EVALUATION OF VIRUCIDAL ACTIVITIES

"Hard surface carrier" methods are mainly used for substantiation of virucidal
efficacy. These methods consist of a non-porous carrier (typically glass) being
inoculated with the selected virus, dried, and then treated with the disinfectant.
Virucidal hard surface carrier methods are quantitative, meaning that percent and
log reductions are calculated by determining the TCID 50 (50% Tissue Culture
Infective Dose) per carrier before and after treatment with the disinfectant. The
disinfectant must demonstrate complete inactivation of the virus down to the limit
of detection of the assay, or (if cytotoxicity is observed) a ≥ 3.00 log 10 reduction
(99.9%).

Observation of Results:

Virus testing is unique within the laboratory because the presence of viruses before
and after product treatment is not determined by observing growth of virus but
rather by observing the damage caused by infection to mammalian host cells.
When virologists analyze individual sets of cells after a study, they use a
microscope to look for where healthy cell layers become damaged. This damage is
known as the cytopathic effects (CPE) of infection. The quantity and quality of
CPE is used to calculate the amount of virus present.

CPE is typically observed as changes to cell appearance and monolayer (the layer
host cells form when they attach to a flask or tray) disruptions. CPE can vary
depending on the virus and host cell line used. Some instances of CPE are distinct,
consisting of severe monolayer disruption and cell rounding. Some CPE can be
subtle, consisting of gradual enlargement of host cells that is only recognizable
when compared to a negative control. In cases where CPE is difficult to
distinguish, special confirmatory assays are used to verify the results of the assay.
Some more common confirmatory assays include Hemagglutination Assays (HA)
and immunofluorescent staining (IF).

Study Preparation and Timeline:

From the laboratory's perspective, a significant amount of work and time is

required to grow and maintain the sterile cell cultures that are needed to propagate
and detect viruses in antimicrobial efficacy studies. From our customer's point of
view, the cell culture requirement means that extra time must be given to the
laboratory to prepare for and execute the study. Most studies take 1-2 weeks to
complete, though some can take 3-4 weeks. The behind-the-scenes cell culture
work and extraordinary expertise necessary to conduct virological assays also
means that virological studies are more expensive than their related bacteriological
assays.

Study Conduct and Parameters:

There are two main viral morphologies - enveloped and non-enveloped. Non-
enveloped viruses consist of genetic material surrounded by a hard protein coat.
Enveloped viruses have an additional lipid layer encompassing their protein coat.
The limited sensitivity of non-enveloped viral components means that these viruses
can persist in an infectious state even when exposed to harsh environmental
conditions - including exposure to UV or relatively high temperatures. When it
comes to testing, this means that one can translate almost any bacteriological study
into a non-enveloped viral assay without changing too many parameters.

Enveloped viruses are another story. Their delicate lipid envelopes leave them
vulnerable to environmental factors like osmotic pressure, low humidity, and high
temperatures. When working with enveloped viruses, certain parameters (like
contact times and contact temperatures) and even general study methods may need
to be modified to accommodate the unique demands of these microbes.

VIRUCIDAL EFFICACY TEST METHODS

1. Preliminary Testing:
i. Preliminary Screening:

Many people are familiar with viruses that infect mammalian cells (e.g. Influenza,
Poliovirus, or Rhinovirus), but there are also viruses capable of infecting bacterial
cells. These viruses are called bacteriophages. Bacteriophages are easy to work
with in the context of disinfectant testing and can provide valuable information
about product efficacy.

MS2 is a bacteriophage that infects "male" Eschericia coli. Male E. coli are
bacterial cells capable of passing a portion of their genetic material (typically in
plasmid form) to other bacterial cells through a structure called a pilus. This
process of horizontal gene transfer is a vehicle of genetic change within bacterial
populations and is particularly well known for spreading antibiotic resistance to
previously antibiotic-succeptible bacterial populations. Morphologically, MS2 is a
non-enveloped, icosohedral virus. MS2's lack of lipid envelope means that it is
generally resistant to chemical disinfectants and is also able to withstand
environmental stressors like temperature changes, dessication, and osmotic
pressure.
Performing preliminary screens with MS2 can allow you to test a large number of
potentially efficacious formulations or evaluate multiple contact times without
waiting weeks for results. In addition, the lack of time-intensive cell passages and
propagations means that MS2 studies are performed at reduced cost relative to
other viruses.
Viral Representative: A disinfection hierarchy for viruses are recognized
currently, which is generally applied when performing bridging or towelette
testing. This hierarchy ranks viruses in the following order:

Small, non-enveloped viruses - most difficult to inactivate

e.g. poliovirus, enterovirus, or rhinovirus

Large, non-enveloped viruses - moderately difficult to inactivate

e.g. adenovirus, rotavirus, or papillomavirus

Enveloped viruses - easy to inactivate

e.g. influenza, herpes virus, or hepatitis virus

The MS2 virions used for this test are 23-28 nm in diameter, putting them in the
category of small non-enveloped viruses like canine parvovirus and human
poliovirus. Literature does exist, however, which indicates that MS2 is more
sensitive than its counterparts to various methods of disinfection, including UVC
light exposure and quaternary ammonium compounds.

