This document discusses several methods used in molecular virology to study plant viruses, including polymerase chain reaction (PCR), sequence analysis, recombinant DNA technology, cell culture, and microarray technology. PCR is used to amplify small amounts of viral genetic material, sequence analysis determines and analyzes viral genomes, recombinant DNA technology creates recombinant viruses, cell culture grows viruses in host cells, and microarray technology studies changes in host gene expression from viral infection. These methods provide insights into viral biology and interactions with host plants.

1. BOT-301
Diversity of Monera

2. Methods in
Molecular Virology

3. Methods in molecular virology
 Molecular virology is a branch of virology that focuses on the molecular biology of
viruses, including their replication, genetic material, and interactions with host cells.
 There are several methods commonly used in molecular virology to study viruses,
including:

4.  Polymerase chain reaction (PCR)
 Sequence analysis
 Recombinant DNA technology
 Cell culture
 Microarray technology

5. Polymerase chain reaction (PCR)
 This method is used to amplify small amounts of viral genetic material, making it
possible to detect and study viruses even in low amounts.
 Plant viruses can be detected using the Polymerase Chain Reaction (PCR)
technique. The following are the steps involved in detecting plant viruses using
PCR:

6. Sample
collection
RNA extraction
Reverse
transcription
PCR
amplification
Gel
electrophoresis
Detection

7.  Sample collection: A plant tissue sample is collected from an infected plant. The
tissue can be leaves, stems, flowers, or other parts of the plant, depending on the
type of virus being tested for.
 RNA extraction: The collected tissue is processed to extract the viral RNA, which
contains the genetic material of the virus.
 Reverse transcription: The extracted RNA is converted into complementary DNA
(cDNA) using reverse transcriptase, an enzyme that synthesizes cDNA from RNA.
 PCR amplification: The cDNA is then amplified using polymerase chain reaction
(PCR), a technique that amplifies specific DNA sequences. The amplification is
performed using specific primers that are designed to target the viral genome.

8. PCR amplification

9.  Gel electrophoresis: The amplified DNA is then separated by size using gel
electrophoresis, a technique that separates DNA fragments based on their size and
charge.
 Detection: The separated DNA fragments are then visualized using a technique
such as ethidium bromide staining, which intercalates into the DNA and causes it to
to fluoresce under ultraviolet light. The presence of a specific band of DNA at the
expected size indicates the presence of the virus.
 It is important to note that PCR is a highly sensitive and specific method for
detecting plant viruses, but it is also important to consider the quality of the
sample and the laboratory protocols used, as these can affect the accuracy of the
test results.

10. Data collection
Data cleaning
Alignment
Phylogenetic analysis
Identification of viral genes
Characterization of viral populations
Prediction of viral resistance
Functional analysis
Sequence Analysis

11. Sequence analysis
 This method is used to determine and analyze the sequence of the viral genome, which
can provide important information about the virus and its evolution.
 Typically includes the following steps:
 Data collection: Obtain the viral sequences from public databases or from laboratory
experiments.
 Data cleaning: Verify the quality of the sequences and remove any contaminants or
errors.
 Alignment: Align the viral sequences to a reference sequence to identify conserved
regions.
 Phylogenetic analysis: Create a phylogenetic tree to infer the evolutionary relationships
among the plant viruses based on the similarities in their nucleotide or amino acid
sequences.

12.  Identification of viral genes: Determine the open reading frames (ORFs) in the viral
genome and use databases or bioinformatics tools to identify the functions of the viral
genes.
 Characterization of viral populations: Use bioinformatics tools to analyze the genetic
diversity and evolution of the viral populations in different geographical regions or
hosts.
 Prediction of viral resistance: Predict the potential of the plant to develop resistance to
the virus based on the analysis of the host's genetic diversity and the evolutionary
relationships between the host and the virus.
 Functional analysis: Experimentally validate the predictions made during the sequence
analysis and determine the molecular mechanisms of virus-host interactions.
 These steps can provide valuable insights into the biology of plant viruses, which can be
used for developing effective control strategies for plant virus diseases.

