Bill B. Barnett

In vitro and in vivo Phlebovirus inhibition by ribavirin.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was markedly inhibitory in vitro to Adames and Balliet strains of Punta Toro virus (PTV), a Phlebovirus related to Rift Valley fever and sandfly fever viruses. By using inhibition of viral cytopathic effect in LLC-MK2 cells with both virus strains, the 50% effective dose was 4 to 10 micrograms/ml and the virus rating was 1.3. The Adames strain of PTV infection in mice was established for evaluation of the in vivo antiviral efficacy of ribavirin. The drug was administered subcutaneously (s.c.) twice daily for 5 to 7 days beginning 4 h pre-virus inoculation, 24 h post-virus inoculation, or 36 h post-virus inoculation, with increased survivors, reduced hepatic icterus, reduction of serum glutamic oxalic acid transaminase and serum glutamic pyruvic acid transaminase, and inhibition of infectious virus from sera and livers of infected mice. The minimum effective dose was 4.7 mg/kg per day, with a maximum tolerated dose of 75 mg/kg per day. When the same treatment schedule beginning 4 h pre-virus inoculation, 4 h post-virus inoculation, or 24 h post-virus inoculation was used, orally administered ribavirin was effective at doses as low as 6.3 mg/kg per day. Single s.c. ribavirin treatments at doses of 175 to 700 mg/kg administered from 4 to 48 h post-virus inoculation were also effective. No effect was seen when ribavirin was administered s.c. to mice infected intracerebrally with the PTV strain Balliet, even though treatment was begun 36 h before virus exposure.

Antiviral activities of nucleosides and nucleotides against wild-type and drug-resistant strains of murine cytomegalovirus

Resistance of human cytomegalovirus to approved antiviral drugs is becoming a problem of increasing concern. In order to further study drug resistance in a related virus, strains of murine cytomegalovirus (MCMV) have been prepared in vitro by extensive adaptation of the virus to increasingly higher concentrations of either ganciclovir, foscarnet, or (S)-9-(3-hydroxy-2-[phosphonylmethoxy]propyl)cytosine (HPMPC). Plaque reduction 50% effective concentrations (EC50) for the above inhibitors increased 9-, 7-, and 23-fold, respectively (against the corresponding virus), compared to wild-type MCMV. Each virus was then evaluated against other known anti-MCMV agents to determine cross-resistance patterns. These compounds included 3-hydroxy-phosphonylmethoxypropyl derivatives of adenine (HPMPA) and guanine (HPMPG), 2-phosphonylmethoxyethyl derivatives of adenine (PMEA) and 2,6-diaminopurine (PMEDAP), cyclobutylguanine, acyclovir, and the methylene phosphonate derivatives of acyclovir (SR3722) and ganciclovir (SR3773). The ganciclovir-resistant MCMV was cross-resistant to foscarnet, HPMPA, HPMPC, HPMPG, SR3722, and SR3773. The foscarnet-resistant virus was also resistant to acyclovir, PMEA, PMEDAP, SR3722, and SR3773. The HPMPC-resistant MCMV was cross-resistant to HPMPA, HPMPG, and SR3773. Changes in susceptibility were from 3- to 22-fold relative to the wild-type virus. Virus yield reduction data correlated with the plaque assay results. Only cyclobutylguanine was approximately equally active against wild-type and the three drug-resistant MCMVs. The patterns of cross-resistance correlated with resistance seen in human cytomegalovirus strains expressing altered DNA polymerase function. The GCV-resistant and HPMPC-resistant viruses were markedly attenuated in their ability to kill severe combined immunodeficient mice.

Treatment of lethal pichinde virus infections in weanling LVG/Lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen

A lethal Pichinde (An 4763 strain) virus infection was produced in 3-week-old random-bred Golden Syrian (LVG/Lak strain) hamsters inoculated intraperitoneally with virus, causing mortality in 6-9 days. High virus titers (> or = 10(7.5) cell culture infectious doses/g) were present in visceral organs, serum, brain and salivary glands near the time of death. Intraperitoneal treatments with ribavirin (10 and 32 mg/kg) and ribamidine (32, 100, and 320 mg/kg) for 10 days starting 24 h after virus challenge significantly decreased mortality and reduced virus titers by 100- to > 10,000-fold in liver, spleen, brain, and serum. Serum alanine aminotransferase (an indicator of liver damage) was also reduced in animals treated with the two compounds (ribavirin at 32 mg/kg; ribamidine at 100 and 320 mg/kg). Intraperitoneal selenazofurin (1-100 mg/kg per day for 10 days) and ampligen (0.5 and 5 mg/kg every other day for 5 injections) treatments provided neither protection from the lethal infection nor increased mean survival times. In fact, selenazofurin was overtly toxic, causing death of uninfected hamsters at 32 and 100 mg/kg. The random-bred LVG/Lak hamster appears to be a viable and cost-effective model for evaluating new therapies for arenavirus infections.

