# Patent

## Title
Influenza C virus and vaccine

## Abstract

A novel influenza C virus with only low homology to any influenza C virus previously characterized. Challenge studies show that the virus can infect pigs and be transmitted between pigs. Additionally, influenza C is commonly thought of as a human pathogen and serological studies have been performed, looking at the incidence of antibodies against this virus in both pigs and humans. Approximately 10% of pigs and 30% of humans have antibodies to this virus. Additional experimental data show that the virus can infect and transmit in ferrets (a surrogate for human infection studies). In a third aspect, the present invention is the partial genome of this novel influenza C virus. In another aspect, the present invention is a method of detection in animals of this novel influenza C virus.

## Background
BACKGROUND OF THE INVENTION
Influenza C is a common pathogen of humans as most individuals are infected as children. Influenza C causes a mild respiratory disease similar to the common cold. There is one report of influenza C being isolated from pigs in China (Yuanji and Desselberger 1984, J. Gen. Virol. 65:1857-72). Additionally, this work demonstrated that porcine influenza C viruses could infect and transmit between pigs. Several other papers have identified antibodies in pigs that react with influenza C, suggesting that either pigs are a reservoir for influenza C or that humans very commonly pass this virus to pigs (Kimura et al. 1997, Virus Res. 48:71-9; Yamaoka et al. 1991, J Gen Virol. 72:711-714; Brown et al. 1995, Epidemiol. Infect. 114:511-20; Ohwada et al. 1987, Microbiol Immunol. 31:1173-80).
SUMMARY OF THE INVENTION
As influenza C has only been reported in one paper, it is rare to find in pigs. The virus that has been isolated and characterized is a very unique variant of influenza C. The overall percent homology of this virus to known influenza C viruses is only about 65%, showing that this is a completely new lineage of virus that has never been described and is only distantly related to described viruses. This low level of homology suggests that vaccines made against typical human influenza C viruses would not protect against C/swine/Oklahoma/1334/2011. The finding that “universal” influenza C primer sets also failed to detect this virus also speaks to its novelty. Additionally, the finding of antibody titers in humans and pigs, as well as the virus' ability to replicate in both pigs and ferrets, suggests that this virus is capable of causing disease in pigs and man. We show that we can create a vaccine that protects pigs from infection and the scientific literature would also support that this vaccine would protect humans.
In one aspect, the present invention is a novel influenza C virus with only low homology to any influenza C virus previously characterized. Challenge studies show that the virus can infect pigs and be transmitted between pigs. Additionally, influenza C is commonly thought of as a human pathogen and serological studies have been performed, looking at the incidence of antibodies against this virus in both pigs and humans. Approximately 10% of pigs and 30% of humans have antibodies to this virus. Additional experimental data show that the virus can infect and transmit in ferrets (a surrogate for human infection studies).
In a second aspect, the present invention is a vaccine to this novel influenza C virus.
In a third aspect, the present invention is the partial genome of this novel influenza C virus.
In another aspect, the present invention is a method of detection in animals of this novel influenza C virus.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is an electron microscopic photograph of the novel influenza C virus of the present invention.
FIG. 2 is a graph showing the HI Titer in vaccinated and non-vaccinated pigs.
FIG. 3 is a graph showing real-time PCR threshold values in vaccinated and non-vaccinated pigs.
FIG. 4 is a graph showing tissue culture infection dose of ferrets challenged with the virus of the present invention.



DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
Isolation of the Virus
Samples submitted to Newport Laboratories were nasal swabs from pigs displaying signs of influenza-like illness. Samples were negative for influenza A by real time reverse transcription PCR. An aliquot of the sample was applied to a confluent monolayer of Swine Testicle (ST) cells to attempt to grow any viruses that were present. After 5 days cytopathic effects were evident, indicating that a virus was growing in the swine testicle cells. Samples of cell culture supernatant were analyzed for influenza A by QPCR and were negative. Cell culture supernatant was analyzed for the ability to hemagglutinate turkey red blood cells (hemagglutination assay). The hemagglutination assay was carried out according to the following protocol (Experiment #1).
EXPERIMENT #1
1.0 Introduction

    
    
        To quantitate Swine Influenza Virus.
    
    


2.0 Materials

    
    
        Reservoir
        96-well microtiter V bottom plates
        Multi-channel pipettor and tips (10-200 μL)
        100-1000 μL pipettor
        10-100 μL pipettor
        Pipette Tips
        Personal Protective Equipment (PPE: lab coat, safety glasses, gloves)
        Biosafety Cabinet
        Cover plate/lid
        Timer
        50 mL falcon tube
        Centrifuge
    
    


3.0 Reagents/Media

    
    
        Turkey Red Blood Cells (TRBC) from Lampire
        1×DPBS
    
    


4.0 Media Formulations
Turkey Red Blood Cell Solution (0.5%)

    
    
        TRBC arrive in Alsevers solution. Centrifuge in 50 mL conical tubes at 2000 rpm for 10 minutes. Aspirate and discard the supernatant.
        Wash the RBC by adding 1×DPBS equal to volume of blood started with. Invert and shake gently. Centrifuge and discard the supernatant. Repeat two more times. Store packed cells at 4° C.
        Add 50 mL 1×DPBS into 50 mL tube. Take out 250 μL 1×PBS. Add 250 μL of Turkey packed RBC. Mix gently. 5 mLs is needed per plate. Store diluted cells at 4° C.
    
    


5.0 Procedure
Preparation

    
    
        Eight samples will run horizontally on a 96 well plate. Figure out how many plates are needed based on samples to be tested and fill out the appropriate paperwork.
        Alternative set-up: Twelve samples can be run vertically on a 96 well plate.

