Journal of Animal Science and Veterinary Medicine

Volume 4(2), pages 52-64, April 2019

Article Number: 46893D142
ISSN: 2536-7099
https://doi.org/10.31248/JASVM2019.129
http://www.integrityresjournals.org/journal/JASVM
Full Length Research

Study of physico-chemical factors affecting the growth

of cell-culture adapted bovine Rotavirus
strain of Pakistan
Wardah Sharmeen Syed*#, Nadia Mukhtar, Nauman Zahid and Tahir Yaqoub#
University of Veterinary and Animal Sciences, Lahore, Pakistan.
*Corresponding authors. Email: [email protected]

#These authors contributed equally to this work.

Copyright © 2019 Syed et al. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 15th January, 2019; Accepted 4th March, 2018

ABSTRACT: Rotavirus (RV) diarrhea is the major cause of death of millions of children in developing countries besides
causing economically significant malady in neonates of many domestic animals. There is a very little information available
for the factors which can affect prevalence of Bovine Rotavirus (BRV) in Pakistan. There is a dire need to propagate BRV
on cell culture and evaluate the physical and chemical control of RV in effective ways for the betterment of human and
livestock welfare. Therefore, the present study aimed to propagate BRV on Madin-Darby bovine kidney (MDBK) cell line.
The screening of the virus was done by using commercially available kit and TCID50 technique. The propagation of BRV
was then led to study its inactivity and infectivity potential using different physical and chemical factors. For this reason, 3
physical factors (Temperature, pH and UV light) and 8 chemical disinfectants were used. The virus got completely inactive
at a temperature range of 75 to 80ºC and 5.00 to 6.00 pH while remained active at 7.04 and 8.00. The virus was inactivated
after exposure to 0.5% Virkon®-S within 30 minutes, 0.5% Bromo-Sept after 30 min, and 1.0% surf excel after 30 min Phenol
within 60 min at a concentration of 1.0% and 0.6% H2O2 after 30 min proved to be virucidal for the virus. These findings can
be helpful for the farmers to keep their farms decontaminated from this virus. Moreover, these results can also be of help in
the prevention of the outbreaks that occur in the hospitals.

Keywords: Bovine Rotavirus, cell culture, chemical factors, culturing of BRV, physical factors.

INTRODUCTION

Rotavirus, a leading source of gastroenteritis in humans infection in the young of nearly every mammalian and avian
and animals, causes diarrhea in young calves (Swiatek et species. They replicate in enterocytes of the intestinal tract
al., 2010). Bovine Rotavirus (BRV) is responsible for which leads to severe gastroenteritis and later viruses shed
neonatal calf diarrhea and it has been perceived in both into the stool (Arnold et al., 2009).
beef cattle and dairy all over the world (Holland, 1990) and In 2007, National Animal Health Monitoring System for
contribute considerably to diarrhea and enteritis in U.S. dairy declared that diarrhea causes 50% of the
competently nurtured neonatal calves (Dhama et al., 2009). mortality in un-weaned calves and most of it occurred in
Neonatal diarrhea represents an imperative hygiene calves of less than 1 month of age. Neonatal calf mortality
problem in livestock production which causes an extremely varies from 8.7 to 64% throughout the world constituting
negative economic influence related to veterinary treatment 84% of the total mortality in the first month of age and is
costs, reduction in weight gain of affected animals and particularly high in the third week (Cho, 2012). Annually
death (Badaracco et al., 2013). It is non-enveloped, Rotavirus kills approximately half a million bovines globally
icosahedral shaped, triple-layered symmetry virus and among these estimated deaths around one-third
belonging to the family Reoviridae (Aiyegbo et al., 2014). (N = 160,000 deaths) occur in the Indian subcontinent
Group A Rotaviruses are intestinal pathogens that cause (Chen et al., 2012). Moreover, three countries Bangladesh,
 Syed et al. 53

