Sick Bees – Part 6: Infection by Multiple Viruses
We beekeepers hear from researchers that our sick bees are full of viruses. Understandably, we want to know what we can do about it. But to most of us, virus infections are a “black box”—a generally invisible, mysterious phenomenon about which we can do little other than to control one of the modes of transmission (via varroa). Our questions to those very researchers are often frustratingly met by more questions. I can assure you that researchers have been working hard to come up with the answers—we gave gained a great deal of understanding these past few years. As Marie Curie once observed: “One never notices what has been done; one can only see what remains to be done.“
Our understanding of viruses and disease has expanded greatly within the lifetime of modern bee researchers. In 1925, the great cell biologist E.B. Wilson wrote: “The key to every biological problem must finally be sought in the cell.” But when Wilson wrote those words the world inside the cell was largely inaccessible. The primary instrument for investigation at the time–the light microscope–was physically incapable of resolving a cell’s finer interior details. The situation encountered by Wilson and his contemporaries could be compared to that faced by astronomers, “who were permitted to see the objects of their interest, but not to touch them; the cell was as distant from us as the stars and galaxies….” .
The development of the electron microscope finally allowed scientists to look deep into the cell—the first virus was directly viewed in 1939 (Fig.1). It wasn’t until 1952 that viruses were first grown in cell culture, finally enabling scientists to isolate strains and observe the cellular effects of infection.
Another huge leap forward occurred in the 1980′s when researchers learned how to amplify DNA by polymerase chain reaction (PCR). This set the stage for in-depth study of the genome; concurrently, the field of epigenetics–the study of the cellular and environmental factors that affect how the genome is expressed–took off. In a mere fifty years, the study of virology went from the macro, to the micro, to the molecular level!
The scope of advancement is apparent in bee research. Most known bee viruses were first isolated and identified (beginning in 1963) by the great bee pathologists Leslie Bailey, Brenda Ball, and RD Woods. They used crude electron microscopy and antisera painstakingly made by injecting viruses into rabbits. Brenda Ball is still active in the field of bee virology, but today has at her disposal such new technologies as real-time PCR, mass spectrometry, and rapid microarray probes. It is amazing to grasp the sheer scope of advances that have taken place in a single career!
Infection by Viruses
Author’s note: I apologize in advance for the rest of this article lacking direct practical applications. However, it is material that I need to cover in order to give you the necessary background before I move on to my next subject, which I suspect will be of more practical interest to beekeepers. That will be an explanation of why we are seeing so many problems in our bees since the arrival of varroa, and in what ways varroa completely changed the dynamics between viruses and bees.
In my previous article in this series, I gave a brief overview of the bee immune response to viruses (you may wish to review the November ABJ before going on). When a virus infects a bee, one of the first things that it does is to trick the bee into producing proteins that suppress various aspects of that bee’s immune response. These immune suppressors may be specific for that virus species, or they may be more generic. So the question then, is what happens in a multiple infection, with each virus producing different immune suppressors? One might assume that the combined effect would invariably lead to virus synergy, but that doesn’t appear to always be the case.
The dynamics of virus-virus interactions are complex and poorly understood. Certain bee viruses may indeed enhance the virulence of other viruses [2, 3]. On the other hand, however, some bee viruses competitively suppress the multiplication of others–KBV suppresses the replication SBV and BQCV ; ABPV interferes with replication of CBPV ; and Bromenshenk’s  data indicate that the iridovirus suppressed DWV (KBV = Kashmir Bee Virus; SBV = Sacbrood Virus; BQCV = Black Queen Cell Virus; CBPV = Chronic Bee Paralysis Virus).
Thus, once a bee is infected by a virus, and that virus’s immune suppression proteins take effect, then that may either open the door for other viruses, or inhibit them. Seeing as how bees in collapsing colonies are often infected by multiple viruses , it looks as though viruses tend to gang up on stressed bees and create serious mayhem.
In some cases an infection by a second virus may cause a dormant virus to start replicating, similar to the way in which a human infected by the flu virus may experience an outbreak of cold sores–virtually every adult human carries herpes virus as an inapparent (not exhibiting symptoms) infection, but you don’t get cold sores unless you become stressed, or contract another virus infection.
