Cultivation of human noroviruses

Patent metadata

Field	Value
Publication number	US-11396645-B2
Application number	US-202017000587-A
Country	US
Kind code	B2
Filing date	Aug 24, 2020
Priority date	Oct 2, 2015
Publication date	Jul 26, 2022
Grant date	Jul 26, 2022

Abstract

Embodiments of the disclosure concern systems, methods, and/or compositions for cultivation of mammalian viruses, including at least human noroviruses and sapoviruses within the Caliciviridae family of viruses. The ex vivo culture systems include intestinal enteroids in combination with bile or a functionally active fraction or component thereof. In specific embodiments, the culture system is utilized to test inactivation compounds for therapeutic or environmental efficacy and to test contaminated comestibles and/or environmental entities for determination of the presence of infectious virus. Furthermore, antiviral compositions may be tested using systems of the disclosure, including drugs, small molecule inhibitors, and biologics such as neutralizing monoclonal antibodies.

First claim

What is claimed is:

1. A method of culturing a virus of the Caliciviridae family, comprising the step of subjecting the virus to a system under suitable conditions, wherein the system comprises:
a) mammalian small intestinal enteroid cultures; and
b) bile or a functionally active fraction or component thereof.

2. The method of claim 1 , wherein the enteroid cultures are plated in a monolayer as differentiated cells prior to exposure to the virus.

3. The method of claim 1 , wherein the bile is combined with the enteroid culture prior to, during, and/or after exposure of the system to the virus.

4. The method of claim 1 , wherein the bile is combined with the enteroid culture prior to, during, and after exposure of the system to the virus.

5. The method of claim 1 , wherein media for the culture comprises one or more growth factors.

6. The method of claim 5 , wherein the one or more growth factors are selected from the group consisting of Wnt3A, nicotinamide, R-spondin-1, noggin, epidermal growth factor, gastrin, laminin-□□1, laminin-□2, an inhibitor of Alk, an inhibitor of p38, fibroblast growth factor 10, and a combination thereof.

7. The method of claim 1 , wherein infection of the culture cells by the virus is monitored by assaying viral nucleic acid(s) levels, identifying nucleic acids, assaying viral protein(s) levels, identifying proteins, and/or assaying cytopathic changes in the enteroids plated in a monolayer.

8. The method of claim 7 , wherein the nucleic acid(s) levels and/or their identity are assayed by quantitative reverse transcription-polymerase chain reaction, hybridization, and/or sequencing.

9. The method of claim 7 , wherein infection of the cultured cells by the virus is monitored by assaying viral protein(s) levels and/or identifying the viral protein(s).

10. The method of claim 9 , wherein the protein(s) levels and/or their identity are assayed by antibody.

11. The method of claim 10 , wherein the antibody is labeled.

12. The method of claim 9 , wherein the protein(s) levels and/or their identity are assayed by electron microscopy, ELISA, western blot, mass spectrometry, or a combination thereof.

13. The method of claim 9 , wherein the viral protein(s) is a structural protein, non-structural protein, or a combination thereof.

14. The method of claim 1 , wherein the virus is obtained from a sample from human clinical samples, samples from non-human mammals, environmental surfaces, comestibles, and/or liquids.

15. The method of claim 14 , wherein the genomic sequence of the virus from the sample is compared to the genomic sequence of a virus cultivated by the method.

16. The method of claim 1 , further comprising the step of obtaining a sample that comprises the virus.

17. The method of claim 1 , wherein the enteroid cultures are derived from tissue from an individual that has a functional fucosyltransferase 2 (FUT2), fucosyltransferase 3 (FUT3), and/or ABH glycans.

18. The method of claim 1 , wherein the virus is GII.4 variant human norovirus.

19. The method of claim 1 , wherein the virus is GII.3, GI.1, GII.4, GII.17, GII.6, GII.8, GII.12, or GII.14 strain human norovirus.

20. A method of culturing a noncultivatable human enteric virus, comprising the step of subjecting the virus to a system under suitable conditions, wherein the system comprises:
a) mammalian small intestinal enteroid cultures; and
b) bile or a functionally active fraction or component thereof.

21. A method of culturing a virus of the Caliciviridae family, comprising the step of subjecting the virus to a system under suitable conditions, wherein the system comprises:
a) mammalian gastroid or colonoid cultures; and
optionally b) bile or a functionally active fraction or component thereof.

Assignees

• Baylor College Medicine

Inventors

• Estes Mary K
• Graham David
• Atmar Robert Legare
• Crawford Sue Ellen
• Ettayebi Khalil
• Murakami Kosuke

Classifications

• A61K31/575 Primary

  substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title

• C12N5/0679

  Cells of the gastro-intestinal tract · CPC title

• C12N2770/16051

  Methods of production or purification of viral material · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title