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VI. The Role of the Gas–Liquid Interface in Protein Virus Inactivation

VI. The Role of the Gas–Liquid Interface in Protein Virus Inactivation

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Virus Inactivation and Protein Denaturation at the Gas–Liquid Interface


Pepsin, renin,


Egg albumin




Influenza A






T1, . . .,T7









P22H5 and



MS-2, φX174


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

prevents inactivation.

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

protein structure.

Inactivation in polypropylene bottles, but not in glass

bottles; inactivation occurs at the triple-phase interface

boundary air–water–solid.


Shaklee and Meltzer (1909)

Bull (1938)

Gorter (1938)

Langmuir and Waugh (1938)

Neurath and Bull (1938)

Grubb et al. (1947)

McLimas (1947)

Adams (1948)

James and Augenstein (1966)

Quinn and Dawson (1970)

Trouwborst et al. (1974)

Trouwborst and de Jong (1973)

Donaldson et al. (1980)

MacRitchie (1987)

ˇ sko (1992)

Bricelj and Siˇ

Tronin et al. (1996)

Thompson et al. (1998)



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

polypropylene surface.

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



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).


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



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).

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