I’ve mentioned the word “stress,” which is a rather generic term. In bees, “stress” may be due to chilling, nutritional deficiency, environmental toxins, or infection. The most insidious form of stress is the targeted suppression of the normal bee immune response by pathogens, notably viruses. This is what makes Human Immunodeficiency Virus (HIV) so devastating—it directly targets one’s immune response, eventually allowing opportunistic pathogens to run rampant, resulting in the syndrome called AIDS (Acquired Immune Deficiency Syndrome). CCD is remarkably like AIDS (CCD could just as well have been called Bee AIDS). And as in human AIDS, in bee collapse events several normally suppressed viruses may explode into active infections.
A number of researchers have found that the mere action of a varroa mite feeding upon a bee may induce or activate the replication of inapparent and normally non pathological virus infections. This physical insult, along with the injection of immune suppressants by the mite [8, 9], and the resultant depletion of bee body protein all contribute to the sort of “stress” that makes varroa-parasitized bees much more susceptible to overt virus infections.
So it may well be that the combination of varroa immune suppression, coupled with additional immune suppression by viruses could throw the bees’ immune response for a loop. Add to that nutritional stress and the energy- and nutrient-robbing effects of a nosema infection, and you could have colonies at the tip point for the initiation of collapse, even though a cursory inspection of the hives would not indicate that anything was amiss!
The bottom line is that we still have a great deal to learn about virus/virus interactions, and more importantly, the complex effects of virus suppression of the bee immune system, especially when more than one virus (and varroa) are involved. Allow me to digress for a moment, after which I will return to this subject.
Adaptation is the heart and soul of evolution. Niles Eldredge
Adaptation is the ongoing process of adjustment of the fitness of an organism to its environment.
A trait is adaptive if it enhances the probability of an organism surviving and reproducing.
The evolution of life forms generally progresses at a relatively sedate pace—over thousands and millions of years. To understand why, imagine taking a perfectly-running complex machine, and randomly changing one part. In general, any random change is going to make the machine run less efficiently. A similar situation exists for all complex organisms—they are the culmination of millions of years of fine tuning (by adaptation) that has resulted in a bunch of parts, elaborate mechanisms, and complex chemistry that must all work in perfect harmony. Any change in any part or process is likely to be nonadaptive. And if a change turns out to indeed be adaptive, then generally a number of other parts or processes must then also change in order to fine tune the modified system. As a result of this restraint on random change, organisms are “cautious” about passing genetic or epigenetic changes to their offspring, as such changes, if nonadaptive, could result in the parent’s germline not surviving into the next generation. This scenario can be applied to all organisms, including parasites.
Viruses, on the other hand, are unlike any other bee parasite, in that they are not really living organisms. Rather, they are (very successful) mindless packets of instructions that depend upon the bee ribosomes to act as copy machines. But that very copying process is very unlike that of a an office copier—any copy may well contain a rewriting of those virus instructions in the form of insertions, deletions, or recombinations of specific instructions, or “collating” errors in that the “pages” (genetic information) of different documents might get randomly inserted into another document (the virion being the final “document”).
RNA viruses are notable for not having any correction mechanism for such random copying (or collating) errors. They can afford to be frivolous in this aspect of their reproduction, since any successful virion, rather than producing only a few offspring, may produce millions! And if some of those offspring are nonadaptive, little is lost. However, if the rare virus mutation happens to be more adaptive, then it may immediately start competing with its “normal” brethren, even in the same cell and same bee! (I strongly suggest that the interested reader download (free) Mike Carter and Elke Genersch’s review  of the genetics of bee viruses).
The viruses themselves are mindless, and have no plan, strategy, or agenda. Those instructions that are successful at getting replicated into the next generation of bees are perpetuated; those that are either unsuccessful at infecting a bee, as well as those that are so virulent that they kill the bee before being transmitted to the next bee (or colony) go into nature’s wastebasket (remember this very important point).