ii. Initial and confirmatory virucidal effectiveness testing for viral

surrogates:

Surrogate viruses are used in place of other viruses for virucidal efficacy testing.
Typically this occurs because a virus is either exceedingly difficult to propagate
and test in vitro or presents such a hazard to scientists performing testing that the
virus is limited to high security laboratories (which generally do not perform
disinfectant efficacy testing).
For instance, duck hepatitis virus is tested in place of human hepatitis B virus, and
feline calicivirus is tested in place of human norovirus. Surrogates are chosen due
to a high level of genetic, morphological, and responsive similarity to the virus
they are selected to represent.
When testing was done on surrogate viruses , two separate efficacy studies, called
"initial" and "confirmatory” are carried out. The only difference between the
initial and confirmatory tests was the number of batches tested. The Initial
Virucidal Effectiveness Test was performed using two lots/batches and the
confirmatory virucidal effectiveness test is performed using one lot/batch.

2. Suspension – based Tests:

i. Standard Test Method for efficacy of Antimicrobial agents against viruses in
suspension – Suspension kill time test for Virus:

 The frozen stock test virus is thawed and diluted to a titer of approximately
6-log10 infectious units per 0.1 ml.
 If requested by the Study Sponsor, the test virus is loaded with organic soil
to the appropriate level. This is typically done when the test product is
intended to be marketed as a one-step disinfectant.
o An organic soil load of 5% is used for a one-step disinfectant claim.
 The test product is prepared to the use-dilution specified by the Study
Sponsor, if applicable. An equal volume of buffered saline or cell culture
media is prepared to serve as a virus recovery control.
 The prepared inoculum is added to the test product and the virus recovery
control media at a ratio of 1 part virus + 9 parts test product (per the
Method).
 Upon closure of the study contact time, the test and recovery suspensions are
neutralized by a method most suitable for the active ingredient(s) present in
the test substance, e.g. dilution into a chemical neutralizer or filtration
through a Sephacryl gel column.
 An aliquot of the use-dilution of the test substance is neutralized in an
identical manner to the test suspension. This is used to generate the
cytotoxicity control to determine the lowest dilution at which, if any, the test
substance causes damage to the host cells.
 An aliquot is removed from the cytotoxicity control and inoculated with a
low-concentration viral suspension. This is used to generate the
neutralization control to confirm the efficacy of the selected neutralization
method.
 The neutralized test, recovery, cytotoxicity control, and neutralization
control suspensions are serially diluted in the appropriate media. Each
dilution is plated in quadruplicate to host cell monolayers in a 24-well tray.
Media is added to each well and the host cell-virus system is allowed to
incubate for the appropriate time.
 At the close of the incubation time, the assay is scored using standard cell
culture methods.
o Each well in the tray is examined under microscope for the presence
of cytopathic effects (CPE) of infection. Cytotoxicity control wells are
examined for damage caused by the test product. Confirmatory assays
are used as necessary.
 o The Spearman-Karber method, or another appropriate statistical
method, is used to quantify the amount of infectious virus present in
the assay.

ii. Quantitative suspension test for the evaluation of virucidal activity of

Chemical disinfectants and antiseptics in the medical area:

 Before test initiation the appropriate conditions for the test product are
selected. Required conditions vary depending on the category of product and
the conditions of use.
 The test substance is prepared according to Study Sponsor directions.
 A 0.8 ml aliquot of test substance is supplemented with 0.1 ml of interfering
substance ("clean" or "dirty" depending on the final use of the test product).
 A 1.4% formaldehyde control is prepared and supplemented with the same
interfering substance as the test substance.
 The test product and formaldehyde control are both inoculated with the viral
suspension and held for the duration of the contact time.
 A virus recovery control is prepared using an inert substance such as
buffered saline or cell culture media in order to determine the initial viral
titer used to challenge the test substance.
 At the conclusion of the contact time the test product and controls are
neutralized, either through dilution into a chemical neutralizer, passage
through a physical matrix (e.g. Sephacryl gel columns), or a combination
thereof.
 Neutralization controls and cytotoxicity controls are also performed to
determine the effectiveness of the selected neutralization method and the
impact of the test product on host cells.
  Once the test product and controls are neutralized, they are serially diluted
and plated onto permissive host cell monolayers
 The host cell-test suspension system is allowed to incubate for the
appropriate time (typically 7 days).
 At the conclusion of the incubation period, the assay is scored using the
Spearman-Karber method to quantify the viral titer, and any cytotoxicity
observed is noted accordingly.

