Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-10-29
2003-06-03
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S353000, C435S235100
Reexamination Certificate
active
06573041
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in general, to neurally-differentiated cells infected with viruses in a manner that supports a long-term non-productive infection for experimentation concerning the reactivation, induction, suppressing of virus latency.
BACKGROUND OF THE INVENTION
Herpes simplex virus types 1 and 2 (HSV-1 and -2) are alphaherpesviruses with similar, but unique molecular (Kieff et. al., 1971, 1972), biological and clinical features (reviewed in Whitley, 1996). The genomes are approximately 150 kb in size and each contains corresponding sets of 74 genes (Dolan et al, 1998). Both viruses infect epithelium, with HSV-1 having a predilection for orofacial sites and HSV-2 preferentially infecting genital surfaces. During the primary infection, HSV invades local nerve endings and travels to sensory ganglia where it can colonize neuronal nuclei and establish a latent state (
Hill
et al, 1972, Stevens and Cook, 1971). Reactivation of HSV from latency occurs intermittently as a result of stressful stimuli (e.g., trauma and heat). Reactivated viruses are responsible for causing recurrent epithelial infections that can occur in up to 89% of infected individuals (Benedetti, et. al. 1994).
The lack of a universally accepted neural cell culture model that supports HSV latency, in particular, HSV-2, restricts our understanding of the molecular events involved in reactivation from latency. Although animal models reproduce certain aspects of HSV-2 latency in humans (Al-Saadi et al, 1988; Bourne et al, 1994; Croen et al, 1991; Krause et al, 1995; Kurata et al, 1978; MacLean et al, 1991; Martin and Suzuki, 1989; Mitchell et al, 1990; Stanberry et al, 1982; Stephanopoulos et al, 1988; Wang et al, 1997; Yoshikawa et al, 1996), limitations in these models make interpretation of reactivation data challenging. Animal models limitations include: (i) latency and reactivation events that are influenced by viral strains with different primary growth phenotypes, (ii) the limited number of neurons latently infected in animal models (Bloom et al, 1996; Hill et al, 1996; Maggioncalda et al, 1996; Mehta et al, 1995; Ramakrishnan 1994; Sawtell, 1997; Sawtell et al, 1998; Thompson and Sawtell, 1997), and (iii) inaccurate quantitation of reactivation events when measuring virus production at the recurrent site as a result of influences of transport, replication in epithelium, and the immune response.
In response to these limitations, tissue and cell culture models of HSV-2 latency have been developed in an attempt to overcome limitations of animal models. A major advantage of tissue and cell culture models includes the ability to observe virus at the single cell level without the overlay of immunological events that modulate the eventual appearance of virus in the host. In addition, tissue culture models derived from neuronal and sympathetic ganglia have properties of the in vivo system including: (i) restricted transcription of the HSV genome (Doerig et al, 1991; Halford et al, 1996; Smith et al, 1992; Smith et al, 1994), (ii) lack of virus production following removal of the inhibitory agent, (Wilcox and Johnson, 1988) (iii) the presence of latency-associated transcripts (LATs) (Doerig et al, 1991; Smith et al, 1994), (iv) impaired reactivation of thymidine kinase negative virus (Wilcox et al, 1992), and (v) inducible reactivation (Halford et al, 1996; Moriya et al, 1994; Smith et al, 1992; Wilcox and Johnson, 1988; Wilcox and Johnson 1987; Wilcox et al, 1990). Nevertheless, tissue culture models have their drawbacks, preparation of dissected ganglia is inconvenient, material is limited, animal use is required, and axotomy introduces traumatic factors that influence reactivation of virus.
Accordingly, development of cell culture models with neuronal characteristics that lack the restrictive requirements of tissue culture models would be advantageous for understanding the molecular mechanisms of the establishment, maintenance and reactivation stages of HSV latency. Cell culture models also allow for an unlimited supply of a defined host cell and the ability to manipulate genetic material.
Over the past 25 years, cell culture systems using fibroblast cultures (Harris and Preston, 1991; Jamieson et al, 1995; O'Neill, 1977; O'Neill et al, 1972; Russell et al, 1987; Scheck et al, 1989; Wigdahl et al, 1982a; Wigdahl et al, 1982b; Wigdahl et al, 1983) and lymphocytes (Hammer et al, 1981; Youssoufian et al, 1982) have enabled the study of HSV-1 during a latent-like state. These models, however required low input multiplicities and/or the use of replication inhibitors such as anti-viral agents, inhibitory temperatures, or the use of a mutant virus, to prevent virus production. A cell line that has neural morphology and physiology, can survive infection and permit viral production, allow establishment of a long term nonproductive viral infection, and support virus in a state suitable for reactivation studies would be more desirable.
More recently, it has been reported that neurally-differentiated PC12 (ND-PC12) cells can harbor HSV-1 in a quiescent, yet reversible state (Danaher et. al., 1999a). These quiescently infected ND-PC12 cultures (QIF-PC12) demonstrate forskolin- and heat stress (HS)-inducible virus production in a high percentage (50-100%) of cultures for up to 8 weeks after infection, whereas mock-induced cultures maintain the quiescent viral state in the majority of infected cultures (Danaher et al, 1999b). In contrast to these cell culture models, the present invention, however, does not require antiviral conditions to maintain and/or establish the latent-like state (Colberg-Poley et al, 1979, 1981; Harris et al, 1989; Kondo et al, 1990; O'Neill, 1977; O'Neill et al, 1972; Russell et al, 1987; Russell and Preston, 1986; Wigdahl et al, 1981; Wilcox and Johnson, 1988; Wilcox et al, 1990; Yura et al, 1986).
The present application demonstrates that ND-PC12 cells permit establishment of an HSV-2 quiescent state, like HSV-1, following transient acycloguanosine (ACV) treatment. Unlike HSV-1, however, antiviral conditions are not required for the establishment of the HSV-2 quiescent state. In addition, the present invention discloses quiescent cultures in the presence of Vero cells, and the presence of Vero cells enhances the sensitivity to detect HSV-2 produced spontaneously and following induction (i.e., forskolin and HS treatment). Thus, the present invention demonstrates that ND-PC 12 cells can harbor HSV-2, like HSV-1, in a cryptic and non-productive state that is reversible, and this model has appealing features for studying gene induction during the establishment and maintenance of virus latency and the activation of HSV-2 from a nonproductive state.
SUMMARY OF THE INVENTION
A primary object of the present application is to provide neurally-differentiated cells infected with viruses in a manner that supports a long-term non-productive infection for experimentation. Another object of the present invention also provides a cell culture research model for HSV-2 quiescent infection in ND-PC12 cells to investigate quiescent and reactivation properties of HSV-2. This model represents an improvement over existing cell culture models for HSV-1. Advantages of this quiescently infected PC12 cell culture model include: (1) establishment and maintenance of a HSV-2 quiescent infection in a high proportion of PC12 cell cultures with and without the transient use of ACV (acyclovir); (2) the ability to produce HSV-2 from a quiescent state in response to forskolin and HS treatment for at lest 4 weeks post infection; and (3) the ability to discriminate between quiescence, spontaneous reactivation and inducible reactivation using a range of multiplicities of infection (MOIs). Thereby, these enhanced features of the invention enable analysis of the establishment and maintenance of latency and the reactivation events of a cryptic HSV genome at the single neural cell level in vitro.
Accordingly, additional objects of the present invention provide for methods of establishing
Danaher Robert J.
Jacob Robert J.
Miller Craig S.
Mosher Mary E.
University of Kentucky Research Foundation
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