In the Vestfold Hills region of Antarctica, a team of researchers have discovered a unique method of genetic exchange happening in a deep lake—so salty it remains unfrozen down to minus 20 degrees. In it, lives members of Haloarchaea, a class of Euryarchaeota that thrive in high salt conditions. These extremophiles are able to thrive by having extremely high rates of horizontal gene transfer, swapping genes with other genera even, to more rapidly evolve. Despite the shockingly high rate of gene sharing, the lake still maintains distinct species with no one dominant species winning out. The lake is incredibly cold, providing very little energy, so the archaea in this area only produce about six generations a year because they have to metabolize and reproduce so slowly.
While people have been aware of the archaeal extremophiles there for some time, it was only recently that anyone noticed their plasmids.
The team discovered plasmids in one strain that were not behaving like regular plasmids. Plasmids are pieces of DNA independent of the host genome which replicate independently and are kept around usually only if they have some beneficial gene to the organism (e.g. antibiotic resistance). What differentiates a plasmid from a virus is the method of transmitting genetic information.
For background, a plasmid typically relies cell-cell contact or “conjugation”–when a sex pilus of a bacterial cell containing the plasmid will combine with another bacterium to transfer the plasmid, or is picked up as naked DNA. Other horizontal gene transfer methods would be transduction (genetic transfer via a virus) or transformation (the collecting of naked DNA from the environment).
Viruses travel, encased in a protein coat, and rely on lysing or budding off their host cell and finding new cells to attach to and enter (or, in the case of retroviruses and lysogens, integrating in the host DNA and being activated later in any of that host’s progeny).
But these plasmids are acting like viruses. The pR1SE (plasmid) encodes proteins that go into the host membrane and allow the membrane to bud into vesicles containing plasmid DNA. These vesicles could then infect more of the archaeal species, plasmid-less members. The plasmids could then replicate themselves in their new host cell.
Besides this just being cool because it’s a way we’ve never seen before of transmitting DNA (and because anything involving archaea in extreme environments is cool), it also may be representative of some early stage of viral evolution.
Viral evolution is a hugely debated topic with no one really agreeing on how viruses came about, whether they evolved many times or once then diversified, which came first in the evolution of life etc., but this does seem to support the idea that at least some groups of viruses may have started as plasmids.
Some people think the obvious answer to viral evolution is gene reduction (a lot of giant virus fans), others think it’s gene addition (what this supports). I think the answer is gradually proving to be both—that viruses have evolved independently many times throughout history, and you could probably find at least a few examples to best support each major theory.
Susanne Erdmann, Bernhard Tschitschko, Ling Zhong, Mark J. Raftery, Ricardo Cavicchioli. A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells. Nature Microbiology, 2017
2. High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake. Ricardo Cavicchioli et al. PNAS. 2013
If, like me, you’ve been reading about the unusually horrific outbreak of cholera in Yemen, you may be wondering how it got so bad. While an understanding of ecology is central to fighting any disease, if feels especially important when discussing cholera, as the current Yemen outbreak is being almost entirely blamed on war resulting in collapsing infrastructure resulting in millions of people losing access to clean drinking water. On top of that, the malnutrition of many children in the area results in them being more susceptible to Vibrio infection.
To add to that, access to rehydration therapy (the common treatment for cholera when intravenous fluids and antibiotics aren’t an option) is low, and the vaccine campaign has been dropped with the justification being that the limited amounts of vaccine would not be as effective in Yemen as they would in areas where less people are infected.
The varieties of Vibrio
The most important vibrio species to human disease are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. Vibrio species have flagella and pili which are important virulence factors–notably the toxin co-regulated pilus. The cell walls of Vibrio contain lipopolysaccharides consisting of lipid A (endotoxin), core polysaccharide, and an O polysaccharide side chain. Vibrio can then be divided into serogroups based on this O polysaccharide (200 serogroups in V. cholerae’s case).
V.cholerae O1 and V. cholerae O139 both produce cholera toxin (which causes a rise in cAMP resulting in the cell losing nutrients, which is why you not only need tons of water, but also need to replenish lost electrolytes). These are the serogroups associated with cholera epidemics. Many strains of V. cholerae do not have this toxin and do not cause epidemics though they may still cause illness.
The O1 serogroup is further subdivided into three serotypes: Inaba, Ogawa, and Hikojima. There are then two “biotypes” of V. cholera O1: Classical and El Tor. These biotypes can be further subdivided but let’s just stop here.
The cholera that were famous for killing lots of people in the 1800s were all of the Classical type. The cholera that is responsible for today’s pandemic is of the El Tor biotype.
The CTXφ phage
V. cholerae secretes cholera toxin. This is the toxin that causes the “rice-water” stool (not diarrhea really, as it’s just mucus and water), resulting in dehydration of the host. Colonization of the small intestine required the toxin co-regulated pilus (coded by the vibrio pathogenicity island).
The genes for cholera toxin are not in the Vibrio genome unless the bacteria has been infected by a CTXphi (CTXφ) filamentous phage, which inserts it’s genome into the V. cholerae genome. The CTXφ can transmit cholera toxin genes from one V. cholerae sstrain to another (via horizontal gene transfer).
Infectious CTXφ particles are produced when V. cholerae infects humans. Phages are then secreted from the infected bacteria without lysing the cell.
Seasonal epidemics inversely correlated with environmental cholera phage presence
Cholera seasons usually make sense as they tend to coincide with monsoon season. But perhaps less obvious (or totally obvious if you’re into viruses) cholera phages have a very dramatic influence on seasonality.
The presence of viruses infecting V. cholerae O1 or O139 inversely correlates with the occurrence of viable V. cholerae in the environment and the number of cholera cases. Both epidemic and nonepidemic serogroups have been shown to sometimes carry lysogenic phages which reproduce and kill epidemic strains. Lysogenic phages integrate into the genome so it replicates with every reproduction of the bacteria
One common O1 phage can use several V. cholerae non-O1/non-O139 strains as alternative hosts.
Having alternative hosts present combined with the lysogenic V. cholerae strains can result in a cholera phage “bloom,” thus lowering the transmission of phage-sensitive, more virulent cholerae strains.
Phage and Vibrio waves controlling epidemics
Cholera outbreaks occur in waves with different serogroups dominating at different times.
The absence of one phage specific for one cholerae type provides an opportunity for that serogroup to begin the seasonal epidemic. However, the phages for that serotype will eventually amplify in the environment and attack this serogroup, ending that epidemic.
A different cholerae serogroup would then be resistant to that first phage or carry it as a prophage in the genome—so it isn’t killing that bacteria. A second epidemic wave from the new dominating serogroup can now occur until phages specific to this new serotype bloom, thus ending the epidemic.
Some serotypes will be resistant to all the phages that were killing the virulent phages in the environment, and these serotypes will occupy the interepidemic periods. These strains usually lack typical virulence factors that would make them particularly good pathogens, but are instead more environmentally adapted than the other more virulent strains.
These resistant serotypes may ALSO harbor prophages—phages integrated in the genome—which kill virulent serogroups and may pick up virulence factors via horizontal gene transfer.
If this happens, new serotypes that were previously not very virulent may emerge as the new epidemic serotype.
Self-limiting seasonal epidemic also probably caused by phage
A naturally occurring lytic phage, JSF4 (lytic meaning it simply lyses the cell), infects and kills Vibrio that are sensitive to it.
In a study from 2005, it was shown that the peak of cholera season was preceded by a peak in V. cholerae presence which was then followed with a peak in JSF4 phage presence as the epidemic ended. JSF4 phages would then also be excreted in the diarrhea of sick cholera patients. So the patients at the end of the epidemic end up ingesting both a lot of V. cholerae as well as JSF4 phage which kills the bacteria. The increase of phage results in the decrease of V. cholerae and the epidemic ends.
This is likely why outbreaks are self-limiting.
V. cholerae O139 spread by turtles
While O1 causes the majority of outbreaks, O139 is confined to Southeast Asia. Recently however, it’s been discovered that soft-shelled turtles in China are big carriers of O139. While a lot of aquatic animals spread cholera, the soft-shelled turtles have been definitively linked to human disease and make excellent hosts as they are unaffected by the bacteria which clings to many of their surfaces and intestines. These turtles are then consumed by people, to spread more cholera to new unsuspecting hosts. If turtles being cute wasn’t a good enough reason to stop eating them, maybe this is?
