Patent Application
20230174953
This invention was made with government support under DARPA agreement number HR00111720023 awarded by the Defense Advanced Research Projects Agency (DARPA).Viruses that possess an RNA genome have polymerases with an intrinsically high error rate. As a consequence of this error prone replication process, these viruses do not only generate full-length viral genomes or genomes with point mutations, but furthermore generate defective genomes. Various types of defective genomes have been described, which include truncations, insertions, deletions, mosaic or rearranged genomes and copyback/snapbacks. While the majority of defective genomes are thought to be dead-end side-products of RNA virus replication, a subset of these defective viral genomes (DVGs) interfere with the generation of infectious progeny virus particles to high numbers.
Along with high mutation rates, recombination is another main driving force of RNA virus evolution. Non-homologous recombination can give rise to truncated and/or rearranged viral genomes that constitute defective viral genomes (DVGs). First described in 1954 by Von Magnus in influenza A virus, DVGs have since been described in all viral families. Because they lack part of their genome or its encoded functions, DVGs must co-infect cells with their parental virus, in order to take advantage of the proteins encoded by the full-length virus. Hence, DVGs that hijack the replication machinery or use proteins encoded by the parental virus, may compete with wild type virus for resources, which can result in inhibition of the parental virus. These types of DVGs are often referred to as defective interfering particles (DIPs). Furthermore, many DVGs have a strong, immunostimulatory potential both in mammals and invertebrates. DVGs were identified in sera from patients suffering from acute dengue virus infection, but their pathophysiological role remains unknown. In humans, their presence correlates with milder disease and better outcome in influenza virus and respiratory syncytial virus infections. All these reasons justify renewed interest in using DVGs as antiviral therapy.
While DVGs have been described to exist for most viruses, DVGs were not considered to be relevant or predominant in in many virus families. Generally, only a few DVGs have been described for any given virus. These previous reports relied on more classic methods of isolation, such as PCR amplification, that select for only one or two DVGs and bias towards the shortest or most abundant DVGs. These DVGs are not necessarily the best candidates in terms of ability to compete with wild type virus. A need thus exists for reproducible and rational methods to identify and generate the best DVG candidates to compete and interfere with wild type virus infection, that could be applied to any virus of interest, especially human pathogens. The inventions disclosed herein meet these and other needs.
Our combined experimental evolution and computational approach identifies thousands of different DVGs for any virus. First, our experimental approach generates all possible DVGs in a virus population. Expectedly, our list includes DVGs that resemble those identified by more classic methods. Second, our computational analysis allows us to predict which DVGs would work best as defective interfering particles, using temporal frequencies estimated from the sequence data. Using this combined approach, we have generated a list of top candidates for a wide range of viruses belonging to different virus families, of which up to 90% have confirmed capacity to interfere with wild type virus replication.
This disclosure provides methods of generating DVGs and also provides new DVGs generated by the methods. DVGs generated from chikungunya virus (CHIKV), Zika virus (ZIKV), enterovirus 71 (EV71), West Nile virus (WNV), yellow fever virus (YFV) and rhinovirus (RV), and coronavirus (CV) (for example, the recently identified novel coronavirus named SARS-CoV-2, 2019-nCov or COVID-19) are provided.
The inventors have identified DVGs using next-generation sequencing methods followed by computational analysis, hotspot analysis identified a variety of DVGs that were deletion DVGs (internal deletions in the viral genomes of CHIKV, EV71, ZIKV as well as RV). Subsequently, these DVGs were genetically engineered and tested for their ability to reduce infectious viral titers of CHIKV, EV71, ZIKV as well as RV. Successful DVG candidates have the potential to be used as therapeutic interfering particles (TIPs), which could be used as a new strategy to treat and limit the severity of virus infections. Furthermore, DVGs for WNV and YFV were also identified. Based on success with other viruses, these DVGs have potential to be used as TIPs.
In a first aspect, a method for producing a defective interfering viral genome (DVG) is provided. The method comprises providing a first set of at least three replicate in vitro cell cultures, comprising a solid support and cells infected with a reference infectious virus at a high multiplicity of infection (MOI); providing a second set of at least three replicate in vitro cell cultures, comprising a solid support and cells infected with the reference infectious virus at a low multiplicity of infection (MOI); culturing the first and second sets of replicate in vitro cell cultures for at least 5 passages under normal growth conditions; collecting a plurality of DVG candidates from the medium of the first and second sets of in vitro replicate cell cultures; deep sequencing the collected DVG candidates; and selecting a subset of DVGs from the plurality of DVG candidates.
In some embodiments the method further comprises providing a third set of at least three replicate in vitro cell cultures, comprising a solid support and cells infected with a reference infectious virus at a high multiplicity of infection (MOI); and/or providing a fourth set of at least three replicate in vitro cell cultures, comprising a solid support and cells infected with the reference infectious virus at a low multiplicity of infection (MOI); and culturing the third and/or fourth sets of replicate in vitro cell cultures for at least 5 passages under mutagenic conditions ; or using a reference infectious virus with a mutator phenotype to perform said passages.
In some embodiments selecting a DVG from the plurality of DVG candidates comprises: comparing the sequences of the DVG candidates with the sequence of the genome of the reference infectious virus; identifying at least one DVG candidate having a genome comprising at least one splicing event where the splicing event is a deletion or a rearrangement; determining the relative frequency of at least one DVG candidate having a genome with at least one splicing event among the population of sequenced DVG candidate genomes; and, selecting at least one DVG for which: a) at least one splicing event is more abundant at high MOI cultures than at low MOI cultures; and/or b) at least one splicing event appears in at least 2, 3, 4, 5, 6, 7, 8 or 9 different cell lines; and/or, c) at least one splicing event is found in at least 3 of at least 12, 24 or 36 independent replicates, after at least 5, 10, 15 or 20 passages; and/or, d) at least one splicing event is a deletion event and the nucleotide size of the deletion is at least 40, 42, 50, 100, 150, 200, 400, 600 or 1000 nucleotides; and/or, e) an open reading frame in the DVG is maintained; and/or, f) the structural domains and functions necessary for viral replication, are maintained in the DVG; and/or g) the DVG comprises at least one mutation that reduces the self-replicative capacity of the reference infectious virus.
In some embodiments the DVG is characterized by an in vitro inhibitory activity of at least 50%, 60%, 70%, 80% or 90% against the reference infectious virus. In some embodiments the reference infectious virus is a chikungunya virus (CHIKV), Zika virus (ZIKV), enterovirus 71 (EV71), rhinovirus (RV), yellow fever virus (YFV) or West Nile virus (WNV). In some embodiments the reference infectious virus is selected from the group consisting of: CHIKV Indian Ocean lineage (GenBank accession no. AM258994), CHIKV Caribbean strain (GenBank accession no. LN898104.1), ZIKV African strain MR766 (KY989511.1), EV71 Sep006 (GenBank: KX197462.1), RV-A01a (NC_038311.1), RV-A16 (L24917.1), RV-B14 (L05355.1), RV-C15 (GU219984.1), West Nile virus Israel 1998 and YFV strain, Yellow Fever Virus Asibi (YF-Asibi) strain and Yellow Fever Virus vaccine (YF-17D) strain. In some embodiments the reference infectious virus is SARS-CoV-2 (Zhu N et al., N Engl J Med., 2020 Jan 24). In some embodiments the cells are a mammalian cell line selected from the group consisting of: a cell line derived from African green monkey kidney cells (optionally Vero, optionally Vero-E6 cell line), a cell line derived from human muscle (optionally RD cell line), a cell line derived from baby hamster kidneys (optionally BHK cell line), a cell line derived from human embryonic kidney cells (optionally HEK293T cell line) a cell line derived from liver carcinoma cells (optionally Huh7 cell line), and a cell line derived from cervical adenocarcinoma (optionally H1-HeLa, optionally HeLa-E8 cell line). In some embodiments the cells are mosquito a cell line selected from the group consisting of: a cell line derived from Aedes aegypti (optionally Aag2 cell line), a cell derived from Aedes albopictus (optionally C6/36 cell line) and a cell line derived from Aedes albopictus (optionally U4.4 cell line). In some embodiments the low MOI for infection is from 0.001 to 0.1 PFU/cell, in particular from 0.01 to 0.1 PFU/cell. In some embodiments the high MOI for infection is from 1 to 100 PFU/cell.
Also provided is a DVG produced by the method of this invention.
