Cancer cousing viruses (carcinogenic viruses)

According to Viral Gene Hypothesis, certain viruses are responsible for the oncogenesis. Virus which are causes cancer called oncogenic virus. First evidence of a relationship between viruses and cancer was obtained by demonstration of transmission of leukemia by cell free filtrate of blood of diseased chickens to healthy chickens.

In 1911, Rous also transmitted chicken sarcoma from diseased chicken to healthy one. The viral etiology of various cancers in animals like fishes, dogs, mice, rats, rabbits, frog, horses etc. is clearly evidenced.

Many viruses are found to be associated with many cancers in humans also. Viruses that are capable of producing cancer are called 'oncogenic viruses' or 'tumor viruses'.

There are two classes of tumor viruses as DNA and RNA tumor viruses. A common characteristic of both these viruses is that the viral genome is some way becomes integrated into the host cell DNA and replicates along with host cell chromosome.

I] DNA Tumor Viruses :

There are five groups of DNA viruses causing cancer in humans. They include -
a. Papova
b. Adeno
c. Pox
d. Herpes and
e. Hepatitis viruses.

Among these, Papova viruses have been received more attention because they are known to cause tumors in a variety of animals.

a) Papova Viruses :

Name papova is derived from first two letters of three viruses as Papilloma, Polyoma and Simian vacuolating 40 virus ( SV 40 virus).
They belong to the papovaviridiae family.
Papovaviridiae family includes two genera - Papiloma viruses and Polyoma viruses.
SV40 virus is now placed in the genus Polyoma viruses.
All papova viruses are small, naked, icosahedral, circular double stranded DNA viruses.
They are commonly found in humans and other mammals.
Their size ranges from 45nm to 55nm in diameter.
They contain 5 to 7 genes. The icosahedral capsid is constructed from 2 to 3 polypeptides

i) Papilloma virus :

Small, non enveloped, icosahedral, circular, ds DNA viruses.
They are about 55 nm in diameter.
Their capsid contains 72 capsomers.
Capsomers made from two types of proteins as L,(Major)and L2(Minor).
These are wart causing viruses.
Natural infection results in formation of benign warts in humans and other animals.
These viruses induce tumor only in natural host.
They transform normal cells into cancer cells in vitro very rarely.
Warts produced by these viruses are usually benign But they may convert into malignant carcinomas.
Also found to be associated with human penile, uterine and cervical carcinomas.
They replicate exclusively in the keratinocytes.
Keratinocytes form the outermost layer of the skin as well as some mucosal surfaces of the genitals, anus and mouth.

ii) Polyoma Viruses :

Name polyoma refers to multiple (poly) tumors (oma) producing viruses.
They are also small, naked, circular, ds DNA viruses, about 45nm in diameter.
Capsid is made from three proteins as - VP1, VP2 and VP3.
These three proteins form pentameric capsomers.
There are about 72 pentameric capsomers.
These viruses also encode a protein called 'T antigen'. The polyoma viruses do not induce tumors in the natural hosts like monkey and mouse.
For example - Simian vacuolating virus (SV 40) replicates in the kidneys of monkeys ( natural host ) without causing any disease.
However, the SV 40 virus cause the sarcomas in the hamster. They also transform the cells in vitro.
The SV 40 virus is most thoroughly studied oncogenic DNA virus.
It is a monkey polyoma virus.
It can't induce tumor in monkey (natural host) but induce tumors in experimental hamsters as well as rodents.
The two other examples of human polyoma virus are JC and BK virus.

JC Virus - It causes progressive multifocal leukoencephalopathy – damage or inflammation of white matter of brain at multiple locations. Symptoms include- weakness, paralysis, loss of vision, impaired speech, coma and death.

BK virus - Associated with renal diseases in kidney transplanted patients.

II] RNA Tumor Viruses :

Oncogenic RNA containing viruses are called "OncoRNA viruses'.
They are classified as 'retroviruses' because the viral RNA produces intermediate DNA by using reverse transcriptase enzyme.
From this DNA copy, viral RNA are produced which are accommodated in viral capsid while DNA copies are integrated into host genome.
Retroviruses have been isolated from many vertebrates like fishes, reptiles, birds and mammals.
They cause leukemia and sarcoma in chicken while lymphoma and mammary carcinoma in mice.

