Fate of enteric viruses and bacteriophages in sequencing batch reactor treating domestic wastewater

VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY
---------------------------------------

NGUYEN HOANG PHUONG THAO

FATE OF ENTERIC VIRUSES AND
BACTERIOPHAGES IN SEQUENCING
BATCH REACTOR TREATING DOMESTIC
WASTEWATER

MASTER'S THESIS

Hanoi, 2018


VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY
------------

NGUYEN HOANG PHUONG THAO

FATE OF ENTERIC VIRUSES AND
BACTERIOPHAGES IN SEQUENCING
BATCH REACTOR TREATING DOMESTIC
WASTEWATER

MAJOR: ENVIRONMENTAL ENGINEERING

SUPERVISORS:
Assoc. Prof. HIROYUKI KATAYAMA

Prof. HIROAKI FURUMAI
Dr. PHAM VAN QUAN
Assoc. Prof. NGUYEN MANH KHAI

Hanoi, 2018


ACKNOWLEDGEMENT
Master thesis is completed at the Vietnam Japan University. A completed study
would not be done without any assistance. Therefore, the author who conducted this
thesis gratefully gives acknowledgment to their support and motivation during the
time of doing this research.
I would first like to express my endless thanks and gratefulness to my supervisor
Associate. Professor Hiroyuki Katayama, Professor Hiroaki Furumai, Associate
Professor Nguyen Manh Khai and Doctor Pham Van Quan. His kindly support and
continuous advices went through the process of completion of my thesis. His
encouragement and comments had significantly enriched and improved my work.
Without his motivation and instructions, the thesis would have been impossible to
be done effectively.
I would like to offer my special thanks to Bac Ninh wastewater treatment plant, I
have had the support and encouragement of them.
I would also like to express my greatest appreciation to Nagasaki Institute for
supporting and facilitating the student's analysis of samples at the laboratory.
I would also like to thank the experts who were involved in the taking sample for
this research: Duong Thu Thuy and Nguyen Tuan Khang. Without their passionate
participation and input, the thesis could not have been successfully conducted.
I wish to receive the contribution, criticism of the professors.

Sincerely thank.

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TABLE OF CONTENTS
INTRODUCTION………………………………………………………………….1
Research purposes and significance ........................................................................1
Scope and objective .................................................................................................2
CHAPTER 1 LITERATURE REVIEW………………………………………….3
1.1

Review on enteric viruses ..............................................................................3

1.1.1.Sources of enteric viruses in the environment ...........................................3
1.1.2.Virus transport and survival in the environment ........................................4
1.1.3.Waterborne enteric viruses and potential human diseases .........................5
1.1.4.The use of microbial indicator ...................................................................9
1.2

Review on bacteriophages ...........................................................................10

1.2.1.The value of phages as surrogates for enteric viruses ..............................11
1.2.2.F-specific RNA bacteriophages ...............................................................12
1.2.3.Detected method for virus and phages .....................................................13
1.3. Review on wastewater treatment plant ........................................................18
1.3.1. State of wastewater treatment plant in the world and in Vietnam ..........18
1.3.2. Sequencing batch reactor treatment ........................................................19
CHAPTER 2 MATERIALS AND METHODS………………………………...23
2.1. Site description ...............................................................................................23
2.2. Sample collection ...........................................................................................24
2.3. Several indicators of analysis .........................................................................26

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2.4. Sample concentration .....................................................................................28
2.5. Detection of E.coli and total coliforms by plate count. .................................28
2.6. Detection of F-RNA phages by plaque assay. ...............................................29
2.7. Method for quantitative genotyping of infectious FRNA phages coupled with
MPN method .........................................................................................................29
2.8. Recovery of viruses from sludge method ......................................................30
2.9. RT-PCR methods ...........................................................................................31
2.10. Statistical analysis ........................................................................................34
CHAPTER 3 RESULTS AND DISCUSSION………………………………….36
3.1. The occurrence of viruses and F-RNA phages ..............................................36
3.2. Reduction of viruses, F-RNA phages and microbial indicators ....................39
CHAPTER 4 CONCLUSION…………………………………………………...45
REFERENCE ...…………………………………………………………………..46

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LIST OF TABLES
Table 1.1. Advantage and disadvantage of culture and molecular methods .............18
Table 2.1. The reaction mixture for RT ....................................................................32
Table 2.2. The thermal condition of RT reaction ......................................................32
Table 2.3. The reaction mix for PCR ........................................................................32
Table 2.4. The thermal condition for PCR ................................................................33
Table 3.1. Log10 mean concentration of F-RNA specific genotype..........................37
Table 3.2. Log10 concentration of E.coli and total coliforms ....................................38

