Bovine viral diarrhea virus (BVDV) is an enveloped, single-stranded, positive sense RNA virus in the genus Pestivirus and the family Flaviviridae. Based on the presence or absence of visible cytopathic effect when susceptible cell monolayers are infected, two pathogenic biotypes of BVDV, referred to as cytopathic and noncytopathic, have been identified. Perdrizet J A in B. P. Smith (ed), Large Animal Internal Medicine, First Edition (Mosby Press, St Louis, 731–737 (1990)). A differentiation is also made between biotypes of BVDV (referred to as biotypes I and II) based on certain viral RNA sequences in the 5′ untranslated region of the genome. Pellerin C, et al., Virology 203, 260–268 (1994); J. F. Ridpath et al., Virology 205, 66–74 (1994).
BVDV may cause acute infection in cattle, resulting in bovine respiratory disease, diarrhea and severe reproductive losses. Clinical symptoms of acute BVDV infection may range from the almost undetectable to the severe. Infection of pregnant cows and heifers may result in breeding problems (e.g., irregular heats), abortion, premature births or the birth of weak or stunted calves. In some cases, temporary damage to an animal's immune system may occur even when the clinical symptoms are not apparent. In addition to the illness caused by the virus itself, infected animals are more susceptible and are more likely to suffer from other diseases, such as pneumonia.
In addition to causing acute disease, BVDV may also establish persistent infections. Potgieter, Vet. Clin. North Am. Food Anim. Pract 11, 501–520 (1995). Persistent BVDV infections are generally established via in utero infection of a developing fetus with a noncytopathic BVDV. The resulting animals are born immunotolerant of the particular BVDV by which they are infected, and may continually shed virus throughout their life span. While some persistently infected animals exhibit congenital malformations due to BVDV infection, many animals persistently infected with BVDV appear clinically normal. Baker, Rev. Sci. Tech 9, 25–41 (1990); Bielefeldt-Ohmann, Vet. Clin. North Am. Food Anim. Pract 11, 447–476 (1995). Persistently infected animals are thought to be the major disseminators of BVDV in the cattle population.
There are more than 140 vaccines against BVDV commercially available in the United States. Bolin, Am J. Vet Res. 46, 2476–2470 (1995). Unfortunately, vaccination does not provide complete protection against BVDV infection, as some vaccinated cattle still become infected with the virus. At present, there is no known cure for BVDV infection. Accordingly, a need exists for an effective treatment for BVDV infection.
In vitro production of embryos has become a useful therapy for increasing reproductive performance of animals and for treating infertility of both, animals and humans. In vitro production of bovine embryos could permit the humane, world-wide transfer of genetic material among cattle while limiting the transmission of many pathogens. However, in vitro-produced bovine embryos are potential vectors for transmission of BVDV. B. Avery et al., Vet Rec 132, 660 (1993); A. Bielanski et al., Theriogenology 46, 1467–1476 (1996); T. Tsuboi et al., Vet Microbiol 49, 127–134 (1996); O. Zurovac et al., Theriogenology 41, 841–853 (1994). BVDV can be introduced into the embryo production system in association with gametes, serum, somatic cells, cumulus oocyte complexes (COCs), and result in contaminated in vitro fertilized (IVF) embryos or cell lines. K. V. Brock et al., J Vet Diagn Invest 3, 99–100 (1991); C. R. Rossi et al., Am J Vet Res 41, 1680–1681 (1980); P. J. Booth et al., J Reprod Fert Abstr Ser Suppl 9, 28 (1992); M. D. Fray et al., Vet Pathol 35, 253–259 (1998); R. Harasawa et al., Microbiol Immunol 39, 979–985 (1995); T. Shin et al., Theriogenology 53, 243 (2000). Association of noncytopathic BVDV with transferred IVF embryos may cause infection of embryo recipients, early embryonic death, abortion or birth of persistently infected offspring.
An analogous hazard exists in human in vitro embryo production. Viral transmission to human embryos and embryo recipients by means of contaminated embryo culture media has been reported. Addition of an anti-viral agent to the culture medium surrounding in vitro-produced embryos could prevent or reduce transmission of virus to the embryo or embryo recipient. P. M. Grosheide et al., Vaccine 9, 682–687 (1991); W. G. Quint et al., J Clin Microbiol 32, 1099–1100 (1994); H. C. van Os et al., Am J Obstet Gynecol 165, 152–159 (1991). Accordingly, an antiviral agent that could be added to both animal and human in vitro embryo production systems may have important applications.
The organization of the portion of the BVDV genome that encodes the proteins used in viral replication is very similar to that of human hepatitis C virus (HCV), another flavivirus. S. W. Behrens et al., J Virol 72, 2364–2372 (1998). It is believed that more than 80% of the individuals infected with HCV will eventually develop a chronic form of the disease. As the disease develops, the liver of the infected subject is progressively damaged, with the symptoms generally being commensurate with cirrhosis and liver failure (e.g., jaundice, abdominal swelling, and finally, coma). The cycle of disease from infection to significant liver damage can take 20 years or more. Liver failure due to HCV is the presently the leading cause of liver transplants in the United States. It is suspected that there are, at present, more than 5 million people in the United States that are infected with HCV, and perhaps as many as 200 million around the world, making HCV infection a significant public health threat.
The development of a vaccine for HCV infection is uncertain, due in part to the high mutation rate of the virus. Recombinant interferon alpha-2b (INTRON A®/Schering) has proved effective in some cases of chronic hepatitis C. However, it has been reported that relapse occurs in at least half the responders after the interferon alpha-2b treatment is discontinued. Additionally, interferon alpha-2b may exacerbate hepatocyte injury caused by autoimmune chronic active hepatitis. J. Y. N. Lau et al., Br Med J. 306, 469–470 (1993). The nucleotide analog ribavirin (VIRAZOLE®/ICN Pharmaceuticals) has been shown to reduce concentrations of hepatitis C viral RNA in an infected subject, although at a slower rate than interferon alpha-2b. As with BVDV infection, a need exists for an effective treatment for HCV infection.