Also provided is a DVG comprising or consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 (DG1 : EV71 DVG 293-390), SEQ ID NO: 2 (DG2 : EV71 DVG 294-404), SEQ ID NO: 3 (DG3 : EV71 DVG 1746-2895), SEQ ID NO: 4 (DG4 : EV71 DVG 1752-2894), SEQ ID NO: 5 (DG5 : EV71 DVG 1752-2896), SEQ ID NO: 6 (DG6 : EV71 DVG 1880-6487), SEQ ID NO: 7 (DG7 : EV71 DVG 1880-6488), SEQ ID NO: 8 (DG8 : EV71 DVG 3513-6516), SEQ ID NO: 9 (DG9 : EV71 DVG 5475-5634), SEQ ID NO: 10 (DG10: EV71 DVG 5610-6876), SEQ ID NO: 11 (DG11 : EV71 DVG 5610-6877), SEQ ID NO: 12 (DG12: EV71 DVG 5746-5821), SEQ ID NO: 13 (DG13 : EV71 DVG 6322-6356), SEQ ID NO: 14 (DG14: EV71 DVG 6322-6358), SEQ ID NO: 15 (DG15: EV71 DVG 6728-6779), SEQ ID NO: 16 (DG16: EV71 DVG 6966-7012), SEQ ID NO: 17 (DG17: EV71 DVG 6966-7014), SEQ ID NO: 18 (DG18: EV71 DVG 7098-7122), SEQ ID NO: 19 (DG19: EV71 DVG 7165-7200), SEQ ID NO: 20 (DG20: EV71 DVG 7165-7201), SEQ ID NO: 21 (DG21: EV71 DVG 7238-7292), SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 23 (ZIKV DVG-B), SEQ ID NO: 24 (ZIKV DVG-C), SEQ ID NO: 25 (ZIKV DVG-D), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 27 (ZIKV DVG-F), SEQ ID NO: 28 (ZIKV DVG-G), SEQ ID NO: 29 (ZIKV DVG-H), SEQ ID NO: 30 (ZIKV DVG-I), SEQ ID NO: 31 (ZIKV DVG-J), SEQ ID NO: 32 (ZIKV DVG-K), SEQ ID NO: 33 (ZIKV DVG-L), SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 45 (CHIKV DVG-CH2), SEQ ID NO: 46 (CHIKV DVG-CH3), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3), SEQ ID NO: 53 (CHIKV DVG-CA4), SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 56 (RV DVG A01a-TIP-02), SEQ ID NO: 57 (RV DVG A01a-TIP-03), SEQ ID NO: 58 (RV DVG A01a-TIP-04), SEQ ID NO: 59 (RV DVG A01a-TIP-05), SEQ ID NO: 60 (RV DVG A01a-TIP-06), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 63 (RV DVG A16-TIP-02), SEQ ID NO: 64 (RV DVG A16-TIP-03), SEQ ID NO: 65 (RV DVG A16-TIP-04), SEQ ID NO: 66 (RV DVG A16-TIP-05), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 90 (RV DVG C15-TIP-02), SEQ ID NO: 91 (RV DVG C15-TIP-03), SEQ ID NO: 92 (RV DVG C15-TIP-04), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 69 (RV DVG B14-TIP-02), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 71 (RV DVG B14-TIP-04), SEQ ID NO: 72 (RV DVG B14-TIP-05), SEQ ID NO: 73 (RV DVG B14-TIP-06), SEQ ID NO: 74 (RV DVG B14-TIP-07), SEQ ID NO: 75 (RV DVG B14-TIP-08), SEQ ID NO: 76 (RV DVG B14-TIP-09), SEQ ID NO: 77 (RV DVG B14-TIP-10), SEQ ID NO: 78 (RV DVG B14-TIP-11), SEQ ID NO: 79 (RV DVG B14-TIP-12), SEQ ID NO: 80 (RV DVG B14-TIP-13), SEQ ID NO: 81 (RV DVG B14-TIP-14), SEQ ID NO: 82 (RV DVG B14-TIP-15), SEQ ID NO: 83 (RV DVG B14-TIP-16), SEQ ID NO: 84 (RV DVG B14-TIP-17), SEQ ID NO: 85 (RV DVG B14-TIP-18), SEQ ID NO: 86 (RV DVG B14-TIP-19), SEQ ID NO: 87 (RV DVG B14-TIP-20), SEQ ID NO:93 (YFV DVG-A), SEQ ID NO: 94 (YFV DVG-B), SEQ ID NO: 95 (YFV DVG-C), SEQ ID NO: 96 (YFV DVG-D), SEQ ID NO: 97 (YFV DVG-E), SEQ ID NO: 98 (YFV DVG-F), SEQ ID NO: 99 (YFV DVG-G), SEQ ID NO: 100 (YFV DVG-H), SEQ ID NO: 101 (YFV DVG-I), SEQ ID NO: 102 (YFV DVG-J), SEQ ID NO: 103 (YFV DVG-K), SEQ ID NO: 104 (YFV DVG-L), SEQ ID NO:105 (WNV DVG-1), SEQ ID NO: 106 (WNV DVG-2), SEQ ID NO: 107 (WNV DVG-3), SEQ ID NO: 108 (WNV DVG-4), SEQ ID NO: 109 (WNV DVG-5), SEQ ID NO: 110 (WNV DVG-6), SEQ ID NO: 111 (SARS-CoV-2 DVG_1), SEQ ID NO: 112 (SARS-CoV-2 DVG_2) and SEQ ID NO: 113 (SARS-CoV-2 DVG_3), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering particle comprising a DVG of this invention.
Also provided are methods of treating a viral infection in a subject, comprising administering an efficient therapeutic amount of at least one DVG or defective interfering particle according to this invention.
In some embodiments the DVG is administered as RNA, naked RNA or RNA formulated with appropriate carriers such as nanoparticles or lipids.
In some embodiments the DVG is administered as a packaged RNA, as a virus-like particle (VLP) or other packaging carrier.
In some embodiments the DVG is transcribed from a DNA under the control of a host-appropriate transcriptional promoter.
In some embodiments the at least one DVG is a defective interfering CHIKV genome. In some embodiments the at least one defective interfering CHIKV genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 45 (CHIKV DVG-CH2), SEQ ID NO: 46 (CHIKV DVG-CH3), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3) and SEQ ID NO: 53 (CHIKV DVG-CA4), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering ZIKV genome. In some embodiments the at least one interfering ZIKV genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 23 (ZIKV DVG-B), SEQ ID NO: 24 (ZIKV DVG-C), SEQ ID NO: 25 (ZIKV DVG-D), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 27 (ZIKV DVG-F), SEQ ID NO: 28 (ZIKV DVG-G), SEQ ID NO: 29 (ZIKV DVG-H), SEQ ID NO: 30 (ZIKV DVG-I), SEQ ID NO: 31 (ZIKV DVG-J), SEQ ID NO: 32 (ZIKV DVG-K) and SEQ ID NO: 33 (ZIKV DVG-L), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering EV71 genome. In some embodiments the at least one defective interfering EV71 genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 (DG1 : EV71 DVG 293-390), SEQ ID NO: 2 (DG2 : EV71 DVG 294-404), SEQ ID NO: 3 (DG3 : EV71 DVG 1746-2895), SEQ ID NO: 4 (DG4 : EV71 DVG 1752-2894), SEQ ID NO: 5 (DG5 : EV71 DVG 1752-2896), SEQ ID NO: 6 (DG6 : EV71 DVG 1880-6487), SEQ ID NO: 7 (DG7 : EV71 DVG 1880-6488), SEQ ID NO: 8 (DG8 : EV71 DVG 3513-6516), SEQ ID NO: 9 (DG9 : EV71 DVG 5475-5634), SEQ ID NO: 10 (DG10: EV71 DVG 5610-6876), SEQ ID NO: 11 (DG11 : EV71 DVG 5610-6877), SEQ ID NO: 12 (DG12: EV71 DVG 5746-5821), SEQ ID NO: 13 (DG13 : EV71 DVG 6322-6356), SEQ ID NO: 14 (DG14: EV71 DVG 6322-6358), SEQ ID NO: 15 (DG15: EV71 DVG 6728-6779), SEQ ID NO: 16 (DG16: EV71 DVG 6966-7012), SEQ ID NO: 17 (DG17: EV71 DVG 6966-7014), SEQ ID NO: 18 (DG18: EV71 DVG 7098-7122), SEQ ID NO: 19 (DG19: EV71 DVG 7165-7200), SEQ ID NO: 20 (DG20: EV71 DVG 7165-7201) and SEQ ID NO: 21 (DG21: EV71 DVG 7238-7292), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering RV genome. In some embodiments the at least one defective interfering RV genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 56 (RV DVG A01a-TIP-02), SEQ ID NO: 57 (RV DVG A01a-TIP-03), SEQ ID NO: 58 (RV DVG A01a-TIP-04), SEQ ID NO: 59 (RV DVG A01a-TIP-05), SEQ ID NO: 60 (RV DVG A01a-TIP-06), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 63 (RV DVG A16-TIP-02), SEQ ID NO: 64 (RV DVG A16-TIP-03), SEQ ID NO: 65 (RV DVG A16-TIP-04), SEQ ID NO: 66 (RV DVG A16-TIP-05), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 90 (RV DVG C15-TIP-02), SEQ ID NO: 91 (RV DVG C15-TIP-03), SEQ ID NO: 92 (RV DVG C15-TIP-04), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 69 (RV DVG B14-TIP-02), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 71 (RV DVG B14-TIP-04), SEQ ID NO: 72 (RV DVG B14-TIP-05), SEQ ID NO: 73 (RV DVG B14-TIP-06), SEQ ID NO: 74 (RV DVG B14-TIP-07), SEQ ID NO: 75 (RV DVG B14-TIP-08), SEQ ID NO: 76 (RV DVG B14-TIP-09), SEQ ID NO: 77 (RV DVG B14-TIP-10), SEQ ID NO: 78 (RV DVG B14-TIP-11), SEQ ID NO: 79 (RV DVG B14-TIP-12), SEQ ID NO: 80 (RV DVG B14-TIP-13), SEQ ID NO: 81 (RV DVG B14-TIP-14), SEQ ID NO: 82 (RV DVG B14-TIP-15), SEQ ID NO: 83 (RV DVG B14-TIP-16), SEQ ID NO: 84 (RV DVG B14-TIP-17), SEQ ID NO: 85 (RV DVG B14-TIP-18), SEQ ID NO: 86 (RV DVG B14-TIP-19) and SEQ ID NO: 87 (RV DVG B14-TIP-20), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering YFV genome. In some embodiments the at least one defective interfering YFV genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:93 (YFV DVG-A), SEQ ID NO: 94 (YFV DVG-B), SEQ ID NO: 95 (YFV DVG-C), SEQ ID NO: 96 (YFV DVG-D), SEQ ID NO: 97 (YFV DVG-E), SEQ ID NO: 98 (YFV DVG-F), SEQ ID NO: 99 (YFV DVG-G), SEQ ID NO: 100 (YFV DVG-H), SEQ ID NO: 101 (YFV DVG-I), SEQ ID NO: 102 (YFV DVG-J), SEQ ID NO: 103 (YFV DVG-K) and SEQ ID NO: 104 (YFV DVG-L), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering WNV genome. In some embodiments the at least one defective interfering WNV genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:105 (WNV DVG-1), SEQ ID NO: 106 (WNV DVG-2), SEQ ID NO: 107 (WNV DVG-3), SEQ ID NO: 108 (WNV DVG-4), SEQ ID NO: 109 (WNV DVG-5) and SEQ ID NO: 110 (WNV DVG-6), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
