BY- K. Sai Manogna (MSIWM014)

Introduction :

Poxviruses are part of the family Poxviridae and can infect humans and animals alike. Smallpox (variola), vaccinia, cowpox, monkeypox, buffalopox, aracatuba, and cantagalo viruses are orthopoxviruses. Orf virus, pseudo cowpox virus, deer poxvirus, bovine papular stomatitis virus, and sealpox virus are parapoxviruses. Yatapoxviruses include the tanapox virus, which is commonly found in Africa. The human poxvirus, the molluscum contagiosum virus, contains mollusc poxviruses. 

Smallpox is unique to humans and molluscum contagiosum. In humans, other viruses cause unusual zoonotic infections. The vaccinia virus used for vaccines will infect humans as well. Molluscum contagiosum is also a human-unique poxvirus. Other human poxvirus infections are caused either by zoonotic exposure to animal poxviruses or scheduled or unintended vaccinia administration. This virus is spread through close contact, often via sexual contact. 

Poxvirus (smallpox) - Bioscience Notes

Recently, laboratory exposures have been reported that have contributed to infection with vaccinia and tanapox viruses, widely used as vectors for experimental vaccines. Due to transmission to contacts, civilian and military personnel’s smallpox vaccination program resulted in multiple infections. 

Incubation Period: Infections with smallpox are caused by inhalational penetration to nasal, oral, or pharyngeal droplets. The incubation period is from 10-14. Viruses of smallpox multiply locally and spread to the nearby lymph nodes. On days 3-4, asymptomatic viremia ensues, spreading to the bone marrow and spleen. On approximately day 8, secondary viremia begins. Generalized signs of fever and a toxic appearance are associated with this secondary viremia. In the dermis blood vessels, the virus in leukocytes then becomes localized. Then the stereotypical smallpox rash emerges. 

The replication: 

1. Generally, poxviruses replicate only in the cytoplasm and can in cells without a nucleus, unlike most DNA viruses. 

2. Either through endocytosis or by a fusion event, the virion enters the cell. 

3. For later replication cases, the viral core reaches the cytoplasm and functions as a scaffolding. 

4. Besides, many necessary enzymes such as viral transcriptase, transcription factors, capping and methylating enzymes, and a poly(A) polymerase are transported by the virus. 

5. Therefore viral DNA transcription is rapidly initiated and about 100 early viral genes, especially enzyme coding genes that are involved in viral DNA replication, are triggered.

6. At the same time as DNA replication, transcription of intermediate and late genes is initiated. 

7. In specific cytoplasm areas, virus assembly and immature viruses can be visualized very quickly. 

8. The Virions travel to the Golgi complex during maturation, where they are enveloped before being released by budding or cell disruption. 

9. Some virions are enveloped, and they may have certain benefits, including cell uptake speed.

Other poxviruses, primarily localized diseases, typically follow the same pattern of evolution. An exception is the outbreak of monkeypox, which progresses to a variola-like clinical syndrome. Monkeypox infections, as in the North American epidemic, can vary from mild infections with few lesions to severe systemic diseases that mimic smallpox. At the inoculation site, the Molluscum contagiosum virus also replicates, but the skin lesions’ character is distinct. 


Human poxvirus infections are obtained from animal reservoirs, except molluscum, which is primarily a human disease. The reservoir is recognized and distributed globally in some cases, as is the case with ovine and bovine parapoxviruses. An occupational hazard of those who interact with the contaminated reservoir hosts is human contamination with these viruses. 

Monkeypox is confined to West Africa, and squirrels are more critical than monkeys as reservoir hosts. The cowpox virus is limited to Europe and the western part of the former Soviet Union. Bovine cowpox is unusual, and the most frequently recorded host is the domestic cat. Lack of definitive knowledge on the cowpox virus reservoir host, but it is presumably tiny wild rodents. Cases occur without established contact with cats or cattle, and it is possible to spread indirectly through barbed wire or brambles. 

Restricted natural person-to-person transmission of monkeypox, but not more than four or five generations, has been observed. Parapox and cowpox diseases spread from person to person occasionally, if ever. Molluscum’s person-to-person spread is historically associated with physical contact sports (e.g., wrestling) and towel sharing. However, there is growing evidence that molluscum sexual transmission is significant. 

