Thank you. I'm not actually going to present an overview, because my field is
not chronic fatigue syndrome. I thank you for the opportunity to speak to you,
because what I have discovered is a mechanism that these lytic RNA viruses
can persist for long periods of time in various tissues of human beings.
I will just give a short recap on some of the things that are known about enterovirus infection
and chronic fatigue, but Dr Chia will probably tell you a good deal more after I've done.
There has been enterovirus RNA protein and infectious particles isolated from cases of chronic fatigue,
and it shares the characteristic seen in persistent enterovirus infections that these
positive strand RNA genomes, these genomes, these viruses normally have much
more of the genomic RNA — which also serves as the mRNA — present, than the
negative anti genome. But in cases of chronic fatigue isolation of enterovirus
they detect negative strand RNA at a much greater level.
Also in some cases when they have isolated virus from intact in vivo samples and culture them,
they don't always find cytopathic enterovirus — another characteristic of persistent enterovirus infection.
My own work has been done largely on human
myocarditis and cardiomyopathy, also using the mouse model of this disease.
Similarly to chronic fatigue syndrome enterovirus RNA is not the only cause;
the frequency of its isolation or detection in cases varies a good deal,
20% to 25% is a frequently given percentage, but it varies from study to study.
It's extremely rare to isolate infectious virus from adult hearts:
even when enteroviral RNA is present in those hearts,
if you do classical cytopathic assays for the virus you don't detect it.
And again, enterovirus RNA in cardiac tissues and in skeletal muscle has been
shown to have a predominance of the anti- genome negative-strand RNA at greater
levels than are seen in lytic infections. And this is odd because these viruses
replicate in the cytoplasm of the cells, they tend to have a predominance of
the positive-strand genomic RNA produced, and they get out of the cells by
actually lysing the cells after infectious particles are generated.
In our own studies we've replicated a finding that a lot of people had with a
murine model of coxsackievirus B3 myocarditis — it's the model for enterovirus myocarditis —
in that you could find at day 4, day 8, day 14 and day 21 after the mice had been inoculated with the
coxsackievirus B3, you can find that their hearts, when passed in culture,
produced a high amount of cytopathic effect. But when you went out to the
chronic myocarditis stage, day 28 and longer, you don't find any cytopathic
effect in those cultures. However if you test these cultures of heart homogenates
you find that enterovirus RNA is present during the chronic stage, even though
it's not killing the cells in culture. And again that was an unexpected finding.
This was found also in cases of polymyositis, as well as chronic myocarditis.
When we looked at these cultures we found that when you
looked at RT-PCR using primers that are specific for the very 5′ terminus
of the positive strand, you did not detect any signal in the later stage
chronic myocarditis samples, although you could detect a signal in the wild-type
virus, and at day 14. And you always got a signal with primers
that were further inset into the into the viral genome, indicating that the
defect in these genomes was actually at the very 5′ end of the genome.
And the reason this was unexpected is that these viruses have a very important
structure in this part of their genome. It wasn't totally unexpected that you
could get such a mutation in that this just results from, this just results from
premature termination of the anti-genome transcription, and it's a relatively
common mutation one would expect. However these viruses would normally be
eliminated very quickly because the wild- type virus replicates so much better.
The reason for that is that this region, if you look at it in poliovirus,
deletions of this region of the negative-strand, which corresponds to the 5′ terminal deletions,
result in a very big change in the ratio of positive- to negative-strand RNA.
Remembering that the positive strand is actually the genome
and is necessary both for viral protein production and for infectious virion.
There is a factor that actually binds to the part of the genome that is deleted
in these defective enteroviruses: heterogeneous ribonucleoprotein C,
which is required for efficient positive strand RNA synthesis.
The deletions that we found in the mouse ranged in size, in this cloverleaf structure, from only
7 nucleotides to 49 nucleotides; we've never found a deletion
that was bigger than 49 nucleotides. This particular stem loop structure in the
RNA genome seems to be necessary for replication even in the defective virus.
However, although these viruses are defective and produce much less positive-
strand viral RNA, they do produce viral proteins, and we have not found any
defects in the genome in the open reading frame in the viral proteins.
If you look at 6 hours in the wild-type virus, you see a nice detection of viral
protein, when you can't see anything in the
defective viruses. If you'd go out for 4 times as long, you can't see
anything with the wild-type virus because all the cells are long since lysed;
but in the defective cultures where you don't see cytopathic effect,
you are seeing viral proteins.
OK, these viruses, although defective, are also
encapsidating infectious particles, and we've been able to show that because you
can actually prevent infection by these terminally-deleted defective viruses
using coxsackievirus B3 neutralizing antibody. They also do a very unusual
thing for this type of virus: they encapsidate the anti-genome nearly as
well as the genome. That also reduces the overall infectivity of these viruses.