In some embodiments the at least one defective interfering genome is a defective interfering CV genome, for example a defective interfering SARS-CoV-2 genome. In some embodiments the at least one defective interfering SARS-CoV-2 genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 111 (SARS-CoV-2 DVG_1), SEQ ID NO: 112 (SARS-CoV-2 DVG_2) and SEQ ID NO: 113 (SARS-CoV-2 DVG_3), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of CHIKV infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 45 (CHIKV DVG-CH2), SEQ ID NO: 46 (CHIKV DVG-CH3), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3) and SEQ ID NO: 53 (CHIKV DVG-CA4), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of ZIKV infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 23 (ZIKV DVG-B), SEQ ID NO: 24 (ZIKV DVG-C), SEQ ID NO: 25 (ZIKV DVG-D), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 27 (ZIKV DVG-F), SEQ ID NO: 28 (ZIKV DVG-G), SEQ ID NO: 29 (ZIKV DVG-H), SEQ ID NO: 30 (ZIKV DVG-I), SEQ ID NO: 31 (ZIKV DVG-J), SEQ ID NO: 32 (ZIKV DVG-K) and SEQ ID NO: 33 (ZIKV DVG-L), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of EV71 infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 (DG1 : EV71 DVG 293-390), SEQ ID NO: 2 (DG2 : EV71 DVG 294-404), SEQ ID NO: 3 (DG3 : EV71 DVG 1746-2895), SEQ ID NO: 4 (DG4 : EV71 DVG 1752-2894), SEQ ID NO: 5 (DG5 : EV71 DVG 1752-2896), SEQ ID NO: 6 (DG6 : EV71 DVG 1880-6487), SEQ ID NO: 7 (DG7 : EV71 DVG 1880-6488), SEQ ID NO: 8 (DG8 : EV71 DVG 3513-6516), SEQ ID NO: 9 (DG9 : EV71 DVG 5475-5634), SEQ ID NO: 10 (DG10: EV71 DVG 5610-6876), SEQ ID NO: 11 (DG11 : EV71 DVG 5610-6877), SEQ ID NO: 12 (DG12: EV71 DVG 5746-5821), SEQ ID NO: 13 (DG13 : EV71 DVG 6322-6356), SEQ ID NO: 14 (DG14: EV71 DVG 6322-6358), SEQ ID NO: 15 (DG15: EV71 DVG 6728-6779), SEQ ID NO: 16 (DG16: EV71 DVG 6966-7012), SEQ ID NO: 17 (DG17: EV71 DVG 6966-7014), SEQ ID NO: 18 (DG18: EV71 DVG 7098-7122), SEQ ID NO: 19 (DG19: EV71 DVG 7165-7200), SEQ ID NO: 20 (DG20: EV71 DVG 7165-7201) and SEQ ID NO: 21 (DG21: EV71 DVG 7238-7292), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of RV infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 56 (RV DVG A01a-TIP-02), SEQ ID NO: 57 (RV DVG A01a-TIP-03), SEQ ID NO: 58 (RV DVG A01a-TIP-04), SEQ ID NO: 59 (RV DVG A01a-TIP-05), SEQ ID NO: 60 (RV DVG A01a-TIP-06), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 63 (RV DVG A16-TIP-02), SEQ ID NO: 64 (RV DVG A16-TIP-03), SEQ ID NO: 65 (RV DVG A16-TIP-04), SEQ ID NO: 66 (RV DVG A16-TIP-05), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 90 (RV DVG C15-TIP-02), SEQ ID NO: 91 (RV DVG C15-TIP-03), SEQ ID NO: 92 (RV DVG C15-TIP-04), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 69 (RV DVG B14-TIP-02), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 71 (RV DVG B14-TIP-04), SEQ ID NO: 72 (RV DVG B14-TIP-05), SEQ ID NO: 73 (RV DVG B14-TIP-06), SEQ ID NO: 74 (RV DVG B14-TIP-07), SEQ ID NO: 75 (RV DVG B14-TIP-08), SEQ ID NO: 76 (RV DVG B14-TIP-09), SEQ ID NO: 77 (RV DVG B14-TIP-10), SEQ ID NO: 78 (RV DVG B14-TIP-11), SEQ ID NO: 79 (RV DVG B14-TIP-12), SEQ ID NO: 80 (RV DVG B14-TIP-13), SEQ ID NO: 81 (RV DVG B14-TIP-14), SEQ ID NO: 82 (RV DVG B14-TIP-15), SEQ ID NO: 83 (RV DVG B14-TIP-16), SEQ ID NO: 84 (RV DVG B14-TIP-17), SEQ ID NO: 85 (RV DVG B14-TIP-18), SEQ ID NO: 86 (RV DVG B14-TIP-19) and SEQ ID NO: 87 (RV DVG B14-TIP-20), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of YFV infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:93 (YFV DVG-A), SEQ ID NO: 94 (YFV DVG-B), SEQ ID NO: 95 (YFV DVG-C), SEQ ID NO: 96 (YFV DVG-D), SEQ ID NO: 97 (YFV DVG-E), SEQ ID NO: 98 (YFV DVG-F), SEQ ID NO: 99 (YFV DVG-G), SEQ ID NO: 100 (YFV DVG-H), SEQ ID NO: 101 (YFV DVG-I), SEQ ID NO: 102 (YFV DVG-J), SEQ ID NO: 103 (YFV DVG-K) and SEQ ID NO: 104 (YFV DVG-L), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of WNV infection to a subject, wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:105 (WNV DVG-1), SEQ ID NO: 106 (WNV DVG-2), SEQ ID NO: 107 (WNV DVG-3), SEQ ID NO: 108 (WNV DVG-4), SEQ ID NO: 109 (WNV DVG-5) and SEQ ID NO: 110 (WNV DVG-6), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a defective interfering genome DVG or defective interfering particle of the invention for use in a method of treatment of a CV infection to a subject, wherein the CV infection is caused by SARS-CoV-2, and wherein the defective interfering genome comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 111 (SARS-CoV-2 DVG_1), SEQ ID NO: 112 (SARS-CoV-2 DVG_2) and SEQ ID NO: 113 (SARS-CoV-2 DVG_3), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is a pharmaceutical, immunogenic, or therapeutic composition, comprising at least one DVG according to the invention, and a pharmaceutically acceptable carrier.
Also provided is a pharmaceutical, immunogenic, or therapeutic composition, comprising at least one defective interfering particle according to the invention, and a pharmaceutically acceptable carrier.
Also provided is a vaccine comprising an immunogenic composition of the invention.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with a target virus of the DVG or defective interfering particle.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with CHIKV.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with ZIKV.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with EV71.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with RV.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with YFV.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with WNV.
Also provided is the use of a DVG of the invention, or a defective interfering particle of the invention, for the preparation of a drug for the treatment of a patient infected with CV, in particular by SARS-CoV-2.
Also provided is a polynucleotide comprising or consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 (DG1 : EV71 DVG 293-390), SEQ ID NO: 2 (DG2 : EV71 DVG 294-404), SEQ ID NO: 3 (DG3 : EV71 DVG 1746-2895), SEQ ID NO: 4 (DG4 : EV71 DVG 1752-2894), SEQ ID NO: 5 (DG5 : EV71 DVG 1752-2896), SEQ ID NO: 6 (DG6 : EV71 DVG 1880-6487), SEQ ID NO: 7 (DG7 : EV71 DVG 1880-6488), SEQ ID NO: 8 (DG8 : EV71 DVG 3513-6516), SEQ ID NO: 9 (DG9 : EV71 DVG 5475-5634), SEQ ID NO: 10 (DG10: EV71 DVG 5610-6876), SEQ ID NO: 11 (DG11 : EV71 DVG 5610-6877), SEQ ID NO: 12 (DG12: EV71 DVG 5746-5821), SEQ ID NO: 13 (DG13 : EV71 DVG 6322-6356), SEQ ID NO: 14 (DG14: EV71 DVG 6322-6358), SEQ ID NO: 15 (DG15: EV71 DVG 6728-6779), SEQ ID NO: 16 (DG16: EV71 DVG 6966-7012), SEQ ID NO: 17 (DG17: EV71 DVG 6966-7014), SEQ ID NO: 18 (DG18: EV71 DVG 7098-7122), SEQ ID NO: 19 (DG19: EV71 DVG 7165-7200), SEQ ID NO: 20 (DG20: EV71 DVG 7165-7201), SEQ ID NO: 21 (DG21: EV71 DVG 7238-7292), SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 23 (ZIKV DVG-B), SEQ ID NO: 24 (ZIKV DVG-C), SEQ ID NO: 25 (ZIKV DVG-D), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 27 (ZIKV DVG-F), SEQ ID NO: 28 (ZIKV DVG-G), SEQ ID NO: 29 (ZIKV DVG-H), SEQ ID NO: 30 (ZIKV DVG-I), SEQ ID NO: 31 (ZIKV DVG-J), SEQ ID NO: 32 (ZIKV DVG-K), SEQ ID NO: 33 (ZIKV DVG-L), SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 45 (CHIKV DVG-CH2), SEQ ID NO: 46 (CHIKV DVG-CH3), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3), SEQ ID NO: 53 (CHIKV DVG-CA4), SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 56 (RV DVG A01a-TIP-02), SEQ ID NO: 57 (RV DVG A01a-TIP-03), SEQ ID NO: 58 (RV DVG A01a-TIP-04), SEQ ID NO: 59 (RV DVG A01a-TIP-05), SEQ ID NO: 60 (RV DVG A01a-TIP-06), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 63 (RV DVG A16-TIP-02), SEQ ID NO: 64 (RV DVG A16-TIP-03), SEQ ID NO: 65 (RV DVG A16-TIP-04), SEQ ID NO: 66 (RV DVG A16-TIP-05), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 90 (RV DVG C15-TIP-02), SEQ ID NO: 91 (RV DVG C15-TIP-03), SEQ ID NO: 92 (RV DVG C15-TIP-04), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 69 (RV DVG B14-TIP-02), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 71 (RV DVG B14-TIP-04), SEQ ID NO: 72 (RV DVG B14-TIP-05), SEQ ID NO: 73 (RV DVG B14-TIP-06), SEQ ID NO: 74 (RV DVG B14-TIP-07), SEQ ID NO: 75 (RV DVG B14-TIP-08), SEQ ID NO: 76 (RV DVG B14-TIP-09), SEQ ID NO: 77 (RV DVG B14-TIP-10), SEQ ID NO: 78 (RV DVG B14-TIP-11), SEQ ID NO: 79 (RV DVG B14-TIP-12), SEQ ID NO: 80 (RV DVG B14-TIP-13), SEQ ID NO: 81 (RV DVG B14-TIP-14), SEQ ID NO: 82 (RV DVG B14-TIP-15), SEQ ID NO: 83 (RV DVG B14-TIP-16), SEQ ID NO: 84 (RV DVG B14-TIP-17), SEQ ID NO: 85 (RV DVG B14-TIP-18), SEQ ID NO: 86 (RV DVG B14-TIP-19), and SEQ ID NO: 87 (RV DVG B14-TIP-20), SEQ ID NO:93 (YFV DVG-A), SEQ ID NO: 94 (YFV DVG-B), SEQ ID NO: 95 (YFV DVG-C), SEQ ID NO: 96 (YFV DVG-D), SEQ ID NO: 97 (YFV DVG-E), SEQ ID NO: 98 (YFV DVG-F), SEQ ID NO: 99 (YFV DVG-G), SEQ ID NO: 100 (YFV DVG-H), SEQ ID NO: 101 (YFV DVG-I), SEQ ID NO: 102 (YFV DVG-J), SEQ ID NO: 103 (YFV DVG-K), SEQ ID NO: 104 (YFV DVG-L), SEQ ID NO:105 (WNV DVG-1), SEQ ID NO: 106 (WNV DVG-2), SEQ ID NO: 107 (WNV DVG-3), SEQ ID NO: 108 (WNV DVG-4), SEQ ID NO: 109 (WNV DVG-5), SEQ ID NO: 110 (WNV DVG-6), SEQ ID NO: 111 (SARS-CoV-2 DVG_1), SEQ ID NO: 112 (SARS-CoV-2 DVG_2) and SEQ ID NO: 113 (SARS-CoV-2 DVG_3), or having at least 70% identity, more preferably at least 80% identity ; more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, or more preferably at least 99% identity with one of these sequences.
