“New Virus Breaks The Rules Of Infection”! No – no, it doesn’t

31 August, 2016

I was prompted to this post by the breathless and much-hyped response to the discovery – the repeated discovery should I say; there was an earlier one that gets glossed over – of a multicomponent flavirus-like virus, this time in mosquitoes.

The actual report was published here: it is a well-done study, describing

“…a genetically distinct, segmented virus isolated from mosquitoes that also exhibits homology to viruses in the familyFlaviviridae and that appears to be multicomponent …, with each genome segment separately packaged into virions”

The authors say

“Although multicomponent genomes are relatively common among RNA viruses that infect plants and fungi, this method of genome organization has not previously been seen in animal viruses [my emphasis]

…which is why there’s all the hype, of course: claiming the virus “…breaks the rules of infection” is simply incorrect, because it is in fact related to very well characterised single-component ssRNA+ viruses of arthropods and mammals – flaviviruses – and infects its mosquito host exactly as these do, except with its genome in separate particles. Which makes it similar to quite a few plant viruses, several of which are, incidentally, probably evolutionarily related to viruses infecting insects – but more later.

Thus, a claim like “…a new study published Thursday is making researchers rethink how some viruses could infect animals” is simply hype.  But it is a sort of hype familiar to plant virologists, who after all showed that multicomponent viruses (=viruses with multipartite genomes packaged in separate particles) existed over 50 years ago – and who also showed that gene silencing was a factor in plant resistance to viruses long before their better-funded animal-researching colleagues got in on the act, but that is another story.

The way in which multicomponency was discovered with plant viruses is interesting: it relied on the fact that plants can respond with local lesions – qualitatively the same as plaques in bacterial or animal cell lawns – to mechanical infection, and that this can be used an an accurate assay of virus titre, as for phages or animal viruses (see here).  It became evident, though, that certain plant viruses produced significantly steeper lesion vs dilution curves than were expected from “one-hit” kinetics, where infection with a single virus particle sufficed to cause a lesion.

This is best shown by a plot like the one below, modified from REF Matthews’ Virology, 3rd Edn, attributed to Lous van Vloten-Doting from 1968.  This shows the curves obtained from accurate and painstaking local lesion assays with the single-component Tobacco necrosis virus (TNV), and the multicomponent Alfalfa mosaic virus (AMV): both are ssRNA+ and have isometric particles, but TNV has a single-component genome, and AMV a tripartite genome packaged in 3 particles.

multicomponent

The insect virus investigators did much the same thing:

“We used a similar approach to assay the nature of segment packaging for GCXV using cell culture plaques instead of leaf lesions. The dose-response curve for GCXV differed significantly from expectations for a single-component virus (i.e., the number of plaques decreased more quickly than expected with dilution of the inoculant)…we used our dose-response curve to estimate the presence of 3.27 ± 0.37 distinct GCXV particles required for plaque formation”

…but with the addition of rapid sequencing techniques not available in 1968, to show that indeed, the different segments were 5 distinct pieces of ssRNA, 3 mono- and 2 tricistronic (=3 ORFs), with the 2 largest monocistronic pieces being similar to flavivirus NSPs and the 3 smallest not encoding anything similar to sequences in the databases.  Four RNAs were essential for infectivity, while the smallest appeared dispensable.  Particles formed during infection of cultured cells were enveloped and 30-35 nm in diameter, considerably smaller than flavivirus virions.

This is a very interesting finding, although not unique: similar viruses were previously found in ticks in 2014, when the authors claimed that:

“To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented [flavi]viral ancestors

They were also wrong: there are a number of viruses for which this could have been said years ago, like the picornavirus superfamily-related comoviruses of plants. These have two-component genomes which both encode polyproteins, one with non-structural and the other with structural ORFs.  In fact, an evolutionary precursor to such viruses could be the more closely picornavirus-related dicistroviruses of insects, which have a classic picornavirus precursor polyprotein ORF split into two, with the structural protein ORF at the 3′ end and the regulatory or non-structural polyprotein at the 5′ end.

I got into this because it irked me mildly that such a fuss was being made of a second group of animal-infecting multicomponent ssRNA viruses, when the multicomponent plant virus precedent and history was VERY well established – but then got more interested when speculation started about what advantage multicomponency could confer on a virus.

I have thought for years that people discussing this generally have it backwards: it’s not that having a divided genome in separate particles offers advantage(s), it’s that it is not a DISadvantage in some circumstances – and particularly where there is no selection against the state.

