TWiV 429: Zika Experimental Science Team

Vincent meets with members of team ZEST at the University of Wisconsin Madison to discuss their macaque model for Zika virus pathogenesis.

You can find TWiV #429 at microbe.tv/twiv, or listen/watch right here.

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Viral RNA is not infectious virus!

Zika RNA and virusA study of sexual transmission of Zika virus among mice (link to paper) demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus.

Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. This observation in itself is very interesting but is not the focus of  my comments.

To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid – by both PCR, to detect viral RNA, and by plaque assay, to detect infectious virus. The results are surprising (see figure – drawn in my hotel room).

Zika virus RNA persisted in semen for up to 60 days – far longer than did infectious virus, which could not be detected after about three weeks.

Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood – it detects nucleic acids, not infectious virus.

Despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection, these mice are not likely to transmit virus after a few weeks. There is a lower limit of detection of the plaque assay – approximately 10 plaque forming units/ml – whether that would be sufficient to transmit infection is a good question.

Why Zika viral RNA and not infectious virus would persist for so long is an important and unanswered question that should definitely be studied.

Recently many papers have been published which demonstrate that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm, both by scientists and by science writers. However, in most cases the assays were done by PCR, not by plaque assay, and therefore we do not know if infectious virus is present. Viral RNA would not constitute a threat to transmission, while infectious virus would.

The lesson from this study is very clear – in novel experimental or epidemiological  studies it is important to prove that any viral nucleic acid detected by PCR is actually infectious virus. Failing to do so clouds the conclusions of the study.

There are few excuses for failing to measure viral infectivity by plaque assays. Please don’t tell me it’s too much work – that’s a poor excuse on which to base selection of an assay. Even if your virus doesn’t form plaques there are alternatives for measuring infectious virus.

If you are wondering how a plaque assay is done, check out my short video below.

TWiV 428: Lyse globally, protect locally

The TWiVsters explain how superspreader bacteriophages release intact DNA from infected cells, and the role of astrocytes in protecting the cerebellum from virus infection.

You can find TWiV #428 at microbe.tv/twiv, or listen below.

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Bacteriophage superspreaders

bacteriophage modelBacteriophages are the most abundant biological entities on Earth. There are 1031 of them on the planet, and they infect 1023 to 1025 bacteria every second. That’s a lot of lysis, and it leads to the release of huge quantities of DNA that can be taken up by other organisms, leading to new traits. It seems that some bacteriophages are very, very good at releasing intact DNA, and they have been called superspreaders (link to paper).

In a very simple experiment, E. coli cells carrying a plasmid encoding ampicillin resistance were infected with the well studied phages T4 and T7 and also with a collection of 20 phages isolated from soil, water, and feces in Miami and Washington DC. After the cells lysed, DNA was extracted from the culture medium and introduced into antibiotic sensitive E. coli. Two phages, called SUSP1 and SUSP2, were thousands of times better at releasing plasmid DNA that readily conferred antibiotic resistance. These phages are superspreaders.

Superspreader phages can promote transformation by different plasmids, so their unique talent is not sequence specific. When these phages lyse cells, intact plasmid DNA is released. In contrast, phage T4 infection leads to degradation of plasmid DNA in the host cell. Superspreader phages lack genes encoding known  endonucleases – enzymes that degrade DNA, possibly explaining why plasmids are not degraded during infection. Other phages that lack such endonucleases, including mutants of lambda and T4, also promote plasmid mediated transformation.

Phages SUP1 and SUP2 don’t just spread plasmids to laboratory strains like E. coli. When crude mixtures of soil bacteria from Wyoming and Maryland were mixed with SUP1 and SUP2 lysates from E. coli, antibiotic resistance was readily transferred. One of the main recipients of plasmid DNA is a member of the Bacillus genus of soil bacteria, showing that superspreaders can move DNA into hosts of a species other than the one they can infect.

With so many bacteriophages on the planet, it is likely that there are many other superspreaders like SUP1 and SUP2 out there. The implication is that massive amounts of intact plasmid DNAs are being released every second. These DNAs can be readily taken up into other bacteria, leading to new phenotypes such as antibiotic resistance, altered host range, virulence, the ability to colonize new niches, and much more.

You might wonder if all that plasmid DNA, floating in the environment, can also enter eukaryotic cells – and the answer is yes. No wonder eukaryotes didn’t invent anything.

TWiV Special: Vincent Munster on MERS-coronavirus and Ebolavirus

At the Rocky Mountain Laboratory in Hamilton, Montana, Vincent speaks with Vincent Munster about the work of his laboratory on MERS-coronavirus and Ebolaviruses.

You can find this TWiV Special at microbe.tv/twiv, or listen below.

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TWiV 427: It was a DURC and UV light

The TWiVoids discuss the March for Science, the GOF moratorium, and a classic virology paper on mapping the gene order for vesicular stomatitis virus.

You can find TWiV #427 at microbe.tv/twiv, or listen below.