Also provided is an expression vector or a plasmid comprising a polynucleotide of the invention.
Also provided is a cell line producing a DVG according to the invention.
The invention will be more fully understood by reference to the following examples, which are non-limiting.
Traditionally, DVGs were observed when virus is passaged at high multiplicity of infection (MOI). Most published work is based on identifying DVGs from few passages and few replicates, resulting in the identification of a single most abundant DVG, generally by methods that biased towards identifying the shortest DVGs with the largest deletions (e.g. by RT-PCR amplification using primers spanning the 5′ and 3′ ends of RNA genomes). This led to the notion that only a few DVG are generated for any given virus. We find that DVGs number is in the hundreds to thousands, in terms of sequence; and that the best candidate DVGs for interference are not necessarily the single most abundant DVG that appears in a single growth condition. In our method, wild-type virus is passaged under both low (control) and high MOI conditions, as well as in mutagenic conditions that we have found to increase the generation of defective genomes. These mutagenic conditions include either mutator variants of viruses that generate more errors than the native wild-type virus, or extrinsic treatments that increase these errors rates (for example, base analogs such as ribavirin, 5-fluorouracil, 5-azacytidine, T705 or other treatments such as increasing temperature or supplementing media with Mn++). Viruses are passaged in high biological replicate numbers (3, 12, 24 or 36) for up to 10 passages. We observed that 5 passages suffice to generate the required data. We also recommend using a variety of cell types or different hosts, since we observe differences in the types of DVGs that are favored between cell lines and hosts.
The viruses from the passage series are deep sequenced to characterize every deletion or rearrangement that occurs across the entire genome. The number of reads describing a specific deletion/rearrangement relative to the total number of reads at that position is used as a proxy of the frequency at which that deletion is present in the entire virus population. Hundreds to thousands of different DVGs are found for any given virus using this method. We use computational tools to identify which of these DVGs occur with high frequency and abundance, and which are redundant between replicates, to select the 10-20 best candidates for further testing.
We use the following criteria and rationale to predict which DVGs would make the best TIP candidates, based on the assumption that a good candidate will have relatively high replication fitness compared to wild-type, will be encapsidated or passaged from cell to cell alongside wild-type, will compete and interfere with wild-type, will retain domains and functions required for propagation, will retain fitness in different cell types and hosts:
DVGs that occur at higher frequency in a population, are most likely to be viable (able to replicate or be replicated by wild-type virus) and have a higher likelihood of competing with wild-type virus.
DVGs that occur across a passage series, are most likely to be able to compete with wild-type, and be encapsidated into VLPs, to infect new cells.
DVGs that occur in more than three biological replicates, are more likely to have a high enough fitness to compete with wild-type, and likely have retained the structural and functional elements required for replication and propagation.
DVGs that occur in more than one cell type or host, are more likely to be viable in different cell conditions.
DVGs that occur at higher frequency in high MOI or mutagenic conditions versus low MOI conditions, are more likely to be true competitors with wild-type virus.
Additionally, we match the nucleotide position of start and stop of the deletion (the breakpoints) with the predicted RNA structure of the genome (See Example 2,
We observed that DVGs may interfere and operate by more than one mechanism. The classic view is that shorter DVGs steal replication machinery from wild-type and outcompete wild-type because their shorter genomes replicate faster. However, additional mechanisms that we have observed include: DVGs that alter the stoichiometry of viral proteins available to wild-type by providing more of some viral proteins than is normally required; DVGs that generate misfolded proteins that induce cellular responses and danger signals; DVGs that encode faulty proteins that result in non-viable wild-type viruses by ‘poisoning’ wild-type replication and assembly; DVGs that induce innate immunity and other cellular responses because they are more readily sensed as danger signals. Ideal DVGs could present one, some or all of these mechanisms or be composed of a cocktail of DVGs each manifesting some of these mechanisms.
Using this approach, we have obtained the total DVG profiles (hundreds to thousands) of the following viruses and strains:
From these lists, using the selection criteria mentioned above, we identified the top candidates for each type of virus. The top sequences of the top candidates are provided in attached information sequence listing. Once the list of top candidates is established, infectious clones of DVGs are made and tested in vitro, and then in vivo. Our results show that this method of identification and down-selection has a very high success rate, where 50-90% of candidates show inhibitory activity against wild-type virus in vitro.
Examples of the method are shown in
A 12-well plate was seeded with Vero cells to reach ~90% confluency the next day. The 12 wells were individually transfected with 500 ng pCAGGS-EV71_Sep006 (EV71 Sep006 - GenBank: KX197462.1), using Lipofectamine 2000 (Life Technologies), to generate infectious virus from a stable background. Subsequently Vero cells were infected with 300 µl of the virus supernatant. Or 12-well plates of Vero or RD cells were infected either with a fixed low volume (3 µl) or a high volume (300 µl). Cells were incubated at 37° C., and 5% CO2 until wells showed >90% CPE. After two freeze-thaw cycles fresh confluent monolayers of cells were infected. This was repeated 12 times using 12 replicates.
The supernatant was processed by centrifugation (13,000 g for 30 min), followed by either RNaseA treatment (1 µl; Thermo; 3 h at 37° C.) or by PEG-precipitation (10 mM HEPES (Sigma), pH 8.0, 0.8% PEG8000 (Sigma), and 50 mM NaCl (Sigma)), followed by another centrifugation of 13,500 x g for 1 h. The RNA was extracted from the cleared supernatant using Trizol (Sigma).
Sample RNA was fragmented using the NEBNext RNA First Strand Buffer (NEB) and heat-fragmentation at 94° C. for 15 min. The RNAseq libraries were generated the NEBNext Ultra II Non-Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions. To allow multiplexing of the samples, NEBNext Multiplex Oligos for Illumina (NEB) were used to generate indexes. The generated libraries were pooled and sequenced on an Illumina NextSeq500 Sequencer using Illumina NextSeq 500/550 Mid Output Reagent Cartridge v2 and Illumina NextSeq 500/550 Mid Output Flow Cell Cartridge v2.5 using 150 cycle single-end reads. The obtained sequences were analyzed using the BBMap suite-based computational pipeline.
Initially identified DVGs were analyzed by reoccurrence between replicates, passages, and cell lines. This yielded a list of 15 DVGs that were taken forward to be analyzed for interference activity and thus, whether these DVGs could be used as TIPs. Of the 15 identified DVGs, 6 resulted in a deletion that would result in a nonsense mutation after the breakpoint. As a control, the deletion length of these 6 DVGs were adjusted to remain the open reading frame. Thus, the candidate list assessed was 21 DVGs.
The deletion candidates, as well as other DVGs, were further examined to determine whether the deleted sequences that were observed, correlated with either specific nucleotide signatures or RNA structures.
The EV71 DGV sequences are listed in the following table.
Preferred EV71 DVGs are selected from the group consisting of: SEQ ID NO: 1 (EV71 DVG 293-390,DG1), SEQ ID NO: 3 (EV71 DVG 17646-2895, DG3), SEQ ID NO: 14 (EV71 DVG 6322-6358, DG14), SEQ ID NO: 15 (EV71 DVG 6728-6779, DG15), SEQ ID NO: 18 (EV71 DVG 7098-7122, DG18), and SEQ ID NO: 21 (EV71 DVG 7238-7292, DG21).
24-wellplates with 293T cells were seeded to reach ~90% confluency the following day. 200 ng of pCAGGS-EV71_Sep006 were transfected either alone, with a control plasmid (pcDNA3-RFP) or the individual DVGs. The other plasmids were mixed with the wild-type EV71 recovery plasmid at the molarity ratios of 1:1, 5:1, or 10:1 (DVG to WT). Transfections were performed in replicates of four. The plasmids were transfected using Lipofectamine 2000 (Life Technologies), media was changed after 6 hours and samples were harvested 4 days post transfection. Virus titers were assessed either by TCID50 or by plaque assay. In brief, for TCID50 96-well plates were seeded with Vero cells and serially diluted virus supernatants, which were freeze-thawed twice, where added and samples were incubated at 37° C., and 5% CO2 for 7 days. For plaque assays, virus was titrated in 24-well plates using VeroE6 cells that were seeded 24 h prior to infections. After 1 h incubation of the 10-fold serial diluted virus samples, the cells were covered with a semi-solid agarose overlay using 2% FBS (LifeTechnologies), MEM (LifeTechnologies), and 0.05% Agarose (Thermo). The plates were incubated at 37° C., and 5% CO2 for 72 h. For both methods cells for fixed with 4% Formalin and plaques were visualized using crystal violet.
Method: The virus used for the identification of DVGs is the African strain MR766 of Zika virus (ZIKV). In order to identify DVGs, ZIKV was passaged at a high multiplicity of infection in Vero, BHK or C6/36 cells. Briefly, cells that were seeded to reach between 70-80% confluence were infected with an initial MOI of 5 PFU/cell. 3 (Vero, BHK) or 5 (C6/36) days post infection, the cell culture supernatant was clarified by centrifugation, and 300 ul of the supernatant used to infect naive cells (passage 2). For low MOI conditions, 3 ul of the viral supernatant was used for infection in the subsequent passage. The passaging was repeated 12 times. RNA was extracted from the cleared supernatant (TRIzol) and used for next-generation sequencing. Libraries prepared using the RNA Library Prep kit (Illumina) were loaded on an Illumina NextSeq500 sequencer, using 150 cycle single-end reads. The reads obtained were analyzed using the computational pipeline described above.
Deep sequencing of samples and computational analysis identified all deletion hotspots across the genome and specific DVGs occurring within the population. In
From these and other analyses, we selected a list of TIP candidates following our criteria for selection. The candidates were named A, B, C, D, E, F, G, H, K and L. The candidates span a variety of sizes and the deletions occur in different places in the genome, representing a wider array of TIP candidates than could be identified using conventional passaging and isolation.