A reason that multicomponency HAS been seen quite frequently with plant viruses could be that mechanically-transmitted viruses can reach VERY high concentrations in infected plants, and even obligately vector-transmitted viruses (eg: the bicomponent ssDNA begomoviruses, multicomponent ssDNA nanoviruses) reach quite high concentrations in the phloem tissue to and from which they are transmitted, compared to viruses in vertebrates.

This is also true for viruses of arthropods compared to vertebrate viruses: dicistroviruses in aphids can reach concentrations that are comparable to those of viruses like TMV in plants, to the point that aphids inject enough virus into plants that our lab originally mistook Rhopalosiphum padi virus for a plant virus. Moreover, plant virus virions often aggregate into quasi-crystalline arrays which can be hard to separate and which are even visible inside insect vectors, thus virtually guaranteeing that >1 virion will be present in any inoculum, even if significantly diluted.

This is most definitely NOT the case for vertebrate viruses, even where the same virus infects both an arthropod and a vertebrate host: the titre in the latter is guaranteed to be orders of magnitude lower, largely due to a more sophisticated immune system keeping viraemia in check. Thus, high inoculum concentrations relative to vertebrate viruses, and a tendency to aggregate, mean there is no DISadvantage inherent in multicomponency.

Having said this, there may be advantages to having a multicomponent genome: one such is presented in a recent article by Sicard et al. (2013), (thanks, @LauringLab and @DiagnosticChick!) in a study of the ssDNA nanovirus Faba bean necrotic stunt virus (FBNSV), which has an 8-component genome of ~1 kb/segment, encapsidated in 8 virions. They proposed:

“…that the differential control of gene/segment copy number may represent an unforeseen benefit for multipartite viruses, which may compensate for the extra costs induced by the low-frequency segments”

Thus, multicomponent viruses may achieve the sorts of gene dosage control only possible in viruses with larger genomes, by virtue of having multiple genome components rather than control elements which add genomic bulk.

Another possible advantage that I recall being touted by plant virological luminaries is the ease of reassortment compared to recombination: this is exemplified by the reo- and orthomyxoviruses, albeit in vertebrates, where they are constrained by having to have all genome components in the same capsid to guarantee infectivity.

I think Vincent Racaniello is correct in the breathless article I quoted in opening, where he is quoted as saying

“There’s so much we don’t know about viruses…We should always expect the unexpected.”

Absolutely. And I think it’s a safe bet that a LOT more multicomponent viruses will be found in arthropods – and even in some vertebrates, to which they will have been transmitted by arthropods. Because that’s the link between many of these viruses: an evolutionary history that involves plants and arthropods, or arthropods and other animals, at an early stage of life on land. Because that’s all there was for advanced eukaryotes, early on: primitive vascular plants, insects that preyed on them and on each other, and protists.

Bunyaviruses in protozoa?

25 August, 2016

A Novel Bunyavirus-Like Virus of Trypanosomatid Protist Parasites

  1. Natalia S. Akopyantsa,
  2. Lon-Fye Lyea,
  3. Deborah E. Dobsona,
  4. Julius Lukešb,c,
  5. Stephen M. Beverleya

ABSTRACT

We report here the sequences for all three segments of a novel RNA virus (LepmorLBV1) from the insect trypanosomatid parasite Leptomonas moramango. This virus belongs to a newly discovered group of bunyavirus-like elements termed Leishbunyaviruses (LBV), the first discovered from protists related to arboviruses infecting humans.

 

OK, seriously interesting viruses – BECAUSE “…The L. moramango virus thus resembles a group of related viruses discovered recently in the closely related human parasite Leishmania”…which I will note, whose phylogenetic affinity to other higher eukaryotes is via a relationship to trypanonosomes, and then – Euglena??  Which is an alga….

Seriously: a bunya-like virus found in a protozoan whose closest affinity to other hosts of bunyaviruses is via algae??
This pushes the possible evolutionary origin of bunyaviruses faaaaaar back, to possibly a billion years or so, when fungi were separating from protists from algae…..
There is another option, however: I note the Leptomonas sp. is a parasite of insects. It is of course worth noting that the Leishmania and other related parasites infecting mammals are also all arthropod-vectored – which could imply an origin in arthropods.
Which makes good sense, considering these beasties date back less than 600 million years or so – which is still pretty good for a virus?!

So: will smallpox come back to kill us, from the melting permafrost??

17 August, 2016
Variola virus, the agent of smallpox.  Image courtesy Russell Kightley Media.

Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.

There has been a lot of tweeting today about how Smallpox Will Come Back From The Grave And Kill Us All: see here, here and here for lurid examples.