Based on the host range and disease type, there are five categories of Oncorna viruses They include -
a) Avian leukosis complex -

e.g. Rous sarcoma virus of chickens.

b) Murine leukosis virus -

e.g. Murine leukemia & sarcoma virus.

c) Mammary tumor virus of Mice -

e.g. Bittner virus that causes breast cancer and multiplies in mammary glands. It is transmitted to the offsprings through the breast milk.

d) Leukosis sarcoma virus -

It produces sarcoma in the animals such as cat, hamster, guinea pigs and monkey.

e) Human T cell lymphotrophic virus ( HTLV) -

It causes T cell lymphoma.
There are two types of HTLV as HTLV type 1 and HTLV type 2.
Type Icauses adult T cell lymphoma and Type 2 causes hairy cell leukemia.
In hairy cell leukemia - the bone marrow has difficulty in producing enough normal cells like WBCs, RBCs and platelets.

Retrovirus :

Retroviruses are icosahedral with envelope. They have diploid genome i.e. they carry two identical segments of RNA genome.

RNA molecules are plus sense single stranded. Consists of 3 important genes- gag, env & pol.
Name retroviruses refers to participation of reverse transcriptase in their multiplication.
Multiplication of these viruses requires the transcription of RNA into DNA and its subsequent integration into a host genome.
Only an integrated viral DNA can be transcribed into viral RNA.

Retrovirus replication

Replication of positive sense ssRNA occurs as shown in figure.
Virus multiplication is not lytic and does not kill the cell.
Integrated genome of the retroviruses replicate along with host genome and therefore, it is present in all cancer cells.
Oncogenic development is due to the expression of a viral gene known as 'src' gene.
Base sequence of viral 'src' gene is similar to that of the host cell gene whose product plays a role in the regulation of growth of the normal cells.
This suggests that the oncogenes of retroviruses are of cellular origin. Thus, the retroviruses cause the cancer as they introduce the new genetic material into host DNA and cause transformation.
Three theories have been proposed which are concerned with the oncogenesis by RNA viruses in human beings.

• These are
a). Oncogene theory
b). Protovirus theory and
c). Provirus theory

a) Oncogene Theory :

Oncogene Theory is Proposed by Huebner & Todaro.

According to this, the genetic information for cancer is present in each of the body cell and it is transmitted from parent to offspring through the germ cells.
Infection of body cells by RNA viruses has occurred millions of years ago during evolution.
Therefore, every cell is assumed to contain oncogene i.e. viral gene responsible for oncogenesis.
This oncogene is normally kept in repressed condition (prevented from functioning by the regulatory genes of the host cell).
When the oncogene becomes derepressed either by a physical, chemical or biological agent, it gets expressed itself by the formation of a transforming protein.
This protein could change the normal cell into a malignant one although no virus could be recovered from it.

b) Protovirus Theory :

Protovirus Theory was Proposed by Temin.

According to this, certain DNA regions of normal cells are transcribed to RNA & reverse transcribed to DNA by reverse transcriptase enzyme.
Such newly formed DNA segments get back integrated into original DNA either at the same site or at different sites.
This leads to the amplification of genetic regions.
In this flow of genetic information from DNA to RNA & back to DNA, the genetic information becomes independent of cell genome.
At this stage with or without effect of mutations, the protoviruses may arise which would direct synthesis of RNA tumor viruses or alternatively the integration of newly formed DNA could produce new gene alignment leading to oncogenesis.
Thus, this theory postulates that the normal back & forth flow of genetic information during amplification process, with or without the effect of mutation or due to alignment of new gene sequences may generate RNA tumor viruses or genes necessary to induce oncogenesis.

c) Provirus theory :

This theory was Proposed by Spiegelman.

It states that some cancers are caused by direct infection with oncogenic viruses.
It further states that tumor viruses are transmitted horizontally from individual to individual entering in provirus states.
They may cause the cancer as a result of chance activation of their transforming genes.
Occasionally, they may be transmitted vertically i.e. from one generation to other.
Thus, this theory is different from the oncogene and protovirus theory which state that the spontaneous mutation in animals and humans cells may cause oncogenic events.
Among these three hypothesizes, the oncogene hypothesis is consistent with number of observations. They include -
Spontaneous intracellular appearance of oncogenic RNA viruses after repeated sub-culturing of cells.
This gave no indication of previous virus infection.
The appearance of oncogenic RNA viruses upon exposure to oncogenic agents in the cells.
The demonstration of the occurrence of oncogenic viral genetic material in the cells by hybridization studies.