The error-prone virus replication process has major implications, as it endows the viruses with enormous genetic “plasticity,” and therefore, the unique ability to quickly adapt to new situations (such as the arrival of varroa, or to any stressor that affects the host bee). We normally think of evolution of a species as occurring over the timescale of tens of thousands of years. Not so with RNA viruses—within mere days, within a single bee pupa, an RNA virus can mutate into a vast pool of novel forms. What then exists in that pupa is a “quasispecies” or “viral swarm” of genetically distinct, although closely related, variants of the virus. So any RNA virus “species” can be more accurately described as a “cloud” of mutants distributed around a generic, well-adapted infectious form (that normally doesn’t kill the bee)(Fig. 2).Now multiply that rapid evolution by every infected bee in the hive! Any particular mutant might be better at infecting a certain bee tissue, avoiding the bee immune response, or be more successful at being vectored by, or reproducing in, varroa mites. Thus, any one of the myriad new forms of the virus has the potential of starting the next virus epidemic in the local bee population! Since the “local” bee population nowadays is homogenized via the migration to almond pollination in California, it is no wonder that new virus epidemics can soon reach every corner of the continent (and also why beekeepers are often cautious about who they set down next to).
Viruses allow us an opportunity to observe the equivalent of “evolution on steroids.” Even during a single season, there is a progression of factors that favor or handicap any of the myriad viral mutations—temperature, nutritional abundance or stress within the colony, rate of growth of the bee population, and mite levels. During spring buildup, for instance, more virulent virus forms are favored, due to the rapid turnover of the bee population. I will return soon to how this affects the dynamics of virus epidemics in bee populations.
Virus Infections are Complex
Virus replication, as I’ve explained, is not merely about cloning an original set of genetic instructions; rather RNA viruses are successful by virtue of their constant evolution in ways that you wouldn’t have dreamed of!
In an infected cell, some parts of the virus RNA strand may replicate into incomplete virus forms. These “defective” virus strands may not be infective in their own right, but may still be able to sneak into newly forming virus capsids (the protective “shell” of a virus) and thus be transmitted to new hosts . In the new host, the defective viruses may be able to continue to replicate by utilizing proteins created by “intact” viruses. These “defective interfering” virus segments may actually suppress the virulence of intact viruses, or may flood the host cells with virus proteins. This makes me wonder what role varroa plays in transmitting not only intact bee viruses, but in also transmitting these defective elements
Or maybe something else is also taking place: “Co–infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources” . The “defective interfering” virus strands can evolve into what are known as “cheater” or “satellite” viruses—viruses that cannot infect or transmit on their own, but are able to do so only when another “helper” virus is present, by “stealing” some of the helper virus’s proteins. An example of the above is Chronic Paralysis Virus Associate (CPVA), which requires the presence of CBPV in order to replicate . These defective viruses can then alter the susceptibility of the host to the intact virus by activating or inactivating the host immune response.
So viruses can act as vectors and accomplices for rogue self-replicating proteins (ever looked into prions?) or defective RNA segments. Can it get any weirder than this?
It Gets Even Weirder
This whole discussion is starting to sound like the Twilight Zone, but I just can’t stop! An intriguing possibility is that a mixture of several defective viruses in combination could mutually complement each others’ defects, and thereby productively infect cells even in the absence of an intact virus . Indeed, López-Ferber  found that a co infection of both a defective virus and an intact virus can be more pathogenetic that the intact virus alone!
Or one virus’s transmission proteins (that allow it to infect additional cells) may act as “chaperones” for the RNA of another virus. This transmission function is of great interest in understanding Deformed Wing Virus (DWV), especially why it normally appears to be relatively benign, but sometimes multiplies wildly in the wings and other tissues [16, 17] (Fig. 3).
The bee picornavirus genomes are composed of “functional modules,” some encoding “structural proteins” (those used to build the virus “shell”—the capsid), and “nonstructural proteins” (which include the enzymes, micro RNA’s, and other proteins involved in the hijacking of the host cell ribosomal and immune functions). What has been recently discovered, is that these functional modules are able to evolve independently of each other, and even more important, are interchangeable between closely-related viruses! Moore/Ryabov  found that in a co-infection of DWV and Varroa Destructor Virus-1 (VDV-1) that over the course of infection that the main forms of virus produced in the bees were hybrids (“recombinants”) between the two viruses!
