IN VITRO PHYSICO-CHEMICAL CHARACTERIZATION OF
NEPHROPATHOGENIC INFECTIOUS BRONCHITIS VIRUS
ISOLATE
KAMRAN SAEED
2015-VA-509
A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE
OF
MASTER OF PHILOSOPHY
IN
MICROBIOLOGY
UNIVERSITY OF VETERINARY & ANIMAL SCIENCES,
LAHORE
2022
To,
The Controller of Examinations,
University of Veterinary and Animal Sciences,
Lahore.
We, the supervisory committee, certify that the contents and form of the thesis submitted
by Kamran Saeed Regd. No. 2015-VA-509 have been found satisfactory and recommend it to
be processed for the evaluation by the External Examiner(s) for the award of the degree.
Supervisor
(Dr. Muhammad Zubair Shabbir)
Member
(Dr. Arfan Ahmad)
Member
(Dr. Muhammad Muddassir Ali)
DEDICATION
To my father who bought
me a pen and to my
mother who taught me
how to write with it.
i
ACKNOWLEDGEMENTS
I bow my head in utmost gratitude before the most Gracious, the most Merciful and
Almighty ALLAH without whose will, I could never have accomplished this endearing task, only He
gave me the strength and power enough to cope up with all the impediments in the way. I, most
modestly, impart my dutiful benefactions to the Holy Prophet Muhammad (Peace Be Upon Him)
who is persistently a torch of guidance and knowledge for the entire mankind.
I deem it as my utmost pleasure to avail this opportunity to express the heartiest gratitude and
deep sense of obligation to my dedicated supervisor Dr. Muhammad Zubair Shabbir, Quality
operation laboratory, Institute of Microbiology UVAS, Lahore for his valuable suggestions, keen
interest, dexterous guidance, enlightened views, constructive criticism, unfailing patience and
inspiring attitude during my studies, research project, and writing of this manuscript. Infect his day
and night pursuance and sincere efforts made this work to a fruitful conclusion.
I gratefully acknowledge invaluable help rendered by Dr. Arfan Ahmad, Institute of
Microbiology UVAS, Lahore for guiding me at every step of the research work. He helped me at
every step in an effective development and conductance of research. Without his untiring efforts, it
would not have been possible for this work to reach its present effective culmination.
I am honored to express my deepest sense of gratitude and profound indebtedness to Dr.
Muhammad Muddassir Ali, Institute of Biochemistry and Biotechnology, UVAS, Lahore, as a
member of my supervisory committee for his support, ever helping behaviour and guidance.
Finally, my unreserved love and thanks to my seniors, fellows and friends especially Mr.
Muhammad Tariq for his interest and appreciated advices in my research project; in fact their
advices will always serve as a beacon of light throughout the course of my life.
Kamran Saeed
ii
CONTENTS
DEDICATION
i
ACKNOWLEDGEMENTS
ii
LIST OF TABLES
iv
LIST OF FIGURES
v
LIST OF ANNEXURES
vi
SR. NO.
CHAPTERS
PAGE NO.
1
INTRODUCTION
1
2
REVIEW OF LITERATURE
5
3
MATERIALS AND METHODS
16
4
RESULTS
25
5
DISCUSSION
30
6
SUMMARY
33
7
LITERATURE CITED
34
iii
LIST OF TABLES
TABLE.NO.
2.1
TITLE
Vaccines available by strain and type for IBV
PAGE NO.
13
3.1
Primers for identification of IBV
19
3.2
Reaction mixtures components along with their
19
concentrations
3.3
Detail of PCR primers with target gene, amplicon size,
20
annealing temperature
3.4
Disinfectants and contact time of each group
23
4.1
Number of dead and live embryos/ 5 inoculated eggs after
28
incubation
iv
LIST OF FIGURES
FIGURE NO.
4.1
TITLE
Embryo showing characteristic IBV symptoms after
PAGE NO.
25
inoculation of IBV virus
4.2
Chicken embryo as negative control at 14 day of age
25
4.3
1.5% agarose gel showing positive PCR product for IBV
26
v
LIST OF ANNEXURES
TITLE
SR. NO.
1
Preparation of 1X TAE buffer from 50X Tris-Acetate-
PAGE NO.
I
EDTA buffer
2
Recipe and preparation of Normal Saline
vi
II
CHAPTER 1
INTRODUCTION
The history of poultry is 150 million years old. In Pakistan, poultry is considered to be
the second-largest industries contributing 1.3 percent to national gross domestic product. The
poultry sector containing over 25,000 poultry farms is involved in employment generation as
well as in food generation such as meat and eggs. In Pakistan, a huge investment is made in the
poultry sector due to its profitability. During the last decade due to outbreaks of many poultry
diseases poultry producers are facing huge economic losses (Umar et al. 2019). Infectious
bronchitis disease is one of the major avian diseases that are prevalent in Pakistan. The disease is
of great economic importance because it causes heavy production losses throughout the life of
the bird although infectious bronchitis disease is more prevalent in birds of young age (Rahim et
al. 2018). Office of International des epizooties listed infectious bronchitis as a notifiable disease
of poultry (Chandrasekar et al. 2015).
Infectious bronchitis is an acute highly contagious respiraotry disease of poultry caused
by Infectious bronchitis virus (IBV) a member of the family Coronaviridae, sub-family
Coronavirinae, genus Gammacoronavirus. Virus has a major impact on the growth and
performance of meat and egg-laying birds, resulting in massive damage to poultry resulting
sector around the world (Xu et al. 2016). IBV is an enveloped virus containing round to
pleomorphic shape lipid envelope around the capsid. The virus has a diameter of approximately
120nm having a crown shape due to the presence of surface spike proteins (Jackwood and De
Wit 2013).
The genome size of the virus is 27.6 kbps. Like other coronavirus IBV genome consists
of Gene 1, the replicase gene, positioned at the 5′ end of the genome, with structural as well as
1
INTRODUCTION
group-specific accessory genes grouped at the 3′ end. The prevailing consensus is that
coronavirus structural and group-specific genes are transcribed via a mechanism of
discontinuous transcription during negative-strand synthesis (Bentley et al. 2013). In virusinfected cells, IBV genome is transcribed into six sub-genomic mRNAs. The mRNA 1 has two
huge overlapping open reading frames that encode the polyproteins 1a and 1b, with 1b being
generated as 1ab protein complex by ribosomal frame-shifting (Ammayappan et al. 2008).
Overall, IBV Genome encodes 15 non-structural proteins from nsp2 – nsp16 as well as four
structural proteins including spike (S) protein, small membrane (E) protein, Membrane (M) and
Nucleoprotein (N) in the following order: 5’ – ORF1 a / b S – E – M – N – 3’(Dent et al. 2015).
