US20190002838A1

US20190002838A1 - Sindbis control virus

Info

Publication number: US20190002838A1
Application number: US15/737,818
Authority: US
Inventors: Russell Garlick; Catherine Huang; Bharathi Anekella; Jonathan Li
Original assignee: Seracare Life Sciences Inc
Current assignee: Seracare Life Sciences Inc
Priority date: 2015-06-19
Filing date: 2016-06-03
Publication date: 2019-01-03
Also published as: WO2016204983A1; EP3310368A1; US20210254020A1; EP3310368A4

Abstract

Disclosed are compositions and methods related to replication deficient Sindbis viruses that are able to function as controls for nucleic acid diagnostic assays (e.g., nucleic acid sequencing based assays and/or nucleic acid amplification based assays).

Description

PRIORITY CLAIM

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/182,104, filed Jun. 19, 2015, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 31, 2016, is named SCX_00325_SL.txt and is 144,758 bytes in size.

BACKGROUND

Regulatory agencies, such as the FDA, CLIA and CAP, generally require developers of nucleic acid-based in vitro diagnostic devices for pathogen detection to include quality controls in their regulatory submissions. Such quality control materials are important tools for the detection of analytical errors, the monitoring of long-term performance of diagnostic test kits, and the identification of changes in random or systematic error. A well-designed laboratory quality control program will generally incorporate at least some form of control that provides added confidence in the reliability of results obtained for unknown specimens.
Whole process controls are needed to monitor the entire analytical process, including sample lysis, nucleic acid extraction, amplification, detection and interpretation of results. Such controls can be natural material derived from infected patients, which have the advantage of behaving very similarly to a clinical sample. However, such natural source controls often have limited and unpredictable availability, concentration and stability. The use of cultured virus to generate positive controls alleviates some of these problems, but virus culture is often unavailable or technologically difficult. In addition, the preparation of large amount of human pathogens caries significant safety risks and is expensive.
Positive controls for amplification and detection are often provided as part of diagnostic test kits. The materials often have a known amount of input copy number and verify the integrity of the reaction components and instrument. However, such controls are not usually taken through the sample lysis or nucleic acid extraction process and are therefore unable to detect errors arising from these steps. Examples of this type of control include a non-infectious DNA plasmid containing the target sequence, purified RNA transcripts, or packaged RNA materials such as Armored RNA. These materials often also suffer from their limited stability at ambient temperatures.
Internal controls contain a non-target nucleotide sequence that is co-extracted and co-amplified with the target nucleic acid. Internal controls confirm the integrity of the reagents (e.g., polymerase, primers, etc.), equipment function (e.g., thermal cycler), and the absence of inhibitors in the sample. The internal control can take the form of a non-target organism that is added to the sample prior to sample lysis and extraction. Alternatively, it could be a non-infectious, non-target DNA or RNA sequence that is added to the sample either prior to or after sample lysis and extraction.
Thus, there is a need for improved compositions able to serve as controls in diagnostic assays.