Influence of Malnutrition and Alterations in Dietary Protein on Murine Rotaviral Disease

Abstract The possible correlation between malnutrition and degree of severity of rotavirus-associated infantile diarrhea which appears to occur in human populations was studied using a mouse model. To determine the effects of general malnutrition or altered levels of dietary protein, female mice were fed throughout pregnancy and infection periods with diets diluted with 0, 300, or 600 g glucose/kg, designated as normal nutrient to calorie ratio (N/C) diet, 70% N/C diet, or 40% N/C diet or with diets containing 75, 150, or 300 g casein/kg, as low-, normal-, or high-protein diets. Murine rotavirus was given by gavage to the 2-day-old offspring of these dams, and the extent of infection determined. Marked increases in severity of diarrheal disease were seen in the infants from dams receiving the 40 and 70% N/C diets and the low-protein diet. Severity of infection was seen as increased deaths, reduced weight gain, and increased passage of diarrheic feces. Intestinal viral levels and intestinal diarrhea scores did not vary appreciably. Serum interferon remained below detectable limits throughout the studies, but serum antibody was determined in dams 30 days post-virus exposure. The latter titers were lower in the infected mice from dams fed the 40 and 70% N/C diets, but were essentially the same in all the protein diet groups. Cross-fostering was done using the 40 and 100% N/C diets, wherein mice from dams fed either diet were placed on mothers fed the opposite diet. Increased severity of infection was again seen when the virus was given 2 days after the exchange, although the greatest infection occurred in animals from dams fed 40% N/C diet which were then fostered by other similarly fed dams. The increased host sensitivity to the rotaviral infection appeared to be a result of both pre- and postnatal dietary effects.

Antiviral immunotoxins : antibody-mediated delivery of gelonin inhibits Pichinde virus replication in vitro

Immunotoxins were produced and evaluated for antiviral activity against Pichinde virus, a member of the family Arenaviridae. Immunoglobulins were conjugated to the ribosome-inactivating protein, gelonin, through a disulfide linkage to form the immunotoxins. Immunotoxins were produced utilizing monoclonal antibodies, immunoglobulin-binding proteins and hyperimmune sera. An immunotoxin consisting of hyperimmune rabbit sera conjugated with gelonin displayed strong antiviral activity against Pichinde virus, as did a protein G-gelonin indirect immunotoxin in combination with nonconjugated hyperimmune sera. Hyperimmune rabbit sera conjugated with gelonin caused no detectable cytotoxicity in non-infected Vero cells as measured by [3H]leucine incorporation. The 50% effective dose for the immunotoxin was 0.018 microM compared with 86 microM for ribavirin.

Bioassay System for Determining Ribavirin Levels in Human Serum and Urine

A simple, sensitive, micromethod was developed to determine levels of ribavirin, or its active metabolic products, in human serum or urine. The procedure utilized the inhibition of measles virus cytopathic effect in BS-C-1 cells. Based upon maximum dilutions of human serum or urine containing ribavirin which inhibited the measles virus, the bioassay detected ribavirin in concentrations as low as 0.006 microgram/ml in serum or 0.03 microgram/ml in urine. Herpesvirus 1, parainfluenza virus 3 and reovirus 1 were also tested for sensitivity to ribavirin. Minimum inhibitory concentrations of ribavirin in serum or urine against these other viruses were no lower 0.32 microgram/ml.

Effects of folic acid malnutrition on rotaviral infection in mice

Abstract A study was undertaken to determine if dietary deficiencies of folic acid would influence rotaviral diarrheal disease in infant mice. Female mice were fed diets containing essentially no folic acid, 25% of a normal quantity of folic acid, or a normally recommended quantity of folic acid, beginning at time of breeding and continuing through periods of gestation and lactation. Two-day-old infants from these dams were exposed to purified murine rotavirus or to sterile virus diluent and the severity of the rotaviral infection monitored. Infants from the low folic acid group had significantly lower folate levels in their livers, indicating a deficiency was achieved, and developed more severe disease manifestations than those infants from the dams receiving the normal folic acid levels in their diet. The infection enhancement was seen as increased incidences of diarrhea and a significantly greater number of mice exhibiting high intestinal rotaviral antigen titers. Serum rotavirus antibody titers were below detectable levels in a significant number of these same infants.