This is alarmism at its insidious best: shouting out a headline, based on flimsy evidence, that says “We’re all going to die!” or similar nonsense.

Really: this IS nonsense.  Some corpses were found in the permafrost in Siberia, that MAY have had smallpox-like lesions on them, and from some of which which smallpox virus DNA could be recovered – presumably by PCR.

This does NOT constitute a threat of live virus being present, or escaping from the corpses even if it WERE there.  I have railed on about this sort of thing before, and I am as unconvinced now as I was then, albeit with SOME reservation about the possibility for smallpox.

"Pithovirus sibericum", from Jean-Michel Claverie and Chantal Abergel

“Pithovirus sibericum”, from Jean-Michel Claverie and Chantal Abergel

I can believe you could get live anthrax: those spores are incredibly tough, and can last for many years in soil, let alone in ice. I could also believe that one could find live megaviruses – the so-called pitho- and molliviruses – in permafrost, because their putative hosts are unicellular protozoans and because they are also seriously stable.

But smallpox? The virus is probably not as stable as the megaviruses mentioned; it relies for infection on its structure, which has membranes integral to it – AND it infects people, who, when they die, don’t cool down very quickly, and whose cells release all sorts of nasty enzymes (lipases, proteases) as they die. Which could be expected to chew up most things, including poxviruses.

Oh, sure, poxviruses CAN survive for years at a pinch – in the form of dried secretions or scabs, which, because they are dehydrated and full of protein, tend to stabilise virus particles. This is how the old variolators and vaccinators (literally: people who used variola or “vaccine” to vaccinate against smallpox) used to preserve their inocula, when they weren’t using fresh material.

Melting tundra is not like that, I will note: bodies with intact virions in them will thaw and rot all over again, and that rotting will reduce what little virus there may be even further.

So I am not a believer in Death From The Permafrost!

And nor should you be.  But it might not hurt for someone qualified to test whether or not there IS live virus in frozen samples, by culturing an extract?

TIJoCV honours World Hepatitis Day

28 July, 2016

IJoCV.2

Graphics by Ian Mackay and Susan Nasif.

A word from the Comics Editor

26 July, 2016

_2__Twitter___Mentions


Renaissance Virology Comics: Get the Facts with Virology Comics!

#VirologyComics (Every Tuesday)

 “The exponential increase in health-related online platforms has made the Internet one of the main sources of health information worldwide. However, online communities with greater freedom of speech have, regretfully, become a powerful platform for anti-vaccine voices and the sharing of defective medical information. Health communicators have not yet taken their responsibilities on digital media as seriously as it should be.” (quoted with a few additions)

Therefore, the main objective of Virology Comics, the winner of the Science Hero Award in 2015, is to raise public awareness, inform readers about routes to prevent the transmission of viral diseases, and to provide basic knowledge and teaching tools for health professionals and educators in an easy, enjoyable, inspirational, and informative way.

Virology Comics’ dream is big, its ability is massive and its greatest strength is determination. We realize that dreams do not become realities without sweat and hard work, but we also know that getting the significant interest & support of like-minded and generous individuals makes a huge difference. I hasten to say that I am not only an artist, but also an artist with a medical background (Ph.D. in Virology). This is a rare combination, which only adds to the uniqueness of my work.

If you enjoy this material, I ask that you consider supporting it: cartooning is expensive, unfortunately, and we need all the resources we can get!  If interested, you can click on the link to the Patreon site to provide a regular monthly financial contribution of as little as $6:  https://www.patreon.com/VirologyComics

I would like you to enjoy, via the Internet Journal of Comprehensive Virology’s site, the outstanding story of Zika Virus Comics straight from The Lancet, published some time ago, and ask you to remain in the loop for more episodes! Learn, Share & Enjoy!

https://www.youtube.com/watch?v=gWjiWUNoNQU

Thank you very much.

Susan Nasif, Ph.D.

Why did you access ViroBlogy today?

25 July, 2016

The Internet Journal of Comprehensive Virology

15 July, 2016

 

See Home Page for details

New developments in a South African HIV vaccine trial

7 June, 2016
HIV life cycle - Russell Kightley Media

HIV life cycle – Russell Kightley Media

Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap 

A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 109 PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4+ T-cell and CD8+ T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4+ T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4+ T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.)

This is a pretty big deal – because it reports an extension of a wholly South African-originated vaccine trial, that consisted of a DNA prime with a subtype C gp150 gene and an artificial Gag-RT-Tat-Nef polyprotein gene, followed by a rMVA boost, that was as immunogenic as anything else trialled around the same time.