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TWiV 426: I’m Axl, and I’ll be your cervid today

The sages of TWiV explain how chronic wasting disease of cervids could be caused by spontaneous misfolding of prion protein, and the role of the membrane protein Axl in Zika virus entry into cells.

You can find TWiV #426 at microbe.tv/twiv, or listen below.

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Communication between virus-infected cells

lysis or lysogenyYou might recall learning in high school biology that bacteriophage infection of a host can lead to either replication and cell lysis, or integration of the viral genome into the host (illustrated). The latter event, called lysogeny, spares the host from virus induced killing. For some phages, the decision between lysis and lysogeny appears to be communicated between cells by a small peptide (link to paper).

Evidence that virus-infected cells produce a substance that can regulate the lysis-lysogeny decision came from the observation that conditioned medium from Bacillus subtilis infected with the bacteriophage phi3T – prepared so that is was virus and cell free – protects cells from lysis. The protective component is destroyed by digestion with a proteinase and hence is a protein. Conditioned medium not only inhibits cell lysis, but increases lysogeny, measured by integration of viral DNA into the bacterial genome.

Examination of the genome sequence of phage phi3T suggested that a six amino acid peptide, Ser-Ala-Ile-Arg-Gly-Ala, was the component in conditioned medium that regulates the lytic-lysogenic decision. Addition of the synthetic peptide to infected cells decreased lysis. The levels of this peptide increase during each cycle of phage infection of the Bacillus host.

The authors call the communication peptide ‘arbitrium’ from the Latin word meaning ‘decision’. The gene encoding the peptide is aimP.

AimP appears to work by entering the bacterium through a transporter protein and binding a protein in the bacterial cell called AimR. The AimR protein in turn binds a sequence in the bacterial genome called aimX. When AimR is bound by the peptide, it cannot bind aimX and lysogeny occurs. In the absence of peptide, AimR binds aimX and lysis proceeds. The product of the aimX gene appears to be a regulatory RNA, but how it promotes lysis is not known.

Different phages of B. subtilis also encode peptides that regulate the lysis-lysogeny decision in a phage-specific manner.

These findings describe a viral communication system that determines whether a bacterial host is lysed or lysogenized. When viruses initially infect a host, the result is lysis because levels of peptide are low. After several cycles of infection the AimP concentrations increase, and upon entry of the peptide into bacteria they lead to lysogeny.

The authors of this work suggest that the arbitrium system is a way for the virus to sense the amount of previous infections to decide whether lysis or lysogeny should occur. If many previous infections have taken place, the host population could be too low to support lytic replication, hence lysogeny occurs.  Because lysogens can divide, the bacterial population can be restored to a level that can sustain virus infection.

Of course, the virus particle cannot sense anything – it is a bacterial protein that  binds AimP and another bacterial gene that controls lysis. In other words, the virus-infected cell, not the virus, can sense the amount of previous infections.

It should be straightforward to search the genome sequences of phages that infect other bacteria to determine if such a communication system is widespread. More interesting is whether viruses that infect eukaryotes also have  communication systems that guide decisions about lytic versus non-lytic or latent infection.

TWiV 425: All picornaviruses, all the time

The TWiVaniellos discuss a thermostable poliovirus empty capsid vaccine, and two cell genes that act as a switch between entry and clearance of picornavirus infection.

You can find TWiV #425 at microbe.tv/twiv, or listen below.

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A viral nucleus

Cell typesA unique feature of eukaryotic cells, which distinguishes them from bacteria, is the presence of a membrane-bound nucleus that contains the chromosomal DNA (illustrated; image credit). Surprisingly, a nucleus-like structure that forms during viral infection of bacteria is the site of viral DNA replication (link to paper).

During infection of Pseudomonas bacteria with the phage 2O1phi2-1, a separate compartment forms in which viral DNA replication takes place. A phage protein, gp105, makes up the outer layer of this compartment, which initially forms near one end of the cell, and then migrates to the center. The migration of the compartment takes place on a spindle made up of the tubulin-like protein PhuZ.

In addition to viral DNA, certain proteins gain entry into this compartment, including viral proteins involved in DNA and mRNA synthesis, and at least one host cell protein. Other proteins, such as those involved in translation and nucleotide synthesis, are excluded. This compartmentalization very much resembles that of the nucleus of eukaryotic cells.

Packaging of the viral DNA takes place on the surface of the viral nucleus. Empty phage capsids form at the bacterial cytoplasmic membrane, then migrate to the compartment where they attach firmly to the surface. By an unknown mechanism, DNA moves from the compartment into the capsid. Then  capsids are released from the surface to further mature in the cytoplasm. The completed phages are released from the cell upon bacterial lysis.

These fascinating observations raise a number of unanswered questions. Does infection with other phages lead to assembly of a viral nucleus? How do molecules selectively move in and out of the structure?

Perhaps the most interesting question relates to the origin of viruses and cells. According to one hypothesis, self-replicating, virus-like nucleic acids might have first appeared on Earth, followed by cells without a nucleus. Was the nucleus a viral invention?