Traditionally, the Vaccinia virus is known as a laboratory virus without a natural reservoir. Although its host reservoir lacks information, the buffalopox virus, now considered a vaccine virus variant, appears to have established itself in India. It is important to note that such strains may become developed in animal populations and interact with genetically related viruses circulating in them due to the possible use of recombinant vaccinia virus vaccines. 


In some instances, a right diagnosis would be made possible by the existence of the lesions and a careful history of interaction with the infected reservoir animal or other infected people; difficulties can occur if no such contact is known. This is perhaps most prominent with human cowpox, as most cases are not linked to a single source, and occasionally an anthrax clinical diagnosis is made. 

An effective means of rapid diagnosis is electron microscopy of the vesicle or scab material; poxviruses and herpesviruses are readily differentiated, and the characteristic morphology of parapoxviruses can be identified. Immunofluorescence of infected cell cultures can distinguish morphologically related poxviruses from various genera for e.g., Orthopoxviruses and Yatapoxviruses. While the molluscum virus has yet to be cultivated in tissue culture and chicken embryos, the other poxviruses are easily isolated. Cultivation then enables biologic and serum neutralization samples to be established. Near antigenic relationships within genera compromise precise recognition through antibody detection, but knowledge of the host and geographic range will validate a presumptive diagnosis. 


Infections of Variola have been globally eradicated. There is a concern, however, about the reintroduction of smallpox through bioterrorism. An international health care emergency would precipitate the reappearance of smallpox. Local and federal public health authorities should be informed of suspected cases of smallpox. 

For smallpox or vaccinia, no known remedies are currently available. Several nucleoside and nucleotide analogues, including variola, vaccinia, monkeypox, cowpox, molluscum contagiosum, and orf, have shown Invitro activity and in vivo antiviral activity against various poxviruses. So far, Cidofovir and a number of its derivatives are the most efficient.



Every time a cell divides, DNA breaks each of its double strands into two single strands. Each of these single strands functions as a template for a new complementary DNA strand. Each new cell, as a result, has its complete genome. This method is known as the replication of DNA. Replication is regulated by the pairing of template chain bases with incoming deoxynucleoside triphosphates and is driven by DNA polymerase enzymes. It is a complicated process involving an array of enzymes, particularly in eukaryotes.

1. DNA biosynthesis begins in the direction of 5′- to 3′. This makes it difficult for both strands to be simultaneously synthesized by DNA polymerases. It is first necessary to unwind a portion of the double helix, and helicase enzymes mediate this.

2. The leading strand is continuously synthesized, but in short bursts of about 1000 bases, the opposite strand is copied as the lagging strand template becomes available. The resulting short strands are called Okazaki fragments.

3. There are at least three distinct DNA polymerases in bacteria: Pol I, Pol II, and Pol III; Pol III is primarily involved in the elongation of the chain.

4. However, DNA polymerases may not initiate de novo DNA synthesis, but require a short primer with a free 3′-hydroxyl group. This is provided by an RNA polymerase (also called DNA primase) in the lagging strand that can use the DNA template and synthesize about 20 bases in length for a short piece of RNA.

5. Pol III can then take over one of the short RNA fragments previously synthesized. Pol I take over at this stage, using it’s 5′- to 3′-exonuclease activity to digest the RNA and fill the DNA gap until it reaches a continuous DNA stretch.

6. This leaves a gap between the newly synthesized DNA 3′-end and the DNA 5′-end previously synthesized by Pol III.

7. DNA ligase, an enzyme that creates a covalent bond between a 5′-phosphate and a 3′-hydroxyl group, aims to fill the gap.


The mechanism of producing an RNA copy of a gene sequence is transcription. This version, called a molecule of messenger RNA (mRNA), leaves the cell’s nucleus and enters the cytoplasm, guiding the protein synthesis that encodes.

One of the fundamental processes that happen to our genome is transcription. It’s the mechanism by which DNA is converted into RNA. And you may have heard of the Central dogma of Molecular biology in which DNA is transcribed to RNA and translated to protein. Well, the first part of moving from DNA to RNA relates to transcription. And in unique locations, we transcribe DNA to RNA.   But there are many other transcribed RNAs, including transfer RNAs and ribosomal RNAs, which do other genomic functions.

Formation Of pre-messenger RNA:

Initiation: There are similarities between the transcription machinery and that of DNA replication. As with DNA replication, before transcription can occur, a partial unwinding of the double helix must happen, and it is the RNA polymerase enzymes that catalyze this process.

