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Employing human adipose-derived stem cells to propagate serum-derived hepatitis c virus and use thereof

A kind of technology of hepatitis C virus and stem cells, which is applied in the system field of multiplying hepatitis C virus

Active Publication Date: 2017-09-26
VANWORLD PHARMA (RUGAO) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the extent to which these data can be extrapolated to actual clinical virus-host interactions remains a concern

Method used

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  • Employing human adipose-derived stem cells to propagate serum-derived hepatitis c virus and use thereof
  • Employing human adipose-derived stem cells to propagate serum-derived hepatitis c virus and use thereof
  • Employing human adipose-derived stem cells to propagate serum-derived hepatitis c virus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1: HCV targets hADSCs in vivo.

[0128] Clinically, an interesting feature of HCV infection is that HCV(+) patients can have excessive fat accumulation in the chronically infected liver, known as hepatic steatosis 46，47 , the severity of hepatic steatosis appears to correlate with the rate of hepatic fibrosis 48 . Recent studies also demonstrate that HCV RNA replication can be stimulated by increased availability of saturated fatty acids and inhibited by polyunsaturated fatty acids or inhibitors of fatty acid synthesis 49，50 . These findings suggest that fat metabolism plays an important role in the life cycle of HCV. We therefore hypothesized that cellular components of adipose tissue may be involved in HCV infection in vivo.

[0129] To test our hypothesis, we harvested subcutaneous adipose tissue from HCV-infected or uninfected individuals (Table 1) and extracted RNA for RT-PCT of HCV-specific 5'-UTR transcripts using HCVser genotype 1b (HCVser-1b) was u...

Embodiment 2

[0142] Example 2: Naive HCV (-) hADSCs are sensitive to HCVser infection and replication in vitro

[0143] To examine whether naive HCV(-) hADSCs are sensitive to HCVser infection and replication in vitro, we prepared hADSCs from HCV(-) individuals and subcultured them. Suspended passage 3 (p-3) or p-4 cells (in Eppendorf tubes) were incubated with HCVser (Table 2) at 0.2 moi in a final volume of 1 ml (i.e. 1x10 5 5'-UTR copy number vs. 5x10 5 hADSC cells).

[0144] Table 2. HCV genotypes and 5'-UTR copy numbers of HCV(+) sera used in this study. All patients had no evidence of HIV or hepatitis B virus infection. They also had no signs of acute infectious disease. Sera were collected from September 2011 to February 2014 and used immediately or stored at -80°C until use. Sera from patient numbers 1-5 were used for figure 2 A-D, and remaining serum for figure 2 E-F, image 3 and Figure 4 .

[0145]

[0146]

[0147] After 3 h, the cells were washed and transfe...

Embodiment 3

[0157] Example 3: The virions produced by hADSCs are true "virions" exhibiting the biological properties of clinical isolates

[0158] To examine the infectivity of hADSC-produced virions (labeled "HCVadsC"), we infected p2 hADSCs from "Donor 1" with HCVser-1b and collected the supernatant on day 21 (labeled "HCVadsc(1) "). After filtration through a 0.22-μm pore filter, HCVadsc(1) was used to infect hADSCs of "Donor 2" to prepare HCVadsc(2), which was subsequently used to infect hADSCs of "Donor 3". The results confirmed that HCVadsc was infectious to naive hADSCs from different donors, with a relatively consistent replication efficiency as seen in the initial infection with HCVser ( image 3 A). Similar observations were also obtained for HCVadsc derived from supernatants of hADSCs initially infected with HCVser-la, -2a and -2b (data not shown).

[0159] We next investigated the permissibility of hADSCs at different passage numbers by infecting p2, p6, p9 and p15 hADSCs w...

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Abstract

Hepatitis C virus replication at extrahepatic sites has been suggested; however, complete viral replication has only been confirmed in hepatocytes. Here we show that human adipogenic DLK-1+stem cells (hADSC) freshly isolated from HCV-infected individuals contained viral transcripts, replication intermediates and viral antigens in vivo, and viral transcripts increased in supernatants upon prolonged ex vivo culture. Furthermore, naive hADSC isolated from HCV(-)individuals support complete replication of clinical isolates in vitro, and the infection is donor-nonspecific for cells and cross-genotypic for viruses. Viral infection / replication is mediated through CD81, LDL-R, SR-B1, EGFR, Apolipoprotein E, occludin, claudin-1, NPC1L1 and diacylglycerol acetyltransferase-1, and can be inhibited by anti-viral drugs. In addition, the physical properties of hADSC-propagated viral particles resemble clinical isolates more than JFH1 / HCVcc, and viruses propagated by in vitro infected hADSC are infectious to primary human hepatocytes. Therefore, hADSC are an in vivo HCV reservoir and represent a novel venue of clinical virus-host interaction. hADSC can also be exploited as a physiologically relevant primary cell culture system to propagate clinical isolates.

Description

field of invention [0001] The present invention relates to systems and methods for propagating hepatitis C virus (HCV) and uses thereof. Background of the invention [0002] HCV is an enveloped positive-sense RNA virus of the family Flaviviridae. It contains a 9.6kb genome that begins with an untranslated region (5'-UTR) followed by coding structural proteins (core, E1 and E2) and nonstructural (NS) including p7, NS2, NS3, NS4 and NS5 The sequence of the protein (for a review, see ref. 1，2 ). Unlike hepatitis B virus (HBV) infection, HCV infection is a multifaceted disease with both intrahepatic and extrahepatic manifestations 3 , and HCV can reside in non-hepatocytes 4-6 , which may play a role in viral persistence and reactivation. However, in vitro systems for studying extrahepatic replication of HCV are severely limited. As far as primary cells are concerned, direct infection with serum-borne HCV (HCVser) has only been demonstrated in human and chimpanzee hepatocyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N7/00
CPCC12N7/00C12N2770/24251G01N33/5767C12N5/0667C12N2502/70C12Q1/707C12Q2600/136G01N33/5073G01N2333/186
Inventor 林成龙
Owner VANWORLD PHARMA (RUGAO) CO LTD
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