And development of which was shut down for political reasons in 2009, but that is old news….
This new development, where a subtype C gp140 (soluble form of Env) was given with MF59 adjuvant to trial participants 2 years after the initial vaccinations, showed that recall responses were strong – in both cellular and humoral arms of the immune system. Moreover, neutralising Ab were elicited.
This is a very promising development in the saga of HIV vaccinology, and it is to be hoped that further trials will be funded.
And both my sister-in-law and my wife are involved B-) What can I say, we’re a virological family!

AIDS: 35 years old this month

6 June, 2016
HIV particle.  Russell Kightley Media

HIV particle. Russell Kightley Media

I was alerted via Twitter this morning to the fact that the CDC’s Morbidity and Mortality Weekly report that reported the first recognition of the syndrome we now know as AIDS, was published on 5th of June 1981.  It appears – sadly – that their archive only goes back to 1982: there’s a missed chance to expose some history, CDC?!

Thirty five years: I was a novice lecturer, just starting out; the Web was still science fiction; HIV and its relatives were still undiscovered – but they had already started to spread out of Africa, after smouldering away in the tropical forests of Gabon and the Congos for decades.

I started an information web page on HIV/AIDS back in 2000 or so, largely in response to the ridiculousness of Thabo Mbeki’s pronouncements on the virus and the disease: thanks to tectonic shifts in the UCT Web policy, these disappeared – but thanks to the invaluable Wayback Machine, can still be found.  If you want a slice of history, and to see how bad I am at designing web pages, go take a look. Still MOSTLY valid, although many of the links are now dead – sic transit the web content, unfortunately!

And here we are in 2016: I’m now an elderly academic, the Honours student who alerted me to the fact the the “GRIDS” syndrome virus may have been identified in 1983 is now a senior Professor and distinguished HIV researcher – there’s a whole career there, Carolyn! – and HIV/AIDS is still with us. And unfortunately, Thabo Mbeki is still being wilfully if not malevolently ignorant, and I am still feeling it necessary to crap on him.

At least the pandemic appears to have peaked in terms of incidence, and ARVs are increasingly good and employed widely; however, we still don’t have a decent vaccine, and people are still being infected. This pandemic will last out my career – but hopefully not those of some of the people I have trained.

New Approaches to Vaccines for Human and Veterinary Tropical Diseases. Or maybe sophisticated safari science?

27 May, 2016

The Keystone Symposia organisation held a meeting entitled “New Approaches to Vaccines for Human and Veterinary Tropical Diseases” in Cape Town this week (May 22-26, 2016).  A summary of the meeting was given as:

Human and livestock vaccines can contribute to improved human welfare and income generation by maintaining human health and meeting the demand for meat, milk and fish in developing countries. All of these factors contribute to the growing importance of improving food safety, availability and nutritional security. An important component of this Keystone Symposia meeting will be to stimulate crosstalk between the human and veterinary vaccine communities by highlighting cross-cutting technical advances and new science and knowledge from laboratory and field research. The meeting will also provide a rare opportunity for scientists from the Northern and Southern hemispheres to interact and pool resources and knowledge in the common fight against tropical diseases.”

It succeeded admirably in a couple of these goals: there were delegates there from 31 African countries, as well as many Europeans, Brits and Americans; the juxtaposition of veterinary and medical talks on similar themes created an excited buzz among folk who hadn’t been exposed to the “other”; there was a wealth of dazzling new tech on display in talks, and intriguing insights into how similar – and sometimes, how different – human and animal responses to vaccines were.  It was obvious that approaches used to develop malaria vaccines could benefit animal vaccinology, and indeed, Vish Nene and colleagues from ILRI in Kenya are following some of the same approaches in their work with the East Coast fever disease organism in cattle.

But, there were a couple of buts.  An important one for me was that while there were many Africans there, they were not much exposed in talks, apart from several South Africans. While amazing results were displayed from deep sequencing of antibody gene repertoires of humans and animals and how these developed with affinity maturation; while grand predictions were made as to how bioinformatics and molecular design would revolutionise vaccinology – this was more of the same kind of thing we have got used to in HIV vaccine meetings over nearly twenty years, where Big Science is always going to provide a solution, but never quite gets to it. Why was there no mention of ZMapp antibody therapy for Ebola, when this (OK, I’m biased) was the single most exciting thing to come out of the Ebola outbreak and the international response to it?