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LIST OF FIGURES
Figure 2.1 Influent tank .............................................................................................23
Figure 2.2 Reaction tank ...........................................................................................23
Figure 2.3. Decanter ..................................................................................................24
Figure 2.4. Effluent area............................................................................................24
Figure 2.5. The cycle of Sequencing Batch Reactor .....................................................
Figure 2.6 Using a long bucket to take the sample ...................................................25
Figure 2.7. Using a syringe to take the sample .........................................................26
Figure 2.8. Thermal cycler ........................................................................................34
Figure 2.9. 7500 fast Real-time PCR system ............................................................34
Figure 3.1. Concentration of E.coli ...........................................................................40
Figure 3.2. The concentration of total coliforms ......................................................40
Figure 3.3. The concentration of F-RNA phages genotype (a – GI, b-GII, c-GIII, dGIV) ..........................................................................................................................41
Figure 3.4. Log 10 reductions of F-RNA phages genotype GI, GII, GIII, GIV and
microbial indicators ...................................................................................................43

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LIST OF ABBREVIATIONS

CFU:

Colony forming unit

EPA:

Environmental Protection Agency

E. coli:

Escherichia coli

GI:

F-RNA Genotype I

GII:

F- RNA Genotype II

GIII:

F-RNA Genotype III

GIV:

F-RNA Genotype IV

MPN:

Most probable number

NoVs:

Norovirus

PCR:

Polymerase chain reaction

PFU:

Plaque forming units

RT-PCR:

Reverse transcriptase polymerase chain reaction

RT-qPCR:

Reverse transcriptase quantitative polymerase chain reaction

WWTP:

Wastewater treatment plant

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INTRODUCTION
Research purposes and significance
Viruses cause harm to humans because they are very small in size and therefore
move in water and can cause disease with very low dose. A group of viral
pathogens, from within the human body, excrete through the feces, and overdisperse the drainage pipes. Currently, researchers have identified several types of
enteric viruses in the domestic effluent (Wong et al., 2012) mainly including
enteroviruses (EVs), rotaviruses (RVs), adenoviruses (AdVs), noroviruses (NVs)
hepatitis A virus (HAV) and astroviruses (AVs). The virus is responsible for some

infectious diseases such as gastroenteritis, conjunctivitis, and respiratory disease,
both developed and developing countries throughout the world. Therefore, studying
the behavioral characteristics, existence or inactivated of the viruses in the water
treatment stages of the plant is the most important.
It is complicated and expensive to analyze all types of virus. At present, many
previous studies have tried to identify suitable viral indicators of wastewater
treatment efficiency. Several studies have proposed F- specific coliphages can be a
good indicator monitoring the virus removal from wastewater (Tree et al., 2003;
Duran et al., 2003), because there have similar morphological characteristics with
many enteric viruses suggesting that they have the same single-stranded RNA,
icosahedral shape, less than 50nm, and they are more persistent than bacteria. FSpecific coliphages are bacteriophages that infect Escherichia coli cells. These
FRNA coliphages include MS-2 in group I, GA in group II, Qβ in group III and SP
in group IV.
GII and GIII F-RNA phage genotypes was found primarily from human feces, while
GI and GIV F-RNA phage genotype are involved in animal waste (Vinjé et al.,
2004). The use of F- specific coliphages as indicators of the presence and behavior
of enteric viruses and animal enteric viruses has always been attractive because of

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the easy of detection and low cost associated with plaque assay and for similarity to
enteric viruses in terms of transport and survival characteristics.
Scope and objective
This research studied the fate of enteric viruses and bacteriophages during the phase
of sequencing batch reactor treatment plant. Sequencing batch reactor (SBR) is a fill
and draws activated sludge system. In this system, each tank in the SBR system is
filled during a discrete period of time and then operated as a batch reactor. It means,
once the reactor is full, it behaves like a conventional activated sludge system, but
without a continuous influent or effluent flow. The aeration and mixing are

discontinued after the biological reactions are complete, the biomass settles, and the
treated supernatant is removed. The reason why this study chooses SBR system
because the hydraulic retention time is time-based so there is no short-circuiting.
The influent and effluent can be pair so it is easy to observe the treatment efficiency
using only a grab sample. In addition, almost no research data in sequencing batch
reactor for viruses’ removal. So SBR system is the good target for an assessment of
virus.
This study has three main objectives:
1. To investigate the removal of enteric viruses and F-RNA phages in SBR
system.
2. To evaluate the concentration of enteric viruses and F-RNA phages in
activated sludge.
3. To find out the relationship between FRNA phage specific group I, II, III, IV
and enteric viruses.