India, and Pakistan in the Indian subcontinent form >30% Cell culture adaptation
(N = 160,000 to 200,000) of all the deaths related to
Rotavirus all over the world (Miles et al., 2012). MDBK cells were grown in the laboratory using Dulbecco’s
Rotaviruses show considerably good growth in Vero and Minimum Essential Medium (DMEM) and F12 supplemen-
MDBK cells, and so testing for them is of particular ted with 10% of Fetal Bovine Serum (FBS). BRV was
importance (Gregersen, 2012). Different studies on BRV propagated in cell culture after treatment with porcine
had been carried out on monkey, mouse, and human cell trypsin 1X (10 µg/ml; Sigma-Aldrich) for 30 to 60 min at
lines (Bugarc̀ić and Taylor, 2006; Kaushik et al., 2008). 37ºC. Two (2) ml of the virus suspension was poured into
Even though a clear information regarding these the flask and let it adsorbed by the cells of the monolayer
heterologous cell culture models might not completely for 2 to 4 hours at 37ºC. Later the suspension was
relate to cattle given known differences in Rotavirus strains, discarded and the cells were filled with 15 to 20 ml of the
cellular receptors, species barriers, and other factors which serum free medium (DMEM w/o serum with 100 µg/ml
are required for Rotavirus infectivity, uptake, pathogenesis streptomycin, 100 I.U/ml penicillin-G and 2.5 µg/ml
and replication (Estes et al., 2001; Lopez and Arias, 2006; amphotericin-B) (Reading et al. 1998, Arias et al. 2002).
Pesavento et al., 2006; Ramig, 2004). For infecting the cells Then, the flask was incubated for a period of 72 hours in
provided, the virus must enter the plasma membrane of the CO2 incubator at 37°C. After visualizing the initial
host cell and this needs good penetration ability. The ability appearance of cytopathic effects (CPEs), namely
to penetrate the cell is increased by the use of proteolytic degeneration, cytoplasmic vacoulation and the
enzymes (Arias et al., 2002). Antigen capture enzyme- development of eosinophilic intracytoplasmic inclusions
linked immunosorbent assay (ELISA), reverse (McNulty, 1978), the flask was re-incubated at 37°C in 5%
transcription-PCR (RT-PCR) and latex agglutination are the CO2 for another 3 to 4 days till complete appearance of
methods which have become standard for the detection CPEs (Figure 1a). At the terminal stage when maximum
and analysis of bovine Rotavirus infections (Maes et al., CPEs were evident as a complete detachment of the cell
2003 and Al-Robaiee and Al Farwachi, 2013). monolayer (Figure 1b), the flasks were placed at -80°C for
Rotaviruses are mainly pervasive in farm animals. 10 min. The flasks were taken out and placed at room
Different viruses vary significantly in liability to treatment temperature till complete thawing of the flask content. The
with high pressure and this distinction can be attributed to flasks were subjected to two freeze-thaw cycles in the
the diversity of the structural morphology of virus (Smelt, similar manner. At the end, the entire harvested medium
1998; Khadre and Yousef, 2002). Heating at 50ºC for 30 was centrifuged at 4000 rpm for a period of 10 min in a
min inactivates 99% of the virus infectivity (Estes et al., refrigerated centrifuge machine at 4°C and supernatant
1979). A number of antibiotics and chemical disinfectants was used as virus source for further experiments.
are commonly used to avert and regulate the dissemination
of viral diseases in hospitals, laboratories and many other
institutional or domestic settings (Höh et al., 1983). Calculation of infectivity titers of BRV
Thus, the objective of the study is to draw an emphasis
towards the study of such physical and chemical factors Biological titration of the harvested BRV as tissue culture
which can help in eliminating Rotavirus from the premises infective dose50 (TCID50) was calculated by using the
of the farms and from the healthy animals and if by chance technique of Reed and Meunch (Swayne et al. 2006).
an animal encounters with rotavirus then the removal of the
virus from the animal body can be done by using easy and Effect of physical factors on the survival of BRV
cheap methods.
The propagated virus particles were treated with three
physical factors namely, Temperature (37, 50 to 80°C), pH
MATERIAL AND METHODS (5.00 to 12.00) and UV light with the wavelength of 253.7
nm for different time intervals. The grown virus was
Bovine Rotavirus (BRV) was collected from a diarrheic calf exposed under the mentioned conditions separately, to
which was injected with the Rotavirus once procured from study the effect of each factor on the growth of the virus.
the seed bank of Quality Operations Laboratory, University Triplicate plates were also used for this purpose.
of Veterinary and Animal Sciences, Lahore. The samples
of intestine and blood of infected calf were used to isolate
the virus. Effect of conventional chemicals on the survival of
BRV

Screening of positive sample For this purpose, 4 conventional (laboratory used)

chemicals were selected which included phenol, ethanol,
The screening of bovine rotavirus antigen from faecal isopropanol and H2O2. Formaldehyde was not used in this
supernatant was done by using commercially available study as it was found to be cytotoxic to the cells. These
antigen capture ELISA kit (Cypress Diagnostics, Belgium). chemicals were used with the concentrations as phenol (0.5
54 J. Anim. Sci. Vet. Med.