Incubation period of infectious bronchitis is very short and is dose-dependent. The birds
show clinical symptoms within 24 – 48 hours but it can be 18 hours in intratracheal inoculation
(Abdel-Moneim 2017). Aerosol, as well as mechanical transmission of virus, is seen between
birds, houses and farms. The virus can spread through large distances by indirect transmission by
the contaminated liter, farm visit, clothing, footwear, utensils, fertilizer and equipment (Raja et
al. 2020). IBV is shed in droppings and tracheobronchial exudates of infected chickens.
Anything that comes in contact with faeces such as drinking water and feed will also get
contaminated with the virus and can be a source of virus transmission (Ramakrishnan and
Kappala 2019).
The virus can replicate in the epithelial cells of both upper and lower respiratory tract and
digestive tract once a bird has been infected. After a brief period of viremia, the virus can
spreads to other systems including tissues of alimentary canal, excretory system and reproductive
tract. The virus is not passed from mother to child through the egg (Bande et al. 2016). The
infection spreads quickly, causing respiratory discomfort in the flock. In simple infections,
2
INTRODUCTION
mortality is normally low; nevertheless, some virus strains have an affinity for the kidneys,
resulting in death from renal failure (Hasan et al. 2020).
Tracheal lesions are usually seen in infection whereas nephropathogenic strains also cause
kidney lesions having 25% mortality in broilers. Mortality rate can be increased by
complications in coinfection with certain other bacteria. Nephropathogenic IBV cause apoptosis
in kidney cells which is a major contributor to the pathogenicity of the virus. Virus also induce
renal endoplasmic reticulum stress in birds (Liu et al. 2017). Infected birds spread the virus
through their droppings and respiratory secretions. Recovery starts after one week in
uncomplicated cases, although flocks may test positive and shed virus for another 15-20 weeks
(Mahana et al. 2019).
After a brief time of incubation (24-48 hours), clinical indications are visible. Avian
infectious bronchitis is mainly a respiratory disease of chicken but damage to the reproductive
system as well as nephritis is also observed. Respiratory signs include sneezing, coughing,
difficulty in breathing, tracheal rales, puffy swollen eyes, congested lungs, depression and
weight loss in two to six-week old chickens (Yan et al. 2019). Reproductive system damage
results in a decline in egg production, weak and broken shelled eggs, low quality of eggs and
oviduct damage in adult hens (Zhang et al. 2020b). Nephropathogenic form of IBV is
characterized by mild respiratory infection followed by diarrheoa, depression, excessive water
intake, reluctance to move, wet liter and death after 5 to 7 days of infections (Najimudeen et al.
2021).
Infection can be prevented using strain-specific vaccination and strict biosecurity standards.
In commercial poultry farms, both killed and live attenuated vaccinations are used to control
3
INTRODUCTION
IBV. Because IBV serotypes do not give cross-protect, a multivalent vaccination with two or
more antigenic variants would provide broad protection (Dhama et al. 2014). Usually
commercially available live vaccinations are administered to day old birds and then at regular
intervals by spray or drinking water to produce local immunity, an important part of effective
mucosal immune response of the bird (Chhabra et al. 2015).
Biosecurity is critical in the control of infectious bronchitis because usually backyard flocks
are unvaccinated. To keep flocks healthy, strict biosecurity controls spanning all elements of the
business are required (Guzmán and Hidalgo 2020). The poultry housing and equipment must
always be sterilized prior to the introduction of fresh birds Pests, rats and insects must also be
kept under control. Equipment, workers, and poultry should not be moved between flocks.
Introducing additional animals to the herd is not a good idea (Shiferaw et al. 2022).
First nephropathogenic IBV infection case was reported in United States in 1960.
nephropathogenic strains of IBV have been the most prevalent strains of IBV in recent years
(Kuang et al. 2021). IBV has a large number of serotypes, owing to the numerous point
mutations and recombination events present in RNA viruses. This study aims to determine the
physico-chemical characterization of newly isolated nephropathogenic IBV isolate. The activity
of commercially used disinfectants (Virkon S, Beloran, Bromosept) against the Infectious
bronchitis Virus will also be determined
4
CHAPTER 2
REVIEW OF LITERATURE
The commercial poultry industry is one of the dynamic and largest agriculture-based
segments of Pakistan established in 1962. Having an investment of 750 billion rupees this sector
provides direct or indirect employment to more than 1.5 million people of Pakistan. According to
economic survey of Pakistan 2020-21 the growth rate of the commercial poultry industry is 1012% per annum producing 21,285 million table eggs and 1809000 tons poultry chicken meat.
The poultry sector has a major contribution of 1.3 % to the national GDP of Pakistan (Ul
Abadeen et al. 2021). Although poultry is a significant sector of Pakistan still evolution of many
avian infectious pathogens is causing a serious threat to the poultry industry. Out of these
infectious diseases of birds, one is infectious bronchitis disease. It is highly contagious and is an
OIE list B disease caused by avian infectious bronchitis virus (Garba et al. 2021). Infected chicks
have serious respiratory problems, irreparable damage to the reproductive system, low egg
production rate and the risk of secondary infections also increase, leading to the death of chicken
(Wu et al. 2022).
2.1. Occurrence:
In 1931 the disease was originally discovered in United States in young chickens. Since
then, the disease has been found in broilers, layers, and breeder chickens all around the world.
Poultry vaccines first were employed in the 1950s against IBV (Butcher et al. 2009). The virus
is found all across the world, Infection can strike animals of any age, although infection in young
birds increases mortality rate. Morbidity rates of 100% are frequent in flocks that have not been
vaccinated (Sid et al. 2015). Mixed infections involving Mycoplasma and E. coli are common
among layers and backyard chickens, and they can make the condition worse (Sid et al. 2015).
5
REVIEW OF LITERATURE
The frequency of infection varies throughout the year, with more cases reported during the
winter time (Gallardo 2021).
2.2. IBV Evolution:
Mutation is the main source of genetic variation and the first essential substratum for
evolution; it produces genotypic (and phenotypic) variation that spread and then become fixed
via genetic drift and natural selection (Valastro et al. 2016). Because of the absence of
proofreading mechanism of RNA dependent RNA polymerase (RdRp) and the lack of RNA
repair mechanisms, RNA viruses have a high mutation rate (about 104 to 105 mis-incorporations
per nucleotide location). This results in around 1 mutation per genome per replication, which is
10-fold higher than that of retroviruses and 10 thousand-fold higher than most DNA viruses.