Test Procedure

        Fill all wells of plates with 50 μL DPBS.
        Put 50 μL of sample #1 into well A1; sample #2 into B1, etc.
        With each test set of plates, run a negative control on the last plate (just DBPS).
        With 50 μL, dilute the plates starting from column 1 through 12 (excluding negative control row), discarding the remaining 50 μL.
        After the plate is diluted, fill all wells with 50 μL of 0.5% TRBC (invert tube to mix blood cells, do not vortex). Tap plates gently, cover and incubate at room temperature up to 2 hours.
        Alternative set-up: To run twelve samples vertically, put 50 μL of sample #1 into well A1; sample #2 into A2.
        With 50 μL, dilute the plates starting from row A through row H (excluding negative control column), discarding the remaining 50 μL.
        After the plate is diluted, fill all wells with 50 μL of 0.5% TRBC (invert tube to mix blood cells, do not vortex). Tap plates gently, cover and incubate at room temperature up to 2 hours.
    
    


6.0 Reading

    
    
        Tilt the plates at 30-45° to read. When reading, the last well without a complete teardrop is the end point. Mark on the corresponding paperwork.
        HA titers are listed on the corresponding paperwork
        Negative control row (or column) should completely tear drop, showing no hemagglutination.
    
    


The unidentified virus had the ability to hemagglutinate crbc's with a titer of 1280. Many viruses contain a gene encoding a hemagglutinin protein which enables a virus to bind cells, including red blood cells. Influenza viruses have the ability to hemagglutinate red blood cells.
EXPERIMENT #2
A flask of ST cells infected with the unknown virus was sent to the University of Minnesota Veterinary Diagnostic Laboratory for electron microscopy. Images of the virus were consistent with the family Orthomyxoviridae (FIG. 1). This family consists of influenza A, B, C and thogoto virus.
EXPERIMENT #3
PCR was attempted with primers designed to specifically detect either influenza A, B or C (Table 1). All PCR reactions were negative. Additionally, a neuraminidase activity assay was performed using the neuraminidase substrate methylumbelliferyl N-acetylneuraminic acid. The virus did not possess neuraminidase activity (both influenza A and B have neuraminidases). An esterase activity assay was next performed with 4-nitrophenyl acetate (Sigma Aldrich N8130). The virus exhibited esterase activity. The ability to hemagglutinate red blood cells and having an esterase activity is characteristic of influenza C and some members of the family Coronaviridae. Based on the hemagglutinin esterase activity and electron microscopy, the virus was preliminarily identified as influenza C although the negative for influenza C by PCR.







TABLE 1







Primers and probes used for real time reverse transcription PCR  


for virus detection. (IABkQ = Iowa black hole quencher)










Virus
Forward Primer
Reverse Primer
Probe





Swine
AGATGAGTCTTCTAACCG
TGCAAAAACATCTTCAA
5′-Cy3-TCA GGC CCC CTC


influenza A
AGGTCG
GTCTCTG
AAA GCC GA-3′-IABkQ


viruses
(SEQ ID NO: 19)
(SEQ ID NO: 20)
(SEQ ID NO: 21 with 5′





Cy3 and 3′ IABkQ)


 


Human
TCCTCAACTCACTCTTCG
CGGTGCTCTTGACCAAA
5′-FAM-CCA ATT CGA


influenza B
AGCG
TTGG
GCA GCT GAA ACT GCG


viruses
(SEQ ID NO: 22)
(SEQ ID NO: 23)
GTG-3′-IABkQ





(SEQ ID NO: 24 with 5′





FAM and 3′ IABkQ)


 


Human
ATTGAGAGCAGGAACGA
TCTTAAAGGCCCAGGAA
5′-FAM-


influenza C
CTG(SEQ ID NO: 25)
ACG(SEQ ID NO: 26)
CCCCTCTGGAAAGAGCC


viruses


ATGCAA-3′-IABkQ





(SEQ ID NO: 27 with 5′





FAM and 3′ IABkQ)