Pasteurization of Milk : Temperature, Types, Advantages and Disadvantages

Pasteurization process developed in 19th century in France by Louis Pasteur, for the preservation of wine. Its application resulted in coining a new term 'Pasteurization'.
Louis Pasteur was pioneer in its use for the preservation of wine Dr. Soxhlet of Germany introduced this process for preservation of milk in 1886.

Definition of Pasteurization

Pasteurization refers to - The process of heating of each and every particle of milk to at least 62.8° C (145°F) for 30 min. or 71.7° C (161ºF) for 15 seconds or to any temperature-time combination, which is equally efficient, in an approved and properly operated equipment.
After pasteurization the milk is immediately cooled to 5°C or below. The term pasteurization is mainly applied to the market milk today.

Objectives of Pasteurization :

To render the milk safe for human consumption by destruction of all pathogenic microorganism.
To improve the keeping quality of milk by destruction of almost all spoilage organisms.
Thus, the pasteurization does not kill all micro-organisms in milk and therefore, it is not a method of milk sterilization but it is a process of disinfection.
Pasteurization is also intended to inactivate some enzymes in the raw milk.

Relationship Between Time & Temperature :

The efficiency of pasteurization depends upon time-temperature relationship.
Initially the pasteurization temperature was set to kill Mycobacterium tuberculosis, which was supposed to be the most heat resistant organism in milk.
This organism gets destroyed at a temperature of 140°F in 10 minutes.
Thus, the initial temperature of pasteurization was set to 143°F (61.6°C) for 30 min.
Later on, it was discovered that another pathogen namely Coxiella burnetii (causative agent of Q fever), also occurs in the milk and it is able to survive at the temperature of 143ºF.
Thus, to ensure the destruction of Coxiella, temperature of pasteurization was raised to 145°F (62.8°C).

Methods of Pasteurization

There are three methods of Pasteurization in common practice.

Low temperature holding (LTH)
The high temperature short time (HTST)
Ultra high temperature (UTH).

1. Low Temperature Holding (LTH) :

This is also called as low temperature long time (LTLT) method or batch or vat pasteurization method.
In this process, the milk is heated to 62.8° C(145°F) for 30 minutes and promptly cooled to 5° C or below.
It is a batch method and thus it takes a little more time. The heating of milk in this method is carried out by three different types of pasteurizers -

i]. Water-Jacketed Vat -

This is double walled tank in which hot water or steam under partial vacuum circulates for heating and cold water for cooling.
The outer wall is usually insulated to reduce heat loss.
The heat exchange takes place through the wall of the inner lining.
The milk is agitated by slowly moving paddles or propellers.
When heating, the vat cover is left open for escape of off - flavours, and when holding, the cover is closed.

ii]. Coil Vat Type -

The heating or cooling medium is pumped through a coil placed in either a horizontal or vertical position.
Such coil is turned through the milk.
The turning of coil agitates the milk.

iii]. Water Spray Type -

A film of hot water is sprayed over the surface of the tank holding the milk.

2. High Temperature Short Time Method (HTST) :

It is the modern method of pasteurization of milk
It is used for large volume of milk.
In this method, the milk is heated to 71.7°C(161ºF)for 15 second and cooled promptly to 5°C or below. This method gives the continuous flow of milk.
Milk is heated either in tubes or in thin metal plates or in heat exchangers by electricity or hot water.
Raw cold milk is allowed to enter in heat exchanger and hot, pasteurized milk is taken out.
It is comparatively faster method. There are different designs of HTST pasteurization system.

Essential Components of a Standard HTST System

1. Raw milk tank : It is a tank where chilled raw milk is stored.
2. Balance tank : It maintains a constant head for the incoming milk.
3. Milk feed pump : It creates suitable pressure that is necessary for efficient flow.
4. Flow control system : It controls the flow rate of milk.
5. Filters and clarifiers : They remove the dirt (if any) from the milk.
6. Homogenizer : It divides fat globules into micro globules to avoid fat separation in standing milk.
7. Plate Heat Exchanger (PHE) with regeneration section, heating section, holding section and cooling sections : It facilitates an efficient pasteurization.
8. Flow diversion valves :

They ensures that all the conditions for pasteurization have been met.
It diverts the milk to head tank if it is not properly heated to pasteurization temperature for reprocessing.
If milk is properly pasteurized milk, it passes forward through the flow diversion valves into the regeneration where it is cooled.