Coronaviruses, including IBV, might be considered an outlier within the RNA virus
group due to the presence of an ExoN domain in the nsp14 gene, which is correlated to host
protein molecule of the DEDD superfamily of exonucleases and engaged in proofreading and
repair activity. Despite this, IBV's predicted mutation rate (104 – 105 substitutions/site/year) is
still impressive indicating that the virus has significant evolutionary potential. (Franzo et al.
2017). Based upon the S1 gene sequencing, the current classification of IBV contains six
genotypes that are further classified into 32 lineages with 30% and 13% pairwise genetic
distance respectively (Lin and Chen 2017).
Using S sequence analysis Gallardo found that the predominate IBV phenotype present in
the vaccine had become a small population with in host, as well as substantial changes in the
prevalence of certain unique IBV population in the studied tissues and fluids. As a result, in
addition to host role, certain tissues may apply some pressure, selecting variations that are better
at replicating in a specific microenvironment. Closely related strains can show significant
6
REVIEW OF LITERATURE
variation in cross-neutralization patterns, indicating that immune evasion is caused by mutations
in specific amino-acid positions (Duffy 2018). At least with in antigenic regions, immune
reaction is predicted to be the dominant selective force on IBV evolution. This hypothesis
appears to be supported by some experimental evidence. Once the field strains were administered
in non - vaccinated and vaccinated chicken groups, just some of vaccinated chickens developed
non-synonymous changes.
Mayr describes two separate and independent mechanisms for viral evolution:

Generation in diversity, genetic variants are generated and used as material for evolution

Selection, in which virion is released into the environment after replication and the survivors
represent as the genetic pool for later generations (Gallardo 2021).
In addition of recombination events, insertions, deletions, and point mutations contribute
to IBV variability. In 1956, Jungherr and colleagues were the first to report viral heterogeneity
amongst IBV isolates. Connecticut and Massachusetss IBV serotypes were distinguished by the
indicated serological differences. IBV's ability to change quickly is what makes it so successful
in the environment and it's why it continues to spread and defy vaccination measures in the
poultry industry. We now know that there
are
dozens
of
IBV
serotypes,
along
with
many genotypes and variations (Hu 2022).
Chemical Composition and Structure:
Infectious bronchitis virus is a member of Coronavirada, order Nidovirales. IBV is a
positive sense RNA enveloped virus having a linear non-segmented genome of 27.6kb
(Cavanagh 2007).The virus is approximately 120nm in size with the crown-like projection of 20
nm in length on the surface of the virus. The virus genome encodes for four structural proteins,
7
REVIEW OF LITERATURE
the spike protein (S) which is cleaved into S1 protein subunit and S2 subunit protein, the small
envelope (E) protein, the membrane M protein and the nucleocapsid (N) protein (Promkuntod et
al. 2015).
The most important structural protein is spike protein or S protein which is cleaved into
amino-terminal S1 (92kDa) and carboxy-terminal S2 (84kDa) proteins after translational
modifications. S1 is a major protein subunit of spike protein that harbours epitopes for virus
neutralization and the S1 protein subunit is also involved in virus entry to the host cell. Any
mutation in the S1 gene will result in the development of new serotypes and genotypes which
give poor protection or no protection against existing vaccines (Fehr and Perlman 2015). S2
subunit of spike protein is involved in virus attachment by anchoring the spike protein.
The small integral` protein, E protein has a role in virus assembly, envelope formation,
apoptosis and budding of virus. M protein helps in virus assembly and also has an interaction
with spike protein and nucleoprotein (Legnardi et al. 2020). Nucleoprotein (N) is a
phosphoprotein that interacts with the viral RNA genome and forms ribonucleoprotein which has
a role in genome replication. N protein has a role in formation of helical nucleocapsid within the
viral genome, it also interacts with envelope protein E and membrane protein M for the assembly
of virus (Villalobos-Agüero et al. 2021).
2.3. Clinical Signs:
IBV is a highly contagious virus causing damage to the respiratory, renal and
reproductive systems. Initially ciliated epithelial cells of the upper respiratory system are
damaged by disease but the virus can spread systematically to many tissues infecting the
epithelial lining of the tissues (Bande et al. 2016). In respiratory damage depression, coughing,
tracheal rales, nasal and ocular discharge and head shake is seen. Tracheitis, cloudy air sacs and
8
REVIEW OF LITERATURE
the inflammation of lungs are major necroscopic findings (Ali et al. 2018). Affected chicks will
appear bewildered and will huddle close to a source of heat. During 36 to 48 hours, all birds in
an infected flock will acquire clinical indications. The clinical illness usually lasts 7 days. Unless
other variables exacerbate the situation, such as Mycoplasma, E.coli, immune suppression,
low air quality, and so on, death rate is usually very low (De Wit et al. 2018)
Damage to the female reproductive tract cause a decrease in production of eggs and
production of poor-quality eggs with weakened eggshells. A reduction of 5 to 10% egg
production rate is seen that lasts 10 to 14 days is common. When aggravating circumstances are
present, eggs productivity decreases as much as 50%. Eggs having thin irregular shells
and thin watery albumen are produced following infection. Pigment loss and brown-shelled eggs
production is quite prevalent (De Wit et al. 2020). Chickens may acquire airsacculitis in severe
circumstances. Chickens that have a severe vaccine reaction after a chick vaccination or a field
infection within the first two weeks after birth may have persistent oviduct injury, results in hens
with limited output (Zhang et al. 2020a).
Infection of nephropathogenic strains can cause serious kidney damage. Affected birds'
kidneys would be pale and enlarged. Urate deposits in the tissue of the kidneys as well as in the
ureters, severe dehydration and swelling of renal organs can be found. High flock mortality and
urine water loss (pollakiuria and polyuria), resulting in damp litter, are the first clinical
indications. The daily death rate is as high as 23 percent. Renal edoema and urate buildup in the
renal tubules
were
prevalent.
Urolithiasis
and
visceral
gout
are less
common.
Lymphoplasmacytic interstitial nephritis, histopathologic changes included typical tubular
epithelial degradation and sloughing is seen (Jackwood 2020).