13. Development of
diagnostic tools
Generation of
recombinant
virus vectors
Generation of
virus-resistant
plants
Study of virus-
host interactions
Development of
viral vaccines
Recombinant DNA technology

14. Recombinant DNA technology
 This method involves creating recombinant viruses by introducing foreign DNA into
the viral genome. This can be used to study viral replication and pathogenesis, and
to develop new vaccines and therapies.
 Development of diagnostic tools: Recombinant DNA technology can be used to
produce specific probes and primers for the rapid and sensitive detection of plant
viruses.
 Generation of recombinant virus vectors: Recombinant DNA technology can be
used to generate recombinant virus vectors that can be used as delivery vehicles
for foreign genes into plants for functional genomic studies or for the expression of
vaccines against plant viruses.

15.  Generation of virus-resistant plants: Recombinant DNA technology can be used to
introduce specific viral genes or silencing constructs into plants to confer virus
resistance.
 Study of virus-host interactions: Recombinant DNA technology can be used to
study the molecular mechanisms of virus-host interactions by producing chimeric
viruses or by introducing mutations in viral genes to study their function.
 Development of viral vaccines: Recombinant DNA technology can be used to
produce viral antigens in large quantities for the development of viral vaccines.

16. Cell culture
Cell line
selection
Cell culture
initiation
Cell
maintenance
Virus
inoculation
Virus
replication
Antiviral drug
treatment
Virus
isolation
Analysis of
virus-host
interactions

17. Cell culture
 This method involves growing viruses in laboratory cell cultures, which can be used to study
 the interactions between viruses and host cells
 and the effects of antiviral drugs.
 The steps involved in cell culture for the study of viruses and their interactions with host cells
include:
 Cell line selection: Select an appropriate cell line that is susceptible to the virus of interest and
can be grown in culture.
 Cell culture initiation: Establish the cell line in culture by transferring cells to a growth medium
in flasks or petri dishes.
 Cell maintenance: Maintain the cell culture by regularly replacing the growth medium and
subculturing the cells to avoid overcrowding and loss of viability.
 Virus inoculation: Introduce the virus into the cell culture by exposing the cells to the virus-
containing medium.

18.  Virus replication: Monitor the replication of the virus in the cell culture and measure its
growth over time.
 Antiviral drug treatment: Test the effectiveness of antiviral drugs by treating the infected
cell culture with various concentrations of the drugs and measuring their effects on virus
virus replication and cell viability.
 Virus isolation: Isolate the virus from the infected cell culture to purify it for further
analysis.
 Analysis of virus-host interactions: Study the interactions between the virus and the host
cells by analyzing the changes in cellular morphology, gene expression, and signaling
pathways.
 These steps can provide valuable insights into the biology of plant viruses, which can be
used for developing effective control strategies for plant virus diseases.

19. Microarray technology
 This method involves using microarrays to study the expression of genes in
response to viral infection. This can provide important information about the host
response to viral infection and the molecular mechanisms of virus-host interactions.
 Microarray technology can be used to detect the response of plants to viral
infection by measuring changes in gene expression. When a plant is infected with a
virus, its cells respond by activating a range of genes involved in defense
mechanisms, such as the production of antiviral compounds or the reinforcement
of the plant cell wall.

21. Gene selection
and array
design
Sample
collection and
RNA extraction
RNA labeling
and
hybridization
Scanning and
data analysis
Interpretation
of results

22.  Here are the steps involved in using microarray technology to detect the response of
plants to viral infection:
 Gene selection and array design: A set of genes known to be involved in the plant's
response to viruses is selected and probes for these genes are designed and placed on
a microarray.
 Sample collection and RNA extraction: Samples of plant tissue are collected from
infected and non-infected plants and total RNA is extracted.
 RNA labeling and hybridization: The extracted RNA is labeled with a fluorescent dye
hybridized to the microarray.
 Scanning and data analysis: The microarray is scanned to measure the fluorescence of
each probe, which provides a measure of the expression level of each gene in the
sample. The data from the infected and non-infected samples is compared to identify
changes in gene expression that are associated with the viral infection.

23.  Interpretation of results: The results are analyzed to identify genes that are
differentially regulated in response to the virus, providing insight into the
mechanism of virus-plant interaction.
 These steps provide a general overview of the microarray process, but the specific
methods and protocols used may vary depending on the type of microarray and
the nature of the experiment.