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VI. The Role of the Gas–Liquid Interface in Protein Virus Inactivation
Virus Inactivation and Protein Denaturation at the Gas–Liquid Interface
T1, . . .,T7
Shaking results in ﬁrst-order denaturation; no denaturation
in absence of interface; denaturation increases with
increasing acidity of solution; no denaturation in full
bottles; in absence of air–water interface; denaturation
not caused by oxidation; different degree of denaturation
for different proteins.
Vigorous shaking results in zero-order denaturation.
Protein unfold at interface and form monomolecular ﬁlm;
stirring creates new interface area.
Spreading of protein at gas–liquid interface results in
denaturation of protein; proteins become insoluble.
Proteins form surface ﬁlm; coagulum formed when
solution is shaken; rate of denaturation dependent on
size of bottle, shaking intensity but not concentration.
Inactivation by bubbling air laden with certain vapors
through virus solution.
Inactivation by shaking in buffered saline solution and by
bubbling gas through solution; inactivation increased as
pH reduced from 7 to 5.
Inactivation by shaking and bubbling air through solution;
inactivation dependent on pH; presence of gelatin
Unfolding of macromolecules largely determined by
interfacial energy; sorption follows Langmuir isotherm.
Irreversible binding of proteins to interface.
EMC virus not affected, bacteriophages T3,T5 only little
affected, T1, MS-2, and Semliki Forest virus inactivated
by bubbling air or nitrogen gas through solution;
inactivation prevented by adding peptone and apolar
carboxylic acids; rate of inactivation dependent on salt
concentration, more sorption at higher salt concentration.
Inactivation of MS-2 at air–water interface.
Denaturation at interface depends on gas–liquid contact
time and surface regeneration rate; denaturation reduced
in presence of surfactants.
Denaturation at interface due to unfolding; insoluble
coagulum formed when solution is shaken.
Denaturation of T7 at the interface due to partial unfolding
of protein structure in aeration and shaking experiments;
P22H5 much more stable than T7.
Denaturation at the interface due to partial unfolding of
Inactivation in polypropylene bottles, but not in glass
bottles; inactivation occurs at the triple-phase interface
Shaklee and Meltzer (1909)
Langmuir and Waugh (1938)
Neurath and Bull (1938)
Grubb et al. (1947)
James and Augenstein (1966)
Quinn and Dawson (1970)
Trouwborst et al. (1974)
Trouwborst and de Jong (1973)
Donaldson et al. (1980)
ˇ sko (1992)
Bricelj and Siˇ
Tronin et al. (1996)
Thompson et al. (1998)
SUBSURFACE VIRUS FATE AND TRANSPORT
Figure 9 Water droplets resting on hydrophobic and hydrophilic surfaces. Adapted with permission from Thompson et al. (1998). Role of the air-water-solid interface in dynamic batch systems.
Appl. Environ. Microbiol. 65, 1186–1190. Copyright American Society of Microbiology.
inactivated. In column transport experiments under unsaturated ﬂow conditions,
Jin et al. (2000a) showed that MS-2 retained in the columns could not be recovered
with beef extract solution, possibly due to inactivation at the AWI, whereas retained
φX174 stayed viable. Thompson et al. (1998) and Thompson and Yates (1999)
suggested that forces associated with the air–water–solid (where the solid is a
hydrophobic surface) interface or triple-phase boundary (TPB), not the AWI alone,
led to the inactivation of MS-2 particles. In batch experiments Thompson et al.
(1998) found that whereas φX174 was resistant toward interfacial inactivation,
MS-2 was inactivated at the air–water–polypropylene interface, but not at the
air–water–glass interface (Thompson et al., 1998). Schematic examples of two
TPB systems are shown in Fig. 9. Thompson et al. (1998) suggested that as a
virus particle adsorbs at the AWI, hydrophobic domains of the virus protein capsid
partition out of the solution and into the more nonpolar gas phase, and such exposed
domains are susceptible to forces at the TPB that are not present at the AWI. The
balance of the forces present at the TPB is inﬂuenced by the surface properties
of the solid (e.g., polypropylene vs glass), mainly the contact angle against water.
They suggest that virus particles partitioned at the TPB experience destructive
forces as a result of the reconﬁguration of water molecules near the hydrophobic
To explain the different behavior between MS-2 and φX174 observed at the
TPB, Thompson (1997) postulated that a greater number of cysteine residues and
disulﬁde bonds within the coat protein of φX174 might give the phage particle
JIN AND FLURY
increased stability over MS-2 (hard vs soft proteins). Another possible explanation
is that φX174 would not be attracted to the AWI or would be only weakly attracted
because it is largely hydrophilic (Thompson and Yates, 1999). In a previous study,
where animal viruses and bacteriophages were ranked on the basis of relative
hydrophobicity (Shields and Farrah, 1987), it was determined that φX174 was
the most hydrophilic, while MS-2 was the most hydrophobic of 15 viruses tested.