Method: DG K-based replicon constructs were designed to assess the ability of the DG to replicate. Nanoluc reporter constructs were generated in which the reporter is inserted in place of the structural proteins, as is normally designed for flavivirus replicons (
The results show that unlike the WT replicon for which an increase in reporter activity is observed over the time course, the DG K replicon showed no increase (
Method: HEK293T cells were transfected with a plasmid encoding wild-type ZIKV or the candidate DVGs in increasing molar ratios (1:10, 1:1 or 10:1 DVG:WT ratio). 4 days post transfection, virus titer was determined by plaque assay.
The candidate TIPs identified above were tested in vitro for their ability to inhibit wild-type Zika virus. Cells were transfected with a plasmid coding for Zika virus, along with plasmids coding for each DVG at increasing molar ratios. The effect of the TIP candidate on WT virus growth was determined by measuring the amount of progeny virus after treatment. The data (
Method in mice: AG129 male mice (4-6 week old) were infected with 104 PFU of Zika virus and co-transfected with 20 ug plasmid encoding DG-E, —H or -K (candidate DVGs that exhibited the highest inhibition effect in vitro), using the in vivojetPEI transfection reagent (Polyplus), in a total volume of 50 ul via a footpad injection (s.c.). Control mice received a control plasmid not coding for any TIP. The ability of TIPs to attenuate infection was monitored by weight loss. Viremia was also measured daily (except day 5). 7 days post infection, mice were euthanized and spleen, brain and tested dissected for determination of virus titer in these organs.
The results show (
Method of VLP packaging : Uninfected or infected Vero cells (producer cells, MOI 1 PFU/cell at 24 h) in 24-well plates were transfected with 100 ng of DVG replicon RNA, WT replicon (positive control) or inactive replicon (negative control), using TransIT-mRNA transfection reagent (Mirus Bio). 48 h p.t. the supernatant endonuclease-treated (25 Units/µl BaseMuncher, Expedeon) and used to infect naive Vero cells in 24-well plates (recipient cells). Replicon activity in recipient cells was assessed 24 h p.i. Donor and recipient cells were lysed in passive lysis buffer (Promega) and luciferase activity measured using the Nano-Glo luciferase assay system (Promega) in a Tecan infinite M200 pro plate reader (Tecan group). HEK-293T cells seeded in a 24 well plate were transfected with a mix of 200 ng of a CPrME-encoding plasmid, 200 ng of E30-NS1, and 200 ng of the DVG-encoding plasmid (LT-1 transfection reagent, Mirus Bio). 72 h p.t., the medium was clarified by centrifugation. N naive recipient cells were infected with producer cell supernatant following endonuclease (Basemuncher, Expedeon) treatment, and successful packaging determined with luciferase measurement when using reporter DVG clones, or by RT-qPCR when using native DVG.
Method of VLP-DVGs in mice: 4-6 week old AG129 or C57BL/6 female mice were given a mix of Ketamine (10 mg/mL)/Xylazine (1 mg/mL) by intraperitoneal injection. Once anesthetized, mice were inoculated by a subcutaneous (footpad) route with vehicle (DMEM media), 104 PFU of Zika virus alone, or complemented with DVG-containing VLPs (TIPs). C57BL/6 mice were treated with 2 mg of an IFNAR1 blocking mouse MAb (MAR-5A3, Euromedex) by intraperitoneal injection one day prior to infection. Weight loss and viremia were monitored at the indicated times after infection. Blood was collected from the facial vein in Microtainer blood collection tubes (BD) and allowed to clot at room temperature. Serum was separated by centrifugation and stored at -80° C. Viremia was quantified either by plaque assay or RT-qPCR on unextracted and diluted serum samples, as described previously19. Infected mice were euthanized by cervical dislocation. Spleen, ovaries, brain and injected footpads were harvested and homogenized with 600 µl DMEM supplemented with 2% FBS in Precellys tubes containing ceramic beads, using a Precellys 24 homogenizer (Bertin Technologies) at 5000 rpm for 2 cycles of 20 seconds. Homogenates were cleared by centrifugation, and the supernatant stored at -80° C. until virus titration or RT-qPCR. Viral burden in organs was measured either by plaque assay (expressed as PFU/g for organs) or by RT-qPCR (expressed as PFU equivalents relative to GAPDH) following RNA extraction using Direct-zol-96 (Zymo). The number of DVG or WT RNA genomes in mouse organs was normalized to GAPDH genomes, derived from a standard curve using mouse GAPDH primers (Supplementary Table 1) and RNA extracted from the respective organ of uninfected mice.
DVG-A can be packaged into virus-like particles (VLPs) for in vivo delivery. To consider DVGs as a potential therapeutic agent, we may require an appropriate delivery method. Thus, we established a packaging system to encapsidate a DVG into virus-like particles (VLPs). We first had to confirm that this DVG could be encapsidated at all. To this end, we transfected uninfected or infected cells with DVG-A reporter or control WT replicon RNA. These cells, named ‘producer’ cells, should (if infected) produce WT virus progeny, as well as virions containing genomes that can be packaged. Transfer of the supernatant derived from infected producer cells to naive (recipient) cells enabled us to assess packaging of reporter-expressing genomes. Significantly higher WT replicon and DVG-A reporter activities were observed in recipient cells in the presence of WT virus, compared to background activity in the absence of virus, confirming that DVG-A was packaged by WT virus (
Zika virus VLP-DVGs reduce infection and virulence in mice. We next assessed the efficacy of these DVGs packaged as VLPs as an antiviral measure in mice. We used mice deficient in α/β and γ receptors (AG129 mice), as these are highly susceptible to Zika virus infection and disease progression. Mice were mock-infected (vehicle alone), infected with 104 PFU of WT virus alone, or with a mixture of WT virus and DVG-A. Approximately 10 DVG-A genome copies per Zika virus infectious virion were used. As an additional control, mice received a mix of WT virus and supernatant from mock conditions of production (‘control TIPs’, generated in the absence of CPrME or NS1) (
Method in mosquitoes: Ae. aegypti mosquitoes were injected with 0.02pmoles RNA using Cellfectin transfection reagent (control RNA, DG H or DG K). 2 days post injection, mosquitoes were fed a bloodmeal containing 7 x105 pfu/mL ZIKV The ability of TIPs to attenuate infection was monitored by dissection of mosquito body parts at 8 or 13 days post infection and determination of viral load. At 13 d p.i. mosquitoes were also salivated to investigate whether transmission could be blocked in the presence of TIPs.
The results show that, in whole bodies of mosquitoes at 13 days p.i. there was a significant decrease in viral load in mosquitoes injected with DG H and DG K, suggesting virus attenuation in the mosquitoes in the presence of TIPs (
The identified ZIKV sequences are listed in
ZIKV DVGs sequences are selected from the group consisting of: SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 23 (ZIKV DVG-B), SEQ ID NO: 24 (ZIKV DVG-C), SEQ ID NO: 25 (ZIKV DVG-D), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 27 (ZIKV DVG-F), SEQ ID NO: 28 (ZIKV DVG-G), SEQ ID NO: 29 (ZIKV DVG-H), SEQ ID NO: 30 (ZIKV DVG-I), SEQ ID NO: 31 (ZIKV DVG-J), SEQ ID NO: 32 (ZIKV DVG-K) and SEQ ID NO: 33 (ZIKV DVG-L).
Preferred ZIKV DVGs sequences are selected from the group consisting of: SEQ ID NO: 22 (ZIKV DVG-A), SEQ ID NO: 24 (ZIKV DVG-C) and SEQ ID NO: 25 (ZIKV DVG-D).
Most preferred ZIKV DVGs sequences are slected from the group consisting of: ), SEQ ID NO: 26 (ZIKV DVG-E), SEQ ID NO: 32 (ZIKV DVG-K), SEQ ID NO: 33 (ZIKV DVG-L) and other possible DVGs within a cluster with the following characteristics; deletion results in in-frame deletion events in which the start site is between positions 500 and 900 nt, and in which the stop site of the deletion is between positions 2800 and 3400 nt of the ZIKV genome.
In this example, we document and characterize naturally occurring DVGs bearing deletions, arising during chikungunya virus infection in vitro and in vivo. From these, we selected the DVGs most likely to have interfering capacity based on their frequency and recurrence between replicates and/or their ability to be carried over multiple passages. We show that natural DVGs have a strong antiviral activity on chikungunya virus both in mammalian and mosquito cells in vitro, and demonstrate that interfering DVGs can be broad-spectrum against other alphaviruses. Finally, we show that DVGs can be introduced into the mosquito vector prior to infection to inhibit arbovirus dissemination.
Vero, Huh7, 293T and BHK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S; Thermo Fisher) and 1% non essential amino-acid (Thermo Fisher) in a humidified atmosphere at 37° C. with 5% CO2. U4.4 and Aag2 cells were maintained in Leibovitz’s L-15 medium with 10% FCS, 1% P/S, 1% non-essential amino acids (Sigma) and 1% tryptose phosphate (Sigma) at 28° C.
The viral stocks were generated from CHIKV infectious clones derived from the Indian Ocean lineage, ECSA genotype (IOL; described in 17) or from the Caribbean strain, Asian genotype (Carib; described in 18). To test DVG interference activity, a CHIKV infectious clone containing the Gaussia luciferase gene under a subgenomic promoter was used (obtained from Andres Merits). In vitro transcription (IVT) with SP6 mMESSAGE mMACHINE kit (Invitrogen) was performed on Not I linearised plasmids prior to transfection in BHK using lipofectamine 2000 (Invitrogen). After one passage in Vero cells, the stocks were titered and kept at -80° C. before use.
Viral titration was performed on confluent Vero cells plated in 24-well plates, 24 hours before infection. Ten-fold dilutions were performed in DMEM alone and transferred onto Vero cells. After allowing infection, DMEM with 2% FCS, 1% P/S and 0.8% agarose was added on top of cells. Three days post infection, cells were fixed with 4% formalin (Sigma), and plaques were manually counted after staining with 0.2% crystal violet (Sigma).
Cells were seeded in 24-well plates to reach approximately 80% confluency the next day. For passage 1, virus was diluted in PBS to obtain a multiplicity of infection (MOI) of 5 PFU/cell (high MOI). Cells were incubated with the viral inoculum at 37° C. for 1 hour. Following virus adsorption, the inoculum was removed, the infected cells were washed with PBS, and replaced with the appropriate cell culture medium containing 2% (v/v) FCS. At 48-72 hour post infection, supernatant was harvested and clarified by centrifugation (12 000 x g, 5 min). The following passages were performed blindly, using a high volume (300 µl) of the clarified supernatant from previous passage to infect naive cells followed by the same procedure. A total of 10 passages were performed. Each passage was titered by plaque assay to determine at which passages DVG accumulation may have resulted in interference. At least three replicates were performed per cell type.