9
REVIEW OF LITERATURE
The degree of disease severity and the body systems affected are influenced by the virus
strain, age of bird, immunological status, and nutrition of chickens exposed to cold
stress. Confection with Mycoplasma gallisepticum, Mycoplasma synoviae, E. coli, and/or
A. paragallinarum can also worsen the condition (Kuang et al. 2021).
2.4. Transmission:
Infectious bronchitis virus can transmit directly by ingestion or inhalation of virus or can
transmit by indirect contact through aerosol, feces and by clothing, shoes and other fomites
which are contaminated with virus (Birhan et al. 2021). Virus replicates in the respiratory and
digestive tracts of infected birds. After a brief period of viremia, the virus spreads to other organs
including gonads and kidneys and multiplies there. The virus is not passed from mother to child
through the egg. After completing a short 24 - 48 hours incubation period, clinical indications
can be seen (Hu 2022). Infected birds shed the virus through their droppings and respiratory
secretions. Recovery usually begins after one week in uncomplicated cases, although flocks may
continue to be tested positive for virus and shed the virus for another 15-20 weeks (Banda and
Yan 2022).
The route of vertical transmission is still not clear but in some studies, vertical
transmission is seen in one day old chicks that got IBV from infected hens which laid infected
eggs between one to six weeks of infection. Recently, male infertility and venereal transmission
of the virus from male to female is also reported. IBV is also recovered in cockerel’s semen
which could infect the oviduct and eggs in the oviduct of hens (Wu et al. 2022).
2.5. Viral Diagnosis:
Clinical symptoms and clinical history might be used to make a presumptive diagnosis.
Clear exudate in sinuses, edema in tracheal mucosa which might be hyperemic with petechial
10
REVIEW OF LITERATURE
hemorrhages, and light foamy airsacculitis that may develop purulent if lesions get contaminated
with E. coli are common necropsy findings (Villarreal 2010). The kidneys will be affected by
infectious bronchitis strains with renal tropism; they may seem pale and swollen, with a
noticeable reticular pattern, leading to severe nephritis. Urates can cause ureters to become
swollen. Follicular regression may occur in the ovary (Yilmaz et al. 2016). Since there are so
many diseases with similar symptoms, confirmation is usually required. If an increase in
antibody levels can be demonstrated, serology can be utilized to make a diagnosis. As a result,
two samples are required: one at the start of the sickness and another 10 days later. Serotype
identification is essential, especially if disease develops in vaccinated animals. To identify
genotype, RT-PCR is advised, which has supplanted serotyping as a method of determining a
strain's identification (Awad et al. 2016).
Diagnostic methods for detection of IBV either focus on detection of the serum
antibodies or detecting and isolating the virus itself. The primary diagnostic approach is based
upon clinical history and characteristic lesions. Inoculation of virus in 9 days old embryos cause
characteristic stunting of growth and curling of embryo (Ennaji et al. 2020). Various antibodybased antigen detection methods such as ELISA, indirect FAT, and virus neutralization test are
being used. Molecular detection of the virus is based upon detection of the viral RNA. The
molecular detection of spike S1 gene by RT-PCR is most preferred virus diagnostic test for IBV
(Bhuiyan et al. 2021).
2.6. Virus Replication:
Replication of IBV mainly occurs in epithelial cells of respiratory tract but virus can also
replicate in the cells of kidney, fallopian tube and in intestinal tract. IBV is an encapsulated virus
containing a positive-sense single-stranded RNA genome of about 27 kb and the genes
11
REVIEW OF LITERATURE
arrangement is as: 5′UTR-1a/1ab-S-3a-3b-E-M-5a-5b-N-3′UTR (Rahim et al. 2018). The
genomic RNA has 3′ poly (A) tail and a 5′ cap and directly works as mRNA for translation, the
replicase polyproteins' coding sections present at the 5′ end occupies the two-thirds of the viral
genome. The two (UTRs) untranslated regions interact with viral replicase enzymes and maybe
other host proteins and play a key role in virus replication (Wang et al. 2020).
The virus attaches to host epithelial cell with the help of S glycoprotein followed by
fusion and entry of virus in cell. After the entry of the virus its genome act as mRNA encoding
Nsp2- Nsp16 which have a role in the formation of RNA dependent RNA polymerase and
regulation of host cell functions (Perlman et al.). Translation of viral RNA occur in the
cytoplasm and results in the formation of structural proteins. S, E and M structural proteins are
inserted in Golgi Membrane, whereas N protein binds with newly form RNA genome of the
virus and results in formation of nucleocapsid. Interaction between M and E protein and
nucleocapsid occur and virus is released by budding from the endoplasmic reticulum. Vesicles
released fuse with plasma membrane and results in the release of the virus particle. The virus can
also release from the cell by cell lysis(Huang et al. 2021).
2.7. Control and Prevention:
An effective biosecurity program is the most effective way to prevent IB. Birds should be
immunized with improved live vaccines as a second line protection mechanism. A large number
of serotypes discovered in the field make developing an efficient vaccination difficult (Awad et
al. 2014). It is critical to identify the most common serotypes in the area and establish the crossprotective capability of existing vaccinations in order to protect chickens against challenge. As
IBV serotypes do not give cross-protection against each other, a multivalent vaccination with
two or multiple antigen types would provide broad protection (Moreno et al. 2017).
12
REVIEW OF LITERATURE
Vaccines that can induce cross protection against multiple genotypes of IBV are crucial
in terms of both economics and practicality. Vaccination application, in addition to vaccine
selection, is critical for preventing variant IBV development. Live attenuated vaccines against
IBV are already available and are employed by drinking water or by spray at first day of birth
and between the age of 10 to 15 days in broilers and in layers at least 3 - 4 times before eggs
laying begins (Jackwood and Lee 2017). IBV vaccination techniques that include a large number
of people are ineffective and frequently result in vaccination failure (Jordan 2016).
Table 2.1: Vaccines available by strain and type for IBV.
Strain
Type of Vaccine
Live attenuated–Live modified–
Massachusetts
Inactivated
Ma5
D274
M41
Live attenuated–Inactivated
H52
CR88121
Delaware
Live modified
H120
4/91
Live attenuated
1/96
13
REVIEW OF LITERATURE
GA-98
Arkansas
1212B
Conneticut
B48
VicS
Armidale
249G
Inactivated
PL84084
2.8. The emergence of new strains with variable Physico-chemical properties:
Various variants of IBV are seen in history. S1 subunit of gene S is very important for the
emergence of new variants of IBV. Any mutation in the S1 gene will result in the emergence of a
new serotype maybe with changed physico-chemical characterization. Classification of virus can
be done by methods of serotyping and genotyping. Cross neutralization, reverse transcriptase
polymerase chain reaction (RT-PCR), gene sequencing techniques and modern bioinformatics
techniques such as Basic Local Alignment Search Tool (BLAST) are some of the basic
techniques used for knowing the emergence of new variants of IBV (Legnardi et al. 2020).
2.9. Statement of Problems:
Poultry is one of the major industries in Pakistan playing a vital role in national growth
domestic production. Due to IBV outbreaks, this sector is facing huge economic losses each year.