Plate Heat Exchanger

The plate heat exchanger is a compact and simple unit.
Its plate may be used for regeneration, heating, holding and cooling.
A space of approximately 3 mm is maintained between plates by a non-absorbent rubber gasket.
These plates are designed to provide a uniform turbulent flow of milk with rapid heat transfer.

Regeneration (heating) -

The raw incoming milk is partially and indirectly heated by the hot outgoing milk.
This reduces the cost of HTST process.

For example -

Milk entering at 4°C
Heated in regenerator to 34°C
Heated in heating section to 74°C
Cooled in regenerator to 44°C
Cooled in cooling section to 4°C
Here, the increase from 4 °C to 34°C is a change of 30°C and the decrease from 74°C to 44°C is also a change of 30°C.
Without regeneration, the milk would need to be heated by hot water/steam from 4°C to 74°C, a difference of 70°C
With regenerative heating, however, hot water or steam need not be used for the temperature change between 4°C and 34°C.
This temperature change is brought about by use of the outgoing hot milk.
Thus, here regeneration saves the heat.
On the other hand, without regeneration the milk would need to be cooled by chilled water from 74°C to 4°C, a difference of 70°C.
With regenerative cooling, however, chilled water need not be used for the temperature change from 74°C to 44°C, a difference of 30°C.
This temperature change is brought about by use of cold incoming milk.
Here also the savings due to regeneration takes place.
The milk to be heated flows across one side of the plate and heating or cooling medium flows across the other side in the opposite direction.

Advantages of Regenerative Heating :

Utilization of the incoming chilled milk to cool the outgoing hot pasteurized milk increases the efficiency of the PHE.
Smaller amount of energy is required to heat the milk to pasteurization temperatures since the heating does not start from 4°C of the chilled milk.
Reduces the amount of time required to pasteurize milk.

Advantages of HTST :

The milk can be pasteurized quickly and effectively
Initial cost of the method is less.
Milk packaging can be started as soon as pasteurization begins.
This permits more efficient utilization of labour for packaging and distribution.
The system can be easily cleaned and sanitized Lower operating cost.
Reduced milk losses. Development of thermophiles is not a problem.

Disadvantages of HTST :

This system is not well adopted for small quantities milk or milk products.
Gaskets require constant attention for possible damage and lack of sanitation.
Complete drainage is not possible.
Raw milk with high thermoduric bacterial count is not efficiently pasteurized as compared to LTH method.

3. Ultra High Temperature Method (UHT) :

Milk is heated at 135ºC to 150°C (average 141°C) for just two seconds or no hold time.
It can be carried out by the direct or indirect means of heat treatment
Direct treatment is either injection of steam into milk or infusion of milk into a steam chamber
Indirect treatment includes the use of heat exchangers.
The success of method depends on immediate aseptic packaging.
The temperature and time combination is much more lethal to bacteria and kills all bacteria.

UHT Pasteurization by Steam Injection Technique :

In this technique, the steam is injected into the milk.
The steam at a pressure higher than that of the pressure of milk flow is injected into the milk stream through a suitable nozzle.
There are many different injector designs.
In all of these, the steam and milk are kept in thermally separate zone until they reach the mixing zone.

UHT Pasteurization by Steam Infusion Technique :

It is very similar to injection system, except for the method of mixing of milk with steam.
The infusion is done by dropping heated milk into a steam pressure vessel with a conical base.
The heating is instant.
The milk is then cooled rapidly by evaporative cooling with exposure to a slight vacuum.
This removes the water that is added to the milk due to the condensation of the steam.
Steam infusion can be used to pasteurize a variety of dairy products like cream and special dairy products but now it is popular for milk.

Pasteurized Milk

Packaging of ultra-pasteurized milk is also carried out under sterile conditions to prevent the recontamination with spoilage bacteria.
Ultra-pasteurization makes milk free of spoilage and harmful bacteria, but it is not considered sterile and, thus, it requires refrigeration.
Ultra-pasteurized milks have more "cooked" flavor as compared to conventionally pasteurized milks.
Average shelf-life of ultra-pasteurized milk products is 30-90 days if held under refrigeration and in packed condition
Once an the product is opened, it may become contaminated with spoilage bacteria. Thus, after opening, ultra-pasteurized milk should be kept well refrigerated and consumed within 7-10 days for best quality and taste.

