Accepted Manuscript

Title: Biophysical characterization of influenza A virions

Authors: Arun Parupudi, Flaviu Gruia, Samuel A. Korman,

Sonia Dragulin-Otto, Kuldip Sra, Richard L. Remmele Jr.,
Jared S. Bee

PII: S0166-0934(17)30156-8
DOI: http://dx.doi.org/doi:10.1016/j.jviromet.2017.06.002
Reference: VIRMET 13271

To appear in: Journal of Virological Methods

Received date: 7-3-2017

Revised date: 3-6-2017
Accepted date: 6-6-2017

Please cite this article as: Parupudi, Arun, Gruia, Flaviu, Korman, Samuel
A., Dragulin-Otto, Sonia, Sra, Kuldip, Remmele, Richard L., Bee, Jared S.,
Biophysical characterization of influenza A virions.Journal of Virological Methods
http://dx.doi.org/10.1016/j.jviromet.2017.06.002

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Biophysical characterization of influenza A virions
Arun Parupudi,a Flaviu Gruia,a Samuel A. Korman,a Sonia Dragulin-Otto,a Kuldip Sra,b Richard
L. Remmele Jr.,a and Jared S. Bee a*
a
Analytical Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD, United States.
b
Manufacturing Science and Technology, AstraZeneca, Liverpool, United Kingdom.
*Corresponding author at: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878,
United States. Tel.: +1 301-398-5912. E-mail address: jaredsbee@gmail.com (Jared S. Bee).
Graphical Abstract

Graphical TOC and Text:

Research

Highlights:
ABSTRACT:

Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains
circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce
intact virions with the surface antigens of the circulating strains. Influenza A typically contains a
large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion
structure, and aggregation are factors that are thought to have an impact on infectivity. However,
these factors are difficult to study because of the intrinsic variability in virion size, shape and
overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for
detailed size/structural analysis are established benchmark techniques for virus characterization.
Other methods may be valuable for certain sample types or circumstances. The aim of this work
is to establish a benchmark comparison between orthogonal biophysical techniques for particle
counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-
MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content.
CryoTEM with size analysis and fraction counting yielded detailed information about the
pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal
AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for
the selection of biophysical tools for the determination of particle counts, aggregation and
pleomorphic characteristics of influenza A virus.
1) NTA provided particle counts and size distributions in LAIV. The results were consistent with
negative stain and cryoTEM respectively.
2) FFF-MALS provided size distributions and aggregates/elongated content.
3) AUC-SV with multi signal detection provided information on the structural integrity of LAIV.
Keywords: influenza vaccine; ; ; ; ; , virion, particle counts, particle size, morphology,
biophysical characterization

1. Introduction

There are millions of cases of severe influenza infections each year worldwide (Webster et al.,

2013). Vaccines against influenza A and B are available in inactive, recombinant, and weakened

(live-attenuated viral) forms (Webster et al., 2013, Shaw and Palese, 2013). New strains emerge

due to ‘antigenic drift’ or ‘antigenic shift’ (Webster et al., 2013, Shaw and Palese, 2013).

Therefore, vaccine production has to be targeted to the strains circulating each year.

Structural variability (pleomorphism) is a feature of influenza A virions, the focus of this

study, which can vary in size and structural integrity and may be spherical, elongated, or even

highly filamentous depending upon the particular strain. Typically, the ratio of total particles

2
(TP) to infective particles (IP) is between about 10:1 and 100:1 for influenza A (Hutchinson et

al., 2010, Fonville et al., 2015, Enami et al., 1991, Wei et al., 2007, Donald and Isaacs, 1954).

The presence or absence of a full complement of the 8 different ribonucleoproteins (RNPs),

virion structure and morphology, and levels of virion aggregation are factors that are thought to

have an impact on infectivity (Hutchinson et al., 2010, Brooke, 2014). However, the dependence

of infectivity on the physical shape and morphology of different classes of virions remains

unclear (Nayak et al., 2009). For these reasons, potential for correlation between structural

features of virions and aspects of either infectivity or pathogenicity is of major interest to the

field (Harris et al., 2006).

Biophysical characterization of influenza virion samples is challenging because of their

intrinsic pleomorphism. The advantages and limitations of different biophysical methods and

how their results compare to established benchmark techniques of TEM and cryoTEM is a

question we investigate in this current work. This information is intended to help with practical

selection of methods for analysis of different influenza samples based on the study goals.

Multiple modern techniques were used to characterize highly purified influenza A H3N2 virus

sample (a live-attenuated version of A/Switzerland/9715293/2013). We evaluated dynamic light

scattering (DLS), nanoparticle tracking analysis (NTA), negative stain transmission electron

microscopy (TEM), cryoTEM, analytical ultracentrifugation sedimentation velocity (AUC-SV),

field flow fractionation with multi-angle laser light scattering (FFF-MALS), and infectivity by

focus forming assay (FFA). We determined the molecular weight of virions by applying the

Svedberg equation to combine AUC-SV and NTA results and independently by FFF-MALS with

refractive index detection, and compared these to a theoretically calculated value based on the

3
reported composition of influenza A virions. The ultraviolet (UV) absorbance spectrum

estimated the ratio of RNA bases to protein content in the virions.