So depending upon the primers used by researchers to identify viruses in bees, they may not even recognize that they are dealing with a virulent hybrid! The Moore/Ryabov team’s paper deserves further elaboration, since it, along with groundbreaking work by the great Israeli virologist Ilan Sela, and intriguing studies by Galacias and Genersche, may have opened a window for us to better understand the dynamics of bee viruses and varroa.
Why would hybrid forms of viruses be more successful at infecting a bee? It appears to boil down to the role of varroa in virus transmission. In order for a virus to multiply in a bee, it must first “recognize” the tissues of certain cells to attempt to gain entry, then avoid or suppress the cellular antiviral response, and finally to manage to be transported (viruses can’t move of their own accord) to uninfected bees. Sometimes a hybrid appears to do some aspect of the above more effectively.
What the authors suggest is that the VDV-1 hybrid may be better at being vectored from bee to bee by the mite, and then the DWV component is then more successful at replicating within the bee. Alternatively, by incorporating VDV-1 RNA, the hybrid may be able to escape the bee RNAi defense against DWV.
This brings up another whole subject—some bee viruses (DWV, KBV) are able to infect other species of bees and wasps, and possibly varroa gut cells. The viruses will quickly evolve to better suit the new host. But what happens when those viruses then reinfect a honey bee? Will the adaptation to the other host then make them (initially) more pathogenic to the bee? And does this go back and forth with each mite-to-bee and bee-to-mite transmission of the virus (assuming that a virus replicates in the mite )?
Bee-Virus Co evolution
If you’re still with me, I commend you! I’d like to return now to the adaptive evolution of bee viruses, as I feel that the study of this will help us to understand why bees are having such a hard time these days.
Professor Ilan Sela of Israel has been on the cutting edge of this field of research, first in plants, and lately in honey bees. He surprised bee virologists when he first discovered that honey bees in Israel had incorporated IAPV sequences into their own genome, thus conferring them with resistance to the virus .
Professor Sela explained to me:
“What we have found is that about 3-10% of bees infected with IAPV are acquiring viral sequences…into their genome. Some of these become virus resistant. However, when we did a field survey (in Israel) we found that about 30% of the bees carried integrated viral sequences, and I suspect that the proportion is increasing. We also found that over 70% of bees in a large USA apiary which suffered significantly from CCD in the last 2-3 years carry viral sequences.
“As to the general question, integrated sequences that are not stabilized within the genome (these are the majority of occurrences) will gradually disappear, and at most (only if you look specifically for them) will be categorized as “junk” DNA, with no evolutionary importance. However, the infrequent occurrences in which the viral sequences have been stabilized (and we have parameters for these) may bring about new phenotypes and might have a great effect on evolution.”
These bee/virus combinations are called “chimeras” (after the mythical lion/goat/serpent creature). But that’s only the half of it; the sequence exchange is reciprocal–Sela also found that viruses can incorporate portions of the bee genome (which may confer the virus resistance to the bee immune response):
Sela continued: “The resultant virus–host chimeras, stabilized, and possibly replicative as “parasites” of the native virus, or even encapsidated within viral particles, can now be transmitted horizontally to other host individuals, to other host species, or even acquire a new host range.”
This phenomenon has stunning implications for our understanding of bee/virus co evolution. Eyal Maori explained to me that the viral capsids can function as “genetic spaceships” that can deliver non-viral DNA or RNA from one organism to another other. Should such a “spaceship’s” contents manage to get into a queen or drone germ cell, then the new genetic material could be passed to the next generation.
In other words, viruses can act as effective vectors of genetic material between different organisms, and can (albeit rarely) transfer genetic material, say, from the varroa mite, bumblebee, or yellowjacket into the honey bee! Their point is that all organisms are in reality “transgenic,” and that viruses play a critical role in vectoring genetic material that can accelerate the evolutionary process!
Varroa Changed Everything! Why have our bee problems so increased these past several years? And a look into the future.