SUMMARY

Provided herein are compositions and methods related to replication deficient Sindbis viruses that are able to function as controls for nucleic acid diagnostic assays (e.g., nucleic acid sequencing based assays and/or nucleic acid amplification based assays).
In certain aspects, disclosed herein is a replication deficient recombinant Sindbis virus comprising a RNA genome comprising (a) an open reading frame (ORF) encoding functional Sindbis non-structural proteins and (b) a heterologous (i.e., non-Sindbis) RNA sequence. In some embodiments, the ORF encoding the functional Sindbis non-structural proteins is located 5′ of the heterologous RNA sequence.
In some embodiments, the ORF encoding the Sindbis non-structural proteins encodes a nsP1 protein, a nsP2 protein, a nsP3 protein and a nsP4 protein. In some embodiments, the ORF encoding Sindbis non-structural proteins has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 1-7648 of SEQ ID NO: 1. These nucleotides encode non-structural Sindbis proteins.
In some embodiments, the RNA genome of the replication deficient Sindbis virus lacks a sequence encoding a functional version of one or more of the Sindbis structural proteins (e.g., Sindbis capsid protein, E3 protein, E2 protein, 6k protein and/or E1 protein),In some embodiments, the RNA genome lacks an RNA sequence encoding any functional Sindbis structural proteins. In some embodiments, the heterologous RNA sequence replaces the ORF encoding the Sindbis structural proteins in the RNA genome.
In some embodiments, the replication deficient recombinant Sindbis virus of claim any one of claims 1 to 8, wherein the RNA genome comprises a 26S subgenomic promoter at the 3′ end of the ORF encoding the Sindbis non-structural proteins.
In some embodiments, the heterologous RNA sequence in the RNA genome comprises a non-Sindbis RNA virus sequence or a retrovirus sequence. In some embodiments, the heterologous RNA sequence includes at least 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 bp of a non-Sindbis RNA virus sequence or a retrovirus sequence. In some embodiments, the heterologous RNA sequence includes 100-300 bp of a non-Sindbis RNA virus sequence or a retrovirus sequence. In some embodiments, the heterologous RNA sequence includes 100-200 bp of a non-Sindbis RNA virus sequence or a retrovirus sequence. In some embodiments, the heterologous RNA sequence is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a non-Sindbis RNA virus sequence or a retrovirus sequence. In some embodiments, the non-Sindbis RNA virus sequence or retrovirus sequence comprises one or more mutations that convey a drug resistant phenotype when present in the non-Sindbis RNA virus or the retrovirus. For example, in some embodiments the non-Sindbis RNA virus sequence or retrovirus sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations that convey a drug resistant phenotype when present in the non-Sindbis RNA virus or the retrovirus.
In some embodiments, the heterologous RNA sequence comprises a non-Sindbis RNA virus sequence. In some embodiments, the non-Sindbis RNA virus sequence is an Ebolavirus sequence, an influenza virus sequence, a SARS virus sequence, a hepatitis C virus sequence, a West Nile virus sequence, a Zika virus sequence, a poliovirus sequence or a measles virus sequence.
In some embodiments, the non-Sindbis RNA virus sequence is an Ebolavirus sequence (e.g., a Zaire ebolavirus sequence, a Bundibugyo ebolavirus sequence, a Reston ebolavirus sequence, a Sudan ebolavirus sequence or a Tai Forest ebolavirus sequence). In some embodiments, the Ebolavirus sequence comprises at least a portion of an Ebolavirus GP gene sequence, an Ebolavirus NP gene sequence or an Ebolavirus VP24 gene sequence. In some embodiments, the heterologous RNA sequence does not encode a functional Ebola protein (e.g., the heterologous RNA sequence encodes truncated Ebola proteins, Ebola proteins with frame-shift mutations and/or Ebola protein sequences lacking a start codon). In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3. SEQ ID NO: 2 is the nucleotide sequence of a GP Ebola target sequence used in an exemplary Ebola Sindbis control virus described in Example 1. SEQ ID NO: 3 is the nucleotide sequence of a NP/VP24 Ebola target sequence used in an exemplary Ebola Sindbis control virus described in Example 1. The portion of the Ebola NP gene consists of nucleotides 1 to 1577 of SEQ ID NO: 3, the portion of the Ebola VP24 gene consists of nucleotides 1578 to 2127 and the sequence of the human RNAse P internal control consists of nucleotides 2128 to 2217.
In some-embodiments, the heterologous RNA sequence comprises a retrovirus sequence. In some embodiments, the retrovirus sequence is an HIV-1 sequence, an HIV-2 sequence, an HTLV-1 sequence, or an HTLV-II sequence.
In some embodiments, the heterologous RNA sequence comprises an HIV-1 sequence. In some embodiments, the HIV-1 sequence comprises one or more mutations that, when present in a HIV-1 virus, conveys a drug resistance phenotype (e.g., resistance to a protease inhibitor, a nucleoside analogue reverse transcriptase inhibitor and/or a non-nucleoside analog reverse transcriptase inhibitor). For example, in some embodiments the HIV-1 virus sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations that convey a drug resistant phenotype. In some embodiments, the one or more mutations, when present in HIV-1 virus, convey resistance to a drug selected from the group consisting of: atazanavir, ritonavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, saquinavir, tipranavir, abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, efavirenz, etavirine, nevirapine or rilpivirine. In some embodiments, the one or more mutations are selected from the group consisting of L24I, D30N, V32I, M46I, I47V, G48V, 150V, I54M,
G73S, L76V, V82A, I84V, N88D, L90M, M41L, K65R, D67N, T69S insert SS, K7OR, L74V, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y, K219Q, L100I, K101E, K103N, V106A, V1081, Y181C, Y188L, G190A, P225H and M230L. In some embodiments, the one or more mutations are selected from the group consisting of L24I (TTA to ATA), D30N (GAT to AAT), V32I (GTA to ATA), M46I (ATG to ATA), I47V (ATA to CTA), G48V (GGG to GTG), I50V (ATT to GTT), I54M (ATC to ATG), G73 S(GGT to GCT), L76V (TTA to GTA), V82A (GTC to GCC), I84V (ATA to GTA), N88D (AAT to GAT), L9OM (TTG to ATG), M41L (ATG to TTG), K65R (AAA to AGA), D67N (GAC to AAC), T69S insert SS (ACT to TCT and insertion of TCC and TCC), K7OR (AAA to AGA), L74V (TTA to GTA), F77L (TTC to CTC), Y115F (TAT to TTT), F116Y (TTT to TAT), Q151M (CAG to ATG), M184V (ATG to GTG), L210W (TTG to TGG), T215Y (ACC to TAC), K219Q (AAA to CAA), L100I (TTA to ATA), K101E (AAA to GAA), K103N (AAA to AAC), V106A (GTA to GCA), V108I (GTA to ATA), Y181C (TAT to TGT), Y188L (TAT to TTA), G190A (GGA to GCA), P225H (CCT to CAT) and M230L (CCT to CAT). In some embodiments, the HIV-1 sequence comprises at least a portion of an HIV-1 gene selected from p7, pl, p6, HIV protease, reverse transcriptase, p51 RNAse, integrase and gp120. In some embodiments, the HIV-1 sequence comprises at least a portion of p7, pl, p6, HIV protease, reverse transcriptase and integrase. In certain embodiments, the HIV-1 sequence comprises at least a portion of 6p120, wherein the portion comprises the V1-V5 variable loops. In some embodiments, the HIV-1 sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 1900 through 5400 and/or 6300 through 7825 of the HXB2 strain of HIV-1 (SEQ ID NO: 4). SEQ ID NO: 4 is the nucleotide sequence of the HIV-1 HXB2 genome. In some embodiments, the HIV-1 sequence is identical to nucleotides 1900 through 5400 and/or 6300 through 7825of the HXB2 strain of HIV-1 (SEQ ID NO: 4) except for the presence of the mutations that convey a drug resistance phenotype.
In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5 and/or SEQ ID NO: 7. SEQ ID NO: 5 is the nucleotide sequence of a 5′ multi-mutant HIV-1 target sequence comprising a number of drug resistance mutations, used in an exemplary multi-mutant HIV-1 control virus described in Example 2. SEQ ID NO: 7 is the nucleotide sequence of a 3′ mutant HIV-1 target sequence used in an exemplary multi-mutant HIV-1 control virus described in Example 2.
In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 and/or SEQ ID NO: 8. SEQ ID NO: 6 is the nucleotide sequence of a 5′ wild-type HIV-1 target sequence in an exemplary HIV-1 control virus, described in Example 2. SEQ ID NO: 8 is the nucleotide sequence of a 3′ wild-type HIV-1 target sequence used in an exemplary HIV-1 control virus, described in Example 2.
In some embodiments, the heterologous RNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to either nucleotides 1-3446, nucleotides 3294-5575, nucleotides 5425-7722, or nucleotides 7542-10272 of SEQ ID NO: 15.
In some embodiments, the RNA genome of the replication deficient Sindbis virus comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
In certain aspects, provided herein is a composition comprising a replication deficient Sindbis virus described herein. In certain aspects, the composition comprises two or more of the replication deficient Sindbis viruses described herein. For example, in some embodiments, the composition comprises a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11 and a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 13. In some embodiments, the composition comprises a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12 and a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14. In some embodiments, the composition further comprises human DNA. In some embodiments, the replication deficient Sindbis virus is in a human bodily fluid. In some embodiments, the human bodily fluid is human plasma (e.g., defibrinated human plasma). In some embodiments, the composition further comprises a preservative, such as sodium azide.
In some embodiments, the composition comprises a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to either nucleotides 1-3446, nucleotides 3294-5575, nucleotides 5425-7722, or nucleotides 7542-10272 of SEQ ID NO: 15.
In certain aspects, provided herein is a nucleic acid molecule encoding the RNA genome of the replication deficient Sindbis virus described herein. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is a plasmid (e.g., a circular plasmid or a linearized plasmid, such as a circular expression plasmid or a linearized expression plasmid). In some embodiments, the nucleic acid molecule is isolated. In certain embodiments, provided herein is a cell comprising a nucleic acid described herein. In some embodiments ,the cell is a BHK cell.
In certain aspects, provided herein is a method of making a replication deficient Sindbis virus. In certain embodiments, the method includes the step of transfecting a cell (e.g., a BHK cell) with a nucleic acid molecule (e.g., an RNA molecule) encoding the RNA genome of a replication deficient Sindbis virus described herein and with a nucleic acid (e.g., an RNA molecule) encoding functional Sindbis structural proteins. In some embodiments, the cell is then cultured under conditions such that the cell produces the replication deficient Sindbis virus into the culture medium. In some embodiments, the method further comprises collecting the replication deficient Sindbis virus (e.g., by collecting the culture supernatant). In some embodiments, the method further comprises filtering and/or heat inactivating the culture supernatant. In some embodiments, the method further comprises determining the titer of the virus (e.g., using real-time PCR).
In certain aspects, provided herein are methods of testing a diagnostic assay by running the diagnostic assay on a composition comprising the replication deficient Sindbis virus described herein. In some embodiments, the diagnostic assay is a nucleic acid amplification based diagnostic assay. In some embodiments, the diagnostic assay is a sequencing based diagnostic assay. In some embodiments the diagnostic assay is an assay for the detection of a RNA virus and/or a retrovirus. In some embodiments, the diagnostic assay is an assay for the detection of Ebolavirus, an influenza virus, a SARS virus, a hepatitis C virus, a West Nile virus, a Zika virus, a poliovirus, a measles virus, an HIV-1 virus, an HIV-2 virus, an HTLV-I virus and/or an HTLV-II virus. In certain embodiments, the heterologous RNA sequence in the RNA genome of the replication deficient Sindbis virus contains the target sequence detected in the diagnostic assay. In some embodiments, the method includes the performance of a sample lysis step on the composition comprising the replication deficient Sindbis virus. In some embodiments, the method comprises performing a nucleic acid extraction step. In some embodiments, the method comprises performing a nucleic acid amplification step (e.g., performing a real-time nucleic acid amplification/detection process). In some embodiments, the method comprises performing a nucleic acid sequencing step. In some embodiments the method comprises performing a nucleic acid detection step.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a schematic depiction of the genomic organization of Sindbis virus. Some of the genes shown encode nonstructural proteins (nsP1-4), which include the helicase and RNA polymerase. Some of the genes are the structural genes, and encode the capsid (C) as well as proteins involved in budding.

