Comparison of genomic sequences of hepatitis C virus in plasma samples. phylogenetic analysis
The Hepatitis C virus (Hepacivirus, Flaviviridae), is a virus with single - stranded RNA genome of positive sign (+ ssRNA) with an RNA-dependent polymerase-RNA (RNAD-RNAP), which lacks correction mechanisms so virus replication is subject to errors. These errors are calculated to occur at a frequency of 2.5 x 10 -5 mutations / nucleotide at each viral genome replication. These errors often result in a virus unable to continue its replication and configuration. However, many of the new replicas if they can continue and lead to a variety of virus within a host (individual), which on one hand favors the escape mechanisms of the immune response, and may also condition the resistance to antivirals. This, of viral diversity within a single host is important to consider in order to understand the comparison of the Hepatitis C virus present in two individuals.
In the nucleotide sequence of the genome of this virus exists an important diversity, but within it, at least 68-79% of the sequence is shared between different strains. However the rest of the sequence 20 to 30% approximately, can differ, and therefore the hepatitis C genotypes differ and in subtypes according to the degree of differences between them. 6 genotypes of hepatitis C (1 to 6), and more than 100 subtypes distributed among the six genotypes are known. Genotypes / subtypes 1a, 1b, 2a, 2c and 3a, they represent more than 90% of hepatitis C virus producers in Europe, North America, South America, Russia, China, Japan, Australia and New Zealand. In North Africa, Egypt, Central Africa and the Middle East, predominant genotype 4; South Africa dominates the genotype 5, and in Southeast Asia genotype 6.
The genome of this virus is subdivided into several genomic regions (5'-NCR, C, E1 and E2, NS2, NS3, NS4A, NS4B, NS5A, NS5B, 3'-NCR). The variability of each of these genomic regions of a virus other does not vary in the same way, and therefore speak of conserved regions (which change little Strains other) of semiconservadas regions (partially change between different strains), and variable or hypervariable (they are changing something or rather between different strains).
The absence of RNA polymerase during virus replication mechanisms correction makes it accumulates this diversification in the nucleotide sequence of the different genomic regions of the virus, which cumulatively has resulted in different genotypes and subtypes. On a smaller scale, ie without having to be as many differences to be considered a genotype or subtype different within the same genotype and subtype, they exist in an infected virus populations that differ in their nucleotide sequence individual, although not so numerous, and we can talk about quasispecies (almost different species but not come to be).
When an individual becomes infected with Hepatitis C virus, it can be done with a strain of genotype / subtype specific, while one or more of the quasispecies that had the infecting individual.
Comparison of strains of hepatitis C virus - infected patients between different
Comparison of genomic RNA sequences of hepatitis C virus, is recommended when it is desired to know whether viruses that infect two or more people are similar.
When considering the comparison must take into account the previously mentioned facts regarding genotype, subtype and quasispecies.
For this reason, the first thing to be checked is whether the virus infecting two or more individuals are of the same genotype and subtype. If different, of course it can not be raised any further comparison. For this reason the first test is indicated genotyping / subtype of the virus. This test is usually done by amplification and sequencing procedures comparing conserved regions or semiconservadas (5'-NCR genomic regions or NS5B, usually), although differences in 5'-NCR may not be sufficient to determine subtypes as a region highly conserved.
Moreover, we must take into account, as mentioned, which is very unusual strains of two individuals that may have infected one from the other, are completely identical, self variability is generating strain infecting virus during replication in both the infecting individual, as in the infected individual. These differences will be greater the longer time has elapsed from the time of infection.
Furthermore, it should be noted that although two strains of hepatitis C virus, obtained from two different individuals and genetically compared, allow establish a similarity between them, the chance that they had been infected from a different source could have given each one and with an identical virus that are present in infected individuals.
For this reason, it is generally accepted that phylogenetic analyzes primarily serve to support other lines of evidence, such as epidemiological, justifying that have given the circumstances, contacts, diagnostic and therapeutic manipulations, justifying that both individuals they have been infected from the same source.
- Knowing that individuals suspected of being infected from the same source, have virus with identical genotype / subtype.
- Perform comparative genetic analysis of the NS5B region, and the corresponding phylogenetic tree construction.
- Perform comparative genetic analysis of the NS3 / 4A and NS5A regions, and the corresponding construction of phylogenetic trees.
- Perform genetic analysis of the hypervariable regions of E1 and E2 (this analysis requires the cloning of virus sequences present in each individual because as we discussed there are many quasispecies).
Tests em IVAM I:
- Test genotype / subtype of hepatitis C virus (if unknown).
- Comparison of the genomic region NS5B (amplification and sequencing).
- Comparison of other genomic regions (NS3 / 4A, NS5A).
- Comparison of the hypervariable genomic regions E1 and E2.
Preservation and shipment of sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: over 2 days.
Cost of testing: