Genetic Virology, In Silico Genome . Manufacturing theoretical viruses and variants, Part 1



All claims of Virus Existence Refuted

The fact of Alignment
Virologists have never isolated a complete genetic strand of a virus and displayed it directly, in its entire length. They always use very short pieces of nucleic acids, whose sequence consists of four molecules to determine them and call them sequences. From a multitude of millions of such specific, very short sequences, virologists mentally assemble a fictitious long genome strand with the help of complex computational and statistical methods. This process is called alignment.
The result of this complex alignment, the fictitious and very long genetic strand, is presented by virologists as the core of a virus and they claim to have thus proven the existence of a virus.



Some background on the Next-generation sequencing technology

Next-generation sequencing technology in clinical virology

Clinical Microbiology and Infection       Volume 19, Issue 1, January 2013, Pages 15-22

So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics.”
“The diagnostic application of NGS is just around the corner.”

 The first commercially available NGS system, developed by 454 Life Sciences, appeared in 2005. Since then, in a relatively short time, several NGS technologies have been developed

Current NGS methods use a three-step sequencing process: library preparation, DNA capture and enrichment, and sequencing/detection.
1. In library preparation, DNA (or cDNA) fragments of appropriate lengths are prepared, by either breaking long molecules, or by synthetically preparing short molecules (i.e. by PCR or cloning)
2. In the DNA capture and enrichment phase, these short molecules are labelled with primers that are used to capture and physically separate each single short fragment, fixing it onto a solid substrate. Each single molecule acts as a template for clonal amplification (single-molecule template principle).
3. The sequencing phase is based on DNA polymerization combined with detection. These steps occur concomitantly on myriads of clonally amplified fragments.

 There are many factors involved in the choice of technology, including cost performance, run time, accessibility, type of application, and convenience.

Virus discovery (metagenomics)

 The term metagenomics designates the analysis of all of the nucleic acid present in a given sample, allowing the exploration of entire communities of microorganisms, and avoiding the need to isolate and culture individual microbial species, and does not need previous knowledge of the sequences.

Whole viral genome reconstruction

 The reconstruction of full-length viral genomes, even in the case of unknown or poorly characterized viruses, is a common application of NGS, starting either from culture-enriched viral preparations, or directly from clinical samples. The assay design can vary from shotgun metagenomic sequencing of random libraries, to random shotgun sequencing of full genome amplicons, to overlapping amplicon sequencing and assembly.

Characterization of intra-host variability

 The highly variable mixture of closely related genomes within a given host, referred to as quasi-species


 The current revolution in microbiology has been primarily driven by advances in technology and, in particular, by the development of parallel sequencing platforms, which have led to a substantial reduction in costs and a substantial increase in throughput and accuracy. Not only does NGS provide knowledge for basic research, but also it affords immediate application benefits, including improved diagnostics, prognostics and therapy monitoring for many viral diseases.

Sequence-independent methods are becoming more important for the identification of emerging viruses in a public health context.

the application of high-throughput NGS methods in viral metagenomics can greatly enhance the chance of identify viruses in clinical samples, including viruses that are too divergent from known viruses to be detected by PCR or microarray techniques.

In addition, the deep investigation of viral quasi-speciesby NGS is substantially increasing our understanding of the dynamics of viral infections

In addition, the possibility of using barcoding systems for the simultaneous analysis of multiple samples can substantially reduce the cost of genotype resistance testing, and may increase the throughput for large-scale population studies

However, although state-of-the-art NGS data analysis tools can provide good precision, continuous improvement is fundamental to solve problems in a number of fields. First, technical errors must be distinguished from real mutations. Indeed, it is known that PCR polymerases typically have error rates of one substitution per 105–106 bases.

As deep sequencing generates millions of reads from a given sample, technology-intrinsic errors might be misinterpreted as mutations or polymorphisms. This requires error identification and correction. Many algorithms have been designed to increase the quality of data (PyroNoise clusters, SHORAH, KEC, ET, etc.), but it is still not possible to obtain data free of error. The use of platforms that do not require PCR amplification prior to sequencing (such as Helicos and PacBio RS) may circumvent some of the problems resulting from the PCR steps, such as primer selection and mutations introduced during amplification. However, due to the lack of PCR, these platforms suffer from low signal strength (Table 1); overall, these methods warrant further validation and investigations of their applicability in virology.

Another problem of data mining is that, at present, there are no perfect alignment tools, and a compromise is generally tolerated between alignment accuracy and time of analysis, where appropriate hardware and software characteristics are crucial.

The persistent problem of DNA contamination and the medical significance of the detection of very low levels of viral nucleic acid will also need to be resolved. This will require close collaboration between different areas of expertise, i.e. bioinformatics and virology, for the development of an efficient pipeline of analysis and for validation of the results.


Genomics and Clinical Virology (Virtual). ( The Future)

Next- generation sequencing (NGS) technologies 

The next-generation sequencing (NGS) technologies is transforming how clinical microbiology laboratories diagnose and manage infectious diseases. Whole genome sequencing (WGS) of hundreds of microbes can be undertaken in hours enabling real time genomics for diagnostics, transmission investigation and infection control.

What will I learn?

The practical part of the course will provide laboratory sessions that will focus on the preparation of sequencing libraries for metagenomics and PCR-based approaches with particular emphasis on how to improve the efficiency of viral NGS by undertaking variations in library preparation techniques such as target enrichment by probe hybridization.



With only a few mouse clicks as well, a program can create any virus by putting together molecules of short parts of nucleic ac- ids from dead tissue and cells with a determined biochemical composition, thus arranging them as desired into a longer gen- otype which is then declared to be the complete genome of the new virus. In reality, not even this manipulation, called “alignment”, can result in the “complete” genetic material of a virus which could then be called its genome. In this process of theoretical construction of the so-called “viral DnA or viral RnA strands”, those sequences that don’t fit are “smoothed out” and missing ones are added. Thus, a RnA or DnA sequence is invented which doesn’t exist in reality and which was never discovered and scientifically demonstrated as a whole.
In a nutshell: From short fragments, theoretically and according to a model of a viral DnA or RnA strand, a bigger piece is also theoretically fabricated, which in reality doesn’t exist.
For example, the “conceptual” construction of the “RnA strand” of the measles virus with its short fragments of cellular particles lacks more than half of the genetic sequences which would represent a complete virus. These are in part artificially created by bio- chemical methods and the rest are simply invented.

 In Part 2 will discuss some of the background and the concept of variants in virology. Why has there been such an increase  in  ‘virus ‘ variants   in the last decades ? 

In the last decades ?

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