14
REVIEW OF LITERATURE
Despite the disinfection of poultry sheds, IBV outbreaks are still being observed regularly.
Among many reasons, one is the development of change in physico-chemical properties of virus
that need to be tested using currently isolated nephropathogenic IBV.
Keeping in view the high mutation rate of IBV, the objectives of the study were:

Nephropathogenic IBV isolate sensitivity and resistance to different physico-chemical
treatments will be known.

Evaluation of disinfection potential of commercially used disinfectants against
nephropathogenic IBV isolate will be made.
15
CHAPTER 3
MATERIAL AND METHODS
3.1. Work Station:
The research work was performed at Quality Operation Laboratory (QOL-WTO),
Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore.
3.2. Study Design:
This study was designed to check effect of different physico-chemical treatments on the
survival of local nephropathogenic IBV isolate and to evaluate disinfection potential of different
commercially available disinfectants against nephropathogenic Infectious Bronchitis Virus
isolate.
3.3. Sterilization of Equipment:
All the glassware’s were sterilized by dry heat in hot air oven at 180 ℃ for about an hour
and all the plastic wares were sterilized in the autoclave at 121 ℃ having 15 PSI pressure for 15
minutes.
3.4. Virus Sample:
One confirmed nephropathogenic infectious bronchitis virus sample will be obtained
from Quality Operation Lab (QOL – WTO), University of Veterinary and Animal Sciences,
Lahore.
3.5. Sample Revival:
The freezed sample was thawed, processed and passaged on 9-days old embryonated
eggs by inoculating 0.2ml of sample through chorioallontoic membrane route.
3.5.1. Preparation of Inoculum:
The frozen infected fluid was thawed and was centrifuged at 3000 rpm for 10 minutes.
Supernatant was transferred to a 15 mL falcon tube and was mixed with Penicillin 1,000 U/ml
16
MATERIAL AND METHODS
and gentamicin 0.05 mg/ml.
3.5.2. Inoculation of embryonated chicken eggs by allantoic cavity method:
9 days old fertile eggs from healthy flocks free of antibody to IBV were obtained. The
eggs were candled to verify their viability and to mark the air-sac. The marked area on the egg
shell was disinfected using pyodine; 10% solution of povidone iodine. A thumb pin was used to
drill a hole in egg shells just above the mark. To avoid contamination of the egg, the tip of the
pin was disinfected before usage. 0.2 ml of the prepared sample was inoculated on allantoic
membranes of 9-days old chicken embryos using a 1-ml syringe and a 25-gauge, 0.5 in. (12 mm)
needle, whereas control chicken embryos were left un-inoculated. After inoculation, wax was
used to seal the hole in the egg shells.
3.5.3. Egg incubation:
At a temperature of 37℃ and a relative humidity level of 50–55 percent,
embryonated chicken eggs were incubated. To ensure healthy embryo development and
to prevent adhesions between the chicken embryo and membranes, eggs were rotated one to
two times per day. For five days, the chicken embryos were incubated and candled every day.
Embryos that died within first 24 hours of being inoculated were discarded.
3.5.4. Collection of infected allantoic fluid:
After incubation infected allantoic fluid from embryos having lesions typical to that of
IBV was harvested. Prior to collecting allantoic fluid, eggs with live embryos were chilled at 4℃
for at least 4 hours, or overnight. Using sterile forceps, the egg shell was broken and removed.
The allantoic fluid was exposed by gently dissecting a sterile forceps through the shell membrane
and CAM. A syringe was used to aspirate allantoic fluid after compressing membranes using
forceps. A sterile 17×120 mm falcon tube was used to collect the fluid.
17
MATERIAL AND METHODS
3.6. Confirmation of sample:
Confirmation of sample was made by using RT-PCR.
3.6.1. Genome extraction Using Qiagen Kit:
Genome of the virus was extracted by using ®QIAGEN RNA kit (SAREYYÜPOĞLU
and Burgu 2017) (QIAMP Viral RNA Mini Kit catalog # 52906). The protocol adopted was as
follows:
i.
600µl AVL Buffer containing carrier RNA was dispensed into a 1.5 mL microfuge tube.
ii.
140µl of sample was poured into the tube containing prepared buffer AVL-carrier RNA.
Then for efficient lysis was mixed properly by pulse-vortex for 15 seconds.
iii.
Incubated at room temperature for 10 minutes.
iv.
To remove drops from the inside of the lid was centrifuged at maximum speed.
v.
Total amount of 560µl of ethanol (96-100%) was added to the tube. Vortexed at 15
seconds and then briefly centrifuged.
vi.
Properly label spin column was attached without wetting the rim. Cap was closed tightly
and then centrifuged at 800 rpm for 1 minute.
vii.
The mini column was placed in a new collection tube and the previous tube containing
filtrate was discarded.
viii.
Step VI was repeated to completely lyse the sample solution. At the end again the filtrate
was discarded and column was shifted to new tube.
ix.
500ul AW1 (wash buffer 1 with ethanol) was added to the column and centrifuged at
8000 rpm for 1 minute. Flow through was discarded.
x.
500µl AW2 (wash buffer 2) was added to the column and centrifuged at 14000 rpm for 3
minutes. Flow through was discarded.
18
MATERIAL AND METHODS
xi.
For complete removal of wash buffer spin column was centrifuged with new collection
tube. The column was placed in a new properly labeled tube meant for elution.
50µl of elution buffer was added to the column and kept for incubation at room
xii.
temperature for 1 minute. Column was centrifuged at 8000 rpm for 1 minute.
xiii.
RNA was extracted.
3.6.2. PCR:
Polymerase chain reaction (PCR) of IBV isolate was performed for the confirmation of
IBV by using primers given in (Table 3.2).
Table 3.2: Primers for identification of IBV.
Gene
Primers sequence (5’-3’)
Product size
Reference
450bp
(Farsang et al. 2002)
5’- TGACTCTTTTGTKTGCACTAT-3’
S1
5’- AAATTATAATAACCACTCTGA-3’
Forward and reverse primers were diluted for the PCR. Then 2µL of each diluted primer
was added to a PCR mixture including 2X master mixure, nuclease free water, and DNA. PCR
mixture components were added in the sequence as given in (Table 3.2).
Table 3.3: Reaction mixtures components along with their concentrations
Components of PCR mixture
Concentration/µL
Nuclease free water
5.5
Master mix
12.5
Forwaed primer
1
Reverse primer
1
RNA template
5
Total volume
25 µl
19
MATERIAL AND METHODS
After PCR mixture components were mixed and droplets from side walls of eppendorf
were removed by spinning and placed in Bio-Rad C1000TM Thermo cycler. The conditions used
for PCR are given in (Table 3.3).