FIG. 2 shows a schematic depiction of the genomic organization of a Sindbis control vector of certain embodiments described herein.

FIG. 3 shows an exemplary schematic for the production of recombinant Sindbis control viruses.

FIG. 4 shows the results of a TaqMan real time quantitation assay of Sindbis control samples unstressed (time=0) or stressed for 1, 3, 5, 11 or 22 days at 37° C.

FIG. 5 shows the results of a TaqMan real time quantitation assay of non-stressed Sindbis control samples or samples stressed through one, two or three Freeze/Thaw cycles.

FIG. 6 shows the results of a TaqMan real time quantitation assay of a Sindbis control virus stored frozen at −20° C., stored refrigerated at 2-8° C. or stored at ambient lab temperature across seven months.

FIG. 7 shows a workflow overview for the production of Sindbis control virus.

FIG. 8 shows a map of the SinRep SC vector. Figure discloses “His8” as SEQ ID NO: 16.

FIG. 9 consists of two maps of the Zika virus genome. The genome was divided into four regions for the construction of four different Zika virus reference materials, and each region is depicted by a rectangle. A first reference material comprises nucleotides 1 to 3446 of the Zika virus from GenBank Accession number EU545988.1, referred to as the “Zika Env Construct” (Construct -1). A second reference material comprises nucleotides 3294 to 5575, referred to as the “Zika NS2/NS3 Construct” (Construct -2). A third reference material comprises nucleotides 5425 to 7722, referred to as the “Zika NS4 Construct” (Construct -3). A fourth reference material comprises nucleotides 7542 to 10272, referred to as the “Zika NS5 Construct” (Construct -4).

FIG. 10A depicts nucleotides 1 to 3446 of the Zika virus from GenBank Accession number EU545988.1, referred to as the “Zika Env Construct,” which includes the NS1 gene. This portion of the Zika virus genome was integrated into a Zika virus reference material.

FIG. 10B depicts of nucleotides 3294 to 5575 of the Zika virus from GenBank Accession number EU545988.1, referred to as the “Zika NS2/NS3 Construct,” which includes the NS2 and NS3 genes as well as a portion of the NS1 gene. This portion of the Zika virus genome was integrated into a Zika virus reference material.

FIG. 10C depicts nucleotides 5425 to 7722 of the Zika virus from GenBank Accession number EU545988.1, referred to as the “Zika NS4 Construct,” which includes the NS4A and NS4B genes as well as a portion of the NS3 gene. This portion of the Zika virus genome was integrated into a Zika virus reference material.

FIG. 10D depicts nucleotides 7542 to 10272 of the Zika virus from GenBank Accession number EU545988.1, referred to as the “Zika NS5 Construct,” which includes the NS5 gene and a portion of the NS4B gene. This portion of the Zika virus genome was integrated into a Zika virus reference material.

FIG. 11A shows stability results of TaqMan real time quantitation of an H7N9 influenza reference material stored at −20° C., 4° C., or room temperature(˜25°) for seventeen months. The results depicted are for reference materials formulated with buffer.

FIG. 11B shows stability results of TaqMan real time quantitation of an H7N9 influenza reference material stored at −20° C., 4° C., or room temperature (˜25°) for seventeen months. The results depicted are for reference materials formulated with human plasma.

FIG. 12 shows stability results of TaqMan real time quantitation of a H7N9 influenza reference material stored at ambient temperature for seventeen months. Each error bar corresponds to 1 standard deviation from the mean.

DETAILED DESCRIPTION

General

Provided herein are compositions and methods related to replication deficient
Sindbis viruses that are able to function as controls for nucleic acid diagnostic assays (e.g., nucleic acid sequencing based assays and/or nucleic acid amplification based assays). In certain aspects, provided herein are Sindbis control virus are useful as whole process controls, positive controls and/or internal controls in nucleic acid diagnostic assays. Such control virus can benefit diagnostics manufacturers by providing a less expensive, consistent and safe source of starting material for controls. The control virus described herein use Sindbis virus, an RNA containing enveloped virus which can be engineered to contain target RNA sequences such as sequences from another virus and/or an internal control sequence. The Sindbis virus coat provides the RNA genome with improved stability. In some embodiments, the recombinant Sindbis virus system described herein results in viral particles that are packaged, so they can be used to evaluate nucleic acid extraction processes that are used before nucleic acid detection. Also provided herein are compositions comprising such viruses, nucleic acid molecules encoding the RNA genome of such control viruses, methods of making such control viruses and methods of using such control viruses.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “biological sample,” “tissue sample,” or simply “sample” each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.
The term “control” includes any portion of an experimental system designed to demonstrate that the factor being tested is responsible for the observed effect, and is therefore useful to isolate and quantify the effect of one variable on a system.
The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
As used herein, the term “heterologous RNA” refers to RNA present in a recombinant Sindbis virus that is not derived from wild-type Sindbis virus. For example, heterologous RNA in a Sindbis virus can be an RNA sequence normally found in a different virus (e.g., a different RNA virus or retrovirus), can be an RNA sequence normally found a non-viral organism, or can be a completely artificial RNA sequence.
The term “isolated nucleic acid” refers to a polynucleotide of natural or synthetic origin or some combination thereof, which (1) is not associated with the cell in which the “isolated nucleic acid” is found in nature, and/or (2) is operably linked to a polynucleotide to which it is not linked in nature.
The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleotide sequences provided herein, U nucleotides are interchangeable with T nucleotides.
As used herein, the term “Sindbis virus” includes viral particles made up of an icosahedral capsid that comprises Sindbis virus capsid, E1 and E2 proteins encompassing a single-stranded RNA genome. The RNA genome can include non-Sindbis RNA (i.e., heterologous RNA) and does not need to include all parts of the wild-type Sindbis genome. For example, in some embodiments the RNA genome does not encode one or more of the Sindbis structural proteins.