EXPERIMENT #4—GENOME SEQUENCING 
Virus was expanded in cell culture to generate 200 mL of cell culture harvest with a hemagglutination (HA) titer of 2560. Cell culture fluids were filtered through a 0.2 micron filter to remove cell debris. The fluids were then centrifuged at 110,000×g for 3 hours to pellet the virus. The virus was then resuspended in 1 mL of phosphate buffered saline and digested with DNase (New England Biolabs M0303S) and RNase (New England Biolabs M0243S) at 37 C for 1 hour. The viral solution was then gently layered on top of 35 mL of 25% sucrose solution and centrifuged for 3 hours at 110,000×g to pellet the virus. Viral RNA was extracted from the viral pellet using a Qiagen[trade] Viral RNA Mini Kit (Qiagen, Inc., 27220 Turnberry Lane Suite 200 Valencia, Calif. 91355) according to the manufacturer's instructions. In brief, the viral pellet was resuspended in 700 μL of buffer AVL containing carrier RNA and incubated for 10 minutes at room temperature. Next, 560 μL of ethanol was added to the sample and then sample was loaded onto a QIAampTM mini column by centrifugation at 6000×g. The column was next washed sequentially with 500 μL each of buffers AW1 and AW2, dried by centrifugation at 14,000×g for 2 minutes and then eluted with 60 μL of water. cDNA was then reverse transcribed from the viral RNA using a Promega Reverse Transcription Kit (Promega Corp., 2800 Woods Hollow Rd., Madison Wis. 53711;) along with enclosed random primers. The GoScriptTM Reverse Transcription System is a convenient kit that includes a reverse transcriptase and an optimized set of reagents designed for efficient synthesis of first-strand cDNA in preparation for PCR amplification. The components of the GoScriptTM Reverse Transcription System can be used to reverse transcribe RNA templates starting with either total RNA, poly(A)+mRNA or synthetic transcript RNA.
The cDNA was next digested for 1 hour at 37C with RNaseH (New England Biolabs M0297S) to remove RNA from the RNA-cDNA hybrid. The single stranded cDNA was made double stranded using the Klenow fragment from DNA polymerase (New England Biolabs M0210S).
Using the BioRuptor® Sonication System (Diagenode Inc. North America, 376 Lafayette Rd., Suite 102, Sparta, N.J. 07871), the double stranded cDNA was sonicated to fragment the viral cDNA. The fragmented cDNA was next used to construct a cDNA library according to the Life TechnologiesTM Ion Plus Fragment Library Kit protocol and described by the following: 50 microliters of the fragmented cDNA was mixed with 108 microliters nuclease-free water, 40 microliters 5× End Repair Buffer, and 2 microliters of End Repair Enzyme. The reaction was incubated at room temperature for 20 minutes. Following incubation, 360 microliters of Agencourt® Ampure® beads were added to the sample, which was then placed on a rotator at 8-10 rpm for 10 minutes. The sample was pulse-spun and placed on a DynaMagTM-2 magnet rack. After the solution cleared, the supernatant was removed and discarded. The sample was then twice washed using the subsequent protocol. 500 microliters of freshly made 70% ethanol were added to the sample without removing it from the magnet. The tube containing the sample was rotated twice on the magnet to move the beads around. After the solution cleared, the ethanol was removed. This wash procedure was repeated. Following the second 70% ethanol wash and removal of the supernatant, the sample tube was pulse-spun and placed back on the magnetic rack. Residual ethanol was aspirated and the sample dried at room temperature for approximately five minutes. 50 microliters of 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) were added to the cDNA and vortexed, pulse-spun, and placed back on the magnetic rack. After the solution cleared, the supernatant containing the eluted cDNA was transferred to a new 1.5-mL LoBind Tube[trade].
20 microliters of 10× Ligase Buffer, 77 microliters Nuclease-Free Water, 50 microliters of Adapters, and 3 microliters DNA Ligase were mixed with the 50 microliters of cDNA. The mixture was incubated at room temperature for 30 minutes. After the solution cleared, the supernatant was removed and discarded. The sample was then twice washed using the subsequent protocol: 500 microliters of freshly made 70% ethanol were added to the sample without removing it from the magnet. The tube containing the sample was rotated twice on the magnet to move the beads around. After the solution cleared, the ethanol was removed. This wash procedure was repeated. Following the second 70% ethanol wash and removal of the supernatant, the sample tube was pulse-spun and placed back on the magnetic rack. Residual ethanol was aspirated and the sample dried at room temperature for approximately five minutes. 30 microliters of 1×TE were added to the cDNA and vortexed, pulse-spun, and placed back on the magnetic rack. After the solution cleared, the supernatant containing the eluted cDNA was transferred to a new 1.5-mL LoBind Tube[trade].
All 30 microliters of cDNA were loaded onto a 2% agarose gel and subjected to gel electrophoresis for 80 minutes at 110V. Following gel electrophoresis, the sample was size-selected for 180-210 base pairs. The size-selected cDNA library was gel purified using the QIAquick[trade] PCR Purification Kit (Qiagen, Inc., 27220 Turnberry Lane Suite 200 Valencia, Calif. 91355) per the manufacturer's directions. The excised cDNA fragment of the gel was weighed and 3 volumes of Buffer QG to 1 volume gel were added. The mixture was incubated on a rotator at room temperature until the gel slice was completely dissolved. Once the gel slice had dissolved completely, the solution was applied to a QIAquick[trade] spin column (Qiagen, Inc., 27220 Turnberry Lane Suite 200 Valencia, Calif. 91355) and provided 2 milliliter collection tube and centrifuged at maximum speed (approximately 16,000 rcf) for 1 minute. The flow-through was discarded and the QIAquick[trade] column was placed back in the same tube. The column containing the cDNA was washed by adding 750 microliters Buffer PE to the column and centrifuging it at maximum speed for 1 minute. The flow-through was discarded and the QIAquick[trade] column was placed back in the same tube and centrifuged for 4 minutes at maximum speed. The QIAquick[trade] column was then put in a clean 1.5 milliliter microcentrifuge tube. 40 microliters of Buffer EB were added to the QIAquick[trade] column membrane and allowed to incubate at room temperature for several minutes before the column was centrifuged for 1 minute to elute the cDNA.
The eluted size-selected cDNA library was nick-translated and amplified according to the Life Technologies[trade] Ion Plus Fragment Library Kit protocol. 40 microliters of size-selected cDNA were mixed with 200 microliters Platinum[trade] PCR SuperMix High Fidelity and 10 microliters of Library Amplification Primer Mix. 125 microliter aliquots were transferred to each of 2 PCR tubes and ran on a thermocycler according to the following parameters:







TABLE 2







PCR Thermocycler Parameters










Stage
Step
Temperature
Time





Holding
Nick Translation
72° C.
20 min


Holding
Denature
95° C.
 5 min


Cycling (10 cycles)
Denature
95° C.
15 sec



Anneal
58° C.
15 sec



Extend
72° C.
 1 min


Holding
—
 4° C.
∞









The samples were pooled in a new 1.5 milliliter microcentrifuge tube and purified by adding 375 microliters of Agencourt Ampure® beads to the sample and incubating it for 10 minutes at room temperature on a rotator. The sample was pulse-spun and placed on a DynaMag[trade]-2 magnet rack. After the solution cleared, the supernatant was removed and discarded. The sample was then twice washed using the subsequent protocol. 500 microliters of freshly made 70% ethanol were added to the sample without removing it from the magnet. The tube containing the sample was rotated twice on the magnet to move the beads around. After the solution cleared, the ethanol was removed. This wash procedure was repeated. Following the second 70% ethanol wash and removal of the supernatant, the sample tube was pulse-spun and placed back on the magnetic rack. Residual ethanol was aspirated and the sample dried at room temperature for approximately five minutes. 50 microliters of 1×TE were added to the cDNA and vortexed, pulse-spun, and placed back on the magnetic rack. After the solution cleared, the supernatant containing the eluted cDNA was transferred to a new 1.5-mL LoBind Tube.
The amount of cDNA was quantified using the Ion Library Quantitation Kit according to the manufacturer's protocol. 5 sequential 10-fold dilutions were prepared from the E. coli DH10B Control Library according to the following table.