Table 4.3: Detail of PCR primers with target gene, amplicon size, annealing
temperature
Tempreture profile
Target
Amplification (35 cycles)
Primary
Gene
Final
cDNA
55ºC/20min
denaturation
Denaturation
Annealing
Extention
Extention
95ºC/2min
94ºC/45sec
55ºC/1min
72ºC/1min
72ºC/7min
After that 1.5 % agarose gel was formed to resolve genus specific PCR products as the
size of bands were 426 bp.
3.6.3. Gel electrophoresis
i. The results of the PCR reaction were visualized using 2% agarose. Agarose gel powder
(0.75 gm) was added to 50 mL of 1X TAE (Tris Acetate EDTA) buffer (pH=7) in a
clean 250 mL glass bottle to give a final concentration of 1.5 percent for gel
electrophoresis.
ii. The agarose powder was mixed by rotating the bottle slightly.
iii. The mixture was heated in the microwave for 1–1.5 minutes until all of the agarose was
dissolved in the buffer.
iv. When the solution had cooled to 55℃, 3µL of ethidium bromide was added and evenly
mixed with a gentle shake of the bottle.
20
MATERIAL AND METHODS
v. The rubber pads were inserted on the edges of the casting tray, and the comb was placed
in the casting tray to complete the gel apparatus.
vi. Poured the solution slowly into the electrophoresis tank to avoid the production of
bubbles. The melted agarose gel was then allowed to solidify for about 30 minutes at
room temperature.
vii. After solidification, the comb was carefully removed, and the gel was placed in a TAE
buffered gel tank (1X).
viii. Using a micropipette, pipette 3µL of 100bp plus DNA ladder into the first well of the
gel.
ix. 4µL of PCR product mixed with 1.5µL of 6X loading dye was placed adjacent to the
ladder's well.
x. The lid was placed over the tank after the samples were loaded, and the terminals were
attached to their appropriate electrodes, and the gel was operated at 110V for 30 minutes.
xi. The results of the gel were visualized using a gel documentation system once it had been
run.
3.7. Physico-chemical Characterization:
Physico-chemical characterization of nephropathogenic Infectious Bronchitis Virus
isolate was carried out by inoculating in 9-days old embryonated egg after applying certain
physico-chemical treatment on the virus.
Following physico-chemical parameters were evaluated:
3.7.1. Heat Stability
Extracted allantoic fluid containing IBV was subjected to heat treatment at 56℃ for
different time intervals of 5, 10, 15, 20 and 30 minutes using a water bath. After each treatment
21
MATERIAL AND METHODS
tubes were removed and placed in an ice bath. Virus viability was determined by titration of heat
treated allantoic fluid in 9-days old embryonated eggs (Jackwood et al. 2010).
3.7.2. pH stability
Virus stability to different pH was determined by mixing IBV infective allantoic fluid in
PBS whose pH was adjusted to 3.0, 7.0, 9.0 and 11.0. The pH was adjusted by adding 1N HC1 or
by adding a small amount of 1 N NaOH. In each case the pH was determined with a glasselectrode pH meter. Following incubation for 180 minutes at 4℃ titration of treated allantoic
fluid was inoculated in 9-days old embryonated eggs. Viral viability was determined (AbdelMoneim et al. 2005).
3.7.3. UV Stability
The nephropathogenic IBV infective allantoic fluid was irradiated by placing it 30cm
under a 30 watt UV lamp to irradiate the virus. After irradiation for 10, 15, 20 and 30 minutes the
viral viability was determined by titration in 9-days old embryonated eggs (Quevedo et al.
2020a).
3.7.4. Chloroform Susceptibility
Chloroform susceptibility was checked by using 4.8 percent reagent-grade chloroform.
IBV infective allantoic fluid was treated with chloroform at a final concentration of 4.8 percent.
Mixture was shaken at 4℃ for 10 minutes and then centrifuged at 500×g for 5 minutes. Topmost
transparent layer was recovered and viral viability was assayed by titrating this layer in 9-days
old embryonated eggs (Fan 2008).
3.8. Evaluation of Disinfectants:
In-vitro efficacy of different routinely used disinfectants in poultry sheds like Virkon-S,
Beloran and Bromosept for virus inactivation was assayed. Evaluation was made by recording
22
MATERIAL AND METHODS
characteristic lesions, stunting of growth, curling or death of embryo / five inoculated 9-days old
embryonated eggs. (Thomrongsuwannakij and Chansiripornchai 2013a). The summary of the
disinfectant and contact time with each disinfectant is given in table 3.4.
Table 5.4: Disinfectant and contact time of each group
Contact Time
Group
1 minute
5 minutes
10 minutes
30 minutes
IBV + PBS
IBV + PBS
IBV + PBS
IBV + PBS
Disinfectant +
Disinfectant +
Disinfectant +
Disinfectant +
PBS
PBS
PBS
PBS
3
IBV + Virkon S
IBV + Virkon S
IBV + Virkon S
IBV + Virkon S
4
IBV + Beloran
IBV + Beloran
IBV + Beloran
IBV + Beloran
IBV +
IBV +
IBV +
IBV +
Bromosept
Bromosept
Bromosept
Bromosept
1
2
5
3.8.1. Efficacy of Virkon S
Virkon S is the most common commercially used disinfectant. It comprises of two
active ingredients; an oxidizing agent potassium monopersulphate and an anionic surfactant
sodium dodecyl benzene sulphonate. 1:100 dilution of Virkon S was made in sterile distilled
water as per the manufacturer’s recommendations. Half ml of 1 × 105 EID50 virus was treated
with 0.5mL of 1:100 diluted Virkon S at room temperature for a time of 30 seconds, 1, 5 and 30
minutes. Efficacy of Virkon S was checked by inoculating this treated mixture in five 9-days
23
MATERIAL AND METHODS
old embryonated eggs.
3.8.2. Efficacy of Beloran
Beloran contains benzalkonium a quaternary ammonium compound working as cationic
surfactant as its active ingredients. As per the manufacturer’s recommendations a dilution of
0.5% beloran was made. Half ml of this dilution was mixed with half ml of allantoic fluid
containing 1 × 105 EID50 virus and was incubated at room temperature for 30 seconds, 1, 5
and 30 minutes. The solutions after completion of specific incubation period were treated in 9days old embryonated eggs and efficacy of beloran was checked.