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CHAPTER 1
LITERATURE REVIEW
1.1 Review on enteric viruses
Viruses are infectious agents that multiply only when they are inside a living
organism of host cells. Viruses can infect all types of organisms, from animals,
plants, bacteria, and archaea. Enteric viruses are the virus that can multiply in the
gastrointestinal of humans and animals.
In general, viruses are smaller in size than bacteria. Most of the viruses studied have
a diameter ranging from 20 to 300 nanometers. The virus is a macromolecular
structure, no energy-producing system, no ribosome, no individual growth, no
division and no susceptibility to antibiotics, contains only one of two types of
nucleic acid (DNA or RNA), living endocytic parasites.

1.1.1. Sources of enteric viruses in the environment
Enteric viruses are viruses that multiply in the intestines of humans and animals and
are emitted by the environment through feces and large numbers excrete from
infected humans. An infected person can emit more than 1 billion viruses/g of feces.
And from human sources, viruses will migrate to other environments through water
pipelines, sewage treatment plants, or direct emissions to the environment.
Usually, enteric viruses are found in surface water and the source can be from
household effluents or urban waste water. Several studies have shown that enteric
viruses such as rotaviruses, HAVs, HEVs, enteroviruses and astroviruses are present
in surface water, groundwater and sewage effluents (Heerden et al., 2005). Sedmak
et al., 2005 has been shown that in the wastewater environment the concentration of
the pathogenic viruses is about 100-10,000 infectious units/L. The concentration of
1 to 10 infectious units/L in surface water. In the groundwater environment, this
amount is in the range of 0-200 infectious virus /100L, according to the US.EPA,

3


2006 a. Some studies have also shown that some enteric viruses are emitted from
animals, but they do not always cause disease in humans (Cox et al., 2005).
1.1.2. Virus transport and survival in the environment
a, Transport
Enteric viruses can be transported in the environment through groundwater,
seawater, rivers, sewage treatment plants, drinking water. Enteric viruses are
transmitted through the fecal-oral route and primarily infect and replicate in the
gastrointestinal tract of the host (Irene and Ziquiang, 2014).
b, Survival
Although the virus can not multiply outside the host cell and cannot grow outside of
the environment, it can live or exist inside the environment for a certain period of
time from 28 to 88 days (Schimdt et al., 1969). The existence of the virus depends

on several factors, such as the characteristic of the virus, water characteristics such
as pH, turbidity, level of disinfection. Most viruses are inactivated at 56 ° C for 30
minutes, or at 100 ° C for a few seconds. The virus is also stable at cold
temperatures and can be stored for up to -70C. Viruses are inactivated by ultraviolet
light and in environments that contain oxidants and reducing agents such as
formaldehyde, chlorine, iodine, and H2O. ß-Propiolactone and formaldehyde are
chemicals used to inactivate viruses in vaccine production (Aimee et al., 2015).
In wastewater, there are some types of pathogenic microorganisms, especially in
areas where there is no proper sanitation system. Microorganisms and viruses often
do not live long in wastewater because they are not suitable environments, but they
exist for a certain period of time depending on the species to infect people, animals,
and plants. Some types of microorganisms and viruses as follows:
-

Intestinal bacteria (E.coli), can live in dirty water for 9-14 days at a
temperature of 20-220C (Elsas et al., 2011).

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-

The Adenovirus, Echo, Coxsackie virus live up to 15 days (Artur and Nigle,
2006).

1.1.3. Waterborne enteric viruses and potential human diseases
Viruses are important pathogens infecting to humans, animals, plants, and
microorganisms. The pathogenic virus only transmits the disease to each specific
host. In almost, viruses infect humans and will not infect animals and plants. In
contrast, viruses that infect animals will not infect humans. Only a few enteric