I hate to be cynical, but seriously: is there one single vaccine in advanced human trial right now that is a result of intelligent molecular design? Has ANYTHING that has been designed from crystallographic evidence or from cryoEM data actually proven useful in animals or people?  Has dissection of the anti-HIV antibody response development actually, really, taught us anything useful about how we should develop vaccines? Even if South Africans were involved?

I told you I was cynical – and my cynicism was reinforced by a couple of displays of “My Ebola vaccine is better than YOUR Ebola vaccine!”, by folk who shall remain nameless – when it was obvious that both ChAdOx and rVSV vaccines have their merits.

Mind you, the tale of how Ebola vaccines were deployed so rapidly, and how what could have been a 15+ year saga was compressed to less than a year for the rVSV-ZEBOV and ChAdOx vaccines was truly inspirational. It is indeed an object lesson in how to respond to an emerging disease that big companies and philanthropic organisations were able to make many thousand doses of different vaccine candidates in just a few months, and that these could be deployed in human “trials” – actually, genuine deployment in ring vaccination for the VSV candidate – almost immediately.  Adrian Hill of Oxford asked the question, albeit outside the meeting at a seminar in our Institute: if this was possible for an Ebola outbreak, why isn’t it possible for everything else?  Why can’t we do it for Zika virus, and for MERS-CoV too?

If there is a Big Lesson to come out of this meeting, why can’t it be – Let’s Make Vaccines Faster!

Oh, there were big plusses too.  There were fascinating parallels to be drawn in the approaches to developing vaccines for malaria and TB and animal parasitic infections; some of the fancier techniques discussed for human vaccines could obviously find applications in veterinary vaccinology; there were even suggestions for vaccine candidates for animals that were drawn from homologous genes in human and animal apicomplexans (=malaria-like organisms).

I was especially impressed by Dean Everett‘s talk, from the Malawi-Liverpool-Wellcome Trust Clinical Research Programme in Malawi, on “Developing Appropriate Vaccines through Bioinformatics in Africa”: they were actually working in under-developed Africa, on pressing local problems, and making significant inroads into the problems.

And yet, and yet: I have railed elsewhere about the J Craig Venter Institute’s grandstanding over their “synthetic” organisms; while the talk here by Sanjay Vashee on “Synthetic Bacterial and Viral Backbones as Antigen Delivery Vehicle” went some way to redeeming my negative impression of the use of this sort of work, I am still left with the impression that there are considerably easier ways of doing what they claim to be able to. Mind you, one of my colleagues was very impressed with the possibility of making Herpesmids (=infectious, engineerable whole genome clones) in yeast, and would love to do it with their poxvirus collection – so maybe I am a touch TOO too cynical.

I also felt that the final address, by Chris Wilson of the Bill & Melinda Gates Foundation, on: “Cross-Disciplinary Science to Accelerate the Discovery of Vaccines for Global, Zoonotic and Emerging Infectious Diseases” exemplified some of the problems inherent in trying to marry up developed and developing world science, especially in vaccinology.  Part of the talk was great: he gave the best description I’ve yet heard of why it could be feasible to inoculate Aedes spp. with Wolbachia, and why it could significantly impact transmission of flavi- and other viruses.  His description of gene drive technology for wiping out selected mosquito populations was also succinct, and masterly – and appropriate for a developing world audience. Then he got on to how dissection of antibody maturation pathways and flavivirus E protein design could provide paths to good vaccines, and the cynicism kicked in again.

We don’t need either technology to get to vaccines for HIV or for flaviviruses that we can test in the near future, and which could have very significant impacts on millions of people.

Really: we don’t. Extant HIV vaccine candidates are almost certainly better than the RV144 Thai trial vaccine components, and they had an efficacy of 60% in the first year. We already have YFV and dengue and JEV live vaccines – why don’t we use one or several of them in combination with an engineered YFV vaccine to protect against ALL epidemic flaviviruses?  Given the Ebola example, we could deploy vaccines for HIV and for flaviviruses in a year or less, and they would have an impact that would tide us over while fancier products were being made. Seriously: we are always waiting for the next best thing; let’s just apply what we know and what we have NOW to make an impact – instead of, like theoretical physicists, perpetually considering the problem of the spherical horse instead of just going out and riding one.

And that should have been one of the Big Lessons, and we missed it. Instead, there was an element of Safari Science, which is what we in Africa call the kind of endeavour which involves people from the global North flying in to sort out our problems – and leaving with our organisms and disease samples.

Which we could do ourselves, given funding. And that’s another lesson for the folk that do Big Science funding….

 


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