(a) (b)

Figure 1. Cytopathic effects of Rotavirus; (a) Early stage of CPEs (Monolayer started
detaching from the cell line) of bovine rotavirus on MDBK cells (b) Terminal stage;
complete detachment of monolayer of rotavirus.

to 2.5%), ethanol (60 to 98.5%), isopropanol (60 to 90%) on the infectivity titers of BRV as recorded at various time
and finally H2O2 (0.2 to 1.0%) for 0, 30, 60, 90 and 120 intervals was checked three times by using the technique
min,respectively. The virus was exposed to these of TCID50 as described in the previous section and shown
chemicals the same way as they were exposed to physical in Table 1 which shows that the virus was able to grow at
factors and then their titer was studied using TCID50. 37°C which was a favourable temperature for its growth,
but the growth of the virus was abolished as the
temperature was raised.
Effect of commercial chemicals on the survival of BRV

In order to study the effect of commercial products on BRV Effect of pH on the survival of BRV
four types of disinfectants were used which includes
Pyodine, Virkon®-S, Bromo-sept and surf excel with the The effect of three pH values 5.00, 6.00 (acidic), 7.40 near
concentrations of 0.5, 1.0 and 2.0% for each and for the to neutral as available in biological system, and 8.00 to
time interval of 0, 30, 60, 90 and 120 min for each of the 12.00 (alkaline) conditions for survival of BRV was
chemical concentration employed. determined and results are given in Figure 2(a, b). The virus
started showing depletion at pH 6 and was found
completely destroyed at pH 5.0 having the mean standard
RESULTS deviation of 3.09±(0.035)b after 120 min. Whereas, BRV
Bovine rotavirus on MDBK cell line showed stunted growth again at pH 12.0 having the mean
standard deviation of 2.75±(0.070)b after 120 min. The virus
The monolayer formed was given infection with the remained unaffected at pH 7.40.
procured and screened bovine Rotavirus by the procedure
described previously. CPEs were observed after 5 days but Effect of UV light on the infectivity of BRV
the terminal stage of the CPEs was obtained after 8 days
of incubation under standard conditions as shown in Fig 1. Exposure of the virus by UV light had been carried out at
From the viral stock, biological titer was calculated by the wavelength of 253.7 nm for different time intervals
using Reed and Muench formula (Swayne et al., 2006) by keeping the same wavelength. UV light showed a very clear
observing the CPEs and scoring the wells positive or decrease in the titer of BRV. BRV started getting inactive
negative according to the effects imposed on the cells by after 30 min with the mean standard deviation value of
the virus. The titer came out to be 106.62/1ml of BRV with 3.47±(0.028)b and was completely destroyed in 3 hours
50% endpoint dilution. exposure of UV light with the mean standard deviation
value of 000±(0.00) as represented by Figure 3.
Effect of physical factors on BRV
Evaluation of conventional chemical factors on the
Effect of temperature survival of BRV
The virus was kept at the normal temperature range (37ºC) Effect of phenol on the infectivity of BRV
and the elevated temperature ranges (50 to 80ºC) using the
electrical water bath. The effect of different temperatures To evaluate the inactivation potential of the phenol to BRV,
 Syed et al. 55

Figure 2(a). Linear regression model showing decrease in titer of BRV after
exposure to pH 5.00-8.00 conditions.

Figure 2(b). Linear regression model showing decrease in titer of BRV after
exposure to pH 9.00-12.00 conditions.

Figure 3. Linear regression model showing decrease in titer of BRV after exposure to UV light.

0.5 to 2.5% solution was prepared and then evaluated for be highly cytotoxic so a much diluted concentrations used
anti-viral activity against BRV. Recommended concentra- on cell culture. The decline growth pattern can be seen from
tion of phenol for disinfection 2.5% and 3.0% were found to Figure 4(a, b, c).
56 J. Anim. Sci. Vet. Med.

Figure 4(a). Linear regression model showing decrease in titer of BRV by

treatment with 0.5% Phenol.

Figure 4(b). Linear regression model showing decrease in titer of BRV by

treatment with 1.0% Phenol.

Figure 4(c). Linear regression model showing decrease in titer of BRV by

treatment with 1.5% Phenol.

Effect of ethyl alcohol on the survival of BRV 80, 90 and 98.5%) were studied. Recommended
concentrations of ethanol 80 and 90% were found cytotoxic
To study the effect of ethanol, five concentrations (60, 70, for BRV for the interval of 30 min so absolute alcohol was
 Syed et al. 57

Figure 5(a). Linear regression model showing decrease in titer of BRV by

treatment with 60% ethanol.

