CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national phase application of PCT/AU2018/050505, filed on May 25, 2018, claiming the benefit of Australian Patent Application No. 2017901985 filed May 25, 2017, both of which are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in the ASCII text file (Name: Substitute Sequence Listing MKPV_ST25.txt; Size: 59,229 bytes; and Date of Creation: Dec. 10, 2021) filed with the application is incorporated herein by reference in its entirety.

FIELD OF THE ART

The present disclosure relates generally to a novel parvovirus that infects mice, typically infecting cells of the kidneys, and to uses of the parvovirus as an animal model of chronic kidney disease, in the screening and identification of therapeutic agents for the treatment of chronic kidney disease, in the development of viral vectors, for the identification of the presence of the virus in animals, and for determining the history of exposure of an animal to the virus.

BACKGROUND

Chronic kidney disease, defined as kidney dysfunction of at least three months duration, affects 3-18% of adults globally. Chronic kidney disease arises from a range of conditions that alter the structure and function of the kidneys, including diabetes mellitus, hypertension, glomerulonephritis and exposure to certain drugs. Irrespective of the underlying source of injury, renal failure in chronic kidney disease occurs as a result of irreversible fibrosis (‘scarring’) of the kidney. The development of effective interventions for renal fibrosis and failure has been hindered by the paucity of animal models of chronic kidney disease.

Viral infections can underlie kidney fibrosis, for example in the setting of kidney transplantation, in which immunosuppression can result in the reactivation of latent viruses within the donor allograft. Viral reactivation may lead to parenchymal cell damage, reactive tissue fibrosis, compromised renal function and eventual loss of the transplant. Polyomavirus-associated nephropathy has emerged as a significant cause of morbidity in transplant patients. The most common infectious agent in this regard is BK virus, a small single-strand DNA virus that propagates within the tubular epithelial cells of the donor kidney. The resultant tubular damage provokes fibrosis and significantly compromises renal function leading to graft loss. Other viruses, such as adenovirus, have also been associated with kidney failure in transplanted patients.

Inclusion body nephritis (tubulointerstitial nephritis) is an idiopathic condition that has been observed in both immunocompetent and immunocompromised mice. Inclusion body nephritis was described by mouse pathologists over 30 years ago. In immunodeficient recombination activating gene (RAG) knockout animals, this disease causes renal failure between 170 and 400 days of life (5-13 months, occasionally younger). The pathological effects of inclusion body nephritis have been previously described in multiple laboratories globally and its high mortality rate in immunocompromised mice makes it particularly devastating to research colonies. While the cause of inclusion body nephritis has remained elusive, it is widely believed to be a degenerative disease, however there is currently no method available to predict or determine susceptible individuals. Methods of detection of the disease in laboratory animals are essential for reliable and reproducible experimentation and to prevent morbidity and mortality of animals. Additionally, due to the chronic and fibrotic state of animals with inclusion body nephritis, such animals may provide a model for human chronic kidney disease.

SUMMARY OF THE DISCLOSURE

Using a metagenomics approach, the present inventors have identified a causative pathogen of murine inclusion body nephritis as a novel virus, termed mouse kidney parvovirus (MKPV), belonging to a previously unclassified genus ofparvoviridae. Based on clinical course, histopathologic features and measurements of biomarkers as exemplified herein, MKPV infection represents a model for chronic tubulointerstitial nephritis in humans. The present disclosure also provides tools deriving from the identification of MKPV, including for the identification of the presence of this virus in mice and other animal species, and for determining the history of exposure to this virus in mice and other animal species. Also provided are uses of MKPV as an animal model of chronic kidney disease, in the screening and identification of therapeutic agents for the treatment of chronic kidney disease, and in the development of viral vectors that use the capsid sequence of, or derived from, MKPV.

According to a first aspect of the present disclosure there is provided a method for detecting the presence of a parvovirus in a sample, comprising detecting one or more nucleic acids or polypeptides derived from the parvovirus, or antibodies against the parvovirus, in the sample, wherein the parvovirus comprises:(i) a gene encoding a non-structural (NS1) protein comprising the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto;(ii) a gene encoding a non-structural (NS2) protein comprising the amino acid sequence set forth in any one of SEQ ID NOs:5 to 7, or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto;(iii) a gene encoding a capsid protein (VP1) comprising the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto; or(iv) the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence comprising at least about 70% sequence identity thereto.