viruses are contagious for both humans and animals.
Viruses infiltrate through many ways, but the main route is the digestive tract, the
virus is excreted in the feces of the infected person, sometimes from the vomit.
More than 140 different types of enteric viruses have been studied that can infect
humans. Several common type of viruses are transmission of the virus such as
noroviruses, hepatitis A virus (HAV), hepatitis E virus (HEV), rotaviruses and
enteroviruses.
a, Norovirus
Norovirus belongs to the family Caliciviridae and has a viral RNA. These viruses
are usually small and have no surrounding envelope, sRNA. Currently, NoVs are
classified into five different groups of genes: GI, GII, GIII, GIV and GV. NoVs are
in the GI, GII and GIV groups, but most human pathogens are GI and GII. The
Norwalk virus was found under a microscope in 1978 from a patient's stool sample
in Norwalk, Ohio. This virus can live very harshly in harsh environmental
conditions as it can live yearly in frozen or hot temperatures of 60 ° C for 30
minutes or in chloride 6.25 mg/liter (Knight et al., 2016).
Norovirus potentially highly infectious. Viruses are present in the stool and in the
vomit liquid. Norovirus can be spread through contacts between healthy people and
infected people, such as sharing eating habits, sleeping, grasping supplies used by
people who carry the disease or using the same ingested or contaminated by
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norovirus. In the United States and Canada, many Norwalk virus outbreaks have
occurred on cruise ships. It is also common in nursing homes, nursing homes for the
elderly, in kindergartens and schools, etc. Older people with weakened immune
systems, pregnant women and children may be more likely to become infected than
other objects. (Knight et al., 2016)
As estimated by the Centers for Disease Control and Prevention 2012, at least 50%
of cases of acute gastroenteritis are attributed to norovirus. The National Institutes

of Health said that between 600,000 and 1 million people in the country are infected
with norovirus annually.
b, Hepatitis virus
Hepatite A virus, belongs to the family Picornaviridae and belongs to type 72 of
Enterovirus and second ranks in foodborne botulism. Compared with other
enteroviruses, HAV is relatively stable at room temperature, at a temperature of 60°
C. The virus is only partially inactivated while boiling for 5 minutes causes
inactivation of the virus. Hepatitis A virus can persist in water for 3 to 10 months.
HAV is resistant to organic solvents such as ether, acid. HAV multiply in the
hepatocyte, the infected cell releases the viral particles infects the bloodstream and
is then released into the stool.
Transmission occurs between humans and humans by eating food and drinking
contaminated water. Hepatitis by HAV is widespread in densely populated areas.
Diseases occur all over the world which usually occurs in late autumn and early
winter. The disease occurs in all ages, children with the benign or asymptomatic
disease, the older the infection the heavier.
Hepatite E virus was found in 1955 during a pandemic in New Delhi, India.
Hepatitis E is found all over the world, but most are in tropical countries near the
equator. These countries include Latin America, Africa, the Indian mainland, the
Middle East, and Asia, especially those in Southeast Asia. Periodic outbreaks,
approximately 5 to 10 years, in heavy rainy seasons, resulting in flooding.
6


Hepatitis E virus is not transmitting by blood, sex, and mother to child, mainly
through gastrointestinal (hepatitis A). Because HEV virus occurs in the feces,
garbage, wastewater, and when the floods happen the virus attached to food and
drinking water. When we eat the food or drink will be infected. A healthy person, if
exposed to the feces of an infected person, is not thoroughly hygienically cleaned,
then he or she may be exposed to a virus or drink unboiled water, the uncooked

meat of infected animals will also be infected. Between 1986 and 1988, more than
120,000 people living in China's Xinjiang region were "poisoned" for hepatitis E.
Even in the United States, hepatitis E is the cause of more than 50% of acute
hepatitis. (Hongwei et al., 2010)
c, Rotaviruses
The complete rotaviruses are about 60-80 nm in diameter, like a short thoracic
wheel. Rotaviruses contain 11 double-stranded RNA, approximately 18,000 pairs of
basalts.
Rotaviruses can survive for several days at 400C, even at 200C in the presence of
CaCl2 rotaviruses that remain infectious for months. At -200C the virus can survive
for many years. Rotaviruses infect small intestinal villi, which alter the structure
and function of the epithelium. Three layers of protein capsid help the virus resist
acid pH in the stomach and digestive enzymes in the intestines. (Bruce et al., 1983)
Diarrhea caused by rotavirus is very contagious, mainly through the fecal-oral and
oral-oral route. The amount of virus excreted in the feces is very sick so easily
spread to other children and caregivers. In addition, rotavirus can survive in the
natural environment such as on contact surfaces of toys, furniture, floors, household
items.
Around the world, about 2 million cases have been hospitalized each year for acute
diarrhea caused by rotavirus. Over the five years (1973-1978), rotaviruses have
been identified as the most common cause of acute diarrhea in infants and young
children around the world, especially children under 5 years of age. Rotavirus
7


