Filovirus contamination of cell cultures.

The filoviruses Marburg and Ebola comprise a newly recognized family of viruses. The first filovirus to be isolated was Marburg virus in 1967. This virus was imported in shipments of African green monkeys from Uganda and infected several cell-culture technicians, with serious illness resulting. The rarity of Marburg and Ebola virus transmission, decreasing use of imported African monkeys, and quarantine efforts have presumably been responsible for the lack of additional episodes until 1989, when a new filovirus related to Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus was imported on multiple occasions from a Philippine supplier of cynomolgus macaques as a consequence of an epidemic of acute infections in the foreign holding facility. While quarantine procedures prevented the use of any of these animals in research and the three human infections that occurred were asymptomatic, this episode emphasizes that these little understood viruses have considerable potential for mischief. The finding of antibodies reacting with Ebola viruses in many biomedically important Old World primates, including colonized monkeys in the U.S., emphasizes the need for more research to understand the specificity of the antibodies, spectrum of filovirus strains in nature, potential hosts, and true distribution of the family. The filoviruses grow well in primary and established cell strains and cell lines, and cytopathogenic effects may be absent or require several days to be manifest, leading to the possibility of occult contamination. The known viruses are readily detected by polyclonal and monoclonal antibody staining of cells and by electron microscopy; nucleic acid probes exist to develop more sensitive techniques if warranted.

Ranking of prophylactic efficacy of poly(ICLC) against Rift Valley fever virus infection in mice by incremental relative risk of death.

The prophylactic efficacy of poly(ICLC) (stabilized, synthetic, double-stranded polyriboinosinic-polyribocytidylic acid) against Rift Valley fever virus infection in Swiss-Webster mice was dependent on the treatment schedule. The treatment schedule was optimized by ranking the results of various treatments by the Cox proportional-hazard model based on the incremental relative risk of death. With this ranking procedure, the schedule of choice was three doses of 20 micrograms each given 5 days apart. This regimen yielded a 90% survival rate. Additional parameters were determined, including the timing of the first and second drug dose, the temporal relationship of these treatments to the day of challenge, and the minimal effective dose (1 microgram per mouse).

Enhanced therapeutic efficacy of poly(ICLC) and ribavirin combinations against Rift Valley fever virus infection in mice.

The therapeutic efficacy of polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose [poly(ICLC)] given alone or in combination with ribavirin was evaluated in Swiss Webster mice infected with Rift Valley fever virus. Four or more 20-micrograms doses of poly(ICLC) given at various intervals beginning 24 h after infection protected all mice against death. On the other hand, a treatment regimen consisting of only three doses of poly(ICLC) given 24 h postinfection resulted in a 50% survival rate. When initiated 48 h postinfection, an extended treatment regimen with the same dose was required to yield 40% survivors. Lower doses (5 micrograms) of poly(ICLC) per mouse were only marginally effective even when six injections were given between days 1 and 9 postinfection. The combined administration of ribavirin and poly(ICLC) initiated as late as 48 h postinfection was effective even when treatment consisted of doses that were ineffective when either drug was used alone.

Development of an attenuated strain of chikungunya virus for use in vaccine production.

An attenuated chikungunya (CHIK) virus clone was developed for production of a live vaccine for human use. CHIK strain 15561 was subjected to 18 plaque-to-plaque passages in MRC-5 cultures before CHIK 181/clone 25 was selected as vaccine seed based on homogeneous small plaque size, suckling mouse avirulence, reduced monkey viraemia and genetic stability. Oligonucleotide mapping demonstrated differences between parent and clone. Vaccine (pilot-lot production) elicited neutralizing antibody and protected mice and rhesus monkeys against challenge. After challenge, viraemias were absent in vaccinated monkeys. Vaccine was then produced and tested in accordance with governmental regulatory requirements of human use.

Comparison of inactivated Newcastle disease viral vaccines containing different emulsion adjuvants.