3. Hard Surface Carrier Test:

i. Standard Test Method to assess Virucidal Activity of chemicals intended for

Disinfection of inanimate, non-porous environmental surfaces:
 The stock virus to be used is thawed and diluted to an appropriate
concentration.
 If requested by the Study Sponsor, the test virus is loaded with organic
soil to the appropriate level. This is typically done when the test product is
intended to be marketed as a one-step disinfectant.
 The EPA requires an organic soil load of 5% for a one-step disinfectant
claim.
 The test product is prepared to the use-dilution specified by the Study
Sponsor, if applicable. An equal volume of buffered saline or cell culture
media is prepared to serve as a virus recovery control.
 The prepared viral inoculum is spread over the entire surface of a glass
Petri dish carrier and allowed to dry.
 At the conclusion of the dry time the test product is applied to the viral
film.
 Typically 2.0 ml of a use-dilution test product will be added. If a spray
product is used it will be applied as 4 sprays at a distance of 6 to 8 inches,
or according to Sponsor directions as appropriate.
 Upon closure of the study contact time, the test and recovery suspensions
are harvested by use of a cell scraper and neutralized by a method most
suitable for the active ingredient(s) present in the test substance.
 Common methods of neutralization include dilution into chemical
neutralizers, filtration through a physical gel matrix (e.g. Sephacryl gel
columns), or a combination thereof.
 An aliquot of the use-dilution of the test substance is neutralized in an
identical manner to the test suspension. This is used to generate the
cytotoxicity control to determine the lowest dilution at which, if any, the
test substance causes damage to the host cells.
 An aliquot is removed from the cytotoxicity control and inoculated with a
low-concentration viral suspension. This is used to generate the
neutralization control to confirm the efficacy of the selected neutralization
method.
 The neutralized test, recovery, cytotoxicity control, and neutralization
control suspensions are serially diluted in the appropriate media. Each
dilution is plated in quadruplicate onto host cell monolayers. Media is
added to each monolayer and the host cell-virus system is allowed to
incubate for the appropriate time. Most assays incubate for a 7 to 10 day
period.
 At the close of the incubation time, the assay is scored using standard cell
culture methods.
  Each well in the tray is examined under a microscope for the presence of
cytopathic effects (CPE) of infection, such as cell rounding, sloughing,
and monolayer degradation. Cytotoxicity control wells are examined for
damage caused by the test product. Confirmatory assays are used as
necessary.
 The Spearman-Karber method, or another appropriate statistical method,
is used to quantify the amount of infectious virus present in the assay.

ii. Germicidal Spray Test, modified for Viruses:

 The first decision that should be made prior to performing this test is the
number of carriers to be evaluated. There are two primary options:
 Ten Individual Carriers: Each carrier receives a 0.01 ml inoculum over a
1"x1" area.
 One 10-in2 Equivalent Carrier: One glass petri dish receives a 0.200 ml
inoculum over its entire surface area.
 Viral stocks are thawed and diluted as appropriate, for a recoverable titer
of 4.00 log10 TCID50 per carrier.
 If the product is to be marketed as a "one step" cleaner/disinfectant, the
test culture is supplemented with organic soil, usually fetal bovine serum.
 Test carriers are inoculated with test culture by evenly spreading it across
the selected surface area(s), then dried.
 Appropriate plate recovery controls are harvested prior to test initiation to
determine pre-test carrier microbial concentrations.
 The dry, contaminated test surfaces are then sprayed, individually, with
test product.
  Contaminated test surfaces incubate in the disinfectant for a specified
contact time.
 After the contact time has elapsed, neutralization of the test substance is
performed and sterile cell scrapers are used to remove the viral films from
the surface of each carrier.
 Appropriate plate recovery controls are enumerated following test
initiation to determine post-test carrier microbial concentrations.
 Dilutions of the harvested test substance-viral suspension are applied to
permissive host cell monolayers, and incubated as required by the test
virus (typically 7 days).

iii. Pre-saturated Towelettes Virucidal Effectiveness Test:

 The use of internationally-recognized standard test methods as the basis

for pre-saturated towelette virucidal efficacy evaluations:
 Two lots/batches of pre-saturated towelettes are evaluated for testing.
Approved surrogate viruses are tested in duplicate, all other viruses are
tested in singlet.
 If requested by the Study Sponsor, the test virus is loaded with organic
soil to the appropriate level.
 The viral inoculum is spread over the carrier surface(s) and allowed to
dry.
 The dried virus films are wiped with the pre-saturated towelette and held
for the specified contact time.
 At the close of the contact time, the test carrier films are neutralized using
a method most suitable for the active ingredient(s) present in the test
substance.
 The test suspensions are assayed for the presence of infectious virus using
established methods.
 Viral plate recovery controls, cytotoxicity and neutralization controls,
stock titer determinations, and blank towelette and sterility controls are
also performed and assayed in parallel with neutralized test suspensions.

CARRIER OPTIONS:

 One of the two following carrier options may be employed per test
lot/batch for single-use towelettes testing:

1. Ten carriers
 Dried virus inoculum area per carrier: 1” x 1”
 Ten carriers treated per lot/batch of test towelettes
 One towelette used to treat ten carriers

2. Two carriers
 Dried virus inoculum area per carrier: 10-in.2 equivalent
 One carrier treated per lot/batch of test towelettes. (Two carriers treated
per lot/batch for surrogate viruses)
 One towelette used to treat each carrier