Selective cytotoxicity towards cytomegalovirus-infected cells by immunotoxins consisting of gelonin linked to anti-cytomegalovirus antibody.

An immunotoxin specific for cells infected with human cytomegalovirus (HCMV) was constructed by attaching the ribosome-inactivating enzyme, gelonin, through a disulfide linkage to polyclonal human immunoglobulin (IgG). In uninfected cells, there was no difference between [35S]methionine incorporation in untreated cultures and those treated with immunotoxin at 100 micrograms/ml. In HCMV-infected cells, there was a significant decrease in [35S]methionine incorporation in the immunotoxin-treated cultures, suggesting a selective cytotoxic effect on the virus-infected cells. An immunotoxin specific for murine cytomegalovirus (MCMV) was prepared by linking gelonin to polyclonal anti-MCMV IgG. Using this same parameter for assay of cytotoxicity, the anti-MCMV immunotoxin had a 50% cytotoxic concentration of 35 micrograms/ml in MCMV-infected cells and greater than 200 micrograms/ml in uninfected cells. MCMV yields measured at 7 days postinoculation were reduced by 2 log10 in cultures treated with immunotoxin at 20 micrograms/ml at 1 day postinoculation. These data suggest immunotoxins may have potential for eliminating CMV-infected cells from the host.

Effects of Monoclonal Antibody Combined with Ganciclovir or (S)-1-[3-Hydroxy-(2-phosphonylmethoxy)-propyl]cytosine against Murine Cytomegalovirus Infections in Cell Culture and in Severe Combined Immunodeficient Mice

The effects of monoclonal antibody used in combination with ganciclovir (GCV) or (S)-1-[3-hydroxy-(2-phosphonylmethoxy)propyl]cytosine (HPMPC) against murine cytomegalovirus (MCMV) were determined in vitro and in vivo, in mice. The antibody and drug were added to cell cultures 24 h after MCMV adsorption so as not to affect the initial infection rate. The antibody (at 1.25-20 micrograms/ml) combined with GCV (0.3-5 microM) or HPMPC (0.008-0.125 microM) caused synergistic inhibition of virus yield in C127I cells. No toxic effect on cell growth in culture was observed at these antibody/drug combinations. The effects of antibody and GCV treatments were studied in MCMV-infected severe combined immunodeficient (SCID) mice. Antibody treatments (2.5 mg/kg/day) given by intraperitoneal injection every 3 days starting 24 h after virus inoculation extended survival time by 1 day relative to placebo-treated animals. Once daily, intraperitoneal treatments with GCV (25 and 50 mg/kg/day) for 7 days starting at 24 h after virus inoculation extended survival time 9-11 days. The combination of antibody plus GCV was only slightly better than GCV alone, indicating an additive interaction.

Combination of antiviral immunotoxin and ganciclovir or cidofovir for the treatment of murine cytomegalovirus infections

The effects of two anti-murine cytomegalovirus (MCMV) immunotoxins used in combination with ganciclovir (GCV) or cidofovir (HPMPC) against MCMV were determined in vitro and in mice. The inhibitors were added to cell cultures 24 or 48 h after MCMV adsorption so as to not affect the initial infection rate. The immunotoxins (0.63, 1.25 and 2.5 micrograms/ml) combined with GCV (1.25, 2.5 and 5 microM) or HPMPC (0.03, 0.06 and 0.12 microM) caused synergistic inhibition of virus yield in C127I cells at most of the combinations tested. No toxic effect on cell growth in culture was observed at these immunotoxin/drug combinations. The effects of immunotoxin and GCV treatment were studied further in MCMV-infected severe combined immunodeficient (SCID) mice. Immunotoxin (1 mg/kg per day) given by intraperitoneal (i.p.) injection on days 1, 4 and 7 of the infection did not extend the mean day to death compared with the placebo group. Once daily i.p. treatment with GCV (50 mg/kg per day) for days starting at 24 h after virus inoculation extended survival time almost 11 days. The combination of immunotoxin plus GCV was better than GCV alone, extending the mean day to death an additional 2 to 3 days, which is suggestive of a synergistic effect.