3.8.3. Efficacy of Bromosept
Bromosept is a twin, containing bromide and a long chain quaternary ammonium
compound showing an efficacy against variety of microorganism. 0.25% dilution 10%
bromosept was made in sterile distilled water as per manufacturer’s recommendations. Half ml
of this dilution was mixed with half ml of allantoic fluid containing 1 × 105 EID50 virus at
room temperature for 30 seconds, 1, 5 and 30 minutes. Efficacy of bromosept was checked by
treating this mixture in 9-days old embryonated eggs.
24
CHAPTER 4
RESULTS
4.1. Sample revival:
Already isolated nephropathogenic infectious bronchitis virus isolate was taken from
cultural bank of Quality Operations Laboratory, Institute of Microbiology at University of
Veterinary and Animal Sciences Lahore. Sample was positive for Infectious Bronchitis Virus
and showed typical characteristic lesions of Infectious bronchitis virus which were stunting of
growth, curling and dwarfism of embryo on inoculation of IBV virus in 9-days old embryonated
egg followed by 5 days incubation period as shown in figure 4.1. A negative control without
virus inoculation was also incubated showing complete growth and no characteristic lesions on
embryo as shown in figure 4.2.
Figure 4.1.: Embryo showing characteristic
Figure 4.2.: Chicken embryo as negative
IBV symptoms after inoculation of
control at 14 day of age
Infectious Bronchitis Virus
25
RESULTS
4.2. Confirmation by PCR:
The sample was processed for RNA extraction followed by PCR. Our universal primers
were of considered to have a size of 322bp and by visualizing the gel we found the band with
the same amplicon size which confirms that the sample is positive for IBV.
Ladder(100bp)
470bp
Figure 4.3.: 1.5% agarose gel showing positive PCR product for IBV
4.3. Physico-chemical Characterization:
Physico-chemical characterization of nephropathogenic IBV isolate was carried out by
inoculation of the treated virus in 9-days old embryonated egg. Following were the results of
the evaluated physico-chemical parameters:
4.3.1. Heat Stability:
Like all corona viruses IBV is also sensitive to heat. Virus viability was reduced by
exposing virus at 56℃ for 5 minutes. The more the exposure time lesser was the viability of the
virus and there were no characteristics lesions on embryo and no embryo death happened even 5
days after the inoculation.
26
RESULTS
4.3.2. pH stability:
Isolated nephropathogenic IBV isolate was relatively resistant to pH= 3, 7 and 9. IBV
withstand low pH and survived in acidic, neutral and slightly basic pH showing characteristics
IBV lesions on embryo. However IBV got inactivated when exposed to pH=11.
4.3.3. UV Stability:
The nephropathogenic Infectious Bronchitis Virus isolate was found highly sensitive to
UV radiations. Virus got killed after being exposed to UV radiation as less as 10 minutes from a
30 cm distance. More exposure time showed the same results and the isolate was found sensitive
to UV radiations.
4.3.4. Chloroform Susceptibility:
The nephropathogenic Infectious Bronchitis Virus isolate was found to be highly
susceptible to chloroform. Virus didn’t survive after being treated with 4.8 percent reagent-grade
chloroform for 10 minutes. No characteristics lesions or mortality was seen in inoculated
embryos.
4.4.
Evaluation of Disinfectants:
Different disinfectants often used in poultry sheds for disinfection, such as Virkon-S,
Beloran, and Bromosept, were tested for their in-vitro effectiveness in inactivating IBV.
Embryos showing characteristic lesions of IBV such as stunting of growth, curling or death of
embryo were considered as survival of virus to disinfection. No characteristics lesions and
survival of embryo was recorded as susceptibility of virus to disinfectant. Results of Infectious
Bronchitis Virus inactivation using several types of disinfectants with exposure durations are
shown in table 4.1.
27
RESULTS
Table.4.1: Number of dead and live embryos/ 5 inoculated eggs after incubation
Disinfectant
Virkon S
Beloran
Bromosept
Dilution
%age
Exposure
Embryo
Embryo
time
died
survived
1 minute
0
5
100%
5 minutes
0
5
100%
10 minutes
0
5
100%
30 minutes
0
5
100%
1 minute
3
2
40%
5 minutes
2
3
60%
10 minutes
1
4
80%
30 minutes
0
5
100%
1 minute
1
4
80%
5 minutes
0
5
100%
10 minutes
0
5
100%
30 minutes
0
5
100%
survival of
embryos
1:100
0.25 %
0.5 %
Positive
-
-
5
5
0%
Negative
-
-
0
5
100%
28
RESULTS
4.4.1. Efficacy of Virkon S
The nephropathogenic IBV isolate showed high sensitivity to Virkon S. Virus got
completely killed when is exposed with 1:100 dilution of Virkon S for 1 minute. No
characteristics lesions on any of the five inoculated embryos were found. Exposure time with
Virkon S for 5, 10 and 30 minutes gave the same results and virus got completely killed.
4.4.2. Efficacy of Beloran
Exposure of the nephropathogenic IBV isolate with 0.5% beloran for different time
intervals gave different result. Two / five inoculated embryos survived after being exposed for 1
minute showing inactivation of the virus. After being exposed for 5 minutes three embryos
survived and after 10 minutes four embryo survived. There was no death of the embryo after
being exposed for 30 minutes which showed complete inactivation of the virus.
4.4.3. Efficacy of Bromosept
The nephropathogenic IBV isolate was found sensitive to 0.25% dilution of bromosept.
4 / 5 embryos survived after virus being exposed for 1 minutes showing inactivation of the virus.
There was complete inactivation of the virus after the virus was being exposed for 5, 10 and 30
minutes as there were no characteristics lesions on any of the inoculated embryo.
29
CHAPTER 5
DISCUSSION
Characterization using physicochemical characters in addition to being a mean of virus
detection also provides details on the appropriate means of virus eradication and pathogenesis
(Wu et al. 2022). In the current study, the effect of various physico-chemical treatments on
viability of the nephropathogenic IBV isolate was investigated. We used the third passage of
PCR confirmed nephropathogenic IBV isolate because it consistently produced characteristic
IBV lesions on embryo lesions that are stunting of growth and curling of the embryo (Banda and
Yan 2022).
The ability of the chicken embryo to survive after being injected with the treated virus
was thought to be sensitivity of the virus to the treatment (Nims and Plavsic 2022). The locally
isolated nephropathogenic strain of IBV is shown to be thermosensitive when heated for five
minutes at 56℃ as reported for IBV (Pohuang and Junnu 2019). Proteins of IBV, like those of
other corona viruses, are unable to tolerate high temperatures (Zhu et al. 2020), and as a result,
the virus was killed and the embryo survived (Jackwood et al. 2010).