Figure 5(b). Linear regression model showing decrease in titer of BRV by

treatment with 70% ethanol.

diluted to the extent of 60% to study its effect on the Effect of Hydrogen Peroxide (H2O2) on the survival of
monolayer of the BRV. As shown in Figure 5(a, b), it can be BRV
seen that a dilution of 60% the virus still had somewhat
smooth growth, whereas it started having stunted growth at The effect of H2O2 was studied on BRV using five
70% after 60 min. concentrations (0.2, 0.4, 0.6, 0.8 and 1.0%). BRV have
been found to get inactivated by the use of H 2O2 with the
concentration of 1.0% (Meng et al. 1987). So, the local
Effect of Isopropanol on the survival of BRV strain of BRV was exposed to varying time intervals to
different concentrations of H2O2. The observations are
In order to study the effect of isopropanol, four shown in Figure 7(a, b). The virus started showing stunted
concentrations (60, 70, 80 and 90%) were studied. It has growth at 0.6% concentration of H2O2 after 90 minutes.
been stated that with the increasing concentration of the
alcohol used, the reduction in the titer of the virus is
significant (Kurtz et al., 1980), so for this reason BRV was Evaluation of commercial chemical factors on the
given trial with the increasing concentration of alcohol with survival of BRV
3 carbon atoms (isopropanol) and studied its impact on the
activity of the virus for varying time intervals. Effect of Pyodine on the survival of BRV
Recommended concentrations of Isopropanol 80 and 90%
were found cytotoxic for BRV for the interval of 30 min so To evaluate the inactivation potential of Pyodine (10%
diluted concentrations were used to study the effect on the solution of povidone-iodine) on BRV, three concentrations
growth as shown in Figure 6(a, b, c). (0.5, 1.0 and 1.5% were prepared of commercially available
58 J. Anim. Sci. Vet. Med.

Figure 6(a). Linear regression model showing decrease in titer of BRV by

treatment with 60% isopropanol.

Figure 6(b). Linear regression model showing decrease in titer of BRV

by treatment with 70% isopropanol.

Figure 6(c). Linear regression model showing decrease in titer of BRV by

treatment with 80% isopropanol.

10% solution). The study was conducted at different time of this chemical (Figure 8a, b). At 1% concentration of
intervals in order to evaluate the cytotoxicity and viral pyodine, a decrease in the infectivity of the virus in the cells
inactivation potential if, however, occurred due to the use was observed after 90 min.
 Syed et al. 59

Figure 7(a). Linear regression model showing decrease in titer of BRV by

treatment with 0.2% H2O2.

Figure 7(b). Linear regression model showing decrease in titer of BRV by

treatment with 0.4% H2O2.

Figure 7(c). Linear regression model showing decrease in titer of BRV by

treatment with 0.6% H2O2.

Effect of Virkon®-S on the survival of BRV by using different concentrations under different time
intervals such as 0.5, 1.0 and 1.5% for the observations
The inactivation potential of Virkon®-Son BRV was studied period of 0, 30, 60, 90 and 120 min. With the increasing
60 J. Anim. Sci. Vet. Med.

Figure 8(a). Linear regression model showing decrease in titer of BRV after
treatment with 0.5% pyodine.

Figure 8(b). Linear regression model showing decrease in titer of BRV after
treatment with 1.0% pyodine.

concentration of the disinfectants, time starts decreasing produced 3 log10 reduction in virus load. The behaviors of
(Figure 9). A magnificent decrease in the infectivity titer can surf excel on the virus in given in Figure 11(a, b). The
be seen at 0.5% concentration of the chemical after 30 min. reduction in viral load can be seen at concentration of 1.0%
after 30 min.

Effect of Bromo-Sept on the survival of BRV

DISCUSSION
The inactivation potential of Bromo-Sept on BRV was
studied by using undiluted bromo-sept for the contact time Neonatal calf diarrhea represents a vital sanitary issue in
of 30 min. It produced 3 log10 (99.9%) reduction in virus load livestock production which leads to an enormous negative
at 0.5% concentration of Bromo-Sept after 30 min. The economic influence related to reduction in weight gain of
behavior of Bromo-sept on the virus is given in Figure 10. affected animals, veterinary treatment costs and death
(Badaracco et al., 2013). The dissemination of disease is
largely contributed by the infected animals which excretes
Effect of Surf Excel on the survival of BRV large quantity of viruses in their fecal material,
contaminating the surrounding and thus the virus spreads
The inactivation potential of Surf excel on BRV was studied to susceptible population which later on ends up in
by using three concentrations of the detergent such as 0.5, outbreaks of gastroenteritis in young calves (Khan et al,
1.0 and 2.0% for the contact time of 30 to 120 min. It 2008).
 Syed et al. 61

Figure 9. Linear regression model showing decrease in titer of BRV after

treatment 0.5% Virkon®-S.