infection is also common in adults, but mild symptoms such as diarrhea and chronic
diarrhea are common. (Bruce et al., 1983)
d, Enteroviruses
Enterovirus is a common name for the virus group Picornaviridae. The common
characteristic of the viruses in the Picornaviridae family is small, containing a

single-stranded RNA, the capsid has a cuboidal shape, with no cover. Enteroviruses
are divided into several groups, including: poliovirus (type 1,2,3); Coxsackie A (24
types), Coxsackie B (6 types); ECHO (34 types); Enteroviruses 68, 69, 70. 71 and
Enterovirus 72.
Viruses are unlikely to infect when heated to 50 ° C. Enterovirus can be stable in
thawing temperatures for decades, at 4 ° C for weeks, and in heat room for several
days. Viruses in Stool Stools at room temperature for at least 4 weeks. When dried,
enterovirus loses its infection capacity. They are also inactive when treated with
ultraviolet light, dye to detect them under the microscope (Chen et al., 2008).
Viruses enter the body through the digestive tract and focal in the stool and
pharynx. The common feature of the viruses in this group is the reproduction and
spread from the carrier to the healthy person through the digestive tract or
respiratory tract. Invasive oncogenes and lymph nodes in the throat. When it
reaches the required amount, the virus enters the bloodstream. Patients will have the
fever (about 1 week after the virus enters the body). Humans are the only reservoir
of Enterovirus. Children are more likely to be infected with Enterovirus than adults.
In the summer of 2000-2001, an outbreak of EV71 occurred in Sydney, with 200
children hospitalized, including nine patients with CNS and five pulmonary
edemata. EV71 has been identified as a causative agent in all patients with
pulmonary edema (Nolan et al., 2003).
In 2009, EV cases in China reached 1,155,525 cases, of which 13,810 (1.2%) were
serious cases and 353 (0.03%) deaths. Cases are widely distributed throughout

8


China. Among EV71 cases, EV71 accounts for 41% of cases, accounting for 81%
of all serious cases and accounting for 93% of deaths (WHO, 1979).
1.1.4. The use of microbial indicator
Contaminated water is mainly caused by feces excrete from human or animal. In the

feces there have many microorganisms are excreted from the intestines or other
parts of the human body, in addition to some of the viruses mentioned above, there
are many other pathogens exist in human and animal feces.
Until now, coliforms bacteria have still considered an indicator for water
contaminant. Coliforms bacteria are organisms that are present in the environment
and are present in the feces of hot-blooded animals and humans. Coliforms bacteria
do not cause disease. Coliforms bacteria have similar characteristics to intestinal
microbes, such as resistant in the environment, morphological characteristics, and
size, so their presence in drinking water indicates that pathogenic organisms may be
present. Testing the presence of coliform bacteria is easy and simple compared to
testing with all possible pathogens.
There are three different groups of coliforms bacteria, each with a different risk
level: total coliform, fecal coliform, and Escherichia coli. To indicate that the
environment is contaminated with feces, E.coli and fecal coliforms are used as an
indicator microorganism, which appears in large numbers in the human intestine
and human feces.
In the past, there also have studies about the relationship between coliforms bacteria
and enteric virus (Zhang et al., 2012). However, the presence of coliform does not
indicate any association with enteric viruses in water. This is because coliform and
enteric viruses are not homogeneous. Viruses are smaller in size than bacteria and
survive longer in environments with disinfectants such as chlorine.
Some requirements to become microorganism’s indicator of enteric viruses
(Grabow, 1986; Kott, 1981). The microorganism’s indicator:

9


-

Need to be present in the water where enteric viruses are present

-

In terms of quantity, directives are equal to or higher than the number of
viruses

-

Have resistance in the environment of antiseptic as viral

-

Does not easy to grow in water

-

Does not cause illness and is proven by easy, quick and cheap methods.

-

Represented in feces or sewage contamination.

1.2 Review on bacteriophages
A bacteriophage is a virus that infects bacteria. This is a group of viruses widely
distributed in nature, first discovered by British scientist Frederick Twort in 1915,
followed by Felix d'Hérelle (1873- 1949) French-Canadian microbiologists have
identified parasitic viruses on the bacillus and called them bacteriophages.
Phage does not cause a disease to humans but causes a disease to bacteria. Each
bacterium can be the host of one or more phages. Currently, phages were used to
study the type of prokaryote, transport of genetic elements in molecular biology

studies, treatment of bacterial diseases, epidemiological investigation ...
Structure and morphology of bacteriophages contain:
-

Nucleic acid molecule (genome): most of the bacteriophages, usually with
twisted strands of DNA, some with single-stranded DNA, non-DNAcontaining phages contain RNA and a sequence of RNA.

-

Capsid: protein coat. The capsid shell is made up of single
molecules called capsomer, which is proteins.

-

Enzymes in the phage's tail contain some enzymes

-

Some bacteriophages contain tails and spikes.

Phages only infect to consist of bacteria. The multiplication of phage in bacteria
usually occurs in the following phases:

10


1) Adsorption and penetration stages: In order to penetrate and multiply in the
bacterium, the phage must first find a specific receptor on the surface of the
bacterial cell. Many studies show that when bacteria mutate, change the
surface properties, the phage is not able to penetrate the bacteria. Once laced

with bacterial cell surface, lysosomes in the phage's tail dissolve the walls of
the bacterial cell, then the tail droops the core of the tail into the bacterium,
followed by the phage's DNA being injected. bacterial cells. The capsid shell
will stay out of the bacteria.
2) Stage of biosynthesis of components: After 2-3 minutes, the phage
deoxyribonuclease (DNase) enzyme breaks down the DNA of the bacterial
cell, mRNA and attaches to the enzyme required for the phage synthesized.
The DNA of the phage formed together with the phage capsid protein is
synthesized in the host cell ribosome. Enzymes needed for phage synthesis.
Phage's DNA is formed together with the phage capsid's protein, which is
synthesized in the ribosome of the host cell. Enzymes needed for phage
synthesis. Phage's DNA is formed together with the phage capsid's protein,
which is synthesized in the ribosome of the host cell.
3) Phase assembly and release: DNA components assembled with proteins
form the phage. New phages are formed after about 12 minutes and new
phage releases usually occur in 25 minutes. On average, each bacterium can
be released from 100 to several hundred phages.
1.2.1. The value of phages as surrogates for enteric viruses
In the world, the use of bacteriophages as a microbiological indicator for enteric
viruses has been investigated by many scientists. Several types of bacteriophage
have been shown to be able to serve as microorganisms’ indicator for enteric viruses
such as somatic coliphages, male-specific F-RNA coliphages, and Bacteroides
fragillis phages. Somatic coliphages and F-RNA coliphages belong to Coliphages
group, which both infect to the host as E.coli.

11


Somatic coliphages are DNA viruses that multiply on the host E. coli by attack cell
wall of the host. Somatic coliphages in river water have a good correlation with

enteroviruses (Stetler, 1984). The analysis of somatic coliphages is also quite
simple, inexpensive and quickly (Grabow et al., 1998).
Bacteroides fragillis is found in the human intestine (109 -1010/ g faeces) and is
associated with enteroviruses and rotaviruses in sewage (Jofre et al., 1989).
However, the presence of Bacteroides fragilis phages in human feces is very rare. In
addition, bacteroids are non-spore with negative gram and are easy to inactivate in
environments with high levels of oxygen, so they are also limited to being the
indicator of enteric viruses.
F-RNA coliphages are the most studied species for use as an indicator of enteric
viruses in aquatic environments. Because there has the size as small as any enteric
virus, while the somatic phage is a bit larger. Havelaa et al., 1993 indicates that the
association of F-RNA bacteriophages and enteric viruses in river and lake water.
However, their existence in natural water is still lower than some types of enteric
viruses (Schaper et al., 2002). According to Vergara et al., 2015 studies on the
relationship between F-RNA coliphages and some enteric viruses such as HAdV,
Norovirus noroviruses, RoV rotaviruses in tropical waters.
1.2.2. F-specific RNA bacteriophages
F-specific RNA bacteriophages infect to male E.coli through the “pili” and belong
to Leviviridae family. This group has linear ssRNA, icosahedral and small. These
viruses are adsorbed by F-pili to cellular receptors. F-RNA infection to the host
usually does not occur at temperatures below 30 °C because E. coli hosts do not
produce F pili at low temperatures and the range of temperatures for 30-37oC
(Franke et al., 2005). Host strains used to detect F-specific coliphages in water are
E.coli resistant to streptomycin and ampicillin and Salmonella enterica senovar
Typhimurium WG49 (Stm WG49). Where WG 49 is the most widely used, a
salmonella strain possesses F-pilus and antibiotic resistance is used for selective
12