Replication Deficient Sindbis Control Viruses

In certain embodiments, provided herein are replication deficient Sindbis control viruses. In some embodiments, such viruses have an RNA genome that includes (a) an open reading frame (ORF) encoding functional Sindbis non-structural proteins and (b) a heterologous (i.e., non-Sindbis) RNA sequence. In some embodiments, the ORF encoding the functional Sindbis non-structural proteins is located 5′ of the heterologous RNA sequence. In some embodiments, the heterologous RNA sequence is a sequence from a different RNA virus (e.g., an Ebolavirus sequence, an influenza virus sequence, a SARS virus sequence, a hepatitis C virus sequence, a West Nile virus sequence, a Zika virus sequence, a poliovirus sequence or a measles virus sequence) or a sequence from a retrovirus (e.g., an HIV-1 sequence, an HIV-2 sequence, an HTLV-1 sequence, or an HTLV-II sequence).
Wild-type Sindbis virus is a member of Alphavirus genus, family Togaviridae. The viral genome is approximately 11,700 nucleotides. As such, Sindbis virus has approximately the same genomic complexity as many human pathogenic viruses, including, for example, HIV-1 (9270 nucleotides), HCV (9700 nucleotides) and Ebola Zaire (18959 nucleotides). This offers a technical advantage over certain other technologies used to package RNA controls, such as Armored RNA, which are based on MS2 bacteriophage technology and produce recombinant RNA molecules as small as 900 bases in length, which in many instances does adequately reflect the complexity or RNA secondary structure of the pathogenic viruses found in patient samples.
As depicted in FIG. 1, wild-type Sindbis virus contains a single-stranded positive sense genomic RNA which encodes both viral structural proteins (for capsid assembly and viral budding) as well as the nonstructural proteins (such as the replication enzymes). Upon entry of the virus into a cell, the RNA is released into cytoplasm and drives production of the viral replicase proteins (non-structural proteins 1-4). These proteins form replication and transcription complexes and are responsible for generating the negative strand of the genomic RNA. Promoters in the negative strand genomic RNA drive transcription of two mRNA species: The full-length genomic RNA encodes the nonstructural proteins and the smaller subgenomic RNA encodes the structural proteins. The 5′ ends of both transcripts are capped with 7-methylguanosine and the 3′ ends are polyadenylated.
In certain embodiments, the recombinant Sindbis control viruses described herein are replication deficient. In some embodiments, any method can be used to render the recombinant Sindbis control virus replication deficient. For example, in some embodiments the Sindbis control virus does not encode one or more functional structural proteins. For example, in some embodiments, the In some embodiments the recombinant Sindbis control virus genome does not encode one or more functional nonstructural proteins. In some embodiments, the Sindbis control virus does not encode a functional nsP1 protein, a functional nsP2 protein, a functional nsP3 protein and/or a functional nsP4 protein.
As described herein, separation of the Sindbis viral genome into two ORF facilitates the manipulation of the viral genome through replacement of the genes coding for the structural proteins with target sequences. This modified genomic RNA can be transcribed in vitro and introduced into cells along with a helper RNA (e.g., encoding structural proteins not encoded for in the modified RNA genome) for the defective virus. In some embodiments, the helper RNA encodes the four structural proteins required for Sindbis Virus packaging. In some embodiments, the helper RNA does not contain a packaging signal, and so does not get incorporated into the assembled viral particles. Thus, in certain embodiments, the viral particles produced therefore contain the target sequences but are replication defective because they do not bear the genetic information to produce the structural proteins. The recombinant viruses produced are effective quality control materials since they bear the selected target sequences, but the design of the recombinant Sindbis system provided herein ensures that the virus particles are safe and are not capable of establishing continuous infection. This is a distinct advantage for these materials over patient sourced or cultured viral materials as controls. FIG. 2 illustrates transcribed RNAs used for assembly of replication defective recombinant viruses.
Assembly of the virus particle occurs at the plasma membrane. A heterodimer of the structural proteins, E1 and E2, inserts into the plasma membrane and the E2 cytoplasmic tail is thought to provide the binding site for the nucleocapsid. This interaction between E2 and the nucleocapsid is thought to initiate the actual budding and release of the virus. When recombinant Sindbis viruses are produced in cultured cells, the virus particles are collected from the culture media, where they typically reach concentrations greater than 1×10⁸viral copies/mL. The budding process results in the recombinant Sindbis virus being enveloped into a lipid bilayer. This is important since the structure of the recombinant virus is thus similar to many other viruses generally classified as RNA-containing enveloped viruses such as HIV-1, HCV, HTLV, Influenza, and SARS. Therefore, the replication deficient Sindbis vectors described herein can be a true whole process control as they undergo sample lysis and nucleic acid processing similar to human pathogenic viruses that may be found in patient samples.
In recombinant Sindbis viruses, the target sequences replace the structural genes. This gives the system great flexibility in the size of the target sequences that can be accommodated and packaged efficiently. Target sequences of less than 100 bp to greater than 4000 bp can be efficiently incorporated in the recombinant viruses. The ability to accommodate large sequences is a distinct advantage, especially when producing controls for multiplexed assays. Multiple target sequences (from different pathogens or from different genes within the same pathogen) can be combined in one recombinant virus to form a multiplex control.
In some embodiments, the Sindbis control viruses described herein comprise HIV-1 sequence and are therefore useful as a control for HIV-1 diagnostic assays. In some embodiments, the HIV-1 sequence in the Sindbis control virus is distinct from naturally occurring HIV-1 virus sequence in that it contains resistance mutations arising from multiple classes of current HIV-1 therapies. Such multiplexed mutations do not occur in nature. In some embodiments, the control virus has the various drug resistance mutations present at the same allelic ratio. This provides users with a clear expectation for their test results. In certain embodiments, stop codons are engineered into the HIV-1 sequences so that no functional HIV-1 proteins are produced.
In some aspects provided herein is an HIV-1 Sindbis control virus that comprises an HIV-1 sequence in its RNA genome. In some embodiments, the HIV-1 sequence comprises one or more mutations that, when present in a HIV-1 virus, conveys a drug resistance phenotype (e.g., resistance to a protease inhibitor, a nucleoside analogue reverse transcriptase inhibitor and/or a non-nucleoside analog reverse transcriptase inhibitor). For example, in some embodiments the HIV-1 virus sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations that convey a drug resistant phenotype. In some embodiments, the one or more mutations, when present in HIV-1 virus, convey resistance to a drug selected from the group consisting of: atazanavir, ritonavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, saquinavir, tipranavir, abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, efavirenz, etavirine, nevirapine or rilpivirine. In some embodiments, the one or more mutations are selected from the group consisting of L241, D30N, V321, M461, I47V, G48V, 150V, I54M, G73S, L76V, V82A, I84V, N88D, L90M, M41L, K65R, D67N, T69S insert SS, K7OR, L74V, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y, K219Q, L100I, K101E, K103N, V106A, V1081, Y181C, Y188L, G190A, P225H and M230L. In some embodiments, the one or more mutations are selected from the group consisting of L241 (TTA to ATA), D30N (GAT to AAT), V321 (GTA to ATA), M461 (ATG to ATA), I47V (ATA to CTA), G48V (GGG to GTG), 150V (ATT to GTT), I54M (ATC to ATG), G73S(GGT to GCT), L76V (TTA to GTA), V82A (GTC to GCC), I84V (ATA to GTA), N88D (AAT to GAT), L9OM (TTG to ATG), M41L (ATG to TTG), K65R (AAA to AGA), D67N (GAC to AAC), T69S insert SS (ACT to TCT and insertion of TCC and TCC), K7OR (AAA to AGA), L74V (TTA to GTA), F77L (TTC to CTC), Y115F (TAT to TTT), F116Y (TTT to TAT), Q151M (CAG to ATG), M184V (ATG to GTG), L210W (TTG to TGG), T215Y (ACC to TAC), K219Q (AAA to CAA), L100I (TTA to ATA), K101E (AAA to GAA), K103N (AAA to AAC), V106A (GTA to GCA), V108I (GTA to ATA), Y181C (TAT to TGT), Y188L (TAT to TTA), G190A (GGA to GCA), P225H (CCT to CAT) and M230L (CCT to CAT). In some embodiments, the HIV-1 sequence comprises at least a portion of an HIV-1 gene selected from p7, pl, p6, HIV protease, reverse transcriptase, p51 RNAse, integrase and gp120. In some embodiments, the HIV-1 sequence comprises at least a portion of p7, pl, p6, HIV protease, reverse transcriptase and integrase. In certain embodiments, the HIV-1 sequence comprises at least a portion of 6p120, wherein the portion comprises the V1-V5 variable loops. In some embodiments, the HIV-1 sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 1900 through 5400 and/or 6300 through 7825 of the HXB2 strain of HIV-1 (SEQ ID NO: 4). In some embodiments, the HIV-1 sequence is identical to nucleotides 1900 through 5400 and/or 6300 through 7825of the HXB2 strain of HIV-1 (SEQ ID NO: 4) except for the presence of the mutations that convey a drug resistance phenotype. In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5 and/or SEQ ID NO: 7. In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 and/or SEQ ID NO: 8.
In some embodiments, the Sindbis control viruses described herein comprise Ebolavirus sequence and are therefore useful as a control for Ebolavirus diagnostic assays. In some embodiments, the Ebolavirus sequence comprises at least a portion of an Ebolavirus GP gene sequence, an Ebolavirus NP gene sequence or an Ebolavirus VP24 gene sequence. In some embodiments, the heterologous RNA sequence does not encode a functional Ebola protein (e.g., the heterologous RNA sequence encodes truncated Ebola proteins, Ebola proteins with frame-shift mutations and/or Ebola protein sequences lacking a start codon). In some embodiments, the heterologous RNA sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3.
The Sindbis control viruses described herein can be generated using any method known in the art. An exemplary method of generating the Sindbis control viruses described herein is illustrated in FIG. 3. In this exemplary method, capped in vitro transcripts of recombinant RNA bearing sequence of interest and the helper RNA are first synthesized. The synthesized RNAs are then electroporated into an appropriate cell, such as a BHK cell. The Sindbis structural proteins are expressed, but since the RNA does not encode replicase enzymes, so no new RNA is transcribed. Recombinant RNA is packaged by the capsid proteins. Viral glycoproteins associate with the nucleocapsid and viral particles bud into the culture medium. The culture supernatant is then collected, filtered and heat inactivated. The viral titer can then be determined using an appropriate method, such as real-time PCR and appropriate quality control tests can be performed to ensure that the RNA is fully encapsulated and there is no contaminating template DNA.