Elongation: Unlike DNA replication, only one strand is transcribed. The strand containing the gene is called the sense strand, while the antisense strand is the complementary strand. A copy of the sense strand is the mRNA produced in transcription but transcribed as the antisense strand.

Termination: Ribonucleoside triphosphates (NTPs), with Watson-Crick base pairing (A pairs with U), align along the antisense DNA strand. For the formation of a pre-m-RNA molecule that is complementary to a region of the antisense DNA strand, RNA polymerase binds the ribonucleotides together. Transcription stops when a triplet of bases read as a “stop” signal enters the RNA polymerase enzyme.

RNA Splicing: Gene expression is regulated at several different stages during transcription and translation to ensure that the right products are produced. The gene includes different sequences in eukaryotes that do not code for protein. The transcription of DNA generates pre-mRNA in these cells. These pre-mRNA transcripts often contain regions known as introns, which are interfering sequences removed by the splicing process before translation. Exons are called the areas of RNA that code for protein. In a process called alternative splicing, splicing can be regulated so that various mRNAs can contain or lack exons. Alternative splicing makes it possible to create more than one protein from a gene. It is a significant regulatory step in deciding which functional proteins are produced from the expression of genes.

Alternative splicing:

Alternative splicing occurs during gene expression and enables multiple proteins (protein isoforms) generated from a single gene coding process. Due to the distinct forms in which an exon can be exempted from or included in the messenger RNA, alternate splicing can occur. It can also occur if parts of an exon are excluded or included, or if introns are included. For example, if four exons (1,2,3 and 4) are present in a pre-mRNA, they can be spliced and translated into many different combinations. It is possible to translate Exons 1, 2, and 3 together or translate Exons 1, 3, and 4 together.

By binding regulatory proteins (trans-acting proteins that contain the genes) to cis-acting sites located on the pre-RNA, the pattern of splicing and production of alternative-spliced messenger RNA is regulated. Splicing activators (that encourage specific splicing sites) and splicing repressors (that minimize the use of particular sites) are among some of these regulatory proteins.

Heterogeneous Nuclear Ribonucleoprotein (hnRNP) and Polypyrimidine Tract binding protein (PTB) include some common splicing repressors. The proteins translated from; spliced messenger RNAs, alternatively differ in their amino acid sequence, resulting in the protein’s altered function. This is one of the reasons, the human genome can encode a broad range of proteins. A typical process in eukaryotes is alternative splicing; most of the multi-exonic genes in humans are alternatively spliced. Unfortunately, the explanation that there are many hereditary diseases and disorders is often irregular differences in splicing.


Messenger RNA splicing is achieved and catalyzed by a macro-molecule complex called the spliceosome. The ligation and cleavage regions are determined by several subunits of the spliceosome, including branch sites and splice sites of 5′ and 3′. Interactions between these sub-units and small nuclear ribonucleoproteins (snRNP) found in spliceosome produce a complex that helps decide which introns to leave out, exons that hold together to bind together. After the introns are cleaved and detached, a phosphodiester bond binds the exons together.

Reverse Transcription: Reverse transcription is a technique used to create a complementary DNA strand from RNA. This mechanism is based on a retroviral mechanism whereby reverse-transcriptase enzymes can reverse transcribe RNA into DNA. This is incredibly helpful when scientists only have tissue and want to study gene sequences. Researchers will separate mRNA from the tissue; in this case, use reverse transcription to generate cDNA.


The translation is the process in which proteins are generated using the information carried in mRNA molecules. The nucleotide sequence in the mRNA molecule provides the code for an essential amino acid sequence to create a protein. A protein is made from many amino acids, similar to how RNA is constructed from many nucleotides. A ‘polypeptide chain’ is considered a chain of amino acids, and a polypeptide chain bends and folds on itself to form a protein.

The RNA strand information is translated from RNA’s language into the language of polypeptides during translation, i.e., the nucleotide sequence is translated into an amino acid sequence.


The smaller and larger subunits of the ribosome bind to the mRNA transcript at its binding site during the translation, in the initiation stage.

When the starting codon AUG is recognized by the tRNA, the process of protein construction begins.


The mRNA triplet codon is “read” during the translation elongation process, and the tRNA is added to the complementary amino acid. Ribosomal RNA catalyzes the whole reaction.


If the termination codon is reached, the polypeptide chain synthesis ends with peptidyl tRNA. Here, the whole process depends on the RNA polymerase’s involvement in the transcription, although no polymerase is involved in the translation. Interestingly, transcription is a method of encoding information in mRNA (messenger RNA), while translation is a decoding method.

