The US Food and Drug Administration does not want Rotarix, the rotavirus vaccine, to be used because it contains porcine circovirus 1 DNA. If complete copies of the circovirus genome were present, would they constitute a potential threat to recipients? Put another way, is circovirus DNA infectious?
Here is the information you need to answer this question.
- The circovirus genome is a circular, single stranded DNA molecule (pictured).
- To infect a cell, the two viral proteins encoded in the DNA must be produced.
- To produce proteins, mRNA must be synthesized from the viral DNA.
- Single-stranded DNA cannot be copied into mRNA; the DNA must be double-stranded.
- The circovirus particle consists of a protein shell surrounding the viral DNA. There are no other components in the virion.
- During infection of cells by circoviruses, the particles enter the nucleus where the viral DNA is released
- If naked DNA is added to cells, a good fraction ends up in the nucleus.
Knowing these facts, can you determine whether introduction of circovirus DNA into cells would lead to viral replication?
Another dose of sanity. Thank you Prof Racaniello.
Why “Single-stranded DNA cannot be copied into mRNA; the DNA must be double-stranded”?
Because the enzyme that copies DNA into mRNA – known as RNA polymerase
II – cannot recognize a single strand of DNA. It will only bind to and
copy a double-stranded DNA copy.
I don't think it would since the viral proteins are needed. Single stranded DNA can be converted to dsDNA but would need a primer, so the viral proteins must be aiding this process (it is circular so it can't fold up on the ends). Without the conversion the viral proteins can't be made so it'd be dead in the water. (Right?)
Sorry for revealing my ignorance, but I've got two questions.
1. Isn't TLR9 directed specifically against ssDNA?
2. How does the replication cycle of this virus proceed if it only delivers ssDNA into the nucleus and has noting but this DNA inside the capsid? Isn’t it supposed to have a primer at least?
Animal circoviruses belong to the Circovirus genus of the Circoviridae family. Nowadays, only swine and birds were identified as circovirus hosts. Circoviruses have a single-stranded circular genome of approximately 2000 nucleotide long. DNA of these viruses possesses : (i) a nonanucleotide sequence essential for replication, flanked by inverted repeat sequences, a palindrome that has the potential to form a stem-loop structure and (ii) two major ORFs, located on the viral and complementary strands, which encode respectively the replication-associated protein (Rep) and the capsid protein (Cap). All the circoviruses described at the present time, except porcine circovirus of type 1, are associated with immunosuppressive or immunodepressive diseases. Histopathological lesions such as cytoplasmic inclusions of virus in histiocytic cells and T and B lymphocyte depletion in lymphoid organs, are commonly noticed. No medical prophylaxis of circovirus infections is currently available. (source : http://www.jle.com/en/revues/bio_rech/vir/e-doc…)
If Rotarix's porcine circovirus can manage to be incorporated using its palyndrome replication site sequence into human cells genome, it can potentially be replicated. Knowing that, last step is to verify if it can be pathogen or not while replicating.
Can´t the origin of replication which in circoviruses are a stem-loop structure be recognized by host RNA polymeras? This would then give the production of the viral proteins needed to continue the replication.
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Now, I am confused. PCV-1 infects pigs but isn't pathogenic in them. So somehow the virus does this. What is different as far as the vaccine and humans are iconcerned? Are we not seeing the whole virus?
Introduction of circovirus DNA certainly could lead to viral infection. If normally the naked genome is released into the nucleus, then that's the only condition that must be met for circovirus replication. Presumably the genome is primed, made double-stranded, then transcribed into infectious mRNA. If indeed the complete genomes are present and these cells are competent for circovirus replication, then there's virus!
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have any experiment been done about tthis?
have any experiment been done about tthis?
Your blog is interesting and is a hot potatoes currently!
some studies have demonstrated that PCV1 can be infectious on PK15 cells (porcine cells) –
the best infectivity test is with SPF pigs with serological assays by Elisa and Q-PCR
of course Q PCR is the most sensitive test for the moment, but it detects only DNA, not infectious particles
Human cell lines can be infected with porcine circovirus type 1&2. Infection of PCV1 was observed with HEK 293 & Hela cells. PCV1 replicates well in PK15 cells. PCV1 can persist in cell lines without causing any visible changes, while PCV2-transfected cells can show a cytopathogenic effect. PCV contamination could possibly occur from the use of porcine trypsin in a cell lines history. Since most cell lines have been exposed to this reagent, the screening of all such cell substrates used for manufacture of clinical material is recommended
I am afraid that I do not agree with the statement that: “single-stranded DNA cannot be copied into mRNA”, IN THE CASE OF PORCINE CIRCOVIRUS type 1 or TYPE 2. Please refer to replication mechanism of porcine circoviruses for details.
I also think, naked-intact porcine circovirus genome could be uptaken into the cell, and what will happen?
Single stranded DNA must be made double stranded before it can be
copied into mRNA. DNA-dependent RNA polymerases do not recognize ssDNA
templates.
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