Advantages of Pasteurization :

Pasteurized milk is safe for consumption.
Pasteurization is designed to decrease microbial count.
Pasteurization has little effect on the nutritive value of milk.
There is some loss of vitamin C and B group vitamins, but this is insignificant.
With Pasteurization, keeping quality of milk remains unaltered. 6. Pasteurization does not reduce the fat content of milk.

Disadvantages of Pasteurization :

Cooked taste may be developed, for which consumer may complain.
Pasteurization reduces cream layer of the milk.
Vitamin content may get changed.

Properties of Immunogenicity

Immunogenicity is an substance that induces an immune response and antigen is any substance that is capable of binding specifically to the components of the immune system such as to the antibodies.
Nowadays the terms antigen and immunogens are used interchangeably by immunologists.

Properties of a substance which determine its immunogenicity

Immunogenicity of a substance is determined by following four main properties.

Foreignness
Molecular size
Chemical complexity and
Stability or degradeability

1). Foreignness

By the term foreign we mean non-self. Our immune system is able to distinguish between self and non-self. To be immuno genic the substance should be genetically foreign to the host. It should be different from the hosts own substances.

Immunogen's are mostly peptides or proteins in chemical composition. If the peptide of immunogens have similar nature of the peptides of host organism, there will be no immune response against the antigen.

For example bovine serum albumin (BSA). It is a protein which is found in all cows. So if BSA is injected from one cow into another there will be no immune response, because the protein is identical and for the immune system of every cow.

This BSA is a self molecule but if the BSA is injected into a rabbit an immune response is induced. This is because BSA is foreign to rabbits immune system. So for a molecule to be immunogenic it must be recognised as foreign by the immune system. More foreign and molecule is more is its immunogenicity.

2) Molecular size

Second property which determines the immunogenicity of a substance is its molecular size. It has been found that relatively small substances have decreased immunogenicity whereas large substances have increased immunogenicity.

The most potent immunogens are molecules with molecular weight above 10,000 Dalton's.
Highly immunogenic molecules have a molecular weight of 1 lakh Dalton's or more.
Molecules with weight than 10,000 Dalton's are weakly immunogenic or not immunogenic at all.

So greater the molecular weight more immunogenic it is.

3). Chemical Composition and Molecular Complexity

Antigens can be carbohydrates, lipids, nucleic acids and proteins chemically but their immunogenicity varies.

Carbohydrates are immunogenic only if they have relatively complex polysaccharides structure or when associated with protein carriers. For example blood group antigens.

Lipids and Nucleic acids are not immunogenic by themselves but they become immunogenic when they are conjugated to protein carriers. Proteins are generally the powerful immunogens and this is because of their molecular complexity in size.

The molecular complexity of proteins is a consequence of the variety of units known as amino acids of which proteins are made of. Besides having number of different amino acids proteins have four levels of protein structure.
- Primary structure
- Secondary structure
- Tertiary structure and
- Quaternary structure.
This also contributes to the immunogenicity of a molecule.

Let's take an example, A homo polymer of an amino acid which has a molecular weight of 30,000 Dalton's. Now if we look at its molecular weight it should be immunogenic but this is not the case because of the absence of chemical complexity. Now a copolymer which is composed of different amino acids in its molecular weight is more than 10,000 Dalton's. So it is immunogenic.

Thus molecules with complex nature are more immunogenic when compared to simple molecules.

4). Stability or Degradeability

To stimulate immune response a substance should bind with B-cell receptors or T-cell receptors. B cells interact with the substance on its own that is via its B-cell receptors.

But T-cell receptors do not interact with the substance as a whole they interact with a distinct portion of the substance which are short peptides. These small molecules are result of antigen processing and presentation.

Large insoluble macromolecules generally are more immunogenic than small soluble ones. Because larger molecules are more readily degraded and processed and presented to the immune system.

This also explains why proteins are excellent immunogens on the other hand highly stable and non degradable substances are not immunogenic. For example silicon. Thus to be immunogenic a substance must be degradable, highly stable and non degradable substances are not immunogenic.

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