SARS-CoV, influenza A and syncitial respiratory virus resistance against common disinfectants and ultraviolet irradiation

Abstract

To evaluate the virucidal efficacy of six commonly used chemical disinfectants, and ultraviolet radiation (U.V.) against SARS-CoV and compare it to the resistance of other airborne viruses, such as Influenza and Respiratory Sincytial Virus, a salt solution containing a standard concentration of cell-grown viruses, was mixed with a larger volume of different disinfectants at their use dilution and the mixtures were held for a defined contact time. The virucidal efficacy of disinfectants and U.V. was evaluated by infectivity, detected by inoculation of samples in suitable cell culture and genome integrity, detected by nested RT-PCR. SARS-CoV as well as RSV seem to be sensible to the different disinfectants tested in our study and U.V. radiation, while influenza virus appear to be more resistant in particular to the action of chlorhexidine digluconate and benzalkonium-chloride. In consideration of the possible infectious role of SARS-CoV RNA, sodium hypochlorite 0.1% appear to be the more efficacious disinfectant for surfaces and hands potentially contaminated with respiratory viruses and SARS-CoV.

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Background
In late 2002 a new acute and often severe respiratory
illness (SARS) emerged in Southern China and spread
to distant areas within a very short period of time 1. The
aetiological agent for SARS is a novel Coronavirus
called SARS-CoV, that H has never been described
both in human and animals previously 2-4. CDC in ac-
cording to WHO, reports that a total of 8,098 people in
32 countries became sick with this new illness during
2003 outbreak. Of these 774 died, with a case-fatality
rate of about 10% that reach 50% in > 64 year age
group 5. On 5 July 2003 WHO announced that SARS
epidemic was over. During outbreak the major percent-
age of cases was among health-care workers because
the main way of transmission seems to be by close per-
son to person contact during the peak of viral shedding
that occurs about ten days after onset of symptoms.
Since epidemic was over there have been a number of
laboratory confirmed SARS-CoV infections resulting
from laboratory accidents and from exposure to animal
sources or environmental contamination 6.
In consideration of the high efficiency in nosocomial
transmission and the occurrence of infection in laborato-
ry, to know the resistance of this new virus in environ-
ment and towards common disinfectants appear to be a
fundamental tool to choose the correct control measures.
The aim of this work is to evaluate the virucidal effica-
cy of chemical disinfectants, used to clean surfaces,
and ultraviolet radiation (U.V.) against SARS-CoV and
compare it to the resistance of other airborne enveloped
viruses, such as Influenza and Respiratory Sincytial
Virus (RSV).
Materials and methods
STUDY DESIGN
A known quantity (1ml) of salt solution containing a
standard concentration of cell-grown virus, was mixed
with a larger volume (2 ml) of different disinfectants
under evaluation at their use dilution. The mixtures
were held for a defined contact time (30 seconds, 1
minute, 2 min, 5 min, 15 min, 30 min) at controlled tem-
perature (18°C) and humidity (40%). The virucidal ac-
tivity of the test products was arrested immediately by
adding a neutralizer (Sodium thiosulphate for sodium
hypoclorite; sodium hydroxide for acid peracetic) or di-
luting the germicide-mixture. For evaluate the physical
effects of U.V. irradiation (40mW/cm2), the salt solu-
tions containing viruses, were distributed on a plate of
20 cm2 and exposed to U.V. irradiation for above-men-
tioned contact time. A positive control containing a
standard concentration of cell-grown virus without dis-
infectant was used in every run. Preliminary tests were
made to evaluate the sensibility of cells against disin-
fectants and neutralizer/disinfectant solution for select-
ing the right dilution that did not damage the cells.
DISINFECTANT
In this study SARS-CoV, Influenza virus and RSV
were tested with six common disinfectants at the fol-
lowing dilutions:
–Sodium Hypochlorite 0.01% – 0.05% – 0.1%;
–Ethanol 70%;
–Benzalkonium-chloride 1%;
–Chlorhexidine digluconate 1%;
SARS-CoV, influenza A and syncitial respiratory virus
resistance against common disinfectants and
ultraviolet irradiation
F. ANSALDI, F. BANFI, P. MORELLI, L. VALLE, P. DURANDO, L. STICCHI, S. CONTOS, R. GASPARINI, P. CROVARI
Department of Health Sciences, University of Genoa
JOURNAL OF PREVENTIVE MEDICINE AND HYGIENE 2004; 45: 5-8
Summary
Key words
SARS-CoV • Disinfectants • Virucidal efficacy
To evaluate the virucidal efficacy of six commonly used chem-
ical disinfectants, and ultraviolet radiation (U.V.) against SARS-
CoV and compare it to the resistance of other airborne viruses,
such as Influenza and Respiratory Sincytial Virus, a salt solu-
tion containing a standard concentration of cell-grown viruses,
was mixed with a larger volume of different disinfectants at their
use dilution and the mixtures were held for a defined contact
time. The virucidal efficacy of disinfectants and U.V. was eval-
uated by infectivity, detected by inoculation of samples in suit-
able cell culture and genome integrity, detected by nested RT-
PCR. SARS-CoV as well as RSV seem to be sensible to the dif-
ferent disinfectants tested in our study and U.V. radiation, while
influenza virus appear to be more resistant in particular to the
action of chlorhexidine digluconate and benzalkonium-chloride.
In consideration of the possible infectious role of SARS-CoV
RNA, sodium hypochlorite 0.1% appear to be the more effica-
cious disinfectant for surfaces and hands potentially contami-
nated with respiratory viruses and SARS-CoV.

