Viruses Studies                                                                                                                             July 2009

Many viruses can not be cultured. Most virus studies are carried out using laboratory strains which are
not as hardy as natural wild virus strains.

Plaques have been produced with the three types of poliomyelitis viruses on monolayer tissue cultures of monkey
kidney and monkey testis. The number of plaques was proportional to the concentration of the virus. Each plaque
originates, therefore, from a single virus particle, defined as the virus unit that is unseparable by dilution. The plaques
are due to the specific action of the virus since they are suppressed by type-specific antiserum.
Pure virus lines were established by isolating the virus population produced in single plaques. These derived virus
lines had the same morphological, serological, and pathogenic properties as the parent strain.
High titer virus stocks, with titers up to 7 x 108 plaque-forming particles per ml., were obtained.
Submitted on June 1, 1953
The Journal of Experimental Medicine, Vol 99, 167-182

Viricidal Efficiency of Disinfectants in Water  (1961)

It is apparent, however, that the different viruses vary widely in their susceptibility to free chlorine. The pH exerts
marked effects on the viricidal efficiency of free chlorine. Weidenkopf's tests (12) show that decreasing the pH from 7.0
to 6.0 reduces the required inactivation time by about 50 percent, and the findings of both Weidenkopf (12) and Clarke
and others (14) indicate that a rise in pH from 7.0 to 8.8-9.0 increases the inactivation period about six times.

Data from recent studies of the efficiency of various disinfectants in inactivating enteric viruses in water appear to
support the following summarizations:
1. Different types of enteric viruses vary widely in the degree of resistance to free chlorine. Poliovirus, Coxsackie, and
some ECHO viruses seem to be more resistant than coliform or enteric pathogenic bacteria. The free chlorine
residuals required for inactivation depend on pH, temperature, and contact time.
2. Combined chlorine is considerably less viricidal than free chlorine, requiring higher concentrations or longer contact
periods to achieve comparable inactivation.
3. Iodine is an effective viricide, but requires greater residuals and longer contact than hypochlorous acid.
4. Chlorine dioxide, ozone, and ultraviolet light may be useful disinfectants; however, their efficiency in water in
comparison with that of free chlorine and the quantitative effects of pH and temperature have not been established.

Concentration of viruses from large volumes of tap water using pleated membrane filters.
S R Farrah, C P Gerba, C Wallis and J L Melnick
A method is described for the efficient concentration of viruses from large volumes of tap water in relatively short time
periods. Virus in acidified tap water in the presence of aluminum chloride is adsorbed to a 10-inch (ca. 25.4 cm)
fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in series at flow rates of up to 37.8 liters/min (10
gallons/min). This filter series is capable of efficiently adsorbing virus from greater than 19,000 liters (5,000 gallons) of
treated tap water. Adsorbed viruses are eluted from the filters with glycine buffer (pH 10.5) and the eluate is
reconcentrated using an aluminum flocculation process. Viruses are eluted from the aluminum floc with glycine buffer
(pH 11.5). Using this procedure, viruses in 1,900 liters (500 gallons) of tap water can be concentrated 100,000-fold in
3 h with an average recovery of 40 to 50%.
Appl Environ Microbiol. 1976 February; 31(2): 221-226

Poliovirus Removal from Primary and Secondary Sewage Effluent by Soil Filtration
Adsorption of poliovirus from primary sewage effluent was similar to that from secondary sewage effluent in both batch
soil studies and experiments with soil columns 240 cm long. Virus desorption by distilled water was also similar in a soil
column that had been flooded with either primary or secondary effluent seeded with virus. These results indicated that
adsorption of poliovirus from primary effluent and virus movement through the soil were not affected by the higher
organic content of primary sewage effluent.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 247-251, Vol. 36, No. 2

Virus Inactivation in Wastewater Effluents by Chlorine, Ozone, and Ultraviolet Light (1981)
This study, however, clearly demonstrated that virus recovery techniques need considerable improvement. Field
studies should be undertaken only after a viable measurement technique exists.

Effect of Chlorine Treatment on Infectivity of Hepatitis A Virus (1983)

From 1972 to 1981, about 40,000 to 50,000 cases of hepatitis A and 6,000 to 9,000 cases of
hepatitis B have been reported annually in the United States, but the actual incidence of clinical
disease and subclinical infection is undoubtedly several times greater than the number of
reported cases. The fecal-oral transmission of HAV by contamination of water supplies, food, and drink is
well documented by the many detailed reports (3, 9, 15) of hepatitis A epidemics.