But it also results in a nearly equal amount of positive- and negative-strand RNA.
So our findings in mice indicated that you could have these type of deletions and
they accounted for the persistent virus in the mice. They replicated very slowly
and we're not cytopathic in culture. They do generate viral proteins and you have
a change in the ratio of the positive and negative strands. A very interesting
finding was that if you inoculated mice with these very defective viruses IP [intraperitoneally],
it would go to the heart, and persist for up to 5 months. That and when you
generally generate TDs in these mice by inoculation of wild type virus,
the persistent infection is seen in the immunocompetent mice, despite their
having a strong anti CD3 immunity. We also found this in human beings: this was
a fortuitous case, we were very lucky to have a collaboration with Dr Oka
in Japan, who had a case of fulminant myocarditis. The reason that this was
lucky for us was that this particular individual had an extremely high titer
of virus in his heart; however he sent us large chunks of
formalin-fixed tissue and we were able to show that despite the fact that this
patient died only 14 days after admission to the hospital, he already had no wild
type-virus at the 5′ end, it was all terminally-deleted. So he had already
progressed to a defective virus, despite the fact that this was a case of
fulminant myocarditis. And we found that we could detect the same type of
deletions in the same region in this human case. We were pleased to find that
this was a modern CVB2 strain, it in fact had a closest
relationship to a CVB2 circulating in the same year in the same region of Japan,
and we weren't able to go beyond a third of the genome from this
formalin-fixed tissue, but we were able to show that it was indeed a coxsackievirus B2.
This also showed us that it didn't have to be a prototype
enterovirus strain that has been studied in the laboratory: modern Coxsackie B2
also already had gone to terminal deletion within 14 days. So very similar finding.
However we've now found that you can generate this type of terminal
deletion not only with coxsackievirus B3 but with other human enteroviruses
using primary cultures of pancreatic and cardiac cells.
What we did was to generate primary cultures of cells from the pancreas and from heart
in both mice and humans, and grow those cultures up. When you passage our viral
strain of CVB3 and other human enterovirus B's in these cultures you
end up with the same terminal deletion. This does not happen when you passage
these viruses in human HeLa and in this case a immortalized murine cardiomyocyte-
like line called HL-1. The difference about these cultures, from the standard
cultures that are used for enterovirus detection, are these cultures are
contact inhibited: when you grow them to confluence, they stop dividing.
We also found that if you put in a terminally-deleted defective virus with
a short deletion, you end up selecting larger
deletions in these cultures. And that replicated what we found in the mouse
inoculated with a coxsackievirus B3 defective virus, that in the heart it
became larger during that 5 month persistence. OK when you looked at
these viruses in these cultures you found that not only did you have, in
serial passage, the loss of the very 5′ end (although the virus remained in
replicating in the passages), cytopathic effect was lost during this period of
passage, but you also found the change to generation of high levels of the
negative-strand anti-genome in those cultures as well, indicating that this is
very similar to what is happening in human tissues with persistent
enterovirus infection. We've been looking at the mechanism for this selection, and
we were drawn to the fact that Dr Semler and Dr Flanagan had shown that
this region of the enterovirus genome (they were working with polio) binds
the hnRNPC at the minus strand part of the genome that's actually deleted.
This is a very common factor in human and murine cells, it's a nuclear
factor and it only is present in the cytoplasm during mitosis. When we looked
at whole cells of our HeLas and cardiac cultures you can see that we could
detect this band across the board, but when we went to cytoplasmic extract,
it took a very long exposure to begin seeing it in the cardiac cells, although
it was present at high levels in the HeLa cells. And I assume that this
essential factor is basically limiting the ability of the virus to replicate in
the wild-type form. So to summarize: we know that these viruses have unique
aspects but ones that are very consistent with the kind of persistence
that Dr Chia is going to talk about; they are defective viruses but they still produce
all the viral proteins, including ones
that have been known in the heart to affect the contractility of the cardiomyocyte.
What they do in other cells we don't know; the interesting things that
we found was not only that it accounted for the what we saw, but you could see
that they don't quickly kill infected cells. So you have an opportunity to have
effects upon immune signaling, and cellular function that you would not see normally.
They also persist in the presence of an active immune
response for a very long period of time. Because they are defective viruses
and because they occur basically in quiescent or differentiated cells,
we we think that they may occur in a variety of different tissues. But it probably
requires a high acute infection to actually seed those quiescent cells with
enterovirus in order to have the selection of these viruses. I also wanted
to thank my collaborators: Steven Tracy who began all this work, and is now
working in type 1 diabetes; Dr Kyung-Soo Kim who did a lot of the
early RT-PCR work and developed it; Dr Tapprich who is looking at
the 5′ NTR structure with me now; Dr Drescher at Creighton University who's
working on a TMEV model; and of course Dr Oka who was
very kind to give us the tissue. Thank you.
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