Jiazheng Wang, Meiying Yan, He Gao, Xin Lu, Biao Kan. Colonization of Vibrio cholerae on the Soft-shelled Turtle. Applied and Environmental Microbiology, 2017
Faruque, S. M., I. B. Naser, M. J. Islam, A. S. G. Faruque, A. N. Ghosh, G. B. Nair, D. A. Sack, and J. J. Mekalanos. “Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages.” Proceedings of the National Academy of Sciences 102.5 (2005)
Faruque, S. M., M. J. Islam, Q. S. Ahmad, A. S. G. Faruque, D. A. Sack, G. B. Nair, and J. J. Mekalanos. “Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage.” Proceedings of the National Academy of Sciences 102.17 (2005)
Murray, Patrick R., Ken S. Rosenthal, and Michael A. Pfaller. Medical microbiology. Philadelphia, PA: Mosby/Elsevier, 2016.
Venezuelan equine encephalitis virus (VEEV) is a nasty virus in horses that causes CNS disorders, resulting in a high biphasic fever, decreased appetite, depression, lethargy, ataxia, stumbling, and in severe cases, nonsupperative meningoencephalitis. So basically, it’s a horse farmer’s nightmare. If it does manage to infect people it just causes some flu-like symptoms and then it’s over, but in horses it can spread through a herd and kill them all.
Ecology of VEEV
VEEV continuously circulates by having an enzootic and epizootic strain where the enzootic strain passes through rodents and causes no viremia in horses. It is specific to the Culex mosquito and circles continuously in the rodent population relatively unnoticed by anyone.
But every decade or so, epizootic strains emerge. These are not specific to a particular mosquito and cause viremia in horses, the amplifier for the epizootic strain. Subtypes of VEE and density of mosquito populations, as well as herd immunity determine how fast the disease will spread.
Serotypes IAB and IC are virulent in horses and produce high virus titers (epizootic).
A study that looked at the history of these outbreaks found there has been convergent evolution from the enzootic ID lineage.
Researchers identify key to epizootic VEEV
A study from 2006 (which in science time is forever ago, but which probably didn’t get the attention it deserved with the general public) wanted to identify mutations mediating emergence of epidemic serotype. They suspected that the origin of all epidemic strains involves enzootic strains and that these strains alter their serotype to induce high-titer viremia sufficient to be amplified in equines. Viruses usually have very small genomes which only contain a small number of proteins. Changing an amino acid can dramatically alter the way a protein may fold or interact with a receptor, so viruses that circulate in a reservoir, and then emerge as epidemics, could be the result of a small number of genetic changes.
In this study, they performed phylogenetic analysis of alphaviruses in the VEE complex to determine genetic differences between the most recent ID strain before 1992-1993 outbreak, and the epidemic IC strain from the 1992-1993 outbreak. They then used reverse genetic approaches to recreate hypothetic emergence of a 1992-1993 outbreak and measured viremia titer and virulence.
They found that the key amino acid changes allowing for an epizootic outbreak were a glycine to an arginine at position 193 in the E2 envelope protein, and a threonine to an arginine at position 213 of the E2 envelope protein.
E2 positive charge mutations have accompanied all epidemic emergences, so they tested these two mutations by making a clone of the “enzootic/epizootic border strain” ZPC738 and added either both mutations, or each one separately.
They found the Gly-193à Arg mutation had no effect on mAb reactivity; enzootic-specific Ab still reacted against strain ZPC738. The Thr-213àArg resulted in antigenicity change from enzootic (mAb 1A1B-9-reactive) to epidemic (1A3A-5-reactive). The Combination of both also resulted in IDàIC serotype change. Individual Arg substitutions had little effect on plaque size, but a combination of both resulted in plaques characteristic of epidemic strains
They ZPC738 clone had a low viremia as expected and the Arg-193 viruses did not produce detectable viremia with no disease seen. The Arg-213 virus however, increased virulence and viremia with the neurological disease and viremia titer consistent with epidemic phenotype.
The double mutant (Arg-213 and Arg-193) produced higher viremia 24hrs after infection but lower viremia 36-48hrs post infection.
The takeaway was that epidemic serotype IC VEEV can arise from a single mutation in enzootic serotype ID. Residue 213 responded to positive selection, and Arg substitution induced high-titer equine viremia. The high titer (104 to 106) and high mutation rate (on the order of 10-4 mutants/nucleotide) means RNA viruses likely have a mutation to be more virulent, broaden tropism, broaden host range, etc. at any given time. Which is terrifying to think about.
RNA virus evolution too often ignored in “vaccine wars”
This study shows the barriers for an outbreak are ecological conditions. Enzootic strains circulate continuously, so vaccines should continue to be used even when there’s no outbreak, which unfortunately often doesn’t happen because people don’t see the point in spending money on a vaccine if nobody’s horses are getting sick.