detection of F-phages in the environment. F-phage and somatic salmonella phage

can infect WG49. However, there are quite a few somatic salmonella phages in the
environment and you can judge that all plaques come from F-phages.
This group of F-RNAs is divided into 4 groups GI, GII, GIII, and GIV (Vinjé et al.,
2004). Including MS-2 in group I, GA in group II, Q in group III, and SP group IV.
These phages are all present in the disinfection environment, light and the stages of
water treatment. There have been numerous studies showing that F-specific RNA
genotype II and III are found primarily from human waste, while genotypes I and
IV are involved in animal waste (Vinjé et al., 2004).
In the four types of F-RNA coliphages, GI and GII are known to be phages that
have a longer lifetime in the environment than GIII and GIV. However, their
survival depends on factors such as temperature and pH. At 4° C, genotypic GI, and
GIII F-RNAs are capable of detecting within 110 days, while GIII and GIV will
reduce detectability after 3 weeks or 10 days (Long and Sobsey, 2004).
In 2012, the study of Hata et al., (2012) concluded that GI F-RNA phage may be
used to represent the minimal and GIII F-RNA phage can be representing to
maximal reduction of viruses during wastewater treatment. In the research by
Haramoto et al., (2015), showed that GI F-RNA coliphages have the potential to be
an indicator of the reduction of viruses through wastewater treatment.
1.2.3. Detected method for virus and phages
 Concentrate viruses’ method
Viruses usually appear in small amounts in water, so the virus can be analyzed in a
volume of water that needs to be concentrated before analysis.
The viral concentrate was studied very early by the use of membranes. According to
Eisenach et al., 1991, used a membrane for viruses to attach to the membrane for
the purpose of determining the size of the virus (Ver et al., 1968). By 1952, ion

13


exchange was used to concentrate polyps (Lo et al., 1952) and Kelly has succeeded

in concentrating viruses from the sewage sewer pattern in 1953.
The concentrate viruses’ method base on the adsorption of the particle to the surface
of the membrane which has a charge and elution from that surface by pH-adjusted
solution was developed by Wallis and Melnick, 1967. WHO, 1979 introduced a
method of virus isolation in wastewater. In 1981, the method of concentrate and
virus analysis from wastewater was published in the Journal of Water Resources
and Sanitation (Greenberg and Taras, 1981).
The size of enteric viruses ranged from 30nm (enterovirus) to 100nm (adenovirus)
and have isoelectric points from 2.8 (hepatitis A virus) to 8.0 (rotavirus) (Michen
and Graule, 2010). In the water environment, viruses usually have a charge, so they
often use a filter to carry the charge to adsorp virus. A negative charge membrane is
used by many scientists to use and develop. Elford, (1931) used pH 4 samples and
adsorbed more than 90% of seeded viruses through membranes. Recently,
Katayama et al., (2002) was developed a procedure for concentration enteric viruses
using negatively charged membrane, the recovery yields for poliovirus from 33 to
95 %; Haramoto et al., (2009) was concentrated and recovery NoVs from 250 to
500ml of MilliQ water (186%), tap water (80%), river water (15%) and pond water
(39%) by using electronegative membrane combined with aluminum or magnesium.
 Culture method
In the 1930s, virologists found that embryonic eggs could be used to cultivate
herpes viruses, smallpox, and influenza viruses. Although chicken embryos are
much simpler in structure than rabbit or rat organisms, chicken embryos remain a
complex organ. The use of chicken embryos does not completely solve the
problems encountered in the cytological effect caused by the virus. On the other
hand, bacteria also develop well on chicken embryos, on bacterial embryos, it is
difficult to accurately assess the effects of the virus.