Chickens inoculated with inactivated-virus Newcastle disease vaccines containing different emulsion adjuvants were challenge exposed with viscerotropic velogenic Newcastle disease virus. Adjuvant activity was evident in all of 9 vaccines containing mineral oil emulsion (OE), but was not evident in 2 vaccines which contained a metabolizable lipid emulsion (LE) adjuvant consisting of peanut oil, glycerol, and lecithin. Serologic responses of chickens inoculated with OE vaccines were 10- to 100-fold higher than those of chickens inoculated with LE vaccines. One of 106 chickens given OE vaccine, 12 of 24 given LE vaccine, and all of 24 nonvaccinated control chickens were clinically affected or died after challenge exposure. Five OE vaccines emulsified only by brief vigorous shaking had adjuvant activity similar to 4 OE vaccines emulsified by conventional homogenization.

Plaque assay for Ebola virus.

A plaque assay for Ebola virus is reported. The procedure has real potential for future research, although it is less sensitive than indirect fluorescent-antibody and mouse inoculation tests.

Evaluation of a formalin-inactivated Rift Valley fever vaccine in sheep.

A formalin-inactivated Rift Valley fever (RVF) vaccine prepared in cell culture for human use was immunogenic in sheep. Vaccine was administered as a single dose of diluted (1:5) or undiluted vaccine with or without an adjuvant. Serum-neutralizing antibodies induced by RVF vaccine persisted for at least 7 months. Seven of 11 vaccinated sheep with prechallenge plaque-reduction neutralization (PRN80) antibody titers of less than or equal to 10 were protected against challenge exposure with 10(6) plaque-forming units of Zagazig 501 strain of RVF virus. Challenge exposure induced abortion in 2 of 2 pregnant sheep. Five sheep with PRN80 titers greater than or equal 1:20 were protected from detectable viremia after challenge exposure. Additionally, 5 of 6 lambs (3 months old) were protected (by maternal antibodies) against challenge exposure. Challenge control sheep developed clinical disease and detectable viremia after exposure. Virus was isolated from saliva of 1 challenge control sheep and virus was transmitted by contact exposure to 1 of 4 seronegative contact-control sheep. Immunization of sheep with formalin-inactivated RVF vaccine induced a priming effect against RVF viral antigens. Challenge exposure with RVF virus resulted in significantly higher neutralizing titers in vaccinated sheep than in nonvaccinated sheep.

Identification and characterization of a bovine rhinovirus isolated from Iowa cattle with acute respiratory tract disease.

Viral isolate FS1-43 isolated from a calf with acute respiratory tract disease was characterized as a bovine rhinovirus. This virus was antigenically indistinguishable from rhinovirus strains C-07 and VC-96. The virus replicated in low passage Madin-Darby and Georgia bovine kidney cells. A relative noncytopathogenic replication occurred in epithelial bovine turbinate, bovine fibroblastic turbinate, and bovine lung cells. Ciliary activity of tracheal explants was unaffected during eight serial passages. Viral replication was detected earlier and viral titers were higher at 33 C than at 37 C. Rotation of cultures did not affect viral titers but was necessary to produce a cytopathic effect in Madin-Darby bovine kidney cells.

Evaluation of experimental subunit vaccines for infectious bovine rhinotracheitis.

Subunit infectious bovine rhinotracheitis (IBR) antigens were prepared by solubilization of IBR-virus infected cell cultures with nonionic detergents Triton X-100 and NP-40. When used with Freund's incomplete adjuvant, Triton X-100 and NP-40 subunit antigens induced high serum-neutralization titers in vaccinated calves. Two doses of Triton X-100 and NP-40 subunit vaccines prevented the appearance of clinical signs of disease in all vaccinated calves exposed 30 days after the second vaccinal dose to intranasal challenge exposure with Cooper strain of IBR virus. Furthermore, two doses of NP-40 subunit vaccine prevented shedding of the virus in all vaccinated calves after challenge exposure.