This paper or at least a summary should certainly be shown to more people as it provides direct evidence of just a single amino acid substitution causing an epidemic and is a great argument for continuing to vaccinate, even if a disease is not common anymore (because maybe it’s not common because the vaccine is stopping it from becoming an epidemic in humans). In the vaccine wars amongst humans, RNA virus evolution and ecology as well as RNA zoonosis is weirdly too often left out of the discussion.
Source: Venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation. 2006. Michael Anishchenko, Richard A. Bowen, Slobodan Paessler, Laura Austgen, Ivorlyne P. Greene, and Scott C. Weaver
Animal viruses typically have a non-segmented genome all packaged together in one particle. Sometimes animal viruses, such as influenza, have a segmented genome—where the genome is cut up into separate pieces—but still, it is always packaged together in one viral particle. This means one particle is all that’s needed to infect a cell. In multicomponent viruses, the genome is split up into multiple pieces AND packaged separately. In this scenario, multiple viral particles must infect one cell.
This setup is not uncommon in plants, although usually there’s only two MAYBE three particles to one virus. But members of a newly described group of animal viruses can have five components.
Guaico Culex virus
Guaico Culex virus (GCXV) was isolated from mosquitoes in Central and South America. So far no other animals have been shown to be infected with any multicomponent viruses (a source of apparent confusion on the internet). The close relative, Jingmen tick virus (JMTV) was isolated from a red colobus monkey but it is not actually a multicomponent virus. It is merely a segmented virus with all the segments in a single virion. Both are members of the recently discovered Jingmenvirus group.
GCXV has five particles to it, but the fifth one does not appear to be necessary for infection and actually seems to provide no apparent advantage to fitness or virulence. This is surprising as while producing different amounts of each segment does make sense, if the virus can infect without one of the segments, how would the segment not be lost?
The evolutionary history of Jingmenviruses is an especially surprising part. Phylogenetic analysis has continuously placed them as being closely related to Flaviviruses, which include, among others, Zika, West Nile, and Dengue. Not only does this show multicomponent viruses evolving independently outside of any multicomponent plant viruses, it also may help provide insight into the evolution of RNA virus segmentation in general. There must be different evolutionary paths that allow for convergence on this phenotype, but it’s surprising this was able to ever evolve in an animal at all.
Vector transmission (which is more of a plant thing), seems to be the best/only way these viruses are able to be multicomponent, but animals have plenty of viruses they get from vectors too so why wouldn’t there be potential for a multicomponent virus to eventually spread from a mosquito to a mammal?
Plant viruses are more prone to coinfection and there’s a lot of examples of common helper and dependent virus relationships, which may explain why more multicomponent viruses evolve in plants. Why this is more common in plants and fungi probably has something to do with plants not moving much.
Very little is known about multicomponent viruses because they don’t cause human disease. But studying viral segmentation is pretty important if you consider that Influenza’s segmented genome has allowed it to go from a mild inconvenience one year, to a deadly pandemic in other years. And studying viral evolutionary strategies more commonly used in plants is pretty important considering plant viruses can wipe out an entire species in a season (plants don’t have fancy immune systems like we do). It would be interesting if they looked at how segment combinations work in these multicomponent viruses and what are the genetic limits of compatibility.
Viruses found in more extreme environments seem to be more morphologically diverse than the viruses that infect mesophilic microbes. Furthermore, the diversity of viruses infecting different domains of life varies greatly. Bacteriophages are almost entirely from one order of dsDNA viruses, with about 96% of all of them being head-tail phages (think T4, lambda etc). Meanwhile animals get infected with ssRNA, dsRNA, ssDNA, dsDNA, and retroviruses. Almost no DNA viruses attack plants and fungi, possibly because they have cell walls (which may have even evolved to protect them from viruses?), but then RNA viruses found a way to swoop in and occupy these new niches.
(Nasir et al. 2014) So far, no RNA viruses have been confirmed to infect archaea, and almost all their viruses are double-stranded DNA viruses. RNA is more labile than DNA at high temperatures (DNA is a much more stable molecule than RNA) so the lack of RNA archaeal viruses could indicate either the first archaea were extremophiles or archaea that were extremophiles benefitted by escaping RNA viral infections, causing an increase in extremophile archaea and decrease in mesophilic archaea.
