites ISM-bearing mice (1500 mg per kg body weight) decreased the mitotic activity of the tumour by 66 per cent 2.5 h after administration. Five hours after administration, complete restoration of the mitotic index to the control level was observed. Chalone encapsulated into Bangham liposomes (1000 mg per kg body weight) in otherwise identical conditions produced 44 per cent inhibition after 2.5 h, while at 5 h post-injection the mitotic inhibition was still present increasing up to as much as 50 per cent. No such effect was evident by chalone encapsulated in freeze-thawed liposomes (500 mg per kg body weight). The chalone under study specifically inhibits the cell division of the originating tumour cells and has no effect on its DNA synthesis or on the mitosis of the basal layer of the oesophagus and crypts of the intestinal epithelium. The results obtained provide evidence that liposome-entrapped chalone prolongs the inhibition of tumour cell mitosis in comparison to the 'free' one even in a reduced dose. Some possible applications of this approach concerning cancer research and treatment are discussed.