In the present investigation, we found that the IBV isolate is resistant to both slightly
basic and acidic pH. Virus survived on treatment at pH 3.0, 5.0 and 9.0 for 180 minutes at 4℃,
we consider that IBV strain is resistant to these pH same as other corona viruses (Miyaoka et al.
2021b). But the virus is sensitive to highly alkaline pH as 11.0 as the virus not got inactivated
and the embryo showed the typical lesions of the IBV.
Virus was found sensitive to UV irradiation within 10 minutes when placed 30 cm under
a 60 watt UV lamp. Electromagnetic energy is released by UV light (Santhosh and Yadav 2021)
30
DISCUSSION
and certain photochemical reactions are triggered in RNA of the virus which can inactivate the
virus (Sun et al. 2021). Uracil dimers are produce in RNA, by mutations caused by UV light thus
inactivating the virus and ending the virus ability to replicate (Quevedo et al. 2020b). The same
was found by (Heßling et al. 2020; Pratelli 2008; Yuan et al. 2020). As demonstrated by other
researchers with different IBV strains, the viral strain was found to be sensitive to chloroform
(Fan 2008), indicating that lipid is a key component of Infectious Bronchitis Virus (Bachar et al.
2021).
IBV isolate was found to be sensitive heating at 56℃ for 5 minutes, to chloroform, UV
light within 10 minutes, sensitive to pH 11 but resistance to pH 3, 7 and pH 9 as the embryo
survived and not shown any of the IBV-specific lesions. Our conclusions are consistent
alongside
numerous
other
researchers
and
were
in
disagreement
with the as
he
reported virus insensitivity to 56℃ for 5 minutes as reported by (Abdel-Moneim et al. 2005).
In this study, we looked into the effectiveness of a number of commonly used
disinfectants in Pakistan, such as Virkon S, beloran, and bromosept against a locally isolated
nephropathogenic IBV isolate as IBV is sensitive to various surface disinfectants (Hasan et al.
2022). Disinfectants were used according to the manufacturers' recommendations. The outcomes
showed that the virus must be inactivated with the right concentration and contact duration.
However, a variety of factors, including concentration of disinfectant, contact time, activity in
organic material or protein-containing materials, temperature and quantity, affect disinfection
efficiency (Miyaoka et al. 2021a). The inactivated effectiveness to IBV for Virkon S appeared to
be in the first rank. Virus got inactivated when Virkon S at the concentration of 1:100 is
contacted with IBV for at least 1 minute (Thomrongsuwannakij and Chansiripornchai 2013b).
31
DISCUSSION
IBV inactivated efficacy was held by bromosept whose 0.25% dilution for a contact time
of 5 minutes eliminated the entire virus. Beloran was at third rank as it need more contact time to
eliminate the virus. IBV got inactivated completely when 0.5% concentration was used for 30
minutes. Pathogen decontamination on surfaces of objects is crucial for preventing infectious
infections. IBV can be decontaminated from different surfaces after being exposed to proper
concentration of disinfectant for a proper contact time.
5.1. Conclusion
In the present study already isolated local nephropathogenic IBV isolate was found
sensitive to high temperature of 56℃ , pH of 11, UV radiations and chloroform treatment,
whereas virus resist the acidic pH such as 3 and slight neutral pH 9. Commercially available
disinfectants showed virucidal activity against IBV isolate. The best activity was found in case
of Virkon S which completely eliminated the virus.
Hence we concluded that local
nephropathogenic IBV isolate showed the typical physico-chemical characteristics of corona
viruses and is sensitive to commercially available disinfectants.
5.2. Suggestions
Infectious Bronchitis virus is causing serious economic issues to poultry sector worldwide.
Being a RNA virus it has high mutation rate and the virus is evolving with the passage of time.
This calls for further work for the molecular characterization of the virus and to develop a
homologous IBV vaccine as well as.
32
CHAPTER 6
SUMMARY
Infectious bronchitis virus (IBV) belongs to cronaviridae family and is a gamma
coronavirus having a positive sense RNA genome of 27.6 Kbps. IBV is an important pathogen in
poultry industry causing avian infectious bronchitis, an acute but highly contagious disease
affecting upper respiratory tract. Infected chickens show symptoms of upper respiratory tract,
urinary and reproductive failure which results in low egg production and even death in chickens.
The virus has high mutation rate leading to the development of new variants.
The Physico-chemical characterization of nephropathogenic IBV isolate was made
including Chloroform sensitivity, pH stability, heat stability for 5, 10, 15, 20, and 30 min at 56℃,
UV exposure for 10, 15, 20, and 30 min. IBV isolate was observed to be susceptible to a
temperature of 56℃ for five minutes, UV for ten minutes and chloroform treatment while being
resistant to pH 3 and 9 while sensitive to pH 11.
The second goal of the study was to investigate in vitro the effectiveness of several
commercially used disinfectants against the infectious bronchitis virus (IBV) isolate. For
this three commercially available disinfectants Virkon S, Bromosept, and Beloran were
employed for virus inactivation test. Following IBV challenge for contact time of 1, 5, 10, and 30
minutes, we counted how many infected eggs had died after incubation. Results showed that on
providing suitable dilution of disinfectant for recommended contact period can kill the virus. The
maximum susceptibility is seen in case of Virkon which killed the virus in just 1 minute. Thus
IBV can be killed by using commercially available Virkon S, Beloran and Bromosept after being
used in recommended concentrations for recommended contact time.
33
CHAPTER 7
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Annexure-I
Preparation of 1X TAE buffer from 50X Tris-Acetate-EDTA buffer:
Serial Number
Ingredients
Concentration
1
Tris
40mM
2
Acetic acid
20mM
3
EDTA
1mM
Preparation of 1X TAE buffer:
Prepared 1X buffer from Thermo-scientific 50X TAE Electrophoresis buffer Ref# 849 by
taking 490 mL distilled water in a clean glass flask. 10 mL of 50X TAE buffer was measured
using a graduated measuring cylinder. 10 mL 50X TAE buffer was added in a flask containing
distilled water and was mixed thoroughly. The resulting solution was 1X TAE buffer.
Annexure-II
Recipe of Normal Saline:
Serial Number
Ingredients
Concentration
1
Sodium chloride
0.89 g
2
Distilled water
100 mL
3
Final pH
7.2± 0.2
Preparation of Normal Saline:
Weighed 0.89g of sodium chloride on a well calibrated weighing balance and dissolved into
distilled water to make final volume 100mL and autoclaved at 121℃ , 15 psi pressure for 15 minutes.