Figure 10. Linear regression model showing decrease in titer of BRV after
treatment with 0.5%Bromo-sept.

Environment contributes largely in the survival and sandwich ELISA (Al-Robaiee and Al Farwchii, 2013).
endurance of all types of organisms. Viruses being non- After getting the virus positive by the kit the first aim was to
living particles need a living host system for their adapt the virus on cell culture. For this purpose, the virus
multiplication but their existence and survival chances in was propagated initially on Vero cell line on which after
the environment plays significant role in transmission to successive 12 passages no significant CPEs were
specific host (Mehle and Ravnikar, 2012). There are a observed, so for adapting the virus in cell culture the cell
number of physical (humidity, sunlight, temperature etc.) line was switched from Vero to MDBK cell line. MDBK cells
and chemical factors which affects viral survival inside and are supposed to be the host cells for adapting BRV as these
outside of the host system. Presence of organic matter cells are obtained from bovine kidney cells. Furthermore,
(mucous, saliva, feces, blood etc.), the type of virus MDBK cells bear bovine carbohydrates as ligands so they
(enveloped or non-enveloped), and the time of exposure to neutralized any effect or hindrance occurred during virus
the susceptible damaging agent are some of the additional adaptation (Reading et al, 1998). When the virus was
and comparative features that can influence the existence switched to MDBK cell line a remarkable difference in the
of the virus in the environment (Tang, 2009). CPEs was observed and after 9 successive passages the
In this present study Bovine rotavirus was procured from titer of the virus was found quite sufficient for carrying
the culture bank of Quality Operations Laboratory, UVAS, further study. The titer of the virus was calculated by Reed
Lahore, as fecal sample. Initial screening of the virus was and Meunch method of TCID50 and was found to be
done by using commercially available kit by Cypress 106.62/ml.
diagnostics which employs the technique of indirect The second aim was to study the infectivity and inactivity
62 J. Anim. Sci. Vet. Med.

Figure 11(a). Linear regression model showing decrease in titer of BRV after
treatment with 0.5%Surf excel.

Figure 11(b). Linear regression model showing decrease in titer of BRV

after treatment with 1.0% Surf excel.

pattern of the BRV. In the previous studies, the literature is enveloped virus, BRV is highly resistant to temperature and
concerned only for the inactivation of human rotavirus in the inactivated within 30 min at 80°C (Jiang et al, 2013). It has
hospitals where it becomes the reason of nosocomial been estimated that high temperature ranges denature the
infections among the infants or the literature tells the viral proteins which ends up in the inactivation of virus, this
studies of physical and chemical inactivation of different is true in case of enveloped viruses. As far as non-
viruses (Human simian virus, adenovirus, noro virus etc). In enveloped viruses are concerned, high temperature causes
this present study, the novelty deals with the study of alterations in the structural proteins (basically capsid
inactivation of bovine rotavirus, which is also zoonotic in proteins), which are left unable to retain their basic
nature. By studying different parameters used for the morphology and thus does not support receptor-ligand
inactivation of the above mentioned viruses, a set of interaction and so virus stops attaching to the surface and
physical and chemical factors were devised to conduct the becomes inactive (Sobsey and Meschke, 2003). However,
research on this propagated virus. For this reason, 3 there are some viruses that resist high temperature for a
physical factors i.e, effect of temperature, pH, and UV substantial period of time like enteroviruses and HAV which
radiations was evaluated on the survival of BRV in the persist even at 70°C for 10 minutes and at 60°C for 30
environment. The virus was completely inactivated at 70, minutes as compared to bacteria and parasites
75 and 80°C within the period of 15 minutes, while at 50, (Guardabassi et al., 2003).
55, 60, and 65°C the virus infectivity was still detectable Variation in pH was not found much effective to
after 15, 20 and 25 min, respectively. A wide temperature completely inactivate BRV within 2 hours, but the infectivity
range was selected to have a better insight into the survival titers were reduced at 5.00 and 9.00 as compared to neutral
of BRV at diverse environmental conditions. Being a non- pH. The pH ranges below 5.0 or above 11.0 inactivates the
 Syed et al. 63