Sequencing. RNA of 100 ul of each sample supernatant was extracted using TRIzol reagent (Invitrogen) or ZR viral RNA kit (Zymo) following the manufacturer’s protocol. RNA was eluted in 15-30 µL nuclease-free water. After quantification using Quant-iT RNA assay kit (Thermo Fisher Scientific), RNA libraries were prepared with NEBNext Ultra II RNA Library kit (Illumina). Multiplex oligos (Illumina) were used during library preparation. The quality of the libraries was verified using a High Sensitivity DNA Chip (Agilent) and quantified using the Quant-iT DNA assay kit (Thermo Fisher Scientific). Sequencing of the libraries (diluted to 1 nM) was performed on a NextSeq sequencer (Illumina) with a NextSeq 500 Mid Output kit v2 (Illumina) (151 cycles).
Next generation sequencing data analysis. All analyses were performed using BBTools suite (Bushnell B. - sourceforge.net/projects/bbmap/). First, fastq files generated from sample sequencing were trimmed for low-quality bases and adaptors using BBDuk. Then, alignment was performed using BBmap and the appropriate CHIKV reference sequence (Carib - GenBank accession no. LN898104.1, IOL -GenBank accession no. AM258994). BBMap’s variant caller, CallVariants, reported deletion events, and the overall DVG frequency per sample was calculated as the sum of the number of junction read counts (n) corresponding to each DVG and normalised as 5/3 × n/N, where N denotes the 98th percentile of the coverage per position. Multiplying by 5/3 aims to correct for the detection limit. Indeed, even if reads are 150 nucleotides long, aligned portions are usually only 30 to 120 nucleotides long. This implies that a read starting 30 or less nucleotides upstream of the breakpoint will be aligned on the right side but not on the left side of the breakpoint, and thus, will not be considered as a DVG. Consequently we are missing (30+30)/150 = 60/150 = ⅖ of the deletions, and the counts must be multiplied by 5/3 as a correction factor. Heatmaps illustrate the deletion score per nucleotide position based on deletion events removing that particular position. Specifically, scores were computed as the sum of the number of reads per million reads (RPM) supporting the deletion of a specific nucleotide position. For plotting start/stop breakpoints, deletions with lengths below 10 nucleotides were discarded.
Cloning selected defective genomes. Defective genomes selected from passages were cloned in the CHIKV infectious clones (under SP6 promoter) corresponding to their strain, using the previously described In Vivo Assembly (IVA) method19 or using In-Fusion reagent (Takara Bio Reagent). Primers were designed using SnapGene software, with a melting temperature of 60° C., and obtained from Integrated DNA Technology (IDT). 50 µl PCR using either Phusion high fidelity DNA polymerase (Thermo Fisher) or Q5 DNA polymerase (NEB) was carried out with a melting temperature of 57° C. for Phusion enzyme or 65° C. for Q5 polymerase. 18 cycles were carried out and the PCR products were then Dpn I(Thermo Fisher)-treated for at least 2 hours to remove the plasmid template and purified (Macherey Nagel PCR and gel purification kit). When IVA technique was used, 2 ul of the PCR products were directly transformed in XL10-Gold extra competent cells (Stratagene) according to supplied protocol. When In-Fusion reaction was needed, PCR products were ligated using In-Fusion reagent (Takara Bio Reagent) following supplier instructions and 1 ul was transformed in Turbo cells (NEB).
Luciferase assay to test DG interference activity. IVTs were performed using the SP6 mMESSAGE mMACHINE kit (Invitrogen) from Not I linearized infectious clones of DVG and CHIKV Carib-GLuc (see above). RNA production was quantified by Qubit RNA TS Assay kit (Thermo Fisher) and diluted to be at the indicated molar ratios compared to the Carib-GLuc CHIKV. Mixed DVG and full genome RNA were transfected in 293T or U4.4 cells seeded in 96-well plates with TransIT-mRNA transfection kit (Mirus) following the supplier’s protocol (25 ng of Carib-GLuc CHIKV per well). The medium was changed 4 hours post transfection to avoid cellular toxicity. 48 hours after transfection, supernatant was collected and mixed with coelenterazine (supplied by Y. Janin, Institut Pasteur) at a final concentration of 0.05 µM and luminescence was measured on a Tecan Infinite 200 microplate reader. When tested with non-luciferase expressing viruses, the procedure was followed the same way but the supernatant was titered by plaque assay 48 hours post transfection.
RT-qPCR to test for DVG self-replication. 50 ng of DVG or CHIKV Carib IVT was transfected in 293T cells (seeded the day before in 48-well plates), in triplicate, as described above. 8 hours post transfection, supernatant was removed, and cells were washed 3 times with PBS before adding fresh medium. At 8, 20, 28 and 44 hours post-transfection, 200 ul of lysis buffer from ZR 96 viral RNA kit (Zymo) was added on the cells after supernatant removal and stored at -20° C. until all time points were collected. Cellular RNA was extracted with the ZR 96 viral RNA kit and eluted in 15 µl. TaqMan RNA-to-Ct One-step RT-PCR kit (Applied Biosystems) was used to perform a quantitative RT-PCR spanning the 5’UTR-nsP1 region, with 5′-GAGACACACGTAGCCTACCA-3′ as the forward primer, 5’-GGTTCCACCTCAAACATGGG-3′ as the reverse, and 5’- [6-FAM] ACGCACGTTGCAGGGCCTTCA-3′ as the probe. After 20 min at 50 °, and 10 min at 95 °, 40 cycles were performed (95° C. for 15 seconds followed by 60° C. for 1 minute).
Mosquitoes. Aedes aegypti female mosquitoes belonging to the 7th generation from wild mosquitoes collected in Kamphaeng Phet Province (Thailand) were grown at 28° C., 70% relative humidity and a 12 hour light: 12 hour dark cycle, and fed with 10% sucrose. A day before blood feeding, 6 to 10-day old females were selected and starved for a day. A blood meal containing 106 PFU/ml of CHIKV was offered to a pool of 60 females through a membrane feeding system (Hemotek Ltd) set at 37° C. with a piece of desalted pig intestine as the membrane. Following the blood meal, fully engorged females were selected and incubated at 28° C., 70% relative humidity and under a 12 hour light: 12 hour dark cycle with permanent access to 10% sucrose. After 7 days, the mosquitoes were sacrificed and dissected to collect heads, thorax, midgut, legs and wings (legs/wings), and abdominal wall (body). Each organ was ground in 300 ul of L15 supplemented with 2% FBS with Qiagen TissuLyser 2 machine, then clarified by centrifugation before titration and RNA extraction with TriZol reagent for RNA deep sequencing. One hundred and fifty nanograms of RNA produced by in vitro transcription (as described above) and purified by phenol-chloroform was mixed with Leibovitz’s L-15 medium and Cellfectin II Reagent (Thermo Fisher) following the supplier’s instructions. For each condition, forty 6 to 8 day-old females were injected intra-thoracically with 300nl of this mix with Nanoject III Nanoliter Injector (Drummond scientific company). Two days later, after a night of starvation, mosquitoes were fed with an infectious blood meal of 106 PFU/ml of CHIKV Carib-GLuc as described above. After 5 days, mosquitoes were sacrificed; midgut and carcass were dissected and ground as mentioned before. Each sample was then tested for luciferase activity and titered by plaque assay.
Generation of defective viral genomes (DVG) by in vitro high MOI passage. In order to capture deleted DVGs that may arise in cell culture, the Caribbean strain of chikungunya virus (CHIKV Carib) was serially passaged in triplicate at high MOI in mammalian (Vero, Huh7) and mosquito (Aag2, U4.4) cells. RNA was extracted from the clarified supernatant of each replicate at each passage and RNA deep sequencing was performed. Reads were analyzed with the Bbmap pipeline to describe and quantify DVGs by identifying sequence reads that contain deletions. As expected, the amount of DVGs between the first and last passages increased by between 1 and 5 orders of magnitude in all cell types, and the rates and oscillations differed with each cell type (
Characterization of the DVGs generated in vitro. Next, we pooled the data for all passages and replicates from each cell, to identify potential deletion hotspots in the different cell types. The resulting heat map (
Generation of chikungunya DVGs in mosquitoes in vivo. To see if DVGs were also generated during in vivo mosquito infection, we fed Aedes aegypti mosquitoes an infected blood meal containing 106 PFU/ml of the Caribbean strain of chikungunya virus. After allowing the infection to disseminate throughout the mosquitoes over seven days, we sacrificed and dissected ten of them to collect individual organs: midgut, abdominal wall (body), thorax, heads and legs/wings (
Chikungunya DVG candidates are non replicative. Our goal in the previous sections was to uncover all of the possible DVGs generated during virus infection and to down-select from the hundreds of individual DVGs, those that would most likely and efficiently compete with wild type virus. In other words, to identify which of the total DVGs would be the most potent defective interfering particles. Our rationale was that such DVGs would occur more frequently at high MOIpassage, and would increase to higher frequency over time as the DVG competes with wildtype virus for resources. DVGs were thus selected based on criteria that would be indicative of higher fitness vis à vis the wildtype competitor: having high frequency, being maintained throughout the passage series and occurring in several replicates. In addition to DVG-CM1, carrying a deletion from nucleotide 2695 (middle of nsP2) to nucleotide 5358 (end of nsP3) (
Chikungunya DVGs interfere with wild type virus in vitro. We next tested the interference activity of the 20 DVGs derived from either mammalian cell culture (
To determine whether these mammalian and mosquito cell-derived DVGs could also inhibit virus replication in mosquito cells, we repeated the experiment in Aedes albopictus cells (U4.4). As for 293T cells, no interference was observed at a 1:1 molar ratio; but considerable inhibition with decreases of 1 to 2 log of luciferase activity was observed at 10:1 ratios for all of the DVGs that previously inhibited virus in mammalian cells, except for IV4. As seen in 293T cells, no interference was observed for DVG CV3 in mosquito cells (p>0.05) (
Chikungunya DVGs can be broad-spectrum inhibitors. The chikungunya virus Indian Ocean lineage belongs to the East, South and Central African (ECSA) genotype and has approximately 7% nucleotide divergence with the chikungunya virus Caribbean strain that belongs to the Asian genotype20. In the previous experiments, DVGs derived from either the Indian Ocean Lineage or Caribbean strains were tested against the Caribbean strain virus expressing Gaussia luciferase. Importantly, all of the CHIKV IOL-derived DVGs inhibited the Caribbean strain, showing that DVGs can act broadly within the same virus species (
We next investigated whether CHIKV DVGs could inhibit even more broadly within the alphavirus genus. We carried out the same experiment in 293T cells with O’nyong’nyong virus (ONNV, CHIKV’s closest relative) or Sindbis virus (SINV, a very distant relative) as the targets. While the inhibitory effect was lost for most DVGs, some DVGs still significantly inhibited O’nyong’nyong virus (CV2 and CH1) (
Chikungunya DVGs block viral dissemination in vivo, in Aedes aegypti mosquitoes. After demonstrating that DVGs can be used to limit/inhibit infection in vitro, we tested if their interference activity could be used to block infection or dissemination in the mosquito host. To do so, we injected purified RNA of the CV4, IH1 or CM1 DVGs, a control RNA, or PBS into Aedes aegypti mosquitoes 2 days prior to feeding them with a blood meal containing the CHIKV Carib-Gluc virus. Five days post infection, mosquitoes were sacrificed and midguts were dissected from the rest of the carcass (
Described in nearly all RNA virus families6,21 and often considered a waste product of viral replication, defective interfering particles, and more broadly speaking DVGs, have recently garnered attention for their possible use as antiviral tools15,6,22-26. Indeed, the presence of DVGs in natural human infections correlates to milder disease in clinical studies on respiratory syncytial virus and influenza virus10,13; and influenza defective interfering particles protect mice against lethal challenge22,23,27. In this study, we aimed to characterize, as widely as possible, the DVGs bearing deletions that arise during chikungunya virus infection in vitro in mammalian and mosquito environments and in vivo in its mosquito host. We show that chikungunya DVGs arise in all environments, in vitro and in vivo, but their type and abundance depend on the host and cell type, and on the virus strain, highlighting that both cellular environment and the viral genome bear determinants of DVG generation. For example, cluster D is strongly represented in both Aedes spp. mosquito cells, even though the deletion does not occur at the exact same position, but it is very rare in mammalian cells.