Use of Sindbis Control Vectors in Nucleic Acid Diagnostic Assays

In certain aspects, provided herein are methods of testing a diagnostic assay by running the diagnostic assay on a composition comprising the replication deficient Sindbis virus described herein. In some embodiments, the diagnostic assay is an assay for the detection of Ebolavirus, an influenza virus, a SARS virus, a hepatitis C virus, a West Nile virus, a Zika virus, a poliovirus, a measles virus, an HIV-1 virus, an HIV-2 virus, an HTLV-I virus and/or an HTLV-II virus. In certain embodiments, the heterologous RNA sequence in the RNA genome of the replication deficient Sindbis virus contains the target sequence detected in the diagnostic assay.
In some embodiments, the diagnostic assay is a nucleic acid amplification based diagnostic assay. In some embodiments, the nucleic acid amplification based diagnostic assay includes a sample lysis step, a nucleic acid extraction step (e.g., a magnetic-bead based nucleic acid extraction step), a nucleic acid amplification step and/or a nucleic acid detection step. In some embodiments, the nucleic acid amplification and detection steps are performed simultaneously (e.g., through the use of a real-time detection technology, such as TaqMan probes or molecular beacons). Examples of nucleic acid amplification processes include, but are not limited to, polymerase chain reaction (PCR), LATE-PCR a non-symmetric PCR method of amplification, ligase chain reaction (LCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), self-sustained sequence replication (3 SR), Qβ replicase based amplification, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), boomerang DNA amplification (BDA) and/or rolling circle amplification (RCA).
In some embodiments, the diagnostic assay is a nucleic acid sequencing based diagnostic assay (e.g., a next-generation sequencing based diagnostic assay). In some embodiments, the nucleic acid sequencing based diagnostic assay includes a sample lysis step, a nucleic acid extraction step (e.g., a magnetic-bead based nucleic acid extraction step), a nucleic acid amplification step, and/or a nucleic acid sequencing step. Examples of nucleic acid sequencing processes include, but are not limited to chain termination sequencing, sequencing by ligation, sequencing by synthesis, pyrosequencing, ion semiconductor sequencing, single-molecule real-time sequencing, 454 sequencing, and/or Dilute-‘N’-Go sequencing.

EXAMPLES

Example 1

Production of an Ebola Sindbis Control Virus

Ebola is a Filovirus with a single stranded, negative sense RNA genome. The Ebola virus genome includes the glycoprotein gene (GP) and the nucleoprotein gene (NP); these two genes were the targets of common nucleic acid-based diagnostic assays.
Ebola Sindbis Control virus was generated to serve as a control in such diagnostic assays. Recombinant Sindbis constructs were designed by cloning either about 2 kb of Ebola Zaire GP gene sequence (SEQ ID NO: 9) or about 1.5 kb of NP gene sequence and about 0.5 kb of a third Ebola gene, VP24 (SEQ ID NO: 10) into the Xba I restriction site of a SinRep SC vector (SEQ ID NO: 1). To ensure that no functional Ebola proteins would be produced, the constructs were designed to encode severely truncated GP and NP gene sequences. The GP constructs also lacked the AUG start codon for translation initiation and the NP construct contained a large internal deletion that changes the reading frame. Engineered stop codons were introduced in both constructs. These measures increase the safety of the product, but do not interfere with target detection (primer and probe binding) of the targeted diagnostic assays.
SEQ ID NO: 9 is an exemplary complete GP Ebola Sindbis control virus genome. Nucleotides 1 to 7652 and 9708 to 10080 of SEQ ID NO: 9 are Sindbis gene sequences, and nucleotides 7653 to 9707 of SEQ ID NO: 9 are Ebola GP insert sequences. SEQ ID NO: 10 is an exemplary complete NP/VP24 Ebola Sindbis control virus genome. Nucleotides 1 to 7652 and 9976 to 10348 of SEQ ID NO: 10 are Sindbis gene sequences, and nucleotides 7653 to 9975 of SEQ ID NO: 10 are Ebola NP/VP24 insert sequences.
Capped Ebola Sindbis control virus RNA was transcribed in vitro along with the helper RNA and introduced into baby hamster kidney cells. At 24 hours post-transfection, the cell supernatant was collected and the viral particles were purified and concentrated. Heat treatment was performed using a time and temperature known to inactivate similar RNA viruses as a further safety precaution. After titering the viruses using a TaqMan reverse transcription PCR assay, the viruses were combined and diluted into defibrinated human plasma containing human genomic DNA and 0.09% sodium azide as a preservative.
Three independent lots of the Ebola Sindbis control virus were tested in a real-time nucleic acid amplification based diagnostic assay developed for the detection of Ebola Zaire virus. The control material was processed identically to how an unknown patient sample would be processed. Representative results of this assay are shown in Table 1. In this table, Ct is the Cycle threshold value and SAC is the sample adequacy control (which verifies human source DNA in the sample).

TABLE 1

Ebola Sindbis control virus tested in a Ebola detection diagnostic assay.