Experimental infection of neonatal striped skunks (Mephitis mephitis) with infectious bovine rhinotracheitis virus.

Experimental infection of neonatal skunks (Mephitis mephitis) with infectious bovine rhinotracheitis virus (IBRV) caused fatal systemic infection. Virus isolation and immunofluorescence tests were used to demonstrate a direct association between IBRV and the lesions. Histopathologic studies revealed multiple focal necrosis in the liver and the adrenal glands.

Evaluation of the rabbit as a laboratory model for infectious bovine rhinotracheitis virus infection.

Experimental infection of rabbits with infectious bovine rhinotracheitis virus (IBRV) produced diverse manifestations of disease which included abortion, conjunctivitis, dermatitis, vulvovaginitis, systemic infection, neonatal death and respiratory tract infection. Each disease syndrome was studied using virus isolation, fluorescent antibody examination and histologic examination. Conjunctivitis, dermatitis and vulvovaginitis lesions were characterized by edema, infiltration of mucosa and submucosa with inflammatory cells and ulceration of epithelium. Systemic infection resulted in severe necrosis of liver and adrenal glands with large numbers of cells containing intranuclear inculsions. Pregnant rabbits aborted within 48 hours following inoculation of IBRV. Virus infection and viral lesions were not demonstrated in aborted fetuses.

Clearance and shedding of infectious bovine rhinotracheitis virus from the nasal mucosa of immune and nonimmune calves.

Infectious bovine rhinotracheitis virus was rapidly cleared from the nasal mucosa of calves after intranasal aerosol exposure. Nonimmune calves (experiment 1) cleared 10(9) plaque-forming units (PFU) of virus from the nasal mucosa in less than 4 hours and 10(6) PFU of virus in 1 hour. An eclipse phase followed the clearance of viral inoculum. Replicating virus was first detected at 9 hours. Viral titers increased stepwise until maximum was attained on postinoculation day 4. Virus persisted in the nasal mucus until day 12. Clinical signs of disease corresponded with the shedding of virus. In contrast to nonimmune calves, immune calves (experiment 2; same calves as in experiment 1, but 30 days after initial exposure) cleared 10(9) PFU of virus in 1 hour and 10(6) PFU of virus in less than 5 minutes. An abortive reinfection occurred after exposure of immune calves with 10(9) PFU of virus. Virus was first detected in these calves at 14 hours after exposure and was not detected beyond 24 hours after inoculation. Immune calves given 10(6) PFU of virus did not shed virus after clearance of inoculum. Clinical signs of infection were not observed in immune calves after viral challenge exposure. The date indicated that there was no detectable residual virus beyond 3 hours after the exposure.

Experimental infection of eastern cottontail rabbits Sylvilagus floridanus) with infectious bovine rhinotracheitis virus.

Experimental infection of eastern cottontail rabbits (Sylvilagus floridanus) with infectious bovine rhinotracheitis virus caused acute keratoconjunctivitis and a fatal systemic infection. The clinical syndrome was characterized initially by blepharospasm and ocular discharge. The rabbits were markedly depressed on post-exposure day (PED) 5 and were dead or moribund on PED 6. The virus was readily recovered from liver and adrenal gland tissue on PED 6 and from conjunctival swabs on PED 1 to 6. Histopathologic studies revealed a few necrotic foci in the liver and multiple focal to diffuse necrosis of the adrenal glands. Viral isolation and immunofluorescence tests were used to demonstrate a direct association between infectious bovine rhinotracheitis viral antigens and the lesions.

Isolation of bovine adenovirus type 7 from calves with pneumonia and enteritis.

Bovine adenovirus type 7 was isolated from a 10-month-old calf with fibrinopurulent pneumonia and from 2 newborn calves with pneumoenteritis. The viruses were isolated on calf lung and adrenal gland cell cultures and were identified as serotype 7 by immunoelectron microscopy and serum-neutralization tests.