F. ANSALDI, ET AL.
6
–2-benzil-chlorophenol 2%;
–Peracetic acid 0.035%.
OUTCOMES
To evaluate the virucidal efficacy of disinfectants and
U.V. we considered two parameters: (i) infectivity, de-
tected by inoculation of samples in suitable cell culture,
i.e. Vero E6 for SARS-CoV, MDCK for influenza virus,
HEp2c for RSV 7; (ii) genome integrity, detected by
nested RT-PCR for SARS-CoV and multiplex nested
RT-PCR.
LABORATORY METHODS
All practice of experiment with SARS-CoV were made
in laboratory BSL3 as recommended by WHO 6.
ISOLATION CULTURE
Conventional viral culture was performed by inoculat-
ing 0.3 ml of each specimen (virus-disinfectant mix-
ture) into Vero E6, MDCK and HEp-2 cells for SARS-
CoV, influenza and RSV isolation, respectively 7. Virus
detection was performed by PCR indirect immuno flu-
orescence and hemagglutination test for SARS-CoV
RSV, and influenza, respectively 8.
RNA was extracted using QIA techniques, following
the manufacturer’s instructions (Rneasy Minikit, Qia-
gen, Valencia, CA). Amplification of specific RSV and
Influenza virus sequence was performed by multiplex
RT-nested PCR (Influenza/RSV multiplex, Amplimed-
ical S.p.A., BIOLINE, Italy). SARS-CoV was detected
by a commercial RT-PCR kit (SARS-CoV RNA poly-
merase, Amplimedical S.p.A., BIOLINE, Italy).
Results
The results of preliminary test of citotoxicy showed that
0.2 ml ethanol 70%, benzalkonium-chloride 1%,
chlorhexidine digluconate 1% in 2 ml cell culture medi-
um did not significantly damage the cell monolayer.
Sodium hypochlorite 10% and peracetic acid 0.035% de-
termined a strong cytotoxic effect on cells, that disap-
peared when disinfectant-neutralizer mixture was used.
Also 2-benzil-chlorophenol 2% showed a strong cyto-
toxic effect on cells that declined and disappeared when
a 1:3 and 1:10 dilution was inoculated, respectively. For
this reason after the end of contact time the mixture
virus-disinfectant was diluted 1:10 before inoculation.
In Table I last virus-disinfectant contact times resulting
positive by cell culture and PCR are reported. Peracetic
acid 0.035%, ethanol 70% and sodium hypochlorite
0.05% showed to inhibit viral replication in cell culture
after < 2’contact time, while viral genome seems to be
intact after prolonged exposition (30’).
Sodium hypochlorite 0.1%, 2-benzil-chlorophenol 2%
and U.V. have a stronger virucidal effect: they inhibit
completely viral replication and damage viral genome
after < 2 minutes of exposition. In particular, sodium
hypochlorite 0.1% showed the more rapid action: after
1’of contact, influenza virus, RSV and SARS-CoV
were not able to replicate and their genome integrity
was lost. This data is confirmed by electronic mi-
croscopy: the viral structures appear completely de-
stroyed with lost of spikes and envelope integrity after
1’of exposition (data not shown).
Chlorhexidine digluconate 1% and benzalkonium-chlo-
ride 1% showed a similar pattern of virucidal efficacy:
they inhibited RSV replication after 1’of contact, while
prolonged exposition (30’) did not affect infectivity of
influenza virus. SARS-CoV replication was inhibited
by 5’exposition to benzalkonium-chloride 1%, while
all experimental samples collected at different time re-
sulted culture negative after contact with chlorhexidine
digluconate 1%. Influenza virus, RSV and SARS-CoV
RNA is still detectable after 30 minutes of contact time
Tab. I. Last contact time resulting positive by cell culture and PCR.
Disinfectant Virus
Influenza virus RSV SARS-CoV
Culture PCR Culture PCR Culture PCR
Peracetic acid 0.035% – 30’ – 30’ – 30’
Ethanol 70% 2’ 30’ – 30’ – 30’
Sodium Hypochlorite 0.01% 30’ 30’ 30’ 30’ 30’ 30’
Sodium Hypochlorite 0.05% 30’ 30’ 1’ 2’ 1’ 30’
Sodium Hypochlorite 0.1% – 30î 1’ 1’ – 30î
Chlorhexidine digluconate 1% 30’ 30’ 1’ 30’ – 30’
2-benzil-chlorophenol 2% – – – 2’ – 2’
Benzalkonium– chloride 1% 30’ 30’ 1’ 30’ 5’ 30’
UV irradiation 1’ 1’ 1’ 1’ 2’ 2’
Legend: – negative after 30’ contact time.