TABLE 3







Dilutions for E. coli DH10B Control Library












Nuclease-free



Standard
Control Library
Water
Fold Dilution













1
5 microliters
45 microliters
0.1



(undiluted)


2
5 microliters Std 1
45 microliters
0.01


3
5 microliters Std 2
45 microliters
0.001


4
5 microliters Std 3
45 microliters
0.0001


5
5 microliters Std 4
45 microliters
0.00001









A 1:20 dilution of the sample library in Nuclease-free water was initially prepared. Serial dilutions at 1:2000 and 1:20000 were subsequently prepared as described in the following table







TABLE 4







Dilutions for Sample Library









Dilution
Library
Nuclease-Free Water





1:2000 
1 microliter of 1:20
99 microliters


1:20000
5 microliters of 1:2000
45 microliters









As each qPCR reaction was prepared in triplicate, the master mix was prepared by mixing 250 microliters of Ion Library TaqMan® qPCR Mix 2×, 25 microliters of Ion Library TaqMan® Quantitation Assay 20×, and 100 microliters of Nuclease-free water per qPCR reaction. For each reaction, 15 microliters of master mix was pipetted into a well of the PCR plate. 5 microliters of the diluted control or sample library were added to each appropriate well. 5 microliters of nuclease-free water was used as the no-template control (NTC). The wells were sealed, briefly centrifuged, and subjected to the following real-time PCR conditions.







TABLE 5







PCR Conditions for qPCR Reaction











Stage
Temperature
Time
















Hold
50° C.
2
minutes



Hold
95° C.
20
seconds



Cycle (40 cycles)
95° C.
3
seconds




60° C.
30
seconds










The Template Dilution Factor was calculated using the following equation:

Template Dilution Factor=[(qPCR relative quantity)*(sample library fold dilution)]/0.32

The sample was diluted accordingly and prepared for sequencing using the Ion XpressTM Template Kit v2.0. The Emulsion OilTM was removed from the fridge and mixed. 9 milliliters of Emulsion OilTM were added to a IKA® DT-20 tube. The filled IKA® tube was placed on ice until ready for use. The aqueous PCR mix was generated by combining 582 microliters of Nuclease-free water, 200 microliters 5×PCR Reagent Mix, 100 microliters of 10×PCR Enzyme Mix, 100 microliters of Ion Sphere[trade] Particles (vortexed for 1 minute before addition), and 18 microliters of the diluted sample library. The mixture was vortexed for 5 seconds and set aside while the IKA® DT-20 tube containing the Emulsion Oil was positioned on the IKA® Ultra-Turrax® Tube Drive and locked in place. The adhesive label on the cap of the IKA® DT-20 tube was removed to expose the sample loading port. The START button on the IKA® Ultra-Turrax® was pushed and the entire volume of the aqueous PCR mix was dispensed through the opening in the blue cap of the IKA® DT-20 tube. After mixing for 5 minutes on the IKA® Ultra-Turrax® Tube Drive, the emulsion was placed on ice for approximately 5 minutes. A wide-bore tip was created by cutting approximately 5 mm from a pipette tip to transfer the emulsion. Using an Eppendorf® Repeater® Pipettor fitted with the wide-bore tip, the emulsion was drawn up and dispensed in 100 microliter increments to each well of a 96-well PCR plate until approximately 90 wells were filled. The 96-well plate was capped and loaded onto a thermal cycler according to the following PCR parameters