virus (Anonymous 2009). In comparison to this, the studies less toxic for the living cells up to 1% concentration. The
of non-enveloped viruses showed that complete concentration of 1.5% causes cytotoxicity in the culture
inactivation of most of the viruses occurred at or below pH vessel so this concentration was not suitable for the cells
3 (Nims and Plavsic, 2012). However, there are certain as this and can damage the cells too. The Virkon®-S at the
viruses which are stable at even at highly acidic conditions, dilution of 1:400th destroyed either of the two porcine
i.e, HAV which is resistant even at pH 1.00 or 2.00 viruses, i.e. Classical swine fever virus (CSFV) and PRV
(Guardabassi et al, 2003). The mechanism of viral within the time range of 30 seconds and reduced infectivity
inactivation under the influence of pH is thought to be titer from TCID50 105.25 and 106.5/ml respectively to 0
directly linked with the conformation and symmetry of the (Bunpapong et al., 2011).
outer capsid proteins (Sobsey and Meschke, 2003). Avian Pyodine had been found to be effective against non-
viruses are enveloped viruses and are thought to be more enveloped viruses in previous studies (Anonymous 2009).
active at low pH i.e, 6.00 to 6.4 (Shahid et al., 2009). The In the present study, pyodine at the concentration of 1.0%
inactivation of virus because of the effect of pH might be was evaluated to be effective against BRV which tend to
due to the conformational changes in hemagglutinating reduce the infectivity titer from 106.62 ± 0.11 to 102.87 ±0.11/ml.
proteins (De Benedictis et al., 2007). The concentration of 2% was found to be toxic to the cells.
Ultra Violet (UV) light at a wavelength of 253.7 nm is Bromo-Sept (10%) solution was very efficient in
vicious for BRV like all other bacteria and viruses. The disinfecting BRV and its concentration of 1.0% completely
effect of UV-light on BRV, in this present study, suggests destroyed the viral activity after 60 min. The concentration
that it is detrimental to virus and inactivates it within 20 min. of 1.5% was found out to be cytotoxic.
The wavelength of 253.7 nm normally lies in the range of The antiviral activity of surf excel was very efficient. The
UV-B radiations on the spectrum, whereas, the natural sun concentration of 1.0% of this commercially available
light contains radiations of UV-A category which contains product was proved to be potent for the survival of the BRV.
greater wavelength and low penetration power, hence, they It completely destroyed the virus after 60 min at this
require more time for viral inactivation (Sobsey and concentration.
Meschke, 2003). UV light penetrates deep down the core
of the virus where its genetic material is present and poses
serious threats like dimerization of the genetic material Conclusion
which is an abnormality. After facing such an insult, the virus
no longer remains active. UV rays present in natural light Calf diarrhea has been a major disease problem in the
introduce structural changes in viral proteins and nucleic cattle industry. The influence of this uproar on the economy
acid, leaving the virus inactive (Wilhelm et al., 2002). of the cattle industry is still significant. Though many new
Phenol (99.5%: Riedel-deHaën) at the concentration of strategies (e.g., vaccine, medications and herd
1.0% effectively inactivated BRV within few hours but it was management) have been developed and practiced to
found to be toxicogenic for the living cells at recommended minimize the economic loss. However, physical and
concentration for disinfection. Only the concentration of chemical factors have not been studied to minimize the loss
0.5% was evaluated for results and found to inactivate the of cattle due to BRV. This study had been conducted to
virus completely within 3 hours. So it can be inferred that at introduce some commonly available disinfectants and
virucidal recommended concentration (2%), it is effective to surfactants along with a few physical parameters to
inactivate the virus within minutes (Meng et al, 1987). decrease the loss caused by the virus to the cattle industry.
As far as the alcohols are concerned, it has been reported
that with the increase in carbon number of the alcohols, the
effect of alcohols on viruses become evident. The more the CONFLICT OF INTEREST
concentration, the more virucidal tendencies it induce
(Kurtz et al., 1980). In this present study, 2 alcohols were The authors declare that they have no conflict of interest.
used; the one being ethanol (2 carbon atoms) and the other
being isopropanol (3 carbon atoms). Ethanol (98.5%: LAB- REFERENCES
SCAN) was found to be effective against BRV at the
concentration of 70% and it reduced the viral titer by 3 log10 Aiyegbo, M. S., Eli, I. M., Spiller, B. W., Williams, D. R., Kim, R.,
after 90 min, whereas, isopropanol (95%: Riedel-deHaën) Lee, D. E., Liu, T., Li, S., Stewart, P. L., & Crowe, Jr. J. E.
dropped the infectivity titer to this extent at the (2014). Differential accessibility of a Rotavirus VP6 epitope in
concentration of 60% after 90 min. trimers comprising type I, II, or III channels as revealed by
Hydrogen peroxide (H2O2) (35%: MERCK) had also been binding of a human Rotavirus VP6-specific antibody. Journal of
Virology, 88(1), 469-76.
employed in this study and the concentration of 0.8% had
Al-Robaiee, I. A., & Al-Farwachi, M. I. (2013). Prevalence of
been proved to be fruitful in disinfecting the virus after 60 rotaviral infection in diarrheic neonatal calves in Mosul city, Iraq.
min. The concentration above that was proved to be Veterinary World, 6(8), 538-540.
cytotoxic to the cells. Anonymous (2009). Rinder pest. In: Technical Disease Card
Virkon®-S inactivated BRV within 30 minutes at 0.5% database, (Paris, France: OIE Office International des
concentration, reducing the titer to 3 log10 and proved to be Epizooties/World Organization for Animal Health).
64 J. Anim. Sci. Vet. Med.