Sample	Input				SAC
ID	Volume	Test Result	GP Ct	NP Ct	Ct

Lot
1	250 μL	Ebola GP DETECTED;	29.5	28.5	35.0
		Ebola NP DETECTED
Lot 2	250 μL	Ebola GP DETECTED;	30.6	29.5	34.9
		Ebola NP DETECTED
Lot 3	250 μL	Ebola GP DETECTED;	30.3	29.5	34.4
		Ebola NP DETECTED

Example 2

Stability of an Ebola Sindbis Control Virus

Stability of quality control materials is critical, especially considering that for many automated systems, reagents are loaded onto the instrument and must be stable at ambient temperatures for extended periods. Thus, the stability of the Ebola Sindbis Control virus produced as described in Example lunder various storage conditions was tested.
Vials of the Ebola Sindbis Control virus produced as described in Example 1 were subjected to 37° C. At designated time points, vials were removed from the stress condition and extracted using the Qiagen QIAamp Viral RNA Mini Kit. Testing was performed via a TaqMan quantitative real time PCR assay. Results are shown in FIG. 4 and indicate no loss of stability after 22 days at 37° C. Using a model based on the Arrhenius equation, this stability at 37° C. correlates with a stability at a storage temperature of 2-8° C. of at least a 2 years.
Vials of the Ebola Sindbis Control virus produced as described in Example lwere subjected to multiple rounds of freezing and thawing (F/T). As shown in FIG. 5, subjecting the Sindbis control virus to three freeze-thaw cycles did not have an adverse effect on the stability of the virus.
To test the extended stability of a Sindbis control vector at various temperatures, a recombinant Sindbis virus (bearing 0.8 Kb of target sequence) was diluted into defibrinated human plasma at 5×10⁵copies/mL target concentration. The material was dispensed into vials and vials were stored frozen at −20° C., refrigerated at 2-8° C. or at ambient lab temperature (approximately 25° C.) for up to 200 days. Vials were tested periodically using a TaqMan real time PCR test. No loss of stability was detected across the seven months of storage, even for samples stored at ambient temperatures. This demonstrates that the viral coat proteins and envelop of the Sindbis virus form a stable protective barrier that prevents nucleases in complex clinical matrices such as plasma from degrading the target RNA sequence.

Example 3

Production of HIV-1 Multiplex Drug Resistance Sindbis Control Virus

A Sindbis control virus was generated for use in diagnostic assays for the detection of drug resistant HIV-1 viruses. The Los Alamos National Laboratory HIV Sequence Database was used to generate a “reference sequence” for the control virus. Based on this database as well as the publication Special Contribution Update of the Drug Resistance Mutations in HIV-1: March 2013 by Victoria A. Johnson et al., in Topics in Antiviral Medicine, mutations in the HIV-1 genome that confer resistance to which therapeutic drugs were identified. These mutations and drugs are summarized in Table 2.

TABLE 2

Drug resistant mutations of HIV included in the HIV-1 multiplex drug
resistance Sindbis control virus.

		Resistance	DNA Sequence change
Drug Class	Therapy	Mutations	from reference sequence

Protease Inhibitors	Atazanavir +/− ritonavir	L24I	L24I (TTA to ATA)
	Darunavir/ritonavir	D30N	D30N (GAT to AAT)
	Fosamprenavir/ritonavir	V32I	V32I (GTA to ATA)
	Indinavir/ritonavir	M46I	M46I (ATG to ATA)
	Lopinavir/ritonavir	I47V	I47V (ATA to CTA)
	Nelfinavir	G48V	G48V (GGG to GTG)
	Saquinavir/ritonavir	I50V	I50V (ATT to GTT)
	Tipranavir/ritonavir	I54M	I54M (ATC to ATG)
		G73S	G73S (GGT to GCT)
		L76V	L76V (TTA to GTA)
		V82A	V82A (GTC to GCC)
		I84V	I84V (ATA to GTA)
		N88D	N88D (AAT to GAT)
		L90M	L90M (TTG to ATG
Nucleoside and	Abacavir	M41L	M41L (ATG to TTG)
Nucleotide Analogue	Didanosine	K65R	K65R (AAA to AGA)
Reverse	Emtricitabine	D67N	D67N (GAC to AAC)
Transcriptase	Lamivudine	T69S insert SS	T69S (ACT to TCT and
Inhibitors (NRTI)	Stavudine		insertion of TCC TCC)
	Tenofovir	K70R	K70R (AAA to AGA)
	Zidovudine	L74V	L74V (TTA to GTA)
		F77L	F77L (TTC to CTC)
		Y115F	Y115F (TAT to TTT)
		F116Y	F116Y (TTT to TAT)
		Q151M	Q151M (CAG to ATG)
		M184VL210W	M184V (ATG to GTG)
			L210W (TTG to TGG)
		T215Y	T215Y (ACC to TAC)
		K219Q)	K219Q (AAA to CAA)
Non-Nucleoside	Efavirenz	L100I	L100I (TTA to ATA)
Analogue Reverse	Etravirine	K101E	K101E (AAA to GAA)
Transcriptase	Nevirapine	K103N	K103N (AAA to AAC)
Inhibitors (NNRTI)	Rilpivirine	V106A	V106A (GTA to GCA)
		V108I	V108I (GTA to ATA)
		Y181C	Y181C (TAT to TGT)
		Y188L	Y188L (TAT to TTA)
		G190A	G190A (GGA to GCA)
		P225H	P225H (CCT to CAT)
		M230L	M230L (ATG to CTG)

In addition to the mutations described above, virus entry inhibitor drugs such as Miraviroc are blocked by mutations in the envelop gene. This drug is a CC chemokine receptor 5 (CCR5) antagonists and is only effective for patients with virus that uses the CCR5 co-receptor for viral entry. Viruses that use both CCR5 and CXC chemokine receptor 4 (CXCR4) or only CXCR4 will not respond to treatment with CCR5 antagonists. A virus's ability to use CXCR4 co-receptor is not defined by a single mutation, but instead is determined by the sequence of several variable “loops” in the gp120 envelop gene.
HXB2 strain of HIV-1 is a CXCR4 utilizing virus. HXB2 sequence is available from the Los Alamos National Laboratory HIV Sequence Database. Its sequence was used in the development of the recombinant virus representing the mutant CXCR4 virus. BaL strain of HIV-1 uses exclusively CCR5 co-receptor. Its sequence was obtained from the NCI database and used in the development of recombinant Sindbis virus representing wild type CCR5 virus.
Four DNA sequences were chemically synthesized and cloned into the Xba I restriction site of a SinRep SC Sindbis expression plasmid (SEQ ID NO: 1), which bears genes required for Sindbis virus production. Four Sindbis control viruses were generated, one that contained the 5′ end of a wild-type HIV-1 genome, one that contained the 5′ end of a multidrug resistant HIV-1 viral genome, one that contained the 3′ end of a wild-type HIV-1 genome and one that contained the 3′ end of a multidrug resistant HIV-1 viral genome. The insert sequences for these four control viruses are described in Table 3.

TABLE 3

Description of recombinant virus sequences.

	Genes included in the	HXB2-Nucleotide
Construct Designation	sequence	Positions

5′ multi-mutant (SEQ ID	part of p7, p1, p6, Protease,	Contains continuous
NO: 11)	RT, p51 RNAse and	sequence from nucleotides
	Integrase	1900 through 5400. The
		mutations shown in Table 1
		are incorporated
5′ WT (SEQ ID NO: 12)	part of p7, p1, p6, Protease,	Contains continuous
	RT, p51 RNAse and	sequence from nucleotides
	Integrase	1900 through 5400.
3′ mutant (SEQ ID NO: 13)	A portion of gp120	nucleotides 6300-7825 of
	including V1-V5 variable	HXB2 sequence are
	loops	included
3′ WT (SEQ ID NO: 14)	A portion of gp120	The BaL sequence which
	including V1-V5 variable	corresponds to HXB2 6300-7825
	loops	(as determined by
		BLAST alignment) is
		included