This study examined the effect of chlorine treatment on the infectivity of hepatitis A virus (HAV). Prodromal chimpanzee
feces, shown to induce hepatitis in marmosets (Saguinus sp.), was clarified, and the virus was precipitated with 7%
polyethylene glycol 6000, harvested, and resuspended. The suspension was layered onto 5 to 30% linear sucrose
gradients and centrifuged; the fractions containing HAV were dialyzed, and a 1:500,000 dilution of this preparation
induced hepatitis and seroconversion in 2 of 4 marmosets. A 1:50 dilution of this preparation served as inoculum.
Untreated inoculum induced overt hepatitis and seroconversion in 100% (5 of 5) of marmosets inoculated
intramuscularly. Inoculum treated for various periods (15, 30, or 60 min) with 0.5, 1.0, or 1.5 mg of
free residual chlorine per liter induced hepatitis in 14% (2 of 14), 8% (1 of 12), and 10% (1 of 10) of marmosets,
respectively, and induced seroconversion in 29, 33, and 10% of the animals. Inoculum treated with 2.0 or 2.5 mg of
free residual chlorine per liter was not infectious in marmosets as determined by absence of hepatitis and
seroconversion in the 13 animals tested. Thus, treatment levels of 0.5 to 1.5 mg offree residual chlorine per liter
inactivated most but not all HAV in the preparation, whereas concentrations of 2.0 and 2.5 mg offree residual chlorine
per liter destroyed the infectivity completely. These results suggest that HAV is somewhat more resistant to chlorine
than are other enteroviruses.

Detection of enteric viruses in treated drinking water.
B H Keswick, C P Gerba, H L DuPont and J B Rose
The occurrence of viruses in conventionally treated drinking water derived from a heavily polluted source was
evaluated by collecting and analyzing 38 large-volume (65- to 756-liter) samples of water from a 9 m3/s (205 X 10(6)
gallons [776 X 10(6) liters] per day) water treatment plant. Samples of raw, clarified, filtered, and chlorinated finished
water were concentrated by using the filter adsorption-elution technique. Of 23 samples of finished water, 19 (83%)
contained viruses. None of the nine finished water samples collected during the dry season contained detectable total
coliform bacteria. Seven of nine finished water samples collected during the dry season met turbidity, total coliform
bacteria, and total residual chlorine standards. Of these, four contained virus. During the dry season the percent
removals were 25 to 93% for enteric viruses, 89 to 100% for bacteria, and 81% for turbidity. During the rainy season
the percent removals were 0 to 43% for enteric viruses, 80 to 96% for bacteria, and 63% for turbidity. None of the 14
finished water samples collected during the rainy season met turbidity standards, and all contained rotaviruses or
Appl Environ Microbiol. 1984 June; 47(6): 1290-1294

Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven
water treatment plants.
P Payment, M Trudel and R Plante
Seven drinking water treatment plants were sampled twice a month for 12 months to evaluate the removal of indicator
bacteria and cytopathogenic enteric viruses. Samples were obtained at each level of treatment: raw
water, postchlorination, postsedimentation, postfiltration, postozonation, and finished (tap) water. Raw water quality
was usually poor, with total coliform counts exceeding 105 to 106 CFU/liter and the average virus count in raw water of
3.3 most probable number of cytopathogenic units (MPNCU)/liter; several samples contained more than 100
MPNCU/liter. All plants distributed finished water that was essentially free of indicator bacteria as judged by analysis of
1 liter for total coliforms, fecal coliforms, fecal streptococci, coagulase-positive staphylococci, and Pseudomonas
aeruginosa. The total plate counts at 20 and 35 degrees C were also evaluated as a measure of the total microbial
population and were usually very low. Viruses were detected in 7% (11 of 155) of the finished water samples (1,000
liters) at an average density of 0.0006 MPNCU/liter the highest virus density measured being 0.2 MPNCU/liter. The
average cumulative virus reduction was 95.15% after sedimentation and 99.97% after filtration and did not significantly
decrease after ozonation or final chlorination. The viruses isolated from treated waters were all enteroviruses:
poliovirus types 1, 2, and 3, coxsackievirus types B3, B4, and B5, echovirus type 7, and untyped picornaviruses.
Appl Environ Microbiol. 1985 June; 49(6): 1418-1428

Reovirses in Water Pollution Testing.
The reoviruses were consistently recovered in approximately 5-fold higher concentrations than enteroviruses.
Reovirus infectivity is activated by enzyme treatment in some sewage, but not in other sewage. Some reoviruses
recovered from sewage were inactivated by enzyme treatment.