TABLE 6







PCR Parameters for Sequencing












Stage
Step
Temperature
Time







Hold
Denature
94° C.
 6 minutes



Cycle (40 cycles)
Denature
94° C.
30 seconds




Anneal
58° C.
30 seconds




Extend
72° C.
90 seconds



Cycle (5 cycles)
Denature
94° C.
30 seconds




Extend
68° C.
 6 minutes



Hold
—
10° C.
∞










Following the PCR reaction, as much of the contents of the wells as possible were transferred using a multi-channel pipette to a multi-channel pipette reservoir. Approximately 1.2 milliliters of the emulsion were transferred to each of six 1.5-mL microcentrifuge tubes. All six microcentrifuge tubes were centrifuged for 2 minutes at 15,500×g to collect the emulsion. During centrifugation, the Breaking SolutionTM was created by mixing 2 milliliters Recovery SolutionTM and 6 milliliters of 1-butanol. The Breaking SolutionTM was then vortexed for about 1 minute until a fine white emulsified material formed. After centrifugation of the emulsion, the clear top fraction of oil from each tube was removed. The 6 tubes containing the white emulsion were each treated with one milliliter of the Breaking SolutionTM, vortexed for 30 seconds, and centrifuged for 2 minutes at 15,500×g. After centrifugation, the top organic phase of each tube was removed. Each sample tube received 1 mL of Recovery SolutionTM, was vortexed for 30 seconds, and then centrifuged for 3 minutes at 15,500×g. The supernatant from each tube was removed until only approximately 100 microliters were left. Using the same pipette tip, the pellets in all six tubes were resuspended and transferred to a new 1.5 milliliter microcentrifuge tube. Three of the original tubes were rinsed with a single 200 microliter aliquot of Recovery SolutionTM. After the third tube was rinsed, the solution was transferred to the tube that contained the combined, resuspended pellets. This procedure was repeated on the three remaining tubes.
Recovery SolutionTM was added to the combined tube until the total volume was 1.5 milliliters. The tube was then vortexed for 30 seconds and centrifuged at 15,500×g for 3 minutes. The supernatant was removed until only about 100 microliters were left. The remaining material was resuspended and transferred to a new 1.5 milliliter microcentrifuge tube. 100 microliters of Wash SolutionTM was added to the original tube, rinsed, and then transferred to the new tube containing the sample. The sample was twice washed by adding 1 milliliter of Wash SolutionTMTM, vortexing for 30 seconds, and then centrifuging the tube for 3 minutes at 15,500×g. The supernatant was removed until only 100 microliters remained. This wash procedure was repeated.
The template-positive Ion SphereTM Particles enrichment was performed in accordance with the Ion XpressTM Template Kit v2.0 protocol. The bottle containing the Dynabeads® MyOneTM Streptavidin C1 beads was vortexed. 10 microliters of MyOneTM beads were transferred to a 1.5 milliliter microcentrifuge tube, washed with 70 microliters of Wash SolutionTM, vortexed, and placed on a magnet for 2 minutes. The supernatant was then discarded. The MyOneTM beads were resuspended in 10 microliters of new Wash SolutionTM and then transferred to the sample tube containing the Ion Sphere ParticlesTM (ISPs). To perform the capture, 100 microliters of Annealing BufferTM were also added to the sample tube which was then placed on the rotator for 10 minutes at room temperature. The sample tube was centrifuged and placed on the magnet until the solution was clear. The supernatant was transferred to a tube labeled “Unbound.” The beads were twice washed with 200 microliters of Wash SolutionTM, mixed, and placed back on the magnet before the supernatant was transferred to the “Unbound” tube. A fresh Melt-Off SolutionTM was prepared by combining 200 microliters 1 M NaOH, 16 microliters of 10% Tween® 20 in molecular grade water, and 1.38 milliliters of molecular grade water. To elute the Ion Sphere Particles from the Dynabeads® MyOne[trade] Streptavidin C1 beads, 400 microliters of the Melt-Off SolutionTM was added to the sample tube, mixed, and then placed on the rotator for 7 minutes. The supernatant was removed afterwards and placed into the tube labeled “Enriched-1.” The “Enriched-1” tube was vortexed and then spun at 15,500×g for 4 minutes. All but 100 microliters of supernatant were then discarded. 1 milliliter of Wash SolutionTM was then applied to the “Enriched-1” tube, vortexed, and spun at 15,500×g for 4 minutes. Once again, all but 100 microliters of supernatant was removed. The remaining sample was mixed and the “Enriched-1” tube was placed back on the magnet. The supernatant was removed after several minutes and put in a tube labeled “Enriched-2.”
DNA sequencing was conducted in accordance with the Life Technologies Ion Sequencing Kit v2.0 user's manual. 50 microliters of the sample in the “Enriched-2” tube were transferred to a new 0.2 mL PCR tube. 5 microliters of Control Ion Spheres trade] and 150 microliters of Annealing BufferTM were added and the solution was mixed and centrifuged for 2 minutes at 15,500×g. Supernatant was removed until only 9 microliters remained. 5 microliters of Sequencing PrimerTM were added to the sample and placed on a thermal cycler for a single cycle of 2 minutes at 95° C. and then 2 minutes at 37° C. The sample was then removed from the thermal cycler, mixed with 1 microliter of Sequencing PolymeraseTM, and incubated at room temperature for 5 minutes.
Meanwhile, a new chip was removed from its packaging and placed in the IonTM centrifuge adaptor/rotor bucket. Using a Rainin® SR-L200F pipette tip, 50 microliters of 100% isopropanol were added to the large port of the chip and then aspirated from the other port. The chip was washed two times with 50 microliters of Annealing BufferTM into the large port on the chip which was then aspirated from the other port.
The “Experiment” tab on the main menu of the PGMTM Sequencer was pressed. When prompted, the old chip was replaced with the new one. The barcode scanner was used to scan the chip barcode on the package. After the barcode was entered, the “Chip Check” button was pushed. After Chip Check was complete, the “Next” button was pressed to proceed to chip calibration. Following calibration, the chip was removed, placed back on the Ion centrifuge adaptor/rotor bucket, and washed with 50 microliters of Annealing Buffer into the large port on the chip, which was then aspirated from the other port on the chip. Using the Rainin® Pipette-Lite® LTS-20 pipette with a Rainin® SR-L200F tip, 7 microliters of the sample were deposited to the large port of the chip. The displaced liquid at the other port of the chip was then removed. The Ion ChipTM was then transferred to the centrifuge and spun for 4 minutes. Using a fresh Rainin® SR-L200F tip, the remainder of the sample was deposited to the loading port of the chip. The displaced liquid was removed from the other port and the chip was again centrifuged for 4 minutes. After the final spin was complete, the “Next” button on the PGM screen was pressed and the chip was loaded back onto the Ion Torrent PGMTM machine and the sequencing run started.
Sequence reads were assembled using the DNAStar software SeqmanNexGen® (DNASTAR, Inc., 3801 Regent Street, Madison, Wis. 53705 using the de novo assembly option. Sequence assembly identified seven contigs with greater than 10,000 reads associated each of them. The contigs were trimmed such that they represent the complete open reading frames. The trimmed sequences and corresponding protein sequences are included in the present application as SEQ ID NOs: 4-17. BLASTP analysis of the putative translated open reading frames revealed homology to the seven segments of human influenza C isolates and the isolate was designated C/swine/Oklahoma/1334/2011. The closest homolog for each segment is shown below. The segment noted as encoding the non-structural proteins (NS) is transcribed into mRNA and alternatively spliced to yield two different proteins (NS1 and NS2) as in Table 7 below. The percentage positive represents percent similarity between the isolated influenza C virus and the closest homolog for each segment in the public databases. While PB1 showed moderate homology to the PB1 of the human influenza C isolate C/Johannesburg/1/66 with 85% similarity, all other segments showed lower homology to previously sequenced influenza C, with % similarities for the other segments from 48-71%. The low overall similarity of C/swine/Oklahoma/1334/2011 with previously sequenced viruses demonstrates the uniqueness of this virus.