References and Suggested Reading
The following three papers are free downloads:
Aubert, M (2008) Virology and the Honey Bee. (Google the title) This recent review contains a wealth of information—I highly recommend it for the serious beekeeper.
Obbard, DJ, et al (2009) The evolution of RNAi as a defence against viruses and transposable elements. Phil. Trans. R. Soc. B 364: 99-115.
Lauring AS, Andino R (2010) Quasispecies Theory and the Behavior of RNA Viruses. PLoS Pathog 6(7): e1001005. doi:10.1371/journal.ppat.1001005
 Smith-Sommerville 1995 http://www.rockefeller.edu/rucal/journey/journey.html
 Xi, D, et al (2007) Characterization of synergy between Cucumber mosaic virus and Tobacco necrosis virus in Nicotiana benthamiana. Journal of phytopathology 155(9): 570-573.
 Reviewed in Ribière, M, B Ball and MF Aubert (2008) Natural history and geographical distribution of honey bee viruses in Virology and the Honey Bee.
 Anderson, D. L. & AJ Gibbs (1988). Kashmir bee virus (KBV) occurs in Australia and the United States, but is not reported in Hawaii. Am Bee J 137: 767–768.
 Bailey, L and RG Milne (1969) The multiplication regions and interaction of acute and chronic bee-paralysis viruses in adult honey bees. J. Gen. Virol 4: 9-14.
 Bromenshenk JJ, Henderson CB, Wick CH, Stanford MF, Zulich AW, et al. (2010) Iridovirus and Microsporidian Linked to Honey Bee Colony Decline. PLoS ONE 5(10): e13181. doi:10.1371/journal.pone.0013181.
 Cox-Foster, DL, et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318: 283-287.
 Yang, X. & Cox-Foster, D. L. (2005). Impact of an ectoparasite on the immunity and pathology of an invertebrate: Evidence for host immunosuppression and viral amplification. Proc Natl Acad Sci USA 102: 7470-7475.
 Gregory PG, Evans JD, Rinderer T, de Guzman L. 2005. Conditional immune-gene suppression of honeybees parasitized by Varroa mites. Journal of Insect Science 5:7, available online: insectscience.org/5.7
 Carter, M and E Genersch (2008) Molecular characterization of honey bee viruses, in Virology and the honey bee.
 Maori, E, E Tanne, I Sela (2007) Reciprocal sequence exchange between non-retro viruses and hosts leading to the appearance of new host phenotypes. Virology 362: 342–349.
 Dennehy, JJ and PE Turner (2004) Reduced fecundity is the cost of cheating in RNA virus ϕ6. Proc. R. Soc. Lond. B 271(1554): 2275-2282.
 Ball, B.V., Overton, H.A., Buck, K.W., Bailey, L. and Perry, J.N. (1985). Relationships between the multiplication of chronic bee paralysis virus and its associate particle. J. Gen Virol., 66: 1423-1429.
 Thompson, K and J Yin (2010) Population dynamics of an RNA virus and its defective interfering particles in passage cultures. Virology Journal 7:257
 López-Ferber, et al (2003) Defective or effective? Mutualistic interactions between virus genotypes. Proc. R. Soc. Lond. B270: 2249-2255.
 Yue, C and E Genersch (2005) RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor). Journal of General Virology 86- 3419–3424.
 Moore, J, et al (2010) Recombinants between Deformed wing virus and Varroa destructor virus-1 may prevail in Varroa destructor-infested honeybee colonies. JGV Papers in Press. Published October 6, 2010.
 Boncristiani, HF, G Di Prisco, JS Pettis, M Hamilton and YP Chen (2009) Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera. Virology Journal 2009, 6:221
 Chen, Y, et al (2004) Multiple virus infections in the honey bee and genome divergence of honey bee viruses. Journal of Invertebrate Pathology 87: 84–93.
 G. Palacios, et al (2008) Genetic analysis of Israel Acute Paralysis Virus: distinct clusters are circulating in the United States. Journal of Virology 82(13): 6209–6217. Free download http://www.ask-force.org/web/Bees/Palacios-Genetic-Analysis-2008.pdf