The interaction of a virus particle with an AWI is strongly inﬂuenced by the virus’
amphipathicity, the result of localized hydrophobic and hydrophilic regions on the
surfaces of the capsid proteins. Amphipathic molecules accumulate at AWIs with
the hydrophobic end orienting into the nonpolar air phase, while the hydrophilic
end remains in the aqueous phase (Stumm and Morgan, 1981). This suggests that
φX174, because of its dominant hydrophilicity, will not readily accumulate at
AWIs, while MS-2 will.
Solution ionic strength has been found to inﬂuence virus survival at the AWI.
Thompson and Yates (1999) demonstrated a clear relationship between the inactivation of MS-2 and R17 and solution salt concentration in their batch experiments.
MS-2 and R17 both underwent greater inactivation as solution ionic strength increased. This type of behavior has been observed for various colloidal particles,
such as viruses, bacteria, clay, and polystyrene and latex particles (Trouwborst
et al., 1974; Wan and Wilson, 1992, 1994; Williams and Berg, 1992), as well
as for individual proteins (Song and Damodaran, 1991). As the ionic strength of
the solution increases, the particles are increasingly attracted to the AWI because
of a compression of the electrostatic double layer (Trouwborst et al., 1974; Wan
and Wilson, 1994; Williams and Berg, 1992). Thompson and Yates (1999) also
conclusively showed that viruses must reach the AWI before being inactivated by
relating the amount of surfactant Tween 80 present at the AWI to the level of phage
inactivation. In other words, organic solutes, such as Tween 80, within an aqueous
system compete with virus particles to accumulate at the AWI and result in decreased virus inactivation. This is because the solution surface tension decreases in
proportion to the amount of organic solutes at the interface (Thompson and Yates,
1999). Other investigators have demonstrated a protective inﬂuence from peptone,
amino acids, and various surfactants against phage inactivation upon exposure to
AWIs (Adams, 1948; Trouwborst et al., 1972, 1974).
VII. TRANSPORT OF VIRUSES IN POROUS MEDIA
Virus transport studies have been conducted at both ﬁeld and laboratory scales.
The focus of the ﬁeld studies conducted in the last 25 years has been on the
transport of enteric viruses, as they were thought to have the greatest potential
to be transported due to their relatively small size. There is evidence that certain
SUBSURFACE VIRUS FATE AND TRANSPORT
viruses can move considerable distances through the vadose as well as through the
saturated zones (Rossi et al., 1994; Sinton et al., 1997; Yates and Yates, 1988).
Such ﬁndings have raised concerns about groundwater contamination. Regulations
regarding drinking-water well disinfections (USEPA, 2000) have led to increased
efforts in past years to elucidate the mechanisms of virus transport through soils
and aquifers. Different aspects of virus transport through soil and groundwater
have been reviewed (Gerba and Goyal, 1981; Keswick and Gerba, 1980; Melnick
and Gerba, 1980; Schijven and Hassanizadeh, 2000; Yates and Yates, 1988).
The movement of viruses, as well as other microorganisms, through the subsurface is dominated by advection and dispersion and has been modeled using various
forms of the advection–dispersion equation (ADE).
Virus transport through porous media is inﬂuenced by adsorption, desorption,
and inactivation processes. These processes are in turn affected by the various
factors listed in Table I, many of which have been the focus of virus transport studies
and are discussed in the following. Column studies and experimental conditions
are listed in Table VI.
1. Soil Properties
Virus retention and transport have been found to be affected by soil properties. In
a set of saturated column experiments, the behavior of three viruses (poliovirus 1,
reovirus 3, and φX174) was studied in several different soils (Funderburg et al.,
1981). A high poliovirus concentration measured in the column percolates correlated most favorably with low soil cation-exchange capacity and high organic
carbon and clay content, whereas a high percolate concentration of φX174 was
related to low soil organic carbon content and residence time of liquid within a
column in combination with either high soil pH or percentage of clay. As with
poliovirus, the detection of reovirus in the soil column percolates was negatively
correlated with soil cation-exchange capacity. These experiments were conducted
with ﬁeld soils in laboratory columns, which were packed in such a manner that
they simulated the vertical proﬁle and bulk density of the soil as found in the ﬁeld.
However, the procedures used for saturating the columns and applying input solution were not well controlled, so that complete saturation and constant steady-state
ﬂow conditions were not guaranteed. In addition, because of the complex nature of
the soils used, such experiments do not allow careful evaluation of speciﬁc mechanisms. Because of these limitations, results from most of the earlier studies are
difﬁcult to interpret and quantify (Drewry and Eliassen, 1968; Gerba and Lance,
1978; Landry et al., 1979).