2. Materials and Methods

2.1. Influenza A virus preparation

Influenza A is a genus of the Orthomyxoviridae family and is part of the Baltimore Group V

classification consisting of negative-sense single-stranded RNA (-ssRNA) viruses. A reverse

genetics method using an attenuated master donor virus (MDV) and wild-type

A/Switzerland/9715293/2013 (H3N2) influenza A virus plasmid was used to generate a live-

attenuated influenza A virus strain as previously described (Wei et al., 2007). The MDV strain is

a cold-adapted (ca), temperature-sensitive (ts), and attenuated (att) version of the A/Ann

Arbor/6/60 (H2N2) strain (Ambrose et al., 2008). The gene segments for ca, ts, and att from the

MDV were combined with two segments coding for the hemagglutinin (HA) and neuraminidase

(NA) from the wild-type A/Switzerland/9715293/2013 (H3N2) in the live-attenuated strain.

Influenza A was grown in embryonic pathogen free egg cells (Charles River laboratories,

Wilmington, MA). After inoculation and growth in hen eggs, the crude allantoic fluid was

harvested, filtered, concentrated, and purified by sucrose gradient centrifugation. The final

monovalent viral bulk (MVB) preparation was diluted into a formulation containing 100 mM

potassium phosphate, 0.19 M sucrose and 0.5 mM Ethylenediaminetetraacetic acid (EDTA)

disodium dihydrate at pH 7.1 and stored at -80ºC until analysis. The final buffer was selected

based on the long term stability data.

2.2. Virus particle infectivity by focus forming assay (FFA)

4
Infectivity by FFA was performed as previously described (Wei et al., 2007). In brief, Madin

Darby Canine Kidney (MDCK) cells were incubated with the influenza A virus particles for up

to 20 hr, washed, fixed with 80% acetone, and dried. The cells were then re-hydrated and

incubated with primary anti-HA antiserum. After washing away excess unbound primary

antiserum, a fluorescently labelled secondary antibody was incubated with the cells. After

washing and drying, the cells with budding viruses labelled with both the primary and secondary

antibody detection reagents were counted using a fluorescent microscope. The result was

reported as the average of six replicates after appropriate dilution corrections as focus forming

units (FFU) per mL.

2.3. Dynamic light scattering

Dynamic light scattering (DLS) measures the diffusion coefficient and determines the Z- average

hydrodynamic diameter using the Stokes- Einstein equation. DLS was performed using a

Zetasizer NanoZS instrument (Malvern Instruments, Westborough MA). Twelve accumulations

of 10 s each were collected for each measurement and repeated for a total of eighteen replicate

results. The intensity-weighted ‘z-average’ hydrodynamic diameter was reported.

2.4. Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) determines the number average hydrodynamic diameter by

individually tracking particles. NTA was performed using a model LM10 Nanosight instrument

(Malvern Instruments, Westborough, MA) equipped with a 532 nm diode laser. Samples were

diluted 5000× (from 0.45 mg/mL) to stay within the operating range of the NTA instrument. Five

separate readings of 60 s each were collected and averaged for each result using the NTA version

5
3.1 software using a Detection Threshold = 10. The measurement generated six replicate results

of separate aliquots.

2.5. Transmission electron microscopy

Electron microscopy was performed using an FEI Tecnai T12 electron microscope at 120 keV

using an Eagle 4k×4k CCD camera. Negative stain grids were imaged at room temperature and

vitreous ice grids were imaged on a stage held at a temperature below -170°C. Images were

acquired at a nominal under focus of -5 μm to -2 μm and electron doses of between 5 and 30 e-

/Å2.

Negative stain transmission electron microscopy (TEM) was used to determine virus

particle counts according to the procedure reported previously (Monroe and Brandt, 1970).

Negative stain TEM was not used to assess morphology because the sample preparation

procedure can distort the virion shape. A known concentration of 100 nm diameter polystyrene

beads (Part#3100A, Thermoscientific, Fremont, CA) was used to calibrate the counts. Samples

were imaged over a layer of continuous carbon supported by nitro-cellulose on a 400-mesh

copper grid after staining with phosphotungstic acid. The number of virus particles was

determined according to the formula: virus particles/mL = (9.0E12)×(2/100)×(virus particles

counted/beads counted), where 9.0E12 is the stock calibration bead concentration (9.0 ± 3.2 E12)

and 2/100 is the ratio of dilution of sample to beads. Forty images were analyzed for a total of

2175 virus particles counted.