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Two new findings have led to the development of cell culture methods for
virologists and other scientists. Firstly, the detection and use of antibiotics to
prevent bacterial contamination (Schmidt, 1969). Secondly, biologists have found
that proteolytic enzymes, especially trypsin, can separate cells from surrounding
tissues without harming them. Wash the cells and count them, dilute them and
transfer them to plastic bottles, test tubes or boxes. The cells in the suspension fluid
adsorbed on the surface of the plastic layer, which multiplies and spread to a
cellular layer called the monolayer. This single cell layer can be subcultured.
Subculturing is to bring the cultured cells to culture in new culture media. Large
numbers of subcultures are made up of a single organism template that will be the
homogenized cell sample needed for a series of viral effects studies.
From the 50s to the 60s of the 20th century more than 400 viruses were isolated and
examined. In 1950, Enders et al., 1949 was isolated poliovirus. For phage, it is
possible to use plaque assay to determine the amount of phage present in the sample
through spots.
 Molecular method
To detect RNA viruses by using animal cell line is difficult and expensive. PCR
methods are widely used for detection of various microorganisms and it is rapid and
sensitive.
The polymerase chain reaction (PCR) was discovered by Kary Mullis in 1983 and
was awarded the Nobel Prize in Chemistry in eight years later for its enormous
applications. Thanks to PCR, an inadequate amount of DNA is no longer an
obstacle to DNA-based molecular diagnostics and research.
According to Mullis, the process by which DNA replicates artificially over many
cycles of DNA polymerase replication. DNA polymerase is naturally present in
living organisms, where it functions as a nucleus of DNA when the cell divides. It
works by hooking up to the DNA strands and creating complementary fibers.
According to Mullis's original PCR, the DNA replication enzyme is performed in
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vitro. Double-stranded DNA was split into two strands when heated at 96 ° C.
However, at this temperature DNA polymerase is destroyed so enzymes are
required after each heating period of each cycle. Mullis's original PCR procedure is
not very effective because it takes a long time, requires a large amount of DNA
polymerase, and has to be constantly monitored throughout the PCR process. Then,
the original procedure was developed using DNA-Polymerase derived from
thermophilic bacteria that live in water at temperatures above 110 ° C. The DNA
polymerase from the organism is thermostable and when used in PCR it is not
broken when the mixture is heated to separate the DNA strands. Since there is no
need to add DNA-polymerase at every cycle, DNA replication can be simpler and
more automated.
One of the first heat-resistant DNA-polymerases was isolated from Thermus
aquaticus and was called Taq. Taq polymerase is widely used in PCR experiments
(Pavlov et al., 2004).
As already practiced today, PCR needs a lot of ingredients. The components are:
1. The template DNA contains the piece of DNA that needs to be amplified
2. Primers, to determine the start and end points of the area to be amplified
3. The DNA-polymerase enzyme catalyzes DNA replication.
4. Nucleotides (eg dNTP) are the material for DNA-polymerase to build new
DNA.
5. A buffer solution, providing a chemical environment for DNA-polymerase
PCR is performed during the heat cycle. This is a heater that heats and cools in the
reaction tube at the correct temperature for each reaction. The PCR process consists
of 20 to 30 cycles. Each cycle consists of 3 steps:
(1) The temperature rises to 94-96 °C to separate the two strands of DNA. This step
is called denaturing, which breaks the hydrogen bond between the two DNA
strands. Before the first cycle, DNA is usually denatured to the time of opening

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to ensure that the DNA and primer are fully separated and only single-stranded.
Time: 1-2 minutes.
(2) After the two strands split, the temperature is lowered so that the primer can
attach to the single strand of DNA. This step is called priming. This stage
temperature is dependent on the primer and is generally lower than the
denaturation temperature of 50 °C (45-60°C). Incorrect use of this temperature
leads to the fact that the primer is not fully attached to the sample DNA, or is
attached arbitrarily. Time: 1-2 minutes.
(3) Finally, DNA polymerase attaches to the fiber. It starts to cling to and work
along the DNA strands. This step is called stretching. The long-term temperature
depends on the DNA-polymerase. The timing of this step depends on both the
DNA-polymerase and the length of the DNA fragment to be amplified 1000bp / 1
minute.
Because ribonucleic acid (RNA) cannot play a role as a direct template for PCR,
reverse transcription (RT) should be combined with PCR to allow the RNA to be
converted to complementary DNA (cDNA) and become template suitable for PCR.
When the two techniques are combined, this is called reverse transcription
polymerase chain reaction (RT-PCR). Previously, three stages of RT-PCR were
performed separately, but these three steps are now combined in one trial and are
called one-step RT-PCR. The use of RT-PCR for the detection of RNA in research
and diagnostics provides a fast, effective, sensitive and specific method.
Real-time PCR is different from common PCR in that it is capable of detecting and
quantifying PCR directly after each cycle of the reaction. The implementation of
real-time PCR technique is also based on the usual principle of a chain reaction.
However, the distinctive feature is that the amplified DNA fragment will be
detected at each time the reaction takes place (real-time). Real-time PCR consists of
two simultaneous processes: DNA replication by PCR and fluorescence imaging
proportional to or proportional to the number of DNA fragments.

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