Viruse Removal During Conventional Drinking Water Treatment
Reduction of enteroviruses and rotaviruses averaged 81% and 93%, respectively, for the complete treatment process.
-- The results of this study indicate that finished water having measurable levels of free chlorine [>0.2 mg/L] and
meeting standards for fecal coliform bacteri and turbidity cannot be assumed to be virus free. -- It is also recognized
that naturally occurring viruses may be more resistant to chlorination than prototype laboratory strains. -- Research
should be conducted on water quality and operational treatment conditions that will assure essentially complete
removal of viruses.

Viruses in Source and Drinking Water
Gerba, CP | Rose, JB
IN: Drinking Water Microbiology: Progress and Recent Developments. Springer-Verlag New York, Inc., New York. 1990.
p 380-396, 5 tab, 64 ref.

All warm blooded animals harbor enteric viruses which are excreted in fecal material and can find their way into the
aquatic environment. Human enteric viruses are able to exist for extended periods in the environment and many may
survive conventional water and wastewater treatment. While a direct association with the presence of virus in a water
supply and illness has not been demonstrated for all enteric viruses, the presence of any of these viruses in drinking
water should be considered a major health concern. More than 110 types of human enteric viruses have been
identified, including the enteroviruses, hepatitis A virus, Norwalk virus, reovirus, rotavirus and adenovirus. Enteric
viruses can be expected to occur in any surface water exposed to contamination by human fecal wastes, while the
most frequently reported source of contamination in outbreaks involving groundwater is overflow or seepage of
sewage from septic tanks. The development of field concentration methods was a major factor in the increased
isolation of enteric viruses from drinking water, but laboratory innovations in the propagation and enumeration of
viruses such as rotavirus and hepatitis A virus have also contributed significantly more sensitivity to virus detection.
Most of the enteroviruses are readily isolated and quantitated by either observation for characteristic viral
cytopathogenic effects in the tissue culture or by the observation of plaques, or areas of cell destruction in a
monolayer of cells. If it is assumed that all of the nonbacterial gastrointestinal illness being reported has a viral
etiology, than as much as 64% could have been related to the presence of viruses in water. By conservative
estimations only a small percentage of the waterborne outbreaks are ever detected or reported. Thus there is
probably significantly more illness in the population due to viral contamination than is recognized. (See also W91-
06194) (VerNooy-PTT)

Waterborne rotavirus: A risk assessment
Charles P. Gerba, a, , , Joan B. Roseb, Charles N. Haasc and Kristina D. Crabtreea
A risk assessment approach was used to estimate the public health impacts from exposure to human rotavirus in
drinking and recreational waters. Rotavirus is the major cause of viral gastroenteritis worldwide and several waterborne
outbreaks have been documented. This results in a significant economic impact on society in terms of direct medical
costs, loss of work, quality of life and mortality. The virus is common in domestic wastewater and polluted surface
waters. Dose-response data in human adult volunteers indicate that it is the most infective of all the enteric viruses,
and this was used to develop a microbial risk assessment model to estimate daily and yearly risks of infection,
morbidity and mortality for exposure via drinking and recreational waters using existing information on the occurrence
of rotavirus. The disease is most severe for the very young, the elderly, and the immunocompromised. Case fatality
rates in the United States are 0.01% in the general population, 1% in the elderly, and up to 50% in the
immunocompromised. Analysis indicates that significant risks of disease (5 × 10−1−2.45 × 10−3) could result for
drinking and recreational waters in which rotavirus has been detected. The major limitation in assessing the risks of
waterborne rotaviral infections at present is the lack of data on its occurrence in water and the potential for human
Water Research, Volume 30, Issue 12, December 1996, Pages 2929-2940

Science 7 August 2009: Vol. 325. no. 5941, pp. 660 - 661
News of the Week
Public Health:
Type 2 Poliovirus Back From The Dead in Nigeria
Leslie Roberts
In 1999, the Global Polio Eradication Initiative scored an unequivocal victory: It wiped one of three serotypes of wild
poliovirus, type 2, off the face of the earth, except for samples stored in labs for study or vaccine creation. That
triumph left just two foes to battle, poliovirus types 1 and 3, which have continued to put up quite a fight. But now a
version of type 2 has returned. Springing back to life from a weakened form of the pathogen used in a vaccine,
poliovirus type 2 is causing a runaway outbreak in Nigeria, where types 1 and 3 are also raging. In July, the World
Health Organization issued a global alert warning that type 2 poliovirus in Nigeria posed an "increasing risk of
international spread." It's a stunning setback for the initiative, now already 9 years past its original deadline for
vanquishing the virus.

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