To find DVGs that could be useful as antivirals, we selected the most redundant DVGs, that arose in several conditions and were propagated over passages, which we assumed to be indicative of higher fitness and the ability to be packaged by wild-type virus. When used in higher quantity relative to wild-type virus, most candidates showed promising inhibiting capacity. Of note, because smaller genomes should be replicated more quickly7,8, the conventional dogma proposes that the smallest DVGs would most efficiently hijack the parental virus replication machinery and outcompete wild-type virus. However, in our work, the smallest DVGs identified (two DVGs from cluster B) had no or very little interference activity. This observation implies that strict competition for the replicase and replication speed is not the only mechanism by which DVGs interfere with parental virus, and that their previously described immunostimulatory activity7,9-11,28-33 or the competition for structural protein resources8,34 may also play an important role. Importantly, we used Illumina deep sequencing technology that only generates small reads to identify DVGs, making it close to impossible to identify DVGs with several deletions and/or major rearrangements. In the future, combining this approach with a long-read sequencing technology such as nanopore might generate an even more complete picture of the nature of DVGs generated in the chikungunya virus population and inform in their mode of action.
Of the DVGs generated in Aedes aegypti in vivo, CM1 attracted our attention because it could cross bottlenecks in vivo. During a natural viral infection, after an infectious blood meal, the midgut is the first organ to be infected before the virus reaches the hemocoel to disseminate to all other organs35. Several works in mosquitoes have shown that virus exit from the midgut is the main population bottleneck during mosquito infection with a drastic reduction in population size, followed by egress from the salivary glands, a mandatory step for viral transmission to the mammalian host through infected saliva35-39. CM1 was newly generated in the midguts of two independent mosquitoes and found to disseminate to the body wall, head and legs/wings of these same mosquitoes. The possibility that CM1 was generated de novo in other organs is unlikely because this DVG never appeared in any organs of any other mosquitoes that had not generated it in the midgut. It is not clear how or why CM1 is able to cross the midgut bottleneck. One possibility is that it belongs to a collective infectious viral unit, a structure that simultaneously contains and transports multiple viral genomes to a single cell, such as polyploid virions, aggregates of virions or virion-containing lipids vesicles40,41. The possibility of collective infectious unit containing DVGs has already been proposed. Indeed, Leeks et al. modeled that if the viral population contains many DVGs, the collective infectious unit could become very large. Nonetheless, the presence of interfering DVGs would disfavor transmission within the collective infectious unit compared to free virions42. While CM1 had an inhibiting activity at high molar ratio compared to the parental virus, its frequency in mosquito samples was very low (4 to 176 reads per million).
A valuable characteristic of some of these interfering DVGs is their broad-spectrum activity, with inhibition not only on other chikungunya virus strains but also on other alphaviruses. This cross reactivity had already been reported with influenza and Sendai virus, since their DVGs were shown to be efficient vaccines or vaccine adjuvants in mammalian models not only against the virus from they were derived, but also against unrelated viruses15,22-26. From a therapeutic point of view, this is of particular interest since the risk of worldwide dissemination of arthritogenic alphaviruses is well accepted3,43-47, especially since no antivirals or vaccines against any of these viruses are currently licensed. In this context, any treatment (antiviral or vaccine) that could work across a viral genus or family would be helpful in facing outbreaks to come.
It will be important in the future to test if chikungunya DVGs could function as antivirals in mammalian in vivo models as well; however, a proper delivery system still needs to be developed. In previous work on influenza A and Sendai DVGs, authors isolated DVGs by ultra-centrifugation of a mix of wildtype virus and DVG on sucrose gradients15,11-16. However, we could not successfully separate chikungunya DVGs from wild type virus using these methods. New approaches, such as delivering RNA or DNA in nanoparticles or nanostructured lipids48,49, or attempting to package DVGs in virus-like particles (VLPs) could be explored. Another major hurdle to overcome is the choice of in vivo model. Wild-type C57BL/6 could be used, but as mice develop only a transient, mild infection, it would be difficult to demonstrate an inhibitory effect for even the best DVG candidates. While the most commonly used mouse models rely on interferon α/β knockouts50,51, the induction of innate immunity would be excluded, which may be an important mechanism of action in chikungunya DVGs10,28-31,33.
Finally, when injected in Aedes aegypti mosquitoes two days before an infectious blood meal, three DVGs significantly reduced viral dissemination from the mosquito midgut, thereby shifting the status of mosquitoes from being competent to incompetent vectors for chikungunya virus spread. This result is a proof of principle that DVGs might be useful tools in controlling chikungunya virus infection in the vector population. As releasing incompetent mosquitoes in the wild during an arbovirus outbreak has already been shown to be an effective epidemic control strategy52,53, and despite the need for a simpler delivery system, it is tempting to propose that interfering DVGs could be used as a control strategy not only for chikungunya virus, but for any arbovirus. Furthermore, interfering DVGs are presumably safe antiviral tools because they are inert molecules that cannot self-replicate, and are only active when wild-type virus is present.
In conclusion, this work describes the different types of deleted DVGs generated during chikungunya virus infection in both vertebrate and invertebrate environments in vitro and in vivo in the mosquito vector. Moreover, we identified criteria to down-select the best defective interfering particle candidates able to inhibit wild-type chikungunya virus in vitro in both vertebrate and invertebrate hosts. An interesting observation is the broad-spectrum activity of some interfering DVGs able to interact with related alphaviruses. Finally, we show that pre-exposure to a DVG can modulate viral dissemination in mosquitoes in vivo. These results strengthen the idea that defective interfering particles might be a useful therapeutic tool for chikungunya virus infection as well as an efficient vector control strategy.
CHIKV DVGs sequences are selected from the group consisting of: SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 45 (CHIKV DVG-CH2), SEQ ID NO: 46 (CHIKV DVG-CH3), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3) and SEQ ID NO: 53 (CHIKV DVG-CA4).
Preferred CHIKV DVGs sequences are selected from the group consisting of: SEQ ID NO: 34 (CHIKV DVG-IV1), SEQ ID NO: 35 (CHIKV DVG-IV2), SEQ ID NO: 36 (CHIKV DVG-IV3), SEQ ID NO: 37 (CHIKV DVG-IV4), SEQ ID NO: 38 (CHIKV DVG-IH1), SEQ ID NO: 39 (CHIKV DVG-IU1), SEQ ID NO: 40 (CHIKV DVG-CV1), SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 42 (CHIKV DVG-CV3), SEQ ID NO: 43 (CHIKV DVG-CV4), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 47 (CHIKV DVG-CM1), SEQ ID NO: 48 (CHIKV DVG-CU1), SEQ ID NO: 49 (CHIKV DVG-CU2), SEQ ID NO: 50 (CHIKV DVG-CA1), SEQ ID NO: 51 (CHIKV DVG-CA2), SEQ ID NO: 52 (CHIKV DVG-CA3) and SEQ ID NO: 53 (CHIKV DVG-CA4).
Most preferred CHIKV DVGs sequences are selected from the group consisting of: SEQ ID NO: 41 (CHIKV DVG-CV2), SEQ ID NO: 44 (CHIKV DVG-CH1), SEQ ID NO: 47 (CHIKVDVG-CM1), SEQ ID NO: 38 (CHIKVDVG-IH1) and SEQ ID NO: 43 (CHIKV DVG-CV4).