Arias, C. F., Isa, P., Guerrero, C. A., Méndez, E., Zárate, S., Khan, M. A., Hussain, S. N., Bahadar, S., Ali, A. and Shah, I. A.
López, T., Espinosa, R., Romero, P., & López, S. (2002). (2008). An outbreak of peste des petits ruminants (PPR) in
Molecular biology of rotavirus cell entry. Archives of Medical goats in district Chitral, N.W.F.P., Pakistan. ARPN Journal of
Research, 33(4), 356-361. Agricultural and Biological Sciences, 3.
Arnold, M., Patton, J. T., & McDonald, S. M. (2009). Culturing, Kurtz, J. B., Lee, T. W., & Parsons, A. J. (1980). The action of
storage, and quantification of Rotaviruses. Current Protocols in alcohols on Rotavirus, astrovirus and enterovirus. The Journal
Microbiology, Chapter 15: Unit 15C 3. of Hospital Infection, 1(4), 321-325.
Badaracco, A., Garaicoechea, L., Matthijnssens, J., Uriarte, E. L., Lopez, S., & Arias, C. F. (2006). Early steps in Rotavirus cell entry.
Odeón, A., Bilbao, G., Fernandez, F., Parra, G. I., & Parreño, Current Topics in Microbiology and Immunology, 309: 39-66.
V. (2013). Phylogenetic analyses of typical bovine rotavirus Maes, R. K., Grooms, D. L., Wise, A. G., Han, C., Ciesicki, V.,
genotypes G6, G10, P [5] and P [11] circulating in Argentinean Hanson, L., Vickers, M. L., Kanitz, C., & Holland, R. (2003).
beef and dairy herds. Infection, Genetics and Evolution, 18, 18- Evaluation of a human group a rotavirus assay for on-site
30. detection of bovine rotavirus. Journal of Clinical
Bugarc̀ić, A., & Taylor, J. A. (2006). Rotavirus nonstructural Microbiology, 41(1), 290-294.
glycoprotein NSP4 is secreted from the apical surfaces of McNulty, M. S. (1978). Rotaviruses. Journal of General
polarized epithelial cells. Journal of Virology, 80(24), 12343- Virology, 40(1), 1-18.
12349. Mehle, N., & Ravnikar, M. (2012). Plant viruses in aqueous
Bunpapong, N., Talummuk, S., Chaiyanate, P., Sievert, K., environment–survival, water mediated transmission and
Thanawongnuwech, R. (2011). VirusnipTM and Virkon®S detection. Water Research, 46(16), 4902-4917.
efficacy test on CSFV and PRV. Proc. Asian Pig Vet. Soc. Meng, Z. D., Birch, C., Heath, R., & Gust, I. (1987).
Congress. 5: 140. Physicochemical stability and inactivation of human and simian
Chen, S. C., Tan, L. B., Huang, L. M., & Chen, K. T. (2012). rotaviruses. Applied and Environmental Microbiology, 53(4),
Rotavirus infection and the current status of Rotavirus vaccines. 727-730.
Journal of the Formosan Medical Association, 111(4), 183-193. Miles, M. G., Lewis, K. D., Kang, G., Parashar, U. D., & Steele, A.
Cho, Y-il. (2012). Ecology of calf diarrhea in cow-calf operations. D. (2012). A systematic review of Rotavirus strain diversity in
Graduate Theses and Dissertations. 12642. Iowa State India, Bangladesh, and Pakistan. Vaccine, 30 Suppl 1, A131-
University Capstones, Theses and Dissertations. Available at 139.
https://lib.dr.iastate.edu/etd/12642. Nims, R. W., & Plavsic, M. (2012). Polyomavirus inactivation – A
De Benedictis, P., Beato, M. S., & Capua, I. (2007). Inactivation of review. Biologicals. In Press.
avian influenza viruses by chemical agents and physical Pesavento, J. B., Crawford, S. E., Estes, M. K. and Prasad, B. V.
conditions: A review. Zoonoses and Public Health, 54(2), 51- (2006). Rotavirus proteins: structure and assembly. Current
68. Topics in Microbiology and Immunology, 309, 189-219.