The method may detect the presence of the parvovirus in an environment, an active or latent infection of an animal with the parvovirus or the history of exposure of an animal to the parvovirus.

In an embodiment, the method comprises detecting one or more nucleic acids derived from the parvovirus in the sample. The method may comprise isolating one or more nucleic acids from the sample, amplifying at least one of the nucleic acids; and analyzing the amplified nucleic acids to identify the presence of the parvovirus. The method may comprise polymerase chain reaction-based amplification and analysis. The sample may be a biological sample from an organism or may be an environmental sample. The environmental sample may comprise, for example, environmental air dust.

In an alternative embodiment, the method comprises one or more serological tests or immunoassays to detect one or more antibodies against the parvovirus in the sample.

According to a second aspect, the present disclosure provides an isolated murine parvovirus comprising a gene encoding a non-structural (NS) protein comprising the amino acid sequence set forth in any one of SEQ ID NOs:4 to 7, or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto, wherein said parvovirus is capable of infecting murine kidney cells and causing murine kidney disease.

In one embodiment the parvovirus comprises a gene encoding an NS1 protein comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto. The NS1 gene may comprise the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence at least about 90% identical thereto.

In another embodiment the parvovirus comprises a gene encoding a NS2 protein comprising the amino acid sequence set forth in any one of SEQ ID NOs:5 to 7, or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto. The NS2 gene may comprise the nucleotide sequence set forth in any one of SEQ ID NOs:10 to 12 or a nucleotide sequence at least about 90% identical thereto.

Also provided is an isolated NS1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence comprising at least about 80% amino acid sequence identity to SEQ ID NO:4. Also provided is an isolated NS2 polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:5 to 7 or an amino acid sequence comprising at least about 80% amino acid sequence identity thereto.

According to a third aspect, the present disclosure provides an isolated murine parvovirus comprising a gene encoding a capsid protein VP1 comprising the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence comprising at least about 80% amino acid sequence identity to SEQ ID NO:8, wherein said parvovirus is capable of infecting murine kidney cells and causing murine kidney disease.

Also provided is an isolated VP1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence comprising at least about 80% amino acid sequence identity to SEQ ID NO:8. The VP1 gene may comprise the nucleotide sequence set forth in SEQ ID NO:13 or a nucleotide sequence at least about 90% identical thereto.

According to a fourth aspect, the present disclosure provides an isolated murine parvovirus comprising the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence comprising at least about 70% sequence identity to SEQ ID NO:3, wherein said parvovirus is capable of infecting murine kidney cells and causing murine kidney disease.

In accordance with embodiments of the above aspects, the parvovirus infects mouse kidney cells and causes kidney disease in mice. The murine kidney disease may be a chronic kidney disease. The kidney disease may comprise, or be characterized by, inclusion body nephritis (tubulointerstitial nephritis) or kidney fibrosis.

According to a fifth aspect, the present disclosure provides an isolated host cell infected with the parvovirus of the second, third or fourth aspect, or a host cell comprising a vector comprising one or more nucleotide sequences of said parvovirus.

According to a sixth aspect, the present disclosure provides a murine animal for use as an animal model of kidney disease, wherein the murine animal is infected with the parvovirus as defined in the second, third or fourth aspect.

Typically the animal model displays one or more symptoms of, or histopathologic features characteristic of, or associated with, the kidney disease. The kidney disease may be a chronic kidney disease of humans, optionally immunocompromised humans. The kidney disease may comprise, or be characterized by, human tubulointerstitial nephritis or kidney fibrosis. Symptoms and histopathologic features characteristic of, or associated with, the kidney disease may include tubular epithelial cells with enlarged nuclei (karyomegaly), the formation of eosinophilic intranuclear inclusion bodies in tubular epithelial cells, fibrosis, reduced renal mass, and kidney dysfunction (as determined, for example, by reduced proteinuria, weight loss and/or reduced urinary creatinine levels).