TABLE 7







BLAST Homologies










ORF (amino acids)
Best blast hit (BLASTP)













PB2
762
polymerase 2 [Influenza C virus (C/Ann Arbor/1/50)]




Identities = 397/762 (52%), Positives = 538/762 (71%), Gaps = 2/762 (0%)


PB1
720
polymerase subunit PB1 [Influenza C virus (C/Johannesburg/1/66)]




Identities = 512/708 (72%), Positives = 601/708 (85%), Gaps = 2/708 (0%)


P3
710
polymerase 3 [Influenza C virus (C/Ann Arbor/1/50)]




Identities = 358/722 (50%), Positives = 479/722 (66%), Gaps = 27/722 (4%)


HE
636
hemagglutinin-esterase [Influenza C virus (C/Catalonia/1318/2009)]




Identities = 306/608 (50%), Positives = 399/608 (66%), Gaps = 19/608 (3%)


NP
552
nucleoprotein [Influenza C virus (C/Ann Arbor/1/50)]




Identities = 199/504 (39%), Positives = 297/504 (59%), Gaps = 14/504 (3%)


M
397
unspliced product of M gene [Influenza C virus (STRAIN C/TAYLOR/1233/47)]




Identities = 145/383 (38%), Positives = 221/383 (58%), Gaps = 12/383 (3%)


NS1
243
nonstructural protein 1 (NS1) [Influenza C virus (C/Hiroshima/248/2000)]




Identities = 76/228 (33%), Positives = 110/228 (48%), Gaps = 21/228 (9%)


NS2
168
NS2 [Influenza C virus]




Identities = 53/180 (29%), Positives = 87/180 (48%), Gaps = 15/180 (8%)









Serological Studies to Determine Prevalence
Having established C/swine/Oklahoma/1334/2011 as a novel virus with weak sequence homology to human influenza C, serological studies were performed to determine incidence of infection for both humans and pigs. Approximately 200 random swine sera samples submitted to Newport Laboratories from numerous states were analyzed for antibodies to C/swine/Oklahoma/1334/2011 using the hemagglutination inhibition assay. Approximately 8% of samples were positive in the HI assay with titers from 10-160. Similarly, collaborators at St Jude's Children's Hospital in Memphis, Tenn., performed HI assays on a bank of human sera collected from elderly adults (age 65-95) from Canada. Approximately 28% of the samples had positive HI titers from 10-80. Together, these results demonstrate that both humans and pigs are commonly exposed to C/swine/Oklahoma/1334/2011. These results also suggest that this virus is capable of infecting both humans and pigs
Pig Vaccination and Challenge Experiment
C/swine/Oklahoma/1334/2011 was grown to a high titer (HA=2560) and inactivated with binary ethyleneimine and then 10% Trigen (an oil in water adjuvant) was added to make a killed virus vaccine. 22 pigs that were serologically negative for antibodies to C/swine/Oklahoma/1334/2011 were vaccinated on days 0 and 14 with 2 mL of inactivated virus vaccine delivered intra muscularly. Serum samples were collected on days 0, 14 and 28 and analyzed for HI titers. 28 pigs were also included as non-vaccinated controls. Vaccinated pigs seroconverted by day 28 with an average HI titer=433. Non-vaccinated pigs were negative on the HI assay. Table 8 and FIG. 3 shows the results. Vaccinated pigs showed strong seroconversion. The rule of thumb for influenza A is that a HI titer>40 is protective. The antibodies measured in pig sera in vaccinated pigs prior to challenge suggests protective antibodies were present. This data is consistent with the challenge results







TABLE 8







HI titers of vaccinated and non-vaccinated pigs











Vaccinates,
Vaccinates,
Non-Vaccinates,
Non-Vaccinates,



Direct Challenge
Contact Pigs
Direct Challenge
Contact Pigs
Controls




























day
day


day
day


day
day


day
day


day
day




28
42


28
42


28
42


28
42


28
42



day
(day
(day

day
(day
(day

day
(day
(day
pig
day
(day
(day
pig
day
(day
(day


pig ID
14
0)
14)
pig ID
14
0)
14)
pig ID
14
0)
14)
ID
14
0)
14)
ID
0
0)
14)





























850
40
320

856
20
320

865
0
0

854
0
0

851
0
0



852
20
160

858
40
640

866
0
0

857
0
0

853
0
0


860
10
320

861
0
20

870
0
0

859
0
0

855
0
0


863
10
640

864
80
1280

873
0
0

867
0
0

862
0
0
0


868
10
80

872
20
40

874
0
0

869
0
0

891
0
0
0


876
0
160

875
40
320

878
0
0

871
0
0

896
0
0
0


880
20
20
640
881
20
1280
320
883
0
0
20
877
0
0
40


886
80
320
640
882
80
1280
640
884
0
0
20
879
0
0
40


888
10
640
640
887
10
80
80
889
0
0
40
885
0
0
0


898
80
80
160
890
20
160
160
895
0
0
40
892
0
0
0


899
20
320
320
893
20
640
640
897
0
0
40
894
0
0
0


Average
27
278
480

32
551
368

0
0
32

0
0
16

0
0
0









On day 28, 11 vaccinated and 11 non-vaccinated pigs were challenged intra-nasally with 2 mL of 6.2 log 10 TCID/mL C/swine/Oklahoma/1334/2011. On day 2 post challenge 11 vaccinated and 11 non-vaccinated pigs were added to the room to serve as contact exposure challenge groups. Temperatures were measured every other day from the day of challenge to 14 days post challenge. Table 9 shows the results.