SEQ ID NO: 11 is the DNA counterpart to an exemplary complete 5′ multi-mutant HIV-1 Sindbis control virus genome. Nucleotides 1 to 7646 and 11167 to 11655 indicate Sindbis gene sequences, and nucleotides 7647 to 11166 indicate multi-mutant HIV-1 insert sequences. SEQ ID NO: 12 is the DNA counterpart to an exemplary complete 5′ wild-type HIV-1 Sindbis control virus genome. Nucleotides 1 to 7646 and 11161 to 11649 indicate Sindbis gene sequences, and nucleotides 7647 to 11160 indicate wild-type HIV-1 insert sequences. SEQ ID NO: 13 is the DNA counterpart to an exemplary complete 3′ mutant HIV-1 Sindbis control virus genome. Nucleotides 1 to 7646 and 9187 to 9675 indicate Sindbis gene sequences, and nucleotides 7647 to 9186 indicate mutant HIV-1 insert sequences. SEQ ID NO: 14 is the DNA counterpart to an exemplary complete 3′ wild-type HIV-1 Sindbis control virus genome. Nucleotides 1 to 7646 and 9182 to 9670 indicate Sindbis gene sequences, and nucleotides 7647 to 9181 indicate wild-type HIV-1 insert sequences.
The process used to produce the recombinant HIV-1 Sindbis control viruses is outlined in FIG. 7. Briefly plasmids that contain the target HIV-1 sequences in the SinRep vector were linearized with Not I restriction enzyme. An aliquot was analyzed by agarose gel electrophoresis to ensure that DNA cutting is complete.
Ambion mMessage mMachine SP6 kit was used for in vitro transcription of large amounts of capped RNA using reaction conditions optimized for long transcripts. DHBB is a helper RNA needed for packaging of the replication defective Sindbis virus; this helper RNA was transcribed from a linearized plasmid as well. The integrity and identity of the transcribed RNA was analyzed by denaturing agarose gel electrophoresis. The RNA was treated with DNAse to remove template plasmid DNA and purified using Ambion MegaClear kit.
To ensure optimal cell viability, BHK-21 (Baby Hamster Kidney cells) were amplified in culture for 2-4 passages after revival of frozen stock. Immediately prior to electroporation, the fetal bovine serum in the culture media was reduced, which helps reduce this cell's tendency to form clumps. Preventing cell clumps is desirable to maximize the transfection efficiency during electroporation.
The in vitro transcribed RNA was introduced into the BHK-21 cells via electroporation. The cells were washed at 6 hours post transfection to remove any unincorporated RNA.
The in vitro transcribed RNAs (HIV-1 sequences in SinRep RNA and DHBB helper RNA) were translated within the cells to produce the proteins required for recombinant Sindbis virus assembly and budding. The recombinant viruses were released into the culture media. The culture media was collected at 24 hours post transfection. The crude viral supernatant and the purified viruses were titered by extracting the viral nucleic acids using the Qiagen QlAamp Viral RNA mini kit and then using quantitative TaqMan real time PCR assay which targets a portion of the Sindbis viral vector RNA.

Example 4

Production of a Zika Sindbis Control Virus

Zika virus is a positive-sense, single-stranded RNA molecule of about 10794 bases long, and it codes a single polyprotein that is subsequently cleaved into capsid (C), precursor membrane (prM), envelope (E), and non-structural proteins (NS). Zika virus reference materials were designed based on a 2007 Zika virus strain with GenBank Accession number EU545988.1 (SEQ ID NO: 15). For the Zika Reference Materials, this genome was divided across four different constructs with at least ˜150 bp overlap between constructs and breakpoints at the ends of conserved domains. The overlap design is shown in FIGS. 9 and 10.
There was a 152 bp overlap between the “Zika Env” and “Zika NS2/NS3” construct, 150 bp overlap between “Zika NS2/NS3” and “Zika N54” construct and 180 bp overlap between “Zika N54” and “Zika NS5” constructs. These overlaps are designed to cover any diagnostic assays that target the ends of conserved domains. All four constructs were synthesized and introduced into Sindbis plasmids, which were used to prepare recombinant Sindbis virus.
The recombinant Zika/Sindbis virus were expressed, and high titer stock solutions of the viruses were prepared. The high titer stock solutions of recombinant Zika/Sindbis virus were diluted 1:100 in PBS, and RNA was extracted and eluted into 120 μL of 1:10 diluted AVE buffer. Extracted RNA was assayed by droplet digital PCR using a one-step RT-ddPCR master mix (Bio-Rad, 186-4021) at neat and 1:10 dilutions. Vector specific primer/probe sets were used for quantifying all four constructs as shown in Table 4.

TABLE 4

Quantification of Zika Construct Copy Numbers
in Zika Virus Reference Materials

			Average
	Copies	Copies per	copies per	Copies per	Back
	Per	μL of	μL	mL of	calculated
	20 μL	Extracted	Extracted	Extracted	copies per
Sample	Well	RNA	RNA	Sample	mL stock

Zika Env	3580	716	6.81E+02	5.84E+05	5.84E+07
Zika Env	2800	560
Zika Env	3840	768
Zika Env 1:10	396	79.2	7.84E+01	6.72E+04	6.72E+07
Zika Env 1:10	388	77.6
Zika Env 1:10	392	78.4
Zika NS2/NS3	6040	1208	1.20E+03	1.03E+06	1.03E+08
Zika NS2/NS3	6360	1272
Zika NS2/NS3	5580	1116
Zika NS2/NS3	692	138.4	1.27E+02	1.09E+05	1.09E+08
1:10
Zika NS2/NS3	636	127.2
1:10
Zika NS2/NS3	578	115.6
1:10
Zika NS4	6460	1292	1.29E+03	1.10E+06	1.10E+08
Zika NS4	5380	1076
Zika NS4	7480	1496
Zika NS4 1:10	532	106.4	1.33E+02	1.14E+05	1.14E+08
Zika NS4 1:10	750	150
Zika NS4 1:10	718	143.6
Zika NS5	2528	505.6	5.33E+02	4.57E+05	4.57E+07
Zika NS5	2268	453.6
Zika NS5	3200	640
Zika NS5 1:10	264	52.8	5.13E+01	4.40E+04	4.40E+07
Zika NS5 1:10	196	39.2
Zika NS5 1:10	310	62

Based on the high titer stock concentration, a 35 mL bulk was formulated at 5.0E+05 copies/mL in filtered human plasma (Basematrix) containing 0.09% NaN₃diluent and human genomic DNA (H9 DNA, 50 ng/mL). A Pall Acropak 1000 Filter Capsule (PES RM-1002220) was used for filtering the plasma. To 900 mL of filtered plasma, 810 mg of sodium azide and 45 μg of human genomic DNA was added and mixed for 15 minutes. All four constructs were targeted to 5.0E+05copies/mL in the prepared bulk. Bulk was mixed thoroughly for about 15 minutes, and RNA was extracted in triplicate and assayed using ddPCR with a One-Step RT-PCR master mix from Bio-Rad Laboratories (Catalogue# 186-4021). Assay specific primers/probe were used to quantify each construct. Data is shown in Table 5.