The animal model may be used in the study of pathophysiological features and progression of kidney disease, optionally human kidney disease, including, for example, disease characterized by, or associated with, tubulointerstitial nephritis or kidney fibrosis.

The animal model may be used in the screening of candidate compounds for use in the treatment of kidney disease.

The animal model may be used in the identification of biomarkers of kidney disease.

According to a seventh aspect, the present disclosure provides a method for screening a candidate compound for use in the treatment of kidney disease, comprising: i) administering the candidate compound to a murine animal model infected with the parvovirus as defined in the second, third or fourth aspect, which animal model displays one or more symptoms of, or histopathologic features characteristic of, or associated with, the kidney disease; ii) characterizing the phenotype of the murine animal model after administration of the candidate compound; and iii) selecting the candidate compound that reverses or delays one or more symptoms, or histopathologic features characteristic of or associated with, the kidney disease, as a compound for use in the treatment of kidney disease.

The kidney disease may be a chronic kidney disease of humans, optionally immunocompromised humans. The kidney disease may comprise, or be characterized by, human tubulointerstitial nephritis or kidney fibrosis. Symptoms and histopathologic features characteristic of, or associated with the kidney disease may include tubular epithelial cells with enlarged nuclei (karyomegaly), the formation of eosinophilic intranuclear inclusion bodies in tubular epithelial cells, fibrosis, reduced renal mass, and kidney dysfunction (as determined, for example, by reduced proteinuria, weight loss and/or reduced urinary creatinine levels).

According to an eighth aspect, the present disclosure provides a method for detecting infection of an animal or cell with the parvovirus as defined in the second, third or fourth aspect, the method comprising contacting a biological sample derived from the animal or cell with one or more oligonucleotides specific for at least one target murine kidney parvovirus nucleic acid sequence under conditions sufficient for amplification of at least one target sequence producing a murine kidney parvovirus amplification product.

The cell may be a cell line, such as an immortalized or other laboratory cell line, or may be derived from an animal. In an embodiment, the animal is a murine laboratory animal. Typically the animal is a mouse. In embodiments, the animal may be immunocompromised or immunodeficient. In exemplary embodiments, the biological sample may comprise urine, serum or one or more tubular epithelial cells isolated from the animal.

The at least one target murine kidney parvovirus nucleic acid sequence may comprise NS1, NS2 or VP1 DNA. The at least one primer may comprise a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, or a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. Primers may be designed to amplify a region comprising a sequence encoding the NS1 (SEQ ID NO:4), NS2 (SEQ ID NO:5, 6 or 7) or VP1 (SEQ ID NO:8) protein.

According to a ninth aspect, the present disclosure provides the use of the parvovirus as defined in the second, third or fourth aspect, or one or more viral polypeptides derived therefrom, for the identification of antibodies against the parvovirus in a biological sample.

According to a tenth aspect, the present disclosure provides a vector comprising a nucleic acid molecule comprising a nucleotide sequence as set forth in any one of SEQ ID NOs:9 to 13, or a nucleotide sequence at least about 90% identical thereto.

The vector may be selected from among a plasmid, cosmid, phage, transposon and viral vector. In particular embodiments the vector is a viral vector. Optionally, the vector may further comprise one or more heterologous sequences. The vector may be designed for introduction into kidney cells and to direct or facilitate expression of the one or more heterologous sequences in kidney cells.

According to an eleventh aspect, the present disclosure provides a recombinant virus comprising a capsid protein comprising the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence comprising at least about 80% amino acid sequence identity to SEQ ID NO:8.

In exemplary embodiments, the recombinant virus further comprises one or more heterologous sequences.

According to a twelfth aspect, the present disclosure provides a method for introducing a heterologous sequence into a host cell, comprising contacting a host cell with a vector according to the tenth aspect, wherein the vector comprises the heterologous sequence, or a recombinant virus according to the twelfth aspect, wherein the recombinant virus comprises the heterologous sequence.

In an exemplary embodiment, the host cell may be a kidney cell.

