Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-29
2004-05-25
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S022100
Reexamination Certificate
active
06740490
ABSTRACT:
SEQUENCE LISTING
A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette and a CDROM.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed towards the screening of different pig breeding lines by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CD 151. Selection of lines which are genetically low in or do not possess CD 151 produces offspring which are less susceptible or not susceptible at all to infection by porcine reproductive and respiratory syndrome virus (PRRSV). The present invention also concerns non-invasive methods for screening live pigs and swine germplasm for susceptibility to PRRSV. Additionally, the invention describes the development of cell lines for propagating high titer stocks for making killed and modified live virus vaccines in non-simian cell lines. Moreover, the invention describes a novel plasmid, useful in transforming previously non-susceptible cell lines into susceptible ones for producing the titer stocks used in vaccines. The invention also pertains to providing a tool for discovery of a novel class of anti-viral compounds that will block the interaction between CD 151 and PRRSV 3′-UTR RNA. This novel class of compounds has been termed anti-RNA Entry Proteins (anti-REPs).
2. Description of the Prior Art
Porcine reproductive and respiratory syndrome (PRRS) is a RNA virus which emerged in the late 1980's as an important viral disease of swine. PRRS is the most important swine health problem today. The origin and evolution of PRRSV is not known. Thus, it is a novel emerging virus of pigs. This disease, which has previously been referred to as “mystery swine disease”, “swine infertility and respiratory syndrome”, or “blue ear disease,” is causing heavy losses in breeding herds of the United States and Canada. A similar disease has also appeared in much of Europe and the virus has been detected worldwide. The disease is manifested in two forms, one causing severe reproductive failure in pregnant sows, manifested in the form of premature farrowings, increased numbers of stillborn, mummified and weak-born pigs, decreased farrowing rate, and delayed return to estrus and the other producing respiratory distress in pigs evidenced by lesions that appear in the lungs of infected swine.
The reproductive form of the disease is described by Keffaber, K.K., “
Reproductive Failure of Unknown Etiology
”, American Association of Swine Practitioners Newsletter, 1:109 (1989). The most prominent clinical symptoms of the reproductive form of the disease are spontaneous late-term abortions, premature births (which can be as high as 20-30% of all births) and the farrowing of mummified fetuses, stillborn or sickly piglets. Such clinical symptoms will typically be observed in a herd from 4-16 weeks, or even longer. Stillborn fetuses in affected litters often are in the early stages of mummification, as evidenced by tan-brown discoloration of the skin and post-mortem autolysis. Dome-shaped malformations of fetal skulls is also sometimes seen. The infection of sows may go unnoticed, or may manifest itself by an impaired general condition lasting up to a few days. For example, the sows may go off feed, and experience body temperatures either above or below normal. In the farrowing phase, the sows may exhibit depression, lethargy, pyrexia and occasional vomiting. In some affected herds, up to 75% of all piglets may be lost. The economic consequences of the disease, accordingly, are devastating.
The respiratory form of the disease exhibits clinical signs which are most pronounced in piglets of 3-8 weeks in age, but are reported to occur in pigs of all ages in infected herds. The diseased piglets grow slowly, have roughened hair coats, respiratory distress (“thumping”) and increased mortality (up to about 80% pre-weaning mortality). To combat the problems associated with infection by PRRSV, vaccines have been developed in an attempt to confer immunity to the current PRRSV strains. The present vaccines are only marginally effective and are all produced in cell lines of simian origin which possessed a risk of continuous introduction of primate viruses in swine populations. Findings in preliminary studies of gross and microscopic lesions of piglets affected with the respiratory form of the disease suggest that microscopic lung lesions are an important clinical feature of this disease.
PRRSV belongs within the order Nidovirales in the family Arteriviridae. Other members in the family include equine arteritis virus, lactate dehydrogenase elevating virus, and simian hemorrhagic fever virus. Currently, there is no known human arterivirus. The PRRSV genome is a positive sense RNA about 15.1 kb in length. Untranslated regions (UTR's) of 156-220 nucleotides at the 5′ end and 59-117 nucleotides at the 3′ end flank the viral genome. The viral genome has eight overlapping open reading frames encoding functional and structural proteins. PRRSV grows primarily in the macrophages of infected pigs. In cell culture, the virus is known to grow in CL 2621, MA-104, MARC cell lines and in primary cultures of porcine alveolar macrophages. All of these continuous cell lines are of simian origin and pose a risk of introduction of primate viruses into swine populations. Entry of the virus occurs by receptor-mediated endocytosis and the receptor has been characterized as a heparin-like molecule. However, the mechanisms of how viral RNAs enter after endocytosis into the cell cytoplasm are not known.
Replication of arteriviruses is similar to that of the coronaviruses. Genomic and subgenomic (−) sense mRNAs are formed in the infected cells along with the (+) sense mRNAs. Subgenomic (−) sense mRNAs have been shown to function as the principal templates for mRNA synthesis in coronaviruses. Discontinuous transcription occurs in arteriviruses with the formation of a functionally monocistronic, 3′-coterminal, nested set of mRNAs. The common leader is joined to the coding region by consensus intergenic sequences through the junction sequence UCAACC. In mouse hepatitis virus (MHV), a corona virus, interaction of the leader, intergenic sequence and the body of the RNA involves cis and trans acting elements. The 3′UTR's of MHV, coxsackie-, rhino- and polioviruses were shown to be essential for the transcription of the genome. There is little sequence complementarity between the 3′UTR and upstream regulatory sequences for interaction, therefore it may be mediated through RNA-protein-RNA interactions involving the viral or cellular proteins.
There are numerous studies on the interactions of viral and cellular proteins with 5′ and 3′ UTR's of viruses. Sindbis virus, brome mosaic virus, QB phage and polioviruses require the interaction of certain host cell proteins with viral UTR's for transcription to proceed. Although numerous proteins have been shown to bind to these regulatory regions, only a few of them have been characterized. La protein, poly (rC) binding protein 2, and polypyrimidine tract binding protein are shown to bind to UTR's of poliovirus. Polypyrimidine tract binding protein and heterogeneous nuclear ribonucleoprotein have been shown to bind to the leader sequence of MHV. These proteins are predicted to play a role in mRNA splicing and transportation. Some cytoskeletal and chaperone proteins, like actin, tubulin, and heat shock proteins, are shown to have RNA binding activity. These proteins might play a structural role in viral RNA synthesis or in orienting and transporting the RNA replication complexes to the site of replication.
The study of host cell proteins that interact with viral RNAs is still in the infancy stage and there is a lack of important information regarding this interaction, especially with respect to host-susceptibility factors. Even in arteriviruses, such as PRRSV, the host susceptibility factors have not been stu
Kapil Sanjay
Shanmukhappa Kumar
Hovey & Williams, LLP
Kansas State University Research Foundation
Tung Joyce
Whisenant Ethan
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