Dhama, K., Chauhan, R. S., Mahendran, M., & Malik, S. V. S. Ramig, R. F. (2004). Pathogenesis of intestinal and systemic
(2009). Rotavirus diarrhea in bovines and other domestic rotavirus infection. Journal of Virology, 78(19), 10213-10220.
animals. Veterinary Research Communications, 33(1), 1-23. Reading, P. C., Holmskov, U., & Anders, E. M. (1998). Antiviral
Estes, M. K., Graham, D. Y., Smith, E. M., & Gerba, C. P. (1979). activity of bovine collectins against rotaviruses. Journal of
Rotavirus stability and inactivation. Journal of General Virology, General Virology, 79(9), 2255-2263.
43(2), 403-409. Shahid, M. A., Abubakar, M., Hameed, S., & Hassan, S. (2009).
Estes, M. K., Kang, G., Zeng, CQ-Y., Crawford, S. E. & Ciarlet, M. Avian influenza virus (H 5 N 1); effects of physico-chemical
(2001). Pathogenesis of Rotavirus gastroenteritis, Novartis factors on its survival. Virology Journal, 6(1), 38.
Foundation Symposium. Chichester; New York; John Wiley; Smelt, J. (1998). Recent advances in the microbiology of high
1999. Pp. 82-100. pressure processing. Trends in Food Science and Technology,
Gregersen, J. P. (2012). Decreasing potential iatrogenic risks 9(4), 152-158.
associated with influenza vaccines. EP Patent 2, Pp. 155 & 236. Sobsey, M. D., & Meschke, J. S. (2003). Virus survival in the
Guardabassi, L., Dalsgaard, A., & Sobsey, M. (2003). Occurence environment with special attention to survival in sewage
and survival of viruses in composted human faeces. Volume 32. droplets and other environmental media of fecal or respiratory
(Denmark), Pp. 27-37. origin. Report for the World Health Organization, Geneva,
Höh, H., Presser, W., & Wigand, R. (1983). Nosokomial-Infektion Switzerland, 70.
durch Rotaviren bei Erwachsenen. DMW-Deutsche Swayne, D. E., Glisson, J. R., Reed, W. M., & Pearson, J. E.
Medizinische Wochenschrift, 108(42), 1586-1591. (2006). A Laboratory Manul for the Isolation and Identification
Holland, R. E. (1990). Some infectious causes of diarrhea in of Avian Pathogens, 4 Ed, International Book Distributing
young farm animals. Clinical Microbiology Reviews, 3(4), 345- Company.
375. Swiatek, D. L., Palombo, E. A., Lee, A., Coventry, M. J., Britz, M.
Jiang, B., Glass, R. I., & Saluzzo, J. f. (2013). Thermal inactivation L., & Kirkwood, C. D. (2010). Detection and analysis of bovine
of rotavirus. US Patent, Pp. 20-384. rotavirus strains circulating in Australian calves during 2004 and
Kaushik, R. S., Begg, A. A., Wilson, H. L., Aich, P., Abrahamsen, 2005. Veterinary Microbiology, 140(1-2), 56-62.
M. S., Potter, A., Babiuk, L. A., & Griebel, P. (2008). Tang, J. W. (2009). The effect of environmental parameters on the
Establishment of fetal bovine intestinal epithelial cell cultures survival of airborne infectious agents. Journal of the Royal
susceptible to bovine rotavirus infection. Journal of Virological Society Interface, 6(suppl. 6), S737-S746.
Methods, 148(1-2), 182-196. Wilhelm, S. W., Jeffrey, W. H., Suttle, C. A., & Mitchell, D. L.
Khadre, M. A., & Yousef, A. E. (2002). Susceptibility of human (2002). Estimation of biologically damaging UV Levels in marine
rotavirus to ozone, high pressure, and pulsed electric surface waters with DNA and viral dosimeters. Photochemistry
field. Journal of Food Protection, 65(9), 1441-1446. and Photobiology, 76(3), 268-273.