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SARS-COV, INFLUENZA A AND RSV VS. COMMON DISINFECTANTS AND UV IRRADIATION
with chlorhexidine digluconate 1% and benzalkonium-
chloride 1%.
Discussion
Since when SARS epidemic appeared, SARS-CoV has
shown high efficiency in nosocomial transmission and
high risk of virus spread in laboratory setting. The new
outbreak and the emergence of new respiratory viruses
such as avian influenza virus and human metapneu-
movirus, underline the needs to improve the knowledge
about the resistance of these microorganisms in envi-
ronment and towards common disinfectants for choos-
ing the correct control measures.
Studies on SARS-CoV demonstrated that the virus is
more stable at room temperature than the previously
known human Coronavirus 9 10. Preliminary study of
WHO laboratory network showed that the virus survive
for up to 48 hours on plastic surfaces and up to 4 days in
diarrhoea. Nevertheless, the virus loses infectivity after
exposure to different commonly used disinfectants and
fixatives 9 10. Our study confirmed that SARS-CoV is
quite sensible to common disinfectants. In fact, SARS-
CoV appears to be completely inactivated by disinfec-
tants such as acid peracetic, ethanol 70%, sodium
hypochlorite 0.05% and 0.1%, chlorhexidine digluconate
1% and 2-benzil-chlorophenol 2% after < 1’exposition,
while a longer contact time with benzalkonium-chloride
1% is necessary to inhibit the replication in culture. PCR
findings are of interest, not only to demonstrate the com-
plete destruction of viral structure, but also as RNAs of
some Coronaviruses are infectious 11 12 . As regards as hu-
man infecting SARS-CoV, this biological property has
not been demonstrated yet. Only sodium hypochlorite
0.1% and 2-benzil-chlorophenol 2% are rapidly effica-
cious in destruction of viral RNA at an undetectable lev-
el after < 2’contact time, while prolonged exposition
with other disinfectants seems not alter RNA integrity.
RSV showed a resistance pattern rather similar to
SARS-CoV and appears to be completely inactivated
by disinfectants such as acid peracetic, ethanol 70%,
sodium hypochlorite 0.05% and 0.1%, chlorhexidine
digluconate 1% and 2-benzil-chlorophenol 2% and
benzalkonium-chloride 1% after < 1’exposition. The
RSV weakness was confirmed by on-field studies on
disinfection and survival studies 13 14.
Influenza can not replicate after short exposure to acid
peracetic, ethanol 70%, sodium hypochlorite 0.05%
and 0.1%, and 2-benzil-chlorophenol 2%, while it
showed high stability after contact with chlorhexidine
digluconate 1% and benzalkonium-chloride 1%. The
complete inefficacy of benzalkonium-chloride 1% is
not completely unexpected as quaternary ammonium
were shown to be very effective against herpes virus
but totally ineffective against non-enveloped virus and
influenza virus, as demonstrated also Moldenhauer 15.
On the other hand, chlorhexidine showed to be totally
ineffective against rotavirus, enveloped virus such as
influenza virus 16.
U.V. radiation, instead, damage nucleic acid because
RNA adsorb UV radiation of germicidal wavelengths
and is the major targets of the powerful antimicrobal
effects of these type of electromagnetic radiation. In
according to this, irradiation of UV for few minutes
(1’-2’) on the virus in culture medium resulted in the
destruction of viral infectivity.
In conclusion, SARS-CoV as well as RSV seem to be
sensible to the different disinfectants tested in our
study and U.V. radiation, while influenza virus appear
to be more resistant in particular to the action of
chlorhexidine digluconate and benzalkonium-chloride.
In consideration of the possible infectious role of
SARS-CoV RNA, sodium hypochlorite 0.1% appear
to be the more efficacious disinfectant for surfaces and
hands potentially contaminated with respiratory virus-
es and SARS-CoV.
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Correspondence: Filippo Ansaldi, Department of Health Scien-
ces, University of Genoa, Via Pastore 1, 16132 Genoa, Italy. Tel.
+39 010 3533002. E-mail: filippo.ansaldi@unige.it