TABLE 9







Pig temperatures following challenge


















pig ID
Day 0
Day 2
Day 3
Day 6
Day 8
pig ID
Day 0
Day 2
Day 3
Day 6
Day 8











Vaccinates, Direct Challenge
Vaccinates, Contact Pigs


















850
102.5
102.1
103.4
103.6

856
104.5
102.1
104.1
104.4



852
104.3
105
106.2
104.6

858
102.9
103.3
103.6
103.1


860
103.5
103.3
102.7
102.4

861
102.9
101.8
104.2
102.7


863
101.2
102.5
103
102.7

864
103.1
100
103.6
102.6


868
103.1
103.9
102.2
103.6

872
102.6
103.4
103.5
102.8


876
105.7
104.5
103.2
102.8

875
102.5
102.9
103.4
103.2


880
103
105.2
104.7
103.6
104.7
881
103.4
103
104.7
104.3
104.1


886
103.8
103.9
104.7
104.1
103.8
882
102.8
102.9
103.2
102.7
102.7


888
103.7
103.7
104.1
103
103.6
887
102
102.8
102.7
103.4
103


898
101.9
103.1
104.4
103
103.3
890
103.9
102.2
103.8
103.2
104.4


899
103.6
103.8
103.8
101.9
102.6
893
104.6
102.7
103.6
103.7
103.6


Average
103.3
103.7
103.9
103.2
103.6

103.2
102.5
103.7
103.3
103.6








Non-Vaccinates, Direct Challenge
Non-Vaccinates, Contact Pigs


















865
105.1
104.6
105.2
102.7

854
104.7
103.7
104.2
102.9



866
103.4
102.7
104.4
102.1

857
103.6
103.6
103.9
103


870
104.5
103.9
104
103

859
102.3
103
104.3
101.9


873
103.6
102.9
105.1
102.1

867
104.2
102.8
103
102.4


874
104.6
103.8
105.6
101.8

869
104.7
103.1
104.3
102.9


878
102.1
102.6
102.4
103.5

871
103.3
103.3
103.6
102.3


883
103.9
104.2
104.7
104.4
104.4
877
104.2
104.1
104.8
103.2
103.3


884
102.5
105.3
105
101.4
103.8
879
103.3
102.9
100.9
100.9
103.9


889
103.7
104.2
104.3
103
103.1
885
102.2
103.4
105.1
103.7
103.3


895
103.5
104.3
102.6
101.8
102.8
892
104
103.9
103.8
102.8
103.6


897
103.1
104.8
105
102.4
103
894
103.8
101.6
104.1
103.1
102.3


Average
103.6
103.9
104.4
102.6
103.4

103.7
103.2
103.8
102.6
103.3








Controls














851
103.9
104
103.3
102.6




853
103.1
102.8
102.6
102.7


855
103.1
103.1
103.1
103.1


862
103.7
102.8
102.7
101.8
102.3


891
103.6
104.3
102.7
103.1
103.1


896
104
103.3
103.3
102.8
103.3


Average
103.6
103.4
103.0
102.7
102.9









No difference was observed between treatment groups, indicating that the virus does not cause a fever as influenza typically does. Similarly, nasal swabs were collected every other day from the day of challenge to day 14 post challenge.
Method of Assaying the Presence of the Virus
RNA was prepared from the swabs and the presence of influenza C was assayed by reverse transcription real time PCR (rt-RT-PCR) using the following primers and probes designed based on the genome sequence for C/swine/Oklahoma/1334/2011. The nucleotide sequences of the primers and probe is included in the present application as SEQ ID NOs:1-3, wherein Forward Primer=5′-GCT GTT TGC AAG TTG ATG GG-3′ (SEQ ID NO:1); Reverse Primer=5′-TGA AAG CAG GTA ACT CCA AGG-3′ (SEQ ID NO:2); and the Probe=Cy5-labeled-5′-TTC AGG CAA GCA CCC GTA GGA TT-3′-(SEQ ID NO:3)-IABkQ
The results are shown in Table 10 and FIG. 3. “Ct” stands for cycle threshold. In real time PCR, the genetic material (RNA or DNA) is copied by a polymerase by following cycling the sample/enzyme at various temperatures. Real time PCR uses a fluorescent reporter that binds to the produced DNA product. As cycling progresses and DNA product accumulates, more fluorescence is produced. At some point the fluorescence is detected by the PCR machine. A certain level of fluorescence detection is called the threshold. The number of cycles required to generate fluorescence above the threshold level is referred to as the Ct. Real time PCR can quantify the amount of RNA/DNA in the starting sample such that when more DNA/RNA is present prior to PCR, then fewer cycles/doubling will be required to generate fluorescence above the threshold. Consequently, a lower Ct value equates to higher levels of DNA/RNA in the original sample.







TABLE 10







Real time reverse transcription Ct values for


C/swine/Oklahoma/1334/2011 shedding in nasal swabs




















pig ID
Day 0
Day 2
Day 3
Day 6
Day 8
Day 10
pig ID
Day 0
Day 2
Day 3
Day 6
Day 8
Day 10