CryoTEM was used for particle imaging, size distribution, and native morphology

evaluation. A 3 μL drop of sample was applied to a grid, blotted, and then immediately vitrified

in liquid ethane and stored under liquid nitrogen until transferred to the electron microscope for

6
imaging. The area equivalent diameter (2×√[area/]), circularity (4× area/perimeter2) and

maximum and minimum Feret diameters (largest and smallest projected diameters) were

extracted from images of 273 particles acquired at 21,000× magnification. About 14 individual

virus particles were captured in each image at the 21,000× magnification used for sizing analysis.

Size distribution and fraction counting was performed where virions in‘chains/aggregates’ were

assessed as a single entity (‘whole aggregate’ sizing) in order to compare the distribution to other

methods and also where each virus particle was sized individually irrespective of whether it was

contained in a chain/aggregate (individual virion sizing).

2.6. Analytical ultracentrifugation

Analytical ultracentrifugation sedimentation velocity (AUC-SV) measures the sedimentation

coefficient and can be used with NTA results to determine the molecular weight using Svedberg

equation. AUC-SV generated a differential sedimentation coefficient distribution c(s) profile of

the influenza A virus particles. Samples and reference buffer (100 mM potassium phosphate,

0.19 M sucrose and 0.5 mM EDTA disodium dihydrate at pH 7.1) were filled into 3 mm double-

sector cells with Epon centerpieces and sapphire windows, placed into an An50-Ti rotor, and

installed into a Beckman Proteome Lab XL-I centrifuge set to 20°C. A rotor speed of 3,000 rpm

was used to collect interference and UV scans (at both 280 and 260 nm) at a radial resolution of

0.003 cm over the range 5.9 - 7.2 cm. The Sedfit software program was used to generate c(s)

distribution from the raw data as described by Schuck (Schuck, 2000). The data were fit from 0

to 3000 S at a resolution of 451 using a confidence level of 0.683 with regularization by second

derivative.

The solution density and viscosity were set at 1.03694 g/mL and 1.2858 mPas

respectively and the meniscus position was allowed to float during the data analysis. A partial
7
specific volume of 0.912 mL/g was used in the fitting. The profiles obtained using the integral

sedimentation coefficient distribution ls-g*(s) analysis approach were essentially identical to the

c(s) analysis and the root mean square deviation (rmsd) of the data fit was insensitive to the

frictional ratio (0.003 for f/f0 values ranging from 1.0 to 1.9). This is expected in the case where

diffusion is small compared with sedimentation. Based on multiple runs we used a fixed value of

1.3 for f/f0 for consistency in the analyses.

The c(s) profile was converted to a size distribution using the nanoparticle size formalism

of the Svedberg equation (Harrison et al., 2003), where  is the solvent viscosity, s is the

sedimentation coefficient, d is the particle diameter, and  and 0 are the sedimenting particle

and solution density values respectively:

18𝜇𝑠
Equation 1: 𝑑 = √
(𝜌 − 𝜌0 )

We used the measured diameter result from NTA and the measured sedimentation coefficient

from AUC-SV to solve for the unknown value of the hydrated virus particle density (). The

hydrated density value of 1.10 g/mL (inverse of the partial specific volume 0.912 mL/g) we

obtained is consistent with data contained in prior literature reports (Sharp et al., 1950, Stanley

and Lauffer, 1947).

The refractive index detection system of the AUC can be used as a measure of

concentration that is insensitive to light scattering. Equation 2 gives the relationship between

measured fringes (J) and the refractive index increment (dn/dc), path length (l), laser

wavelength (), and concentration (c). We calculated the value of dn/dc and also the partial

specific volume (𝜈̅ , needed for molecular weight calculations) of influenza A virus particles

8
based on the values of the individual virus particle components (Schuck.P et al., 2015), at the

following ratios as reported in Fields Virology (Shaw and Palese, 2013) 71% protein (dn/dc =

0.185 mL/g and 𝜈̅ = 0.735 mL/g), 20% lipids (dn/dc = 0.16 mL/g and 𝜈̅ = 1.005 mL/g), 8%

carbohydrate (dn/dc = 0.15 mL/g and 𝜈̅ = 0.63 mL/g), and 1% RNA (dn/dc = 0.20 mL/g and 𝜈̅ =

0.508 mL/g). We calculated a value of 0.177 ± 0.001 mL/g for dn/dc and 0.78 ± 0.01 mL/g for 𝜈̅

of influenza virions. The potential compositional variation of virions estimated the error in the

calculation. Our calculated partial specific volume matches previously reported experimental

value of 0.79 ± 0.01 mL/g measured by pycnometer (Lauffer and Stanley, 1944). The dn/dc

value of 0.177 mL/g for influenza A virus particle is equivalent to an interference extinction

value, IF = 2.62 fringes∙mL∙mg-1 cm-1 for a 675 nm laser and 2.70 fringes∙mL∙mg-1 cm-1 for a

655 nm laser.