In eukaryotes transcription occurs in the nucleus, while translation in the cytoplasm. However, the entire process occurs only in the cytoplasm in the prokaryotes.

Rifampicin antibiotics inhibit transcription, while translation is hindered by puromycin and anisomycin. The final transcription product consists of Adenine, Guanine, cytosine, and Uracil messenger RNA. At the leading site, it has the initial codon and, in the end, the termination codon. Due to a long adenine chain, the 3 ‘end of the mRNA is called a poly-A tail. The final translation result is a long amino acid chain, a fundamental building block of a protein called the polypeptide chain. The first amino acid is methionine in the amino acid chain. (Although, in most cases, it is removed).

Transfer RNA (tRNA):

1. Ribonucleic acid transfer (tRNA) is a type of RNA molecule that helps decode a sequence of messenger RNA (mRNA) into a protein.

2. During translation, tRNAs act at specific sites in the ribosome, which is a mechanism that synthesizes a protein from an mRNA molecule.

3. Proteins are formed from smaller units called amino acids, which are specified by codons called three-nucleotide mRNA sequences.

4. A codon represents a specific amino acid, and a particular tRNA is known for each codon.

5. With three hairpin loops forming a three-leafed clover, the tRNA molecule has a distinctive folded structure.

6. A series called the anticodon includes one of these hairpin loops, which can identify and decode an mRNA codon. Each tRNA has attached its corresponding amino acid to its end.

7. The tRNA transfers the necessary amino acid to the end of the increased amino acid chain when a tRNA recognizes and binds to its respective codon in the ribosome.

8. Then, the mRNA molecule begins to decipher the tRNAs and ribosomes until the whole sequence is converted into a protein.

Genetic Code: In the early 1960s, American biochemists Marshall W. Nirenberg, Robert W. Holley, and Har Gobind Khorana completed the deciphering of the genetic code. Genetic code is the term we use for the context in which the four DNA bases — A, C, G, and Ts — are linked together to be read and converted into a protein by the cellular machinery, the ribosome. Each of the three nucleotides in a row counts as a triplet in genetic code and codes for a single amino acid. So, each of the three sequences codes for an amino acid. And often, proteins are made up of hundreds of amino acids. Thus, the code that would make one protein could contain hundreds, sometimes even thousands, of triplets.


DNA: Replication, Transcription and Translation



  • The process of producing identical replicas of DNA from original DNA molecule in known as DNA replication.
  • It is the most important part of inheritance and occurs in all living organisms.
  • DNA consist of two strands which are complementary to each other during the process of DNA replication these tow strand get separated and serve as template for the synthesis of new complementary strand this is known as semiconservative replication.
  • After cellular proofreading and error checking mechanism new DNA molecule is synthesized with one strand from parent DNA molecule and another is fresh.
  • Each cell of a human being contains 3 X 109 base pairs of DNA distributed over 23 pairs of chromosomes.
  • In human genome 95% region do not code for any protein known as introns.
  • According to central dogma DNA makes RNA and RNA makes protein.
  • The process of producing RNA from DNA is called transcription and synthesis of protein from RNA is called translation

Steps Involved:

  • DNA replication
  • Transcription
  • Translation

DNA Replication:

  • When cell divides, the double stranded DNA splits into single strand and makes new copy of the genome which gets transferred into each cell.
  • Base pairing is according to the Watson-Crick paring of the DNA strand.
  • It is a complex process which includes array of enzymes.
  • Synthesis of DNA proceeds in the 5’-3’ direction and for attachment of DNA polymerase first DNA strand has to be unwind which is regulate by the helicase enzyme.
  • During the synthesis of the new strands by the DNA polymerase one strand is synthesized continuously and called leading strand on the other hand the opposite strand known as lagging strand synthesised in to the small fragments called okazaki fragments.
  • In bacteria there are three distinct DNA polymerase: pol I, pol II and Pol III. Pol III largely involve in chain elongation. DNA polymerase itself cannot initiate DNA synthesis it require a short primer with free 3’ hydroxyl group.
  • Once the primer attaches to the DNA strand Pol III then take over and synthesis of new strand begins.


  • The process by which DNA is copied to mRNA is called transcription.
  • The mRNA contains information for the protein synthesis.
  • Transcription occurs in two steps. In first step pre mRNA is synthesized with the help of RNA polymerase, the resultant RNA strand is reverse complement of the original DNA sequence. Then after pre messenger RNA is edited and forms the mRNA molecule by the process of RNA splicing.