TABLE 5

Quantification of Zika Construct Copy Numbers in Zika Virus Reference
Materials Formulated with Human Plasma

		Copies	Copies per
		per	μL of		Average
		20 μL	Extracted	Copies per	Copies per
Sample	Conc.	Well	RNA	mL of bulk	mL of bulk

Zika Env	50.8	1016	203.2	1.74E+05	1.72E+05
Zika Env	48.6	972	194.4	1.67E+05
Zika Env	50.7	1014	202.8	1.74E+05
Zika NS2/NS3	33.6	672	134.4	1.15E+05	1.22E+05
Zika NS2/NS3	37.3	746	149.2	1.28E+05
Zika NS2/NS3	36.2	724	144.8	1.24E+05
Zika NS4	125.6	2512	502.4	4.31E+05	4.07E+05
Zika NS4	117.2	2344	468.8	4.02E+05
Zika NS4	113.1	2262	452.4	3.88E+05
Zika NS5	207	4140	828	7.10E+05	6.88E+05
Zika NS5	197	3940	788	6.75E+05
Zika NS5	198	3960	792	6.79E+05

An Altona Realstar Zika RT-PCR assay was performed on the extracted RNA from prepared bulk. The Altona Zika RT-PCR assay is a qualitative assay that gives a Positive or Negative result as shown in Table 6. Data is shown for both Zika and internal control analytes. The internal control (IC Zika (JOE)) should be detected in all negative and positive wells for a valid result, whereas Zika signal (Zika (FAM)) should be detected only in Positive wells. Bulk was tested in five replicates with Ct values around 28. Negative control was undetermined as expected, and the positive control Ct was 32.

TABLE 6

Altona Realstar Zika RT-PCR Assay Performed on Zika Virus Reference
Materials Formulated with Human Plasma

Well	Sample Name	Detector	Task	Ct	Result

A1	Zika Bulk	Zika (FAM)	Unknown	28.4665	POSITIVE
A1	Zika Bulk	IC Zika (JOE)	Unknown	30.7525	VALID
A2	Zika Bulk	Zika (FAM)	Unknown	28.5619	POSITIVE
A2	Zika Bulk	IC Zika (JOE)	Unknown	30.9062	VALID
A3	Zika Bulk	Zika (FAM)	Unknown	28.5517	POSITIVE
A3	Zika Bulk	IC Zika (JOE)	Unknown	30.9627	VALID
A4	Zika Bulk	Zika (FAM)	Unknown	28.7069	POSITIVE
A4	Zika Bulk	IC Zika (JOE)	Unknown	31.0635	VALID
A5	Zika Bulk	Zika (FAM)	Unknown	28.6494	POSITIVE
A5	Zika Bulk	IC Zika (JOE)	Unknown	31.0911	VALID
C1	Negative control	Zika (FAM)	NTC	Undetermined	NEGATIVE
C1	Negative control	IC Zika (JOE)	NTC	30.8181	VALID
C2	Positive Control	Zika (FAM)	Unknown	32.0931	POSITIVE
C2	Positive Control	IC Zika (JOE)	Unknown	30.809	VALID

6 mL of prepared bulk was sent to a commercial laboratory for bioburden testing. The bioburden result was 0 cfu/mL for bacterial growth and the Zika reference materials passed the acceptance criteria (<100cfu/mL or No growth).
Extracted viral RNA from recombinant Sindbis virus was sequence-verified by Sanger sequencing. All four constructs were PCR amplified at the beginning and end of the insert, and each nucleotide sequence displayed 100% sequence homology with the EU545988.1 sequence used to design the constructs (SEQ ID NO:15).

Example 5

Stability of an Influenza Sindbis Control Virus

An influenza reference material comprising an 800-nucleotide sequence of the H7N9 influenza virus was constructed using methods similar to those described above. The influenza reference material was diluted into aqueous buffer or defibrinated human plasma at 5×10⁵copies/mL in a commutable matrix. The material was dispensed into vials and stored at −20° C., 4° C., or room temperature (˜25° C.). Vials were tested periodically using a laboratory developed H7N9 TaqMan real time PCR test. As shown in FIGS. 11 and 12, the influenza reference material stored at ambient temperature for 500 days was stable as only ˜15% loss of signal was observed. This stability profile suggests that the viral coat and envelope proteins form a stable protective barrier that prevents nucleases in complex clinical matrices (such as plasma) from degrading the target RNA sequence.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A replication deficient recombinant Sindbis virus comprising a RNA genome comprising:

an open reading frame (ORF) encoding functional Sindbis non-structural proteins; and

a heterologous RNA sequence.

2. The replication deficient recombinant Sindbis virus of claim 1, wherein the ORF encoding functional Sindbis non-structural proteins is located 5′ of the heterologous RNA sequence.

3. The replication deficient recombinant Sindbis virus of claim 1, wherein the ORF encoding Sindbis non-structural proteins has a nucleotide sequence that is at least 90% identical to nucleotides 1-7648 of SEQ ID NO: 1.

4. (canceled)

5. The replication deficient recombinant Sindbis virus of claim 1, wherein the RNA genome lacks a sequence encoding a functional version of one or more of the Sindbis structural proteins.

6.-9. (canceled)

10. The replication deficient recombinant Sindbis virus of claim 1, wherein the heterologous RNA sequence comprises a non-Sindbis RNA virus sequence or a retrovirus sequence.

11.-16. (canceled)

17. The replication deficient recombinant Sindbis virus of claim 10, wherein the heterologous RNA sequence comprises a non-Sindbis RNA virus sequence.

18. The replication deficient recombinant Sindbis virus of claim 17, wherein the non-Sindbis RNA virus sequence is an Ebolavirus sequence, an influenza virus sequence, a SARS virus sequence, a hepatitis C virus sequence, a West Nile virus sequence, a Zika virus sequence, a poliovirus sequence, or a measles virus sequence.

19. The replication deficient recombinant Sindbis virus of claim 18, wherein the non-Sindbis RNA virus sequence is an Ebolavirus sequence.

20.-26. (canceled)

27. The replication deficient recombinant Sindbis virus of claim 10, wherein the heterologous RNA sequence comprises a retrovirus sequence.

28. The replication deficient recombinant Sindbis virus of claim 27, wherein the retrovirus sequence is an HIV-1 sequence, an HIV-2 sequence, an HTLV-1 sequence, or an HTLV-II sequence.

29. The replication deficient recombinant Sindbis virus of claim 28, wherein the retrovirus sequence is an HIV-1 sequence.

30.-65. (canceled)

66. A composition, comprising a replication deficient Sindbis virus of claim 1.

67. The composition of claim 66, wherein the replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 90% identical to SEQ ID NO: 11; and a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 90% identical to SEQ ID NO: 13.

68. (canceled)

69. The composition of claim 66, wherein the replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 90% identical to SEQ ID NO: 12; and a replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 90% identical to SEQ ID NO: 14.

70. (canceled)

71. The composition of claim 66, wherein the replication deficient Sindbis virus comprising a RNA genome comprising a sequence that is at least 90% identical to either nucleotides 1-3446 of SEQ ID NO: 15, nucleotides 3294-5575 of SEQ ID NO: 15, nucleotides 5425-7722 of SEQ ID NO: 15, or nucleotides 7542-10272 of SEQ ID NO: 15.

72.-77. (canceled)

78. A nucleic acid molecule encoding the RNA genome of the replication deficient Sindbis virus of claim 1.

79. The nucleic acid molecule of claim 78, wherein the nucleic acid molecule is an RNA molecule.

80.-82. (canceled)

83. A method of making a replication deficient Sindbis virus comprising:

(a) transfecting a cell with the RNA molecule of claim 79 encoding the RNA genome of the replication deficient Sindbis virus and a helper RNA molecule encoding functional Sindbis structural proteins;

(b) culturing the transfected cell of step (a) under conditions such that the cell produces a replication deficient Sindbis virus comprising the mRNA genome; and

(c) collecting the replication deficient Sindbis virus.

84. (canceled)

85. A method of testing a diagnostic assay, comprising performing the diagnostic assay on a composition of claim 66.