Method: Rhinovirus (RV) types used for the identification of defective viral genomes (DVGs) include RV-A01a (NC_038311.1), RV-A16 (L24917.1), RV-B14 (L05355.1), and RV-C15 (GU219984.1). Briefly, 12-well plates were seeded with 3.5 x 105 (350,000) H1—HeLa cells/well in complete media (DMEM, 10% FBS, 1% penicillin/streptomycin) and incubated overnight at 37° C./5% CO2 to reach ~90% confluency. In the case of RV—C15 virus, the HeLa-E8 cell line was used. Next day, the cells were infected with wild type RV-A01a, RV-A16, RV-B14, and RV-C15 viruses at high (20) and low (0.1) multiplicity of infection (MOI). Following an hour of incubation at 34° C./5% CO2, the virus was removed and the cells were washed with phosphate buffered saline (PBS) solution. Then, PBS was aspirated and one mL of infection media (DMEM, 2% FBS, 1% penicillin/streptomycin) was added. Cells were incubated at 34° C./5% CO2, until complete cytopathic effect (CPE) was observed. Upon CPE, viruses were harvested by three repeated freeze-thaw cycles, and cell debris was removed by centrifugation (17,000 x g for 10 min at 4° C.). The viral supernatant was aliquoted and stored at -80° C. until ready to use. The supernatant was used to passage the virus into fresh cells. This process was repeated until passage 10 (
Considering the presence of numerous RV types, we conducted our experiments with RV-A01a, -A16, -B14, and -C15, to make sure that all the RV species are represented within our dataset. These viruses were passaged up to 10 times at low and high-MOI in H1—HeLa cell line as described above (
The selection of TIP candidates was done based on the presence of hotspots, location along the genome, size of the deletions, and the frequency of appearance for each deletion within/across passages. The TIP candidates’ names include the RV type followed by the TIP number (e.g. B14-TIP-01) (
Method: Prior to transfection, 96-well plates were seeded with 2.0 x 104 H1-HeLa cells/well in complete media (DMEM, 10% FBS, 1% penicillin/streptomycin) and then incubated for 24 hrs at 37° C./5% CO2 to reach ~90% confluency. The complete media was replaced with infection media (DMEM, 2% FBS, 1% penicillin/streptomycin) and the cells were co-transfected with wildtype and the selected TIP candidates at indicated molar ratios using the TransIT-mRNA transfection reagent. All the co-transfections were done in 1:1, 1:5, and 1:10 (WT:TIP) molar ratios and in triplicates (n = 3). In the case of WT RNA alone, the amount for “1” equals 25 ng of RNA. The transfected cells were incubated at 34° C./5% CO2 for 48 hrs. Following the incubation, the virus was harvested by three repeated freeze-thaw cycles, and the cellular debris was removed by centrifugation (17,000 x g for 10 min at 4° C.). The titers of infectious virus were determined by plaque assay. As a control, WT RNA was co-transfected with control RNA (pTRI-Xef: Xenopus elongation factor 1α), which resulted in similar titers as WT alone. Additional controls, including the control RNA alone, TIP candidates alone, and a mock transfection were also tested, which yielded no infectious virus, as expected. Panrhinoviral activity of the best performing TIP candidate was tested the same way using WT RNA from RV-A01a and RV-A16.
Results indicate that from this select set of TIP candidates, B14-TIP-03 candidate performed the best by lowering the WT titers by almost 100-fold (
Our in vitro results indicate that antiviral feature of B14-TIP-03 is panrhinoviral. Co-transfecting B14-TIP-03 with RV-A01a and RV-A16 reduce infectious viral titers significantly. Further enforcing the potential of B14-TIP-03 to be used as a broad-spectrum antiviral against multiple RV types.
Method : Prior to transfection, 96-well plates were seeded with 2.0 x 104 H1-HeLa cells/well in complete media (DMEM, 10% FBS, 1% penicillin/streptomycin) and then incubated for 24 hrs at 37° C./5% CO2 to reach ~90% confluency. The complete media was replaced with infection media (DMEM, 2% FBS, 1% penicillin/streptomycin) and the cells were co-transfected with WT and B14-TIP-03 at a 1:10 (WT:TIP) molar ratio using the TransIT-mRNA transfection reagent. The amount for “1” equals 25 ng of WT RNA. All the co-transfections were in triplicates (n = 3). The transfected cells were incubated at 34° C./5% CO2 for 3 hrs and then washed with thee times with phosphate buffered saline solution in order to remove the input RNA and prevent it from interfering with RT-qPCR experiments. Then, the transfected cells were incubated at 34° C./5% CO2 for 48 hrs. Following the incubation, the virus was harvested by three repeated freeze-thaw cycles, and the cellular debris was removed by centrifugation (17,000 x g for 10 min at 4° C.). To isolate the viral RNA, the Direct-zol RNA Miniprep Kit (Zymo Research) was used following the manufacturer’s recommended protocol. The total RNA was then reverse transcribed using SuperScript IV (ThermoFisher) reverse transcriptase enzyme and random hexamer primers following manufacturer’s recommended protocol. Then, 2 uL of 10-fold diluted cDNA was amplified using Power SYBR™ Green PCR Master Mix using gene-specific primers against retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), interferon (IFN)- α1, IFN- β1, and IFN-λ1 genes. The relative fold-change calculations were done using the ΔΔCt analysis against mock transfected cells with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as a control.
Results : Based on qPCR results, B14-TIP-03 is a potent stimulator of antiviral innate immunity. The co-transfection of RV-B14-WT and control RNA (pTRI-Xef: Xenopus elongation factor 1α) resulted in mild induction of all the genes tested. On the other hand, the co-transfection of RV-B14-WT and B14-TIP-03 RNAs resulted in a significant increase in the gene expression of pattern recognition receptors (PRRs), such as RIG-I and MDA5. Additionally, the B14-TIP-03 also induced the gene expression of IFN-β1 and IFN-λ1, but not IFN-α1. These results indicate the major mechanism of action for the B14-TIP-03.
The identified DVGs are listed in
RV DVGs Sequences are selected from the group consisting of: SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 56 (RV DVG A01a-TIP-02), SEQ ID NO: 57 (RV DVG A01a-TIP-03), SEQ ID NO: 58 (RV DVG A01a-TIP-04), SEQ ID NO: 59 (RV DVG A01a-TIP-05), SEQ ID NO: 60 (RV DVG A01a-TIP-06), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 63 (RV DVG A16-TIP-02), SEQ ID NO: 64 (RV DVG A16-TIP-03), SEQ ID NO: 65 (RV DVG A16-TIP-04), SEQ ID NO: 66 (RV DVG A16-TIP-05), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 90 (RVDVG C15-TIP-02), SEQ ID NO: 91 (RVDVG C15-TIP-03), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 69 (RV DVG B14-TIP-02), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 71 (RVDVG B14-TIP-04), SEQ ID NO: 72 (RV DVG B14-TIP-05), SEQ ID NO: 73 (RV DVG B14-TIP-06), SEQ ID NO: 74 (RV DVG B14-TIP-07), SEQ ID NO: 75 (RV DVG B14-TIP-08), SEQ ID NO: 76 (RV DVG B14-TIP-09), SEQ ID NO: 77 (RV DVG B14-TIP-10), SEQ ID NO: 78 (RV DVG B14-TIP-11), SEQ ID NO: 79 (RV DVG B14-TIP-12), SEQ ID NO: 80 (RV DVG B14-TIP-13), SEQ ID NO: 81 (RV DVG B14-TIP-14), SEQ ID NO: 82 (RV DVG B14-TIP-15), SEQ ID NO: 83 (RV DVG B14-TIP-16), SEQ ID NO: 84 (RV DVG B14-TIP-17), SEQ ID NO: 85 (RV DVG B14-TIP-18), SEQ ID NO: 86 (RV DVG B14-TIP-19) and SEQ ID NO: 87 (RV DVG B14-TIP-20).
Preferred RV DVGs sequences are selected from the group consisting of: SEQ ID NO: 55 (RV DVG A01a-TIP-01), SEQ ID NO: 62 (RV DVG A16-TIP-01), SEQ ID NO: 89 (RV DVG C15-TIP-01), SEQ ID NO: 68 (RV DVG B14-TIP-01), SEQ ID NO: 70 (RV DVG B14-TIP-03), SEQ ID NO: 73 (RV DVG B14-TIP-06) and SEQ ID NO: 77 (RV DVG B14-TIP-10).
Most preferred RV DVGs sequences are selected from the group consisting of: SEQ ID NO: 70 (RV DVG B14-TIP-03) and SEQ ID NO: 73 (RV DVG B14-TIP-06).
Generation and characterization of DVGs occurring within yellow fever virus passaging
Yellow Fever virus Asibi (YF-Asibi) strain and vaccine (YF-17D) strain were blindly passaged between 10 and 20 times in 12 biological replicates at high and low MOI in mammalian (SW13, Vero) and mosquito (C6/36) cell lines.
Computational analysis on sequenced passages identified all deletion hotspots across the genome and overall DVGs occurring within the population.
The most conserved DVGs, schematically represented in Figure XX4, have been selected for de-novo synthesis of TIPs.
YFV DVGs sequences are selected from the group consisting of: SEQ ID NO:93 (YFV DVG-A), SEQ ID NO: 94 (YFV DVG-B), SEQ ID NO: 95 (YFV DVG-C), SEQ ID NO: 96 (YFV DVG-D), SEQ ID NO: 97 (YFV DVG-E), SEQ ID NO: 98 (YFV DVG-F), SEQ ID NO: 99 (YFV DVG-G), SEQ ID NO: 100 (YFV DVG-H), SEQ ID NO: 101 (YFV DVG-I), SEQ ID NO: 102 (YFV DVG-J), SEQ ID NO: 103 (YFV DVG-K) and SEQ ID NO: 104 (YFV DVG-L).
West Nile virus Israel 1998 strain (WNV) was passaged at a high multiplicity of infection in BHK or C6/36 cells. Briefly, cells that were seeded to reach between 70-80% confluence were infected with an initial MOI of either 0.1 or 10 PFU/cell. Two days post infection, 3 µl or 300 µl of the infected cell supernatant (respectively) was used for infection in the subsequent passage. The passaging was repeated 10 times.
Altogether, the data show the importance of using more than one cell line or host to determine the total breadth and overall fitness of DVGs in different conditions. These data highlights that there are also many differences between cell types, yet reveals a cluster of deletions that emerges as the passage series progresses (suggesting high fitness of these DVGs).
From these and other analyses, we selected a list of DVG candidates. The candidates, named numbered from 1 to 6, are shown in
WNV DVGs sequences are selected from the group consisting of: SEQ ID NO: 105 (WNV DVG-1), SEQ ID NO: 106 (WNV DVG-2), SEQ ID NO: 107 (WNV DVG-3), SEQ ID NO: 108 (WNV DVG-4), SEQ ID NO: 109 (WNV DVG-5) and SEQ ID NO: 110 (WNV DVG-6).
Coronavirus SARS-CoV-2 strain (BetaCoV/ France/IDF0372/2020) was passaged at a high multiplicity of infection in Vero E6 cell. Briefly, cells were infected with an initial MOI of 5 x 10 \^5 PFU/cell. 3 days post infection, the infected cell supernatant was used for infection in the subsequent passage. The passaging was repeated 10 times. The overall process included passaging at high-MOI, followed by viral RNA purification, deep sequencing, and data analysis to identify defective viral genomes (DVGs).
SARS-CoV-2 sequences are selected from the group consisting of: SEQ ID NO:111 (SARS-CoV-2 DVG_1), SEQ ID NO: 112 (SARS-CoV-2 DVG_2) and SEQ ID NO: 113 (SARS-CoV-2 DVG_3).
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A part of the invention was made with United States Government Support under Agreement No.: HR0011-17-2-0023 awarded by the Defense Advanced Research Projects Agency (DARPA). The United States Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/000231 | 3/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63000998 | Mar 2020 | US |