𝑑𝑛 𝑙
Equation 2: ∆𝐽 = ( ) 𝑐 = 𝜀 𝐼𝐹 𝑙𝑐
𝑑𝑐 𝜆

2.7. Field flow fractionation with multi-angle laser light scattering

Field flow fractionation with multi-angle laser light scattering (FFF-MALS) separates species

based on the size, shape and diffusion coefficients and MALS determines the weighted average

molecular weight and the radius of gyration of the species as they elute from the channel. FFF-

MALS fractograms were collected on a Wyatt Eclipse system with a DAWN EOS detector

(Wyatt Technology Corp., Santa Barbara, CA) equipped with either a 13 cm or 27 cm channel

with a regenerated cellulose membrane and a 350 m spacer. The mobile phase composition was

0.1 M sodium phosphate with 0.1 M sodium sulfate at pH 6.8. Sample was introduced over 5

min and then focused for 15 min using 0.2 mL/min opposing flows and 0.4 mL/min crossflow.

Separation was then achieved with a 1 mL/min lateral flow and a cross flow gradient of 0.4
9
mL/min to 0 mL/min over 35 min and then continued for another 10 min with a cross flow of 0

mL/min. The measurement generated six replicate fractograms.

The Wyatt Astra V software was used to process the light scattering data to obtain the

root mean square (RMS) and geometric radius values. The divergence point of the RMS and

geometric diameters at 156 nm was used to define the cutoff in the integration of UV data used

to calculate percent monomeric spherical particles and percent elongated particles and/or particle

chains/aggregates.

The raw Raleigh ratio data was exported from the Astra software in order to apply

corrections for counting of aggregates according to the procedure described by McEvoy et al.

(McEvoy et al., 2011). In brief, the normalized inverse Rayleigh ratio’s (1/Rθ) were plotted

𝜃
against 𝑠𝑖𝑛2 and fitted to a second order polynomial to calculate experimental form factors at
2

all the angles. The experimental form factors and the geometric volumes of aggregated particles

were used with the Rayleigh ratio to calculate the particle concentrations for the monomer and

aggregate regions. The refractive index (RI) of influenza A virus particles was set to 1.44 based

on measurements of minimum 90° scattering of 658 nm light for dilutions of influenza virus

particles in solutions with different refractive index values according to the procedure described

by Zölls et al. (Zölls et al., 2013).

2.8. Ultraviolet (UV) spectrum of virus particles

An Agilent 8453 UV spectrophotometer (Santa Clara, CA) was used to obtain a UV spectrum of

the influenza A virus particles without dilution using a 3 mm path length cell. Corrections for

light scattering were made using a log-log extrapolation of the region 330 to 350 nm as described

by Ksenofontov et al. (Ksenofontov et al., 2006).

10
3. Results

3.1. Particle count and size distribution results

Figure 1 shows a comparison of the size distributions obtained using DLS, NTA, cryoTEM

image analysis of ‘whole aggregates’, and AUC. The particle counts and size distributions of the

influenza A virus preparation are summarized in Table 1. The average size reported ranged from

about 124 nm (cryoTEM whole aggregates sizing) to 153 nm (FFF-MALS). The peak or mode

size was in closer agreement between methods, ranging from 114 nm by cryoTEM to 127.6 nm

and 129.5 nm for NTA and FFF-MALS respectively.

3.2. cryoTEM

The cryoTEM images analysis showed that the viruses were fully and evenly spiked, with

dense interiors, and with intact outer layers about 8 nm thick. Figure 2 is a representative image

obtained at 52,000× magnification. Additional images are provided in supplementary Figure S1.

Very few viruses appeared to be only partially spiked, of irregular in shape, or lacking integrity

of the outer layer. Most particles were spherical or slightly elongated/bean-shaped. The mean

value of circularity was 0.9 and about 73% of the virions had a circularity value greater than

0.85. The mean value of the minimum and maximum Feret diameters of the individual virus

particles were 96 nm and 139 nm respectively. Particles elongated by a factor greater than 2 were

a minor fraction at about 11%. The level of extracellular vesicles particles in this particular

sample was about 13%. This was determined by fraction counting of particles in images and an

example image is shown in supplementary Figure S3. Some infrequent instances of dense

proteinaceous material were observed. Triangular particles that resembled free spikes of length 3

nm were also noted and presumably related to the processing and sample handling. Fraction

11
counting of 292 virus particles in randomly selected images revealed that about 38.6% of the

individual virus particles by number were contained in chains/aggregates. The aggregates were

mostly dimer or trimer chains. The size results for both individual virions and for ‘whole

aggregates’ is given in Table 1 and the distribution in Figure 1 is shown for ‘whole aggregates’

for direct comparison to the other sizing methods. Supplementary Figure 2 shows the size

distribution, circularity, and elongation (max/min Feret diameter) of virus particles sized

individually.

