Los Alamos National Laboratory

In-vitro Evolution of Influenza-A Virus Under Artificial Pressure

RNA viruses cause many significant diseases worldwide, such as

Dengue and Yellow fevers, the Flu, AIDS, etc. These viruses evolve very

rapidly and few antiviral therapeutics and vaccines exist to treat them. The

exact mechanism for viral evolution remains poorly understood.

We are studying the evolution of RNA viruses with the aim to answer

important questions about the prediction of virus phenotype from its

genotype, protein mutations and function, and functions of structural RNAs.

Although we are currently focused on the human Influenza-A (IAV) virus, we

hope our model can be applied to other RNA viruses.

Our experimental design creates an artificial environment for viral

infection, and our hypothesis is that we will be able to direct the viral

evolution in a specific and predictable way. We have engineered host cells to

express proteins that the IAV naturally produces (supplemented cells). After a

series of infections within the supplemented cell lines, we will study the effect

of this supplementation on viral evolution.

We expect that the viral population will evolve to efficiently infect the

supplemented cells and lose the ability to infect the wild type (natural) cells. If

our hypothesis is correct, this approach may provide novel viral therapeutic

approaches.

Abstract Producing IAV Protein - Supplemented Cells

RT-qPCR Assay Optimization

In-vitro Viral Evolution Model

LA-UR-15-25466

O. M. Ishak, C. Margiotta, A. Rooney, A. Weibel, A. Hatch, R. Toma, J. F. Harris, M. Wolinisky, J. Gans, and M.

Vuyisich. *Corresponding : vuyisich@lanl.gov, 505-695-3431

RNA based viruses are a substantial public health threat. For example, in

2014, 130–150 million people globally have chronic hepatitis C infection and

350,000 to 500,000 people die each year from hepatitis C-related liver

diseases (+ sense, SS., RNA based virus).

To evade the immune system, RNA viruses express less proteins than

DNA viruses. Having relatively smaller genomes and viral RNA polymerases

lacking proofreading capabilities, RNA viruses exhibit substantially higher

mutation rates than their DNA counterparts; (DNA viruses have mutation

rates between 10−6 to 10−8 mutations per base per generation, and RNA

viruses have mutation rates between 10−3 to 10−5 per base per generation).

Because of the rapid mutation rates seen in RNA viruses, our immune

systems are not as efficient when it comes to countering their attacks, as well

as our capacities to produce efficient therapeutics against them are

drastically reduced. This rapid evolution makes RNA viruses much more

dangerous because they are always changing.

Our research project focuses on the evolution of RNA viruses, how to

better understand the ways they evade the immune response and to

ultimately use their own strategies against them.

Background

After conducting multiple literature searches, we selected 5 proteins that

are expressed by Influenza-A virus. The genomic sequences of these

proteins have been optimized and inserted into a eukaryotic plasmid. A V5-

epitope tag was also added to allow for the detection of the viral IAV proteins.

Supplemented cells were made by transfecting IAV viral proteins into wild

type (WT) Madin-Darby canine kidney (MDCK) cells (Figure 1). Cells were

further sub-cloned in order to select varying levels of viral protein expression

(i.e. high, medium and low).

Our goal (Figure 2) was to develop an artificial environment in which the

virus (IAV) does not have to produce one of it’s proteins in order to replicate

(viral-protein supplementing [VP] cells).

During successive cycles of host cell infections (supplemented & WT),

the evolution of the virus was monitored using RT-qPCR (Figure 3), next

generation sequencing, comparative genomics, and bioinformatics.

Methodology

Figure 1: WT MDCK cells are transfected with 1 of the 8 IAV viral proteins to produce new IAV protein -

supplemented cells. Supplemented cells are distinct from WT MDCK cells in terms of morphology,

metabolism, and trypsin-EDTA sensitivity.

Figure 2: This figure illustrates the workflow for carrying out in-vitro evolution. From the top of the

figure you can see that one parent influenza strain (H1N1, Calif. 2009) was used for both viral

populations. Infection of supplemented and WT MDCK cells occurred simultaneously. WT MDCK cells

were infected in parallel to serve as a control. Both viral populations are harvested for sequencing and

other downstream functions.

Sample Viral Infection (PB1C1- Generation 1)

Next Steps• Continue the in-vitro evolution process for other clones (HAC7 & HAC9,

PB1C1 & PB1C2).

• Carry out G4 and G5 in-vitro evolution process for M1 clones and verify

end results with RT-qPCR and sequencing.

• Explore using different cell lines for the same in-vitro evolution protocol

(i.e. A549s, BEAS-2Bs) and for making new supplemented cells.

Figure 3: After much trail and error we were able to get an influenza based Taqman assay to work. The

above comparison of two different RT-qPCR assays illustrate that Flu-2 shows greater nonspecific

amplification (i.e. primer dimer) as compared to Taqman; this can be seen in the difference in CT’s

between the two assays. For our purposes we chosen the Taqman assay because of its increased

specificity (primers + probe) vs. the previous Flu-2 assay that was SYBR green based (relied solely on

primers).

Figure 4: These are sample cell morphology and Taqman RT-qPCR results from the previous in-vitro

evolution workflow for PB1C1 clones. Unlike previous clones (HA, and M1) that we have tested, the

PB1C1 clones had a substantially longer infection time of 144 hours (HA – 72hrs, M1 – 65hrs). In

addition, there were alive cells still attached and growing at the time the virus was harvested (144 hr. PI).

For our next PB1C1 generation (G2) we will be monitoring CPE in 12 hour intervals and waiting until

majority of the cells have been infected / killed.

Observations / Recent Findings• We have optimized cell growth & infection rates for supplemented cells.

Different clones (HA, PB1, M1) exhibit different rates in terms of

metabolism, growth, CPE, and cell turnover.

• We have also finalized an influenza based Taqman assay for RT-qPCR

(vs. old Flu 2 assay that was SYBR green). The new Taqman assay will

help solve our previous issues with nonspecific binding and primer

dimers.

• Over the course of maintaining the growth of the viral protein

supplemented cells, we encountered a problem with the cells taking

months at a time to reach confluence. To mitigate this issue we ended up

adjusting the concentration of Geneticin (G418) from 0.30 to 0.15 mg/mL.

This adjustment has helped decrease supplemented cell growth from

months to 1-2 weeks.

• After the first round of in-vitro evolution for PB1C1 supplemented clones,

we have found measureable ΔCT and CPE results, that suggest

successful viral replication within these clones.

• We have conducted up to generation 3 for M1 supplemented clones. The

results have been somewhat discouraging because of the lack viral

replication in later generations (G2 and G3). We are hypothesizing that

the supplemented viral M1 protein might be interfering with competent

virion packaging.