RNA Splicing:

  • The pre-messenger synthesized contains introns and exons in it, introns are the sequence which does not code for any protein hence it is not required in protein synthesis.
  • Thus the pre- messenger RNA is chopped up and introns are removed from it which synthesise messenger RNA contain only exons.


  • The mRNA synthesised in the transcription process is now transported outside the nucleus into cytoplasm to the ribosome.
  • Messenger RNA does not directly involve in the protein synthesis transfer RNA is required the process of protein synthesis is called translation.
  • The mRNA contains three base stretch called codon and each codon contains information for a specific amino-acid. While the ribosome passes through the mRNA each transfer RNA molecule anticodon interacts with codon of mRNA.
  • The transfer RNA contains amino acid at 3’ end which is added to the growing protein chain and the t-RNA expelled from the ribosome.

Transfer RNA:

  • Transfer RNA is tertiary structure which is represented in two dimensions as cloverleaf shape.
  • Each amino acid has its own unique t-RNA like t-RNA for phenylalanine is different from that of histidine.
  • Attachment of amino acid is at 3’- OH group of t-RNA.


DNA replication


  • Introduction
  • Types of replication
  • Model of DNA replication
  • Enzymes involved in DNA replication


  • It is the process of producing two identical copy of DNA from one original DNA molecule.
  • DNA replication is one of the most essential or important properties exhibited by DNA.
  • Evolution of all morphologically complex form of life is based on the replication.

Types of Replication

  • There are possibly three modes of replication:
  • Dispersive
  • Conservative
  • semi-conservative

Semi Conservative

  • In this mode of replication, two  old parental strands serve as template for synthesis of new daughters strands and each new DNA contains one strand from the parent and one from newly formed progeny.
  • The evidence of semi conservative replication of DNA was first presented by Meselson and Stahl in 1958.


  • The old DNA molecule would break into several pieces, each fragment would replicate and the old and new segment would be combined to yield two progeny DNA molecules; each progeny molecule contains  both old and new strands along its length.


  • According to the conservative replication, the old parental strands remains together and newly formed daughter strands are also together.

Model of DNA Replication

  • The brief discussion of DNA replication model is as follows:
  • DNA applications initiate at certain unique and fixed point called origin.
  • There is unwinding of complementary strands of DNA duplex with the help of two enzymes DNA gyrase and DNA helicase, this process is called Melting.
  • Single strand binding protein are attached to two single stranded region so that they not join to form duplex.
  • Due to melting in the origin region it produces two Forks(Y), in the DNA duplex and one fork is located at the end of the melted region. Generally, both the fox are involved in replication and become Replication Fork.
  • After formation of replication fork ,Primase enzymes initiate transcription of the strand in the 3’-5’ direction. This results in the generation of 10-60 long primer RNA.
  • The free 3’-OH of the  RNA primer provide initiation point for DNA polymerase for the sequential addition of deoxyribonucleotide.
  • The replication of the second strand (5’-3’) DNA is discontinuous, Hence 3’-5’strand of DNA is termed as the Leading strand and 5’-3’ is termed as Lagging strand.
  • Lagging strand generates small polynucleotides fragment called Okazaki fragments, during replication. This fragment is about 1000-2000 nucleotides long in E.coli.

Enzymes Involved in the DNA Replication.

  • There are some important enzymes which are involved in the replication of DNA :
  • DNA polymerase
  • Primase
  • Polynucleotide ligase
  • Endonuclease
  • Helicase
  • Single stand binding protein (SSB).

DNA polymerase

  • This enzyme synthesizes new strand on a template DNA strand.
  • It is also known as DNA replicase.
  •  Its activity was first explained by Kornberg in 1956.
  • In prokaryotes, there are five types of DNA polymerase:
DNA polymeraseGeneFunctions
1polAMajor repair enzyme
2polBMinor repair enzyme
3polCDNA replication
4dinBSOS repair
5umuD2’CSOS repair


  • It involves in the synthesis of RNA primer, which are required for initiation of DNA replication. It is RNA polymerase that are used only to synthesize primer during replication.


  • This involves in the formation of phophodiester linkage and joining of two newly formed DNA strands.


  • It produces an internal cut in a DNA molecule, while Restriction Endonucleases are those that cuts at only specific site or sequences.

Single strand binding protein

  • It prevents from forming duplex by binding to single strand DNA.