3.3. Analytical ultracentrifugation sedimentation velocity (AUC-SV)

AUC-SV analysis of the influenza virus particles gave a c(s) distribution with a peak at 473 S

and a weighted-average sedimentation coefficient value of 565 S. The three replicate normalized

distributions from the interference signal and the UV at 260 and 280 nm all overlaid very closely

(Figure 3). Using the interference signal, we determined the concentration of virions to be 0.45 ±

0.02 mg/mL. The concentration was converted to counts using a molecular weight of 306 MDa

to give a log10 particle count value of 11.95, which was similar to the negative stain TEM and

NTA count results (Table I).

3.4. Field flow fractionation with multi-angle laser light scattering (FFF-MALS)

The FFF-MALS fractogram profile of the influenza A sample is shown in Figure 4. Virus

particles that eluted between 29 and 37.7 min had mean RMS and geometric diameters of 129.4

nm and 133.0 nm respectively, consistent with unresolved elution of spherical and bean-shaped

influenza A virions. Quantitation of the UV peak (n = 6) showed that the sample contained about

72.7 ± 2.6% spherical or bean-shaped monomeric virions and 27.3 ± 2.6% elongated particles

and/or chains/aggregates on a mass-basis. The total particle counts of the sample were found to

12
be log10 = 12.12 ± 0.04 based on the procedure described by Matt McEvoy et al. (McEvoy et al.,

2011).

3.5. Molecular weight of influenza A particles

The Svedberg equation (Equation 3) was used to calculate the molecular weight (M) using the

sedimentation coefficient (s) measured by AUC, the diffusion coefficient(D) measured by NTA,

the solution density (, and the partial specific volume of the ‘dry’ (unhydrated) particle (𝜈̅𝑉𝑃 )

(Schuck.P et al., 2015). The diffusion coefficient (D) measured by the NTA instrument is

converted to hydrodynamic diameter (d) using the Stokes-Einstein equation where the viscosity

(), and the Boltzmann constant (kb) are known: D = kbT/(3d). Using the mean sedimentation

coefficient from AUC-SV (565 S) and the mean particle diameter from the NTA (143.1 nm) and

a value of 0.78 mL/g for the partial specific volume, we determined that the average molecular

weight of our slightly aggregated preparation of influenza A particles was 306 MDa (Table II).

The peak of the NTA and AUC-SV distributions were at 127.6 nm and 473 S respectively,

resulting in an estimated value of 229 MDa which may more closely represent the monomer

molecular weight.

𝑠𝑅𝑇 3𝑠𝜋𝜇𝑑𝑁𝐴
Equation 3: 𝑀 = =
𝐷(1 − 𝜈̅𝑉𝑃 𝜌0 ) (1 − 𝜈̅𝑉𝑃 𝜌0 )

As an orthogonal measure, the Zimm equation (Wyatt, 1993) was applied to an FFF-

MALS fractogram profile collected with a higher sample injection (60 uL) on a longer channel

(27 cm). The higher loading and longer channel were used to help improve the signal from the

refractive index detector. Even with a higher loading the refractive index signal was still quite

noisy and the signal from elongated particles and/or chains/aggregates was quite small (Figure

5). The weight average molecular weight from the light scattering and refractive index (RI) for
13
the distribution shown in Figure 5 (for normalized RI signal > 0.25) was found to be 265 ± 4

MDa (Table 2).

Table 2 also lists a benchmark estimate of the molecular weight that we calculated based

on the mass of each of the major proteins in a virus particle. For this calculation, we used the

protein composition for a nominal intact virus particle with a full set of 8 RNP complexes as

reported by Hutchinson et al. (Hutchinson et al., 2014). The GISAID EpiFlu database and

Influenza Research Database were used to obtain the sequences needed for the calculations

(http://platform.gisaid.org/epi3/frontend#1262f2,

https://www.fludb.org/brc/home.spg?decorator=influenza ). We used the reported value that

virions contain 71% protein and an estimated additional 29% of mass content from lipids,

carbohydrates and RNA to determine the final total molecular weight (Shaw and Palese, 2013).

Details are provided in supplementary data.

In addition to molecular weight, we were able to use the NTA and AUC-SV data to

determine the water content, B1 = 1.5 g/g, for hydrated virions using Equation 4 (Schuck.P et al.,

2015). Influenza virus particles contain a relatively large amount of water inside the virus capsid

which can vary as a function of solution osmotic pressure (Sharp et al., 1950, Lauffer and

Stanley, 1944).

𝜈̅𝑉𝑃 + 𝐵1 𝜈̅1
Equation 4: 𝜈̅ℎ𝑉𝑃 =
1 + 𝐵1

3.6. Estimated average ratio of RNA to protein in virions

We estimated the weighted average UV extinction of the virion proteins at 280 nm based on the

relative proportions of proteins in an intact virus particle as reported by Hutchinson et al.

14
(Hutchinson et al., 2014). We calculated that the average overall extinction coefficient for

influenza virion proteins at 280 nm in this particular strain to be about 53,016 M-1cm-1 with an

SD of about 14% based on the error in the reported composition weighted by each protein’s

extinction coefficient. An assessment of 8 additional strains gave an average value of the

calculated average protein extinction coefficient 53,521.3 M-1cm-1 ± 15% based on a the

propagated error in composition (14%) and variation in strain properties (estimated at 4%).

Additional details are provided in supplementary data.

The influenza virion composition for an idealized intact virion with a full set of 8 RNPs

reported by Hutchinson et al. (Hutchinson et al., 2014) is equivalent to a molar ratio of between

about 4.1 to 5.7 RNA bases per ‘average’ virion protein. We then used the method described by

Porterfield et al. (Porterfield and Zlotnick, 2010) to simultaneously determine the protein and

RNA concentrations using light-scattering-corrected UV absorbance values at 260 nm and 280

nm. We used the average extinction coefficient at 280 nm that we calculated based on the virion

composition and a relative value of 0.6 at 260 nm for protein. For RNA we used an extinction of

8000 M-1cm-1 at 260 nm and 4000 M-1cm-1 at 280 nm. The molar ratio of RNA bases per

‘average’ virion protein calculated from the UV spectrum in this way for our sample was 3.6 ±

0.5.

4. Discussion

In generating live-attenuated influenza virus, attention must be given to assess total particle

counts, size distributions, aggregation states and structural integrity. In this work several

biophysical techniques were utilized to assess the ratio of the total particles to the infective

particles along with size distributions and structural integrity.

15
In the context of live-attenuated virus manufacturing, the virus titer is monitored and controlled

during dilution and formulation/blending unit operations. In this work, the FFA assay determined

infective particles per unit volume (FFUs per mL) of the bulk intermediate sample. Infective

particles typically represent less than between 1/10 to 1/100 of the total particles (Hutchinson et

al., 2010, Fonville et al., 2015, Enami et al., 1991, Wei et al., 2007). The total particle count is

therefore needed for estimation of the ratio of total to infective particles in a sample.

The total particle counts determined by NTA, FFF-MALS, and the historically

established method of negative stain TEM were similar when the variability of methods is

considered. We found that NTA was a rapid and simple method for counting total particles while

also providing additional size distribution information in a single measurement. A similar

conclusion was made by Kramberger et al (Kramberger et al., 2012) in their comparison of total

particle counts of from the NTA but with counts from a Hemagglutination assay (HA). This

might be useful for process monitoring where a rapid turnaround of many samples is needed.

Although the total particle count from FFF-MALS light scattering data was also in good

agreement our assessment was that the highly complex data analysis for counting particles was

not well suited for routine use. The refractive index signal from AUC-SV is insensitive to light

scattering (unlike UV absorbance) and determined counts of adenoviruses (Berkowitz and Philo,

2007). Application of this approach to the influenza A sample in the current work gave count

results also consistent with the other methods, providing confirmation that the values of the

dn/dc and molecular weight calculated in this work were reasonable estimates of the properties

of this influenza A virus sample. The calculated total particles to infective particles (TP/IP) ratio

using the TEM total counts and FFA infectivity result was 160 ± 88. Alternatively, using the

NTA total count the calculated total particles to infective particles (TP/IP) was 276 ± 118. These

16
values are similar to the typical benchmark of 100 to 1 that has been reported for influenza A

virions (Hutchinson et al., 2010, Hutchinson et al., 2014).

The size distribution and aggregate levels are potential factors that may impact

infectivity. It has been proposed that aggregation of non-infective virions can complement their

defects (e.g. missing RNA segments) thereby resulting in a productive infection (Brooke, 2014).

The size distribution and aggregation levels may be influenced by manufacturing unit operations

such as filtration and freeze-thaw. Aggregation can be caused by binding of surface HA and NA

proteins on different virions, a property that may vary for different strains (Rudneva et al., 1993,

Palese et al., 1974). Nikitin et al. (Nikitin et al., 2015) compared the size distributions of

icosahedral viruses determined using negative stain TEM, DLS and NTA and concluded that

larger difference in diameters between NTA and DLS compared to TEM may be due to electrical

double layer effects on hydrodynamic diameter. However, negative stain TEM cannot be utilized

for assessment of the size distribution of influenza virions since virus sample fixing and drying

alters the morphology.

In this work, we also compared the capabilities of several techniques to probe the size

distribution and aggregation of virions in solution. Although simple and rapid, DLS could not

resolve aggregates or elongated particles. Like DLS, AUC was also unable to resolve additional

distinct peaks. The DLS resolved size distributions were different than those generated by NTA

or cryoTEM. The inherent lack of resolution in DLS measurements is generally associated with

the data analysis procedure. The autocorrelation function is converted into size distribution

information through an inverse Laplace transform and generally the least detailed distribution

that is consistent with the data is chosen by the fitting algorithms.

17
 However, a small aggregate shoulder was resolved by both NTA and by cryoTEM. This

demonstrates that particle counting techniques provide specific advantages, namely increased

resolution when compared against bulk measurements. Fraction counting of the cryoTEM

images determined that about 39% of virions were contained in aggregated chains. The FFF-

MALS provided a very good separation of the elongated particles/aggregated chains, which

estimated at about 27%. The lower value of the FFF-MALS compared to the cryoTEM fraction

counting might represent changes that occur during the FFF separation, such as some dis-

aggregation of very weakly associated chains. It is also possible the cryoTEM images may give a

higher estimate due to sample size statistics or because particles that are not physically

aggregated are counted as aggregates because they are adjacent in the image.

Estimation of molecular weight is another alternative approach to characterize the ‘size’

of virus samples. We found that the two independent approaches (NTA/AUC or FFF-MALS) we

used in this work gave results (229 and 249 MDa respectively) for the monomer peak that were

broadly consistent with a theoretical benchmark monomer value we calculated (206 MDa).

These value also compare fairly well with prior literature (174 MD by TEM (Ruigrok et al.,

1984); 180 MDa by neutron scattering (Cusack et al., 1985); and 360 MDa by dry weight

(Horace, 1998).

Structural integrity and morphology may influence infectivity. For example, empty

capsids form because of repeated freezing and thawing (Tyrrell and Horsfall Jr., 1954).

Morphology can vary from strain to strain (Nayak et al., 2009) and can be influenced by the host

cells (e.g. MDCK vs. chicken eggs) (Ewan et al., 2015). Using cryoelectron tomography, Harris

et al found that elongated particles contained an ordered parallel arrangement of 8 RNPs,

whereas a more random arrangement of RNPs was noted in larger spherical virions (Harris et al.,
18
2006). Some of the smaller spherical particles contained fewer than a full complement of 8 RNA

segments. In this work, very few viruses that appeared to be only partially spiked or slightly less

intact in overall shape and no empty capsids were observed in cryoTEM images. The purified

and concentrated MVB sample in this work, with log10 particles of about 12, gave about 14

virus particles per image. This technique may therefore not be as practically feasible for samples

with lower particle counts such as crude allantoic fluid. Only a single peak with a consistent ratio

of 280 and 260 nm signals was detected by AUC, consistent with the absence of empty capsids.

Structural composition may also impact infectivity. A full complement of 8 RNPs is

required for a productive infection from a single influenza virion. If 8 RNPs were packaged

randomly then the total particle to infective particle ratio is predicted to be 416 to 1 (Enami et al.,

1991). If virions are assumed to contain more than 8 RNPs then the probability that a full set is

included is higher and the predicted total particles to infective particles (TP/IP) ratio decreases

accordingly (Enami et al., 1991). However, the random packaging model is inconsistent with

cryotomography and electron microscopy observations where an ordered 7+1 arrangement of

RNPs have been observed (Harris et al., 2006, Noda et al., 2006, Noda, 2015). Infectivity losses

can potentially occur during each stage of the lifecycle from infection, replication, budding,

release. It has also been reported that many virions are missing at least one RNP (Brooke, 2014)

and this could represent the case where the RNPs are ordered but with less than perfect fidelity

leading to incomplete sets of RNPs in virions that contribute to the non-infective particle content.

A measure of the relative amounts of RNA per protein may provide some insight into the

composition of a sample. We expect that larger virions with an elongated structure, yet still with

only 8 RNPs, would contain proportionally more protein per capsid (due to the increased protein

content in the capsid wall) relative to the 8 RNPs in the core of the capsid. However, the ratio of

19
RNA to protein for smaller virions with an incomplete set of RNPs may also be lower, so

interpretation of this overall average ratio in terms of infectivity is not necessarily clear unless

populations of capsids of different sizes could be isolated and compared separately. The c(s)

distribution by AUC using UV signals at 260 and 280 nm and interference detection overlaid for

the sample studied in this work, indicating that the composition of the virions (protein, RNA,

lipid) was relatively uniform for smaller and larger virions. Using the method of Porterfield et al.

(Porterfield and Zlotnick, 2010) we simultaneously determined the RNA and protein

concentration and their ratio based on the scattering-corrected UV spectrum of the sample and

found it to be a little lower than the ratio reported for the relative virion composition

benchmarked at a complete set of 8 RNPs reported by Hutchinson et al. (Hutchinson et al.,

2014).

5. Conclusions

This work evaluated the suitability and the value of several biophysical methods in determination

of the particle counts, aggregation and pleomorphic characteristics of an influenza A virus

sample. NTA was found to give similar total virus particle counts to negative stain TEM. The

NTA also provided additional size distribution information that was consistent with the

cryoTEM size distribution. FFF-MALS was able to well-resolve monomeric spherical and bean-

shaped virus particles from elongated particles. The aggregate content determined by FFF-

MALS was lower than by cryoTEM fraction counting analysis. Based on our observations we

recommend using NTA for routine particle counts and size distributions, especially for lower

concentration samples where a rapid turnaround time is needed. DLS does not provide counts

and its resolution is lower than NTA. FFF-MALS has value for assessment of the size

distribution and estimation of potential aggregate levels at a moderate throughput. NTA and

20
FFF-MALS were relatively rapid measurements that might be used to determine total counts,

size, and size distributions in routine manufacturing and process development studies. FFF-

MALS could also be explored as a tool for fractionation and isolation of different populations for

further studies of their properties. AUC-SV with multi-signal detection (UV at 260, 280 nm and

interference) provided information on the structural integrity of the sample as a whole. AUC-SV

is low throughput and less commonly available but shows promise for assessment of structural

integrity (such as empty capsids and so on). We suggest exploration of less intact samples than

that studied in this work might be a good application of AUC-SV. CryoTEM is an established

benchmark method for virus characterization and provided additional detailed information about

the structural integrity, morphology and virus particle aggregate content. This technique has the

limitation that the samples must be concentrated, the turnaround is relatively slower, and

resource required is higher than the other methods.

Future studies to compare strains, process intermediates, or size fractions with different

infectivities might provide more information and insight into possible links between measurable

biophysical properties and infectivity of influenza virus samples.

Conflict of interest disclosure

Medimmune, a member of the AstraZeneca group of companies, manufactures Influenza

Vaccine Live, Intranasal.

Acknowledgments

Electron microscopy was performed by NanoImaging Services, Inc. and manuscript review was

provided by Anette Schneemann and Joyce Sung. Peter Schuck generously provided AUC-SV

21
data analysis advice. We thank Kun Yao, Christian McLarnon-Riches, Helen Bright, Venkat

Raghavan, Michael Washabaugh, Gautam Sanyal, and Xiangyang Wang for supporting this work

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25
Figure 1. Size distributions of influenza virus particles determined using: Dynamic Light
Scattering (DLS) (a); Nanoparticle Tracking Analysis (NTA) (b); cryoTEM image analysis
sizing (c); and Analytical Ultracentrifugation (AUC) (d).

26
Figure 2. Selected cryoTEM image of influenza A at a magnification of 52,000× (scale bar =100
nm). Spherical, ‘bean-shaped’, ‘chains/aggregates’, and free spikes are labelled. Additional
images are provided in supplementary Figure S1.

27
Figure 3. Overlaid normalized c(s) distributions (n = 3 for each signal) of influenza virus A
particles generated from interference data (blue-dash-dot line), and UV at 260 nm (red dashed
line) and 280 nm (black solid line) data.

Figure 4. Individual fractograms of influenza A virus particles (n = 6) showing the normalized

Raleigh ratio light scattering signal overlaid with the mean values (n = 6) for the calculated Root
Mean Square (RMS) (solid circles) and geometric diameters (open diamonds).

Figure 5. Fractogram of influenza A virus particles showing the normalized Refractive Index
(RI) signal (solid line, left axis) and the molecular weight values (red solid circles, right axis).

28
Table 1. Summary of size distribution and count results for influenza A virus particles
determined by FFA infectivity assay (n = 6), DLS (n = 18), NTA (n = 6), cryoTEM (n = 273
particles), negative stain TEM (n = 2177 particles), AUC-SV (n =3), and FFF-MALS (n = 6).

Particle counts
Measurement Diameter (nm) (log10 number/mL)
Infectivity assay (FFA) NA 9.9 ± 0.2
DLS 135.2 ± 1.3 (z-average mean) NA
127.6 ± 4.0 (peak)
NTA 12.34 ± 0.03
143.1 ± 3.8 (mean)
114 ± 1.4 (individual virion sizing)
cryoTEM NA
124 ± 2.0 (‘whole aggregate’ sizing)
negative stain TEM NA 12.1 ± 0.2
135.3 ± 0.9 (peak)
AUC-SV a 11.95 ± 0.02b
143.2 ± 1.5 (mean)
129.5 ± 0.8 (peak)
FFF-MALS 12.12 ± 0.04
152.6 ± 2.0 (mean)
a
Calculated using a hydrated particle density of 1.10 g/mL
b
Calculated using interference signal with dn/dc = 0.177 mL/g and M = 306 MDa

Table 2. Summary of influenza A virus molecular weight estimates

Measurement Molecular weight (MDa)

Mean = 306 ± 36
Application of Svedberg equation using NTA diameter
Peak = 229
and AUC-SV sedimentation coefficient
Distribution SD = 147
Mean = 265 ± 4
Application of Zimm light scattering equation to FFF-
Peak = 249
MALS and RI signal data using dn/dc of 0.177 mL/g
Distribution SD = 66
Calculated monomer value based on virion composition Nominal = 206

29