Vaccinates, Direct Challenge
Vaccinates, Contact Pigs




















850
37.1
37.1
37.1
37.1


856
37.1
37.1
37.1
37.1




852
37.1
37.1
37.1
37.1


858
37.1
37.1
37.1
37.1


860
37.1
37.1
37.1
37.1


861
37.1
37.1
37.1
37.1


863
37.1
37.1
37.1
37.1


864
37.1
37.1
37.1
37.1


868
37.1
37.1
37.1
37.1


872
37.1
37.1
37.1
37.1


876
37.1
37.1
37.1
37.1


875
37.1
37.1
37.1
37.1


880
37.1
37.1
37.1
37.1
33.6
37.1
881
37.1
37.1
37.1
37.1
37.1
37.1


886
37.1
37.1
37.1
37.1
37.1
37.1
882
37.1
37.1
37.1
37.1
37.1
37.1


888
37.1
37.1
37.1
37.1
37.1
37.1
887
37.1
37.1
37.1
37.1
37.1
37.1


898
37.1
37.1
37.1
37.1
37.1
37.1
890
37.1
37.1
37.1
37.1
37.1
37.1


899
37.1
37.1
37.1
37.1
37.1
37.1
893
37.1
37.1
37.1
37.1
37.1
37.1


Average
37.1
37.1
37.1
37.1
36.4
37.1

37.1
37.1
37.1
37.1
37.1
37.1








Non-Vaccinates, Direct Challenge
Non-Vaccinates, Contact Pigs




















865
37.1
37.1
34.53
37.1


854
37.1
37.1
37.1
37.1




866
37.1
37.1
37.1
37.1


857
37.1
37.1
37.1
37.1


870
37.1
37.1
37.1
37.1


859
37.1
37.1
37.1
37.1


873
37.1
37.1
37.1
37.1


867
37.1
37.1
37.1
37.1


874
37.1
37.1
35.2
28.6


869
37.1
37.1
37.1
37.1


878
37.1
37.1
37.1
31.32


871
37.1
37.1
37.1
37.1


883
37.1
37.1
35.05
34.85
34.23
37.1
877
37.1
37.1
37.1
37.1
37.1
34.98


884
37.1
37.1
37.1
29.65
24.21
29.45
879
37.1
37.1
37.1
37.1
37.1
40.52


889
37.1
37.1
34.69
35.54
35.85
29.37
885
37.1
37.1
37.1
37.1
37.1
32.49


895
37.1
37.1
35.5
30.26
29
37.1
892
37.1
37.1
37.1
37.1
37.1
27.35


897
37.1
37.1
32.8
29.76
28.11
37.1
894
37.1
37.1
37.1
37.1
36.53
37.1


Average
37.1
37.1
35.8
33.5
30.3
34.0

37.1
37.1
37.1
37.1
37.0
34.5








Controls















851
37.1
37.1
37.1
37.1





853
37.1
37.1
37.1
37.1


855
37.1
37.1
37.1
37.1


862
37.1
37.1
37.1
37.1
37.1
37.1


891
37.1
37.1
37.1
37.1
37.1
37.1


896
37.1
37.1
37.1
37.1
37.1
37.1


Average
37.1
37.1
37.1
37.1
37.1
37.1









The Ct values in the above chart show viral RNA levels in the nasal swabs. 37.1=negative. Any value less than 37.1 is positive for viral RNA (hence viral shedding). This data is also graphed in FIG. 3. The non-vaccinated challenge group viral RNA shedding numbers are significantly different from the vaccinates. We ran a Student's t-test on the data and the non-vaccinates that were directly challenged shed virus at higher levels than all other groups (P<0.001). Consequently, the vaccine protected pigs. Virus was only detected in one vaccinated pig following challenge, either direct or by contact exposure. In contrast, non-vaccinated pigs began to shed virus on day 3 post challenge (directly challenged) or on day 6 post exposure (contact challenge). This experiment demonstrates the virus is capable of infecting pigs and can replicate in them and is shed to the environment and contact animals. Additionally, pigs vaccinated with a homologous vaccine are fully protected from infection.
Ferret Challenge
Ferrets are commonly used as a surrogate for humans in influenza research as human influenza viruses typically replicate well in ferrets. As influenza C in normally thought of as a human pathogen, C/swine/Oklahoma/1334/2011 was used to challenge ferrets to determine if this virus is a likely human pathogen. Three ferrets were challenged intranasally with 6.0 log10 tissue culture infectious dose 50 per ml (TCID50/mL) of C/swine/Oklahoma/1334/2011. On day 1 post challenge, three ferrets were added to the pen to serve as contact animals. Additionally, 3 ferrets were housed in a separate pen to serve as aerosol only exposure animals. Table 11 and FIG. 4 shows the results.







TABLE 11







TCID50/mL of virus detected in nasal swab washes for ferrets


infected with C/swine/Oklahoma/1334/2011 by direct


inoculation, direct contact or aerosol contact









ferret

days post inoculation (TCID50/mL)













id

0
3
5
7
10
















55
direct

3.3
2.3
0.0
0.0



challenge


56
direct

3.9
0.0
0.0
0.0



challenge


57
direct

2.8
0.0
0.0
0.0



challenge


mean
direct
0.0
3.3
0.8
0.0
0.0



challenge


58
direct

0.0
0.0
2.0
4.5



contact


60
direct

0.0
0.0
0.0
4.2



contact


61
direct

0.0
0.0
2.2
4.1



contact


mean
direct
0.0
0.0
0.0
1.4
4.3



contact


62
aerosol

0.0
0.0
0.0
0.0



contact


63
aerosol

0.0
0.0
0.0
0.0



contact


65
aerosol

0.0
0.0
0.0
0.0



contact


mean
aerosol

0.0
0.0
0.0
0.0



contact









Virus was detected in nasal swabs by titration of ST cells. Virus was detected by day 3 post challenge in intranasally challenged animals and day 6 post exposure in contact challenge ferrets. No virus was detected in aerosol exposure ferrets. This data demonstrates C/swine/Oklahoma/1334/2011 is capable of infecting ferrets exposed either by direct challenge or contact with challenged animals. However, the virus does not appear to spread via aerosol transmission. This suggests that C/swine/Oklahoma/1334/2011 is likely capable of infecting humans.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety to the extent allowed by applicable law and regulations. In case of conflict, the present specification, including definitions, will control.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore desired that the present embodiment be considered in all respects as illustrative and not restrictive, reference being made to the appended claims rather than to the foregoing description to indicate the scope of the invention.

## Claims

1. A vaccine for protecting a mammal against infection by an influenza C virus, comprising an acceptable carrier, an immunologically effective amount of a binary ethyleneimine-inactivated swine influenza C virus and an oil in water adjuvant; and
wherein the virus comprises at least one nucleic acid molecule encoding eight influenza C polypeptides (PB1, PB2, P3, HE, NP, M, NS1 and NS2) having the sequences as set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17 and 18.

2. The vaccine of claim 1, wherein the isolated strain of influenza C virus was isolated from a pig.

3. The vaccine of claim 2, wherein said isolated strain of influenza C virus is capable of infecting a ferret.

4. The vaccine of claim 2, wherein said vaccine protects pigs against infection by said isolated strain of influenza C virus.

5. The vaccine of claim 1, wherein the oil in water adjuvant is present in an amount of about 10%.

6. The vaccine of claim 1 or 5, wherein the adjuvant is present in an amount of 10%.

7. The vaccine of claim 1, wherein the nucleic acid molecule comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or combinations thereof.

8. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 4.

9. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 6.

10. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 8.

11. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 10.

12. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 12.

13. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 14.

14. The vaccine of claim 7, wherein the nucleic acid molecule comprises SEQ ID NO: 16.