Replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells.

UNLABELLED: Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following the identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the classical Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts--if any exist besides boid snakes themselves--are not yet known. In this report, we use University of Helsinki virus (UHV; a virus that we isolated from a Boa constrictor with BIBD) to show that BIBDAV can also replicate effectively in mammalian cells, including human cells, provided they are cultured at 30°C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transferring infected cells from 30°C to 37°C ambient temperature resulted in progressive declines in IB formation and in the amounts of viral NP and RNA, suggesting that BIBDAV growth is limited at 37°C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30°C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature, such as snakes. IMPORTANCE: The newly discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in captive boid snakes, but their possible reservoir host(s) have not yet been identified. Here we show, using University of Helsinki virus as a model, that these viruses are able to infect mammalian (including human) and arthropod cells. Our results provide in vitro proof of the considerable ability of arenaviruses to cross species barriers. However, our data indicate that BIBDAV growth occurs at 30°C but is inhibited at 37°C, implying that crossing of the species barrier would be hindered by the body temperature of mammalian species.

Photochemical inactivation of viruses with psoralens: an overview.

In the presence of longwave ultraviolet light, psoralen derivatives photoreact with the nucleic acids within intact viruses and cells. This photoreaction can leave protein antigens and other surface components relatively unmodified, while eliminating the infectivity of a wide range of infectious agents. The kinetics of inactivation differ among RNA and DNA viruses photoreacted with different derivatives of psoralen. The inactivation kinetics are nonlinear as a result of photodegradation of psoralens and the unexplained biphasic inactivation of some viruses. In spite of these complexities, the photoreaction is capable of generating broad safety margins in the disinfection of microbial products under gentle, physiologic conditions. The psoralen photoreaction provides a potential method for inactivating both known and unknown viruses in active blood products. Psoralen-inactivated viruses have already proven useful as noninfectious antigens for use in immunoassays and as successful experimental vaccines.

Sensitivity of lymphocytic choriomeningitis and Tacaribe viruses to several physical factors.

A comparative study was performed as regards the behaviour of lymphocytic choriomeningitis virus (LCMV)--strains Fo-2 and 522--and of Tacaribe virus (strain Tr 11573) at different temperatures and pH values. All the three arenavirus strains had a high thermosensitivity and were rapidly inactivated in an acid medium. LCMV was more sensitive to UV irradiation than Tacaribe virus. The effect of sonication was also investigated.

Interactions of Junin and Tacaribe viruses during mixed infections.

The interaction between Junin virus (JV) and Tacaribe virus (TACV) during mixed infections of RK13 cells was examined. The effects of a prior infection with JV upon TACV replication depended on the time between the two inoculations. Simultaneous infection of RK13 cells with TACV and JV did not alter the plaquing efficiency of TACV; but if there was a 1- to 24-hour delay between JV preinfection and TACV superinfection, a variable increase of TACV replication was observed. The enhancement of TACV replication by preinfection with JV was dependent on several factors, such as the MOI of both viruses and the integrity of the JV genome. This effect was also highly specific, as the plaquing efficiencies of the arenavirus Pichinde and the unrelated vesicular stomatitis virus were not affected by preinfection with JV at any multiplicity assayed. The majority of the progeny formed in cells superinfected with TACV 1 or 24 h after JV infection was partially neutralized by antisera to both viruses. This suggested that phenotypic mixing, with JV or TACV genomes enclosed within an envelope containing TACV and JV glycoprotein, had occurred.

Response of cells persistently infected with arenaviruses to superinfection with homotypic and heterotypic viruses.

Vero cell cultures persistently infected with the arenaviruses Junin, Pichinde, Tacaribe, and Tamiami were established and designated Vero-Jun, Vero-Pic, Vero-Tac, and Vero-Tam, respectively. Two types of carrier cultures could be easily distinguished: Vero-Jun and Vero-Tac systems were characterized by a lack of infectious virus production after a few cell transfers, whereas a more productive state with continuous release of virus was observed in Vero-Pic and Vero-Tam cultures. These differences appeared to be related to resistance of the culture to viral superinfection. In fact, Vero-Jun and Vero-Tac cultures totally excluded only the replication of the serologically more closely related arenaviruses Amapari, Junin, or Tacaribe, while the refractoriness of Vero-Pic and Vero-Tam cultures was extended to most of the virus group members. The resistance of Vero-Jun cells to superinfection by Junin or Tacaribe virus could be ascribed to the production of specific uv-resistant Junin interfering particles, which showed a specific range of interference against Junin and Tacaribe viruses. Interfering particles against homotypic and heterotypic arenaviruses were isolated from Vero-Pic cultures. However, the degree of interference developed by these Pic-interfering particles was not enough to fully explain reinfecting virus exclusion from Vero-Pic cultures. Viral susceptibility of persistent cultures is proposed as a useful tool to examine relationships of members of the arenavirus group.

Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation.

Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with Co60 gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of CO60 radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose per rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. We found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents.