Virucidal activity of immobilized quaternary ammonium compounds (IQACs) coated onto glass and plastic surfaces was tested against enveloped influenza A (H1N1) virus and nonenveloped poliovirus Sabin1. The IQACs tested were virucidal against the influenza virus within 2 min, but no virucidal effect against poliovirus was found in 6 h

Beumer, R.R.

Duizer, E.

Fundeanu, I.

Koning, M.C., de

Tuladhar, E.

Wageningen Yield 

  Published Ahead of Print 27 January 2012. 
10.1128/AEM.07738-11. 
2012, 78(7):2456. DOI:Appl. Environ. Microbiol. 
Beumer and Erwin Duizer
Era Tuladhar, Martijn C. de Koning, Irina Fundeanu, Rijkelt
 
Coatings on Poliovirus and Influenza Virus
Hyperbranched Quaternary Ammonium 
Different Virucidal Activities of
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Different Virucidal Activities of Hyperbranched Quaternary
Ammonium Coatings on Poliovirus and Influenza Virus
Era Tuladhar,a Martijn C. de Koning,c Irina Fundeanu,c Rijkelt Beumer,b and Erwin Duizera
Laboratory for Infectious Diseases and Screening, Center for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the
Netherlandsa; Laboratory of Food Microbiology, Wageningen University, Wageningen, the Netherlandsb; and TNO Earth, Environmental and Life Sciences, Department of
Chemical Toxicology, Lange Kleiweg, Rijswijk, the Netherlandsc
Virucidal activity of immobilized quaternary ammonium compounds (IQACs) coated onto glass and plastic surfaces was tested
against enveloped influenza A (H1N1) virus and nonenveloped poliovirus Sabin1. The IQACs tested were virucidal against the
influenza virus within 2 min, but no virucidal effect against poliovirus was found in 6 h.
Enteric and respiratory viruses can be transmitted directly fromperson to person but also indirectly via contaminated surfaces
(1, 3, 4). Key factors in successful transmission are the quantity of
infectious virus particles shed, the minimal infectious dose, the
stability in the environment, and the resistance to disinfection (2).
Consequently, cleaning and disinfection are means to control out-
breaks and transmission of viruses; however, proper cleaning and
disinfection can be laborious, and for nonenveloped enteric vi-
ruses such as noroviruses, the effectivity of currently practiced
cleaning and disinfection procedures is not clear (5, 7). Therefore,
self-decontaminating surfaces may be helpful in preventing trans-
mission in health care settings, in food production areas, and in
general from frequently touched and contaminated fomites. Ap-
plying antimicrobial coatings to produce self-decontaminating
surfaces could help to interrupt the indirect transmission of
pathogens and reduce the labor and time required for adequate
cleaning and disinfection.
In this study, we coated plastic and glass surfaces with quater-
nary ammonium compounds (QACs). The immobilized QACs
(IQACs) are comprised of hyperbranched polymers functional-
ized with tertiary amines, which are quaternized by alkylation with
an alkylhalide. Various types of Hybrane hyperbranched polymers
(HA1690, HA5290, DA17395, and DA33295, all from DSM,
Heerlen, The Netherlands) were quaternized with heptylbromide
(C7H15Br) to obtain various IQACs using the following proce-
dure: a solution consisting of 10 g polymer, 7.1 g K2CO3, 70 ml
tert-amylalcohol, and 12 ml heptylbromide was mixed and stirred
for 24 h at 96°C. After the solids were removed by filtration, the
polymer was precipitated by pouring the solution in a large excess
of hexane or diethyl ether. After the polymer was washed with
hexane or ether, the polymer was dried under vacuum. The poly-
mer was dissolved in dry acetone, and the resulting solutions were
used to form transparent films on glass slides (76 by 26 by 1 mm)
and plastic wells (24-well plate; Corning, Germany) by applying a
thin layer of fluid on the surfaces followed by drying under vac-
uum for 24 h. The polymers are very poorly water soluble and can
be extracted only from the surface using organic solvents. These
positively charged IQACs interact with the negatively charged
outer surface of bacteria and change the permeability of the bac-
terial membrane. In this exploratory study, we determined the
virucidal properties of several IQACs against the enveloped (lipo-
philic) respiratory influenza virus and nonenveloped (hydro-
philic) enteric poliovirus.
Viruses used for the test were poliovirus Sabin1 (vaccine
strain) and influenza A (H1N1) virus (clinical isolate, Hu/influ-
enza A/266/2008/Netherlands [H1N1]). Virus stocks were pre-
pared as described before (10). The poliovirus stock contained
1.6 107 50% tissue culture infective doses (TCID50/ml), and the
influenza A (H1N1) virus stock contained 4.0  106 TCID50/ml.
Fifty microliters of poliovirus or influenza virus were dispensed
and spread at the center of each surface. Environmental persis-
tence of both viruses was studied for 10 days on uncoated plastic
and glass. The poliovirus was left on the coated surfaces for 1 and
6 h and influenza virus for 2 min and 1 h, respectively. The viruses
were removed from the glass slides by swabbing with rayon swab
sticks (Copan) as described before and from the plastic wells by
rinsing with 1 ml cell culture medium (10). Visual inspection of
the coatings after virus removal did not show any damage to the
coat. Enumeration of the viruses and quantitative real-time re-
verse transcription (RT)-PCR was performed as described previ-
ously (10). Data for environmental resistance are presented as
total infective viruses recovered from the surfaces (at t 0 h, the
suspension is not yet dried), and data for virucidal activity of the
IQACs are presented as reduction relative to uncoated glass and
plastic carriers. Recoveries of infective viruses after 1 h from the
control carriers were 1.3%  0.0% and 45.0%  7.3% for influ-
enza virus from glass and plastic, respectively, and 56.6% 9.2%
and 89.7% 14.5% for poliovirus from glass and plastic, respec-
tively. All experiments were performed at room temperature in
duplicate, and the outcome is from two experiments. Statistical
analysis was done using t test.
The persistence of poliovirus was high, with less than 1 log10
decay in 10 days (Fig. 1). Influenza virus showed fast decay the first
24 h, probably due mostly to drying, followed by slower decay up
to day 5 and again faster decay after day 5 (Fig. 1). The decay in
infective virus that could be retrieved from the coated carriers
differed widely for poliovirus and influenza virus (Fig. 2). After 1
h, no significant reduction of infectious poliovirus was measured
Received 6 December 2011 Accepted 15 January 2012
Published ahead of print 27 January 2012
Address correspondence to Erwin Duizer, Erwin.Duizer@rivm.nl.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.07738-11
2456 aem.asm.org 0099-2240/12/$12.00 Applied and Environmental Microbiology p. 2456–2458
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for any of the coatings, while for influenza virus the reduction of
infective virus was complete: no infective influenza viruses could
be retrieved from the coated surfaces, except from the nonquater-
nized HA5290 polymer, for which no reduction was found. Iden-
tical levels of inactivation of influenza virus were already achieved
after 2 min, while even after 6 h no significant reduction of polio-
virus could be detected (data not shown). Remarkable was the
complete inactivation of influenza virus by the quaternized and
the nonquaternized HA1690 polymer. The reduction in infective
influenza virus by quaternized HA1690 was concomitant with a
significant reduction in PCR units (PCRU), while this was not true
for the nonquaternized HA1690 (Fig. 3).
Even though quantitative data are missing, transmission of en-
teric and respiratory viruses via hard surfaces is a concern in health
care settings, individual houses, and food production facilities. In
this study, we found that no infective influenza viruses could be
retrieved from the coated surfaces, indicating that the environ-
mental survival of these enveloped respiratory viruses was reduced
from over 5 and 10 days for glass and plastic, respectively, to less
than 2 min on IQAC-coated surfaces. The virucidal mechanism of
QACs has been described for the lipophilic enveloped viruses to
involve disruption or detachment of the viral envelope, and envel-
oped viruses, such as herpes simplex virus, have been found to be
very sensitive (8, 9). However, data on influenza viruses and QAC
resistance are less unambiguous. In suspension tests, avian influ-
enza H5N1 strains were not completely inactivated by 0.02% ben-
zylalkonium chloride (manufacturers’ recommended concentra-
tion) after 10 min (11), while in general QACs are considered
good disinfection agents for influenza viruses (http://www.cdc
.gov/h1n1flu/guidelines_labworkers.htm). We confirm virucidal
effects of the IQACs tested against influenza viruses in our carrier
tests. Further studies will be needed to determine how many times
the coated surfaces can be cleaned and maintain this activity and if
activity remains when surfaces are filthy or dusty.
The finding that no infective influenza viruses could be re-
trieved from the HA1690 base polymer-coated slides might be due
to the length of the lipophilic tails and the high density of func-
tional end groups (tertiary amines) which are available to be quat-
ernized in the HA1690 base polymer (both tail length and density
are higher for HA1690 than for HA5290). The full recovery of
influenza virus RNA in the HA1690 base polymer shows that the
infectivity reduction is not the result of a profound effect on the
viral genome but indicates a primary effect on the virus envelope.
QACs have been reported to show low virucidal activity against
hydrophilic nonenveloped viruses, such as picornaviruses. Infec-
tive poliovirus was reduced by 1.1 to 2.3 log10 by a QAC (alkyldi-
methylbenzyl ammonium chloride plus EDTA) in a suspension
test (450 ppm, 10 min, 20°C) (6), while less than 1 log10 reduction
was reported for a similar QAC in a comparable exposure in the
presence of 20% blood but over 2.4 log10 in the absence of inter-
fering substances (12). We report the lack of significant virucidal
activity of the IQACs tested against poliovirus after up to 6 h of
exposure. To the best of our knowledge, this is the first report on
FIG 1 Environmental decay of poliovirus and influenza virus at room tem-
perature. PV-P is poliovirus on plastic, PV-G is poliovirus on glass, Inf-P is
influenza virus on plastic, and Inf-G is influenza virus on glass. Data are from
2 experiments with duplicate samples per experiment, and error bars indicate
standard deviations (SD).
FIG 2 Reduction of infective poliovirus and influenza virus by different IQACs after 1 h of exposure at room temperature. Data are from 2 experiments with
duplicate samples per experiment, and error bars indicate SD.
FIG 3 Reduction of PCRU of influenza virus by different IQACs after 1 h of
exposure at room temperature. Data are from 2 experiments with duplicate
samples per experiment, and error bars indicate SD.
Virucidal Efﬁcacy of Immobilized QACs
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virucidal activity of IQACs against poliovirus and influenza virus,
and we show that hyperbranched QAC coating is effective against
influenza A (H1N1) virus but not effective against poliovirus Sa-
bin1.
ACKNOWLEDGMENTS
We thank Duurt Alkema for preparing the QACs and coating the carriers.
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Different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus

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