Acidianus two-tailed virus
So far the only virus that’s been discovered to grow outside of cells. Acidianus two-tailed virus was isolated from a 87-93 ºC, acidic hot spring in Italy. This is an enveloped lemon-shaped virus with 72 open reading frames and tails at both ends. When they infect Acidianus archaea at 85 ºC, they lysogenize. However in stressful conditions such as cold shocking them at 75°C (yeah this is chilly for them) the virus actually lyses the cells releasing virions four days later. This is actually very unique in archaeal viruses, the other ones tend to lysogenize.
Unique life cycle
The virions don’t finish growing though before lysis. They actually emerge as little lemon-shaped, tailess virions and then grow the tails outside the cell. The tail growth seems to be temperature dependent, with them growing tails at 75 ºC but taking over a week, and then growing tails very quickly if the temperature is closer to 85 degrees. While the two tails grow, with NO CONTACT TO THE CELL, the virions shrink. The weird viral morphology of having two long tails may help the virus find hosts.
Something very strange is that these viruses have a gene containing an AAA ATPase domain, something found in motor proteins like dynein and kinesin. But scientists don’t know what it’s for, and while it might make sense for a giant virus to have collect some strange genes it may not need, most viruses tend to want to limit their genome size.
We need to know more
Archaea viruses are understudied and so far, almost a total mystery. Almost all of their genomes have no homologs to anything else discovered. Questions regarding virus evolution, early life evolution, viral ability to cross domains, when did viruses diversify, and how did the first archaea and then eukaryotes come about are still unresolved, so studying archaea viruses would be far from useless.
Archaeal viruses and bacteriophages: comparisons and contrasts, Maija Pietilä-Tatiana Demina-Nina Atanasova-Hanna Oksanen-Dennis Bamford – Trends in Microbiology – 2014
Häring M, Vestergaard G, Rachel R, Chen L, Garrett RA, & Prangishvili D (2005). Virology: independent virus development outside a host. Nature, 436
The Wonderful World of Archaeal Viruses. David Prangishvili – Annual Review of Microbiology – 2013
R. Danovaro et al., “Virus-mediated archaeal hecatomb in the deep seafloor,” Science Advances, doi:10.1126/sciadv.1600492, 2016.
Figure: Consider something viral in your research. Forest Rohwer & Merry Youle, Nature Reviews Microbiology 9, 308-309(May 2011). doi:10.1038/nrmicro2563
Encodes aminoacyl transfer RNA synthetases with specificities for all 20 amino acids. Evolutionarily, probably just a gene collector.
Published in April 7, 2017’s Science, researchers have discovered another group of giant viruses—the Klosneuviruses. They were discovered in Austria, at a Klosterneuburg waste water treatment plant biomass, and ranged from .86 Mb (Indivirues) to 1.57 Mb (Klosneuvirus).
Klosneuvirus has a 1.57 Mb genome coding 1272 genes, 335 or so of which are shared eukaryotes. Like other giant viruses, Klosneuviruses seem to infect protists–Cercozoans and the marine flagellate, Cafeteria specifically. Surprisingly they do not seem to infect Amoebas (which are thought of as the typical host for giant viruses and therefor often used as bait when hunting for new viruses).
Klosneuviruses have managed to evolve complex translation system components including “25 tRNAs with anticodons for at least 14 different amino acids, as well as more than 40 translation-related proteins, including 19 aminoacyl tRNA synthetases (aaRSs) with distinct amino acid specificities, 11 translation initiation and elongation factors, a peptide chain release factor, and several tRNA modifying enzymes.”
The researchers performed a maximum likelihood phylogeny from a concatenation of five conserved genes nucleocytoplasmic large DNA viruses (NCLDVs) and found that Klosneuvirus is evolutionarily most closely related to Mimiviruses but still very distinct due to it’s sophisticated translational system components.
The discovery of Klosneuviruses supports the hypothesis that giant viruses more likely evolved from smaller viruses and their large genome is largely the result of accumulation from host genes. There’s been a lot of different hypotheses floating around since the relatively recent discovery of giant viruses, notably that they may be a fourth domain of life. This discovery makes that seem less likely. Giant virus research will probably be huge in helping to tackle questions regarding viral evolution, the origin of life, and deep evolutionary relationships between bacteria, archaea, and eukaryotes.
Schulz, Frederik, Natalya Yutin, Natalia N. Ivanova, Davi R. Ortega, Tae Kwon Lee, Julia Vierheilig, Holger Daims, Matthias Horn, Michael Wagner, Grant J. Jensen, Nikos C. Kyrpides, Eugene V. Koonin, and Tanja Woyke. “Giant viruses with an expanded complement of translation system components.” Science 356.6333 (2017): 82-85.