Hantavirus and leaky vessels

I once saw a video in which the lung of a Bluetongue Virus (BTV) infected sheep was cut open at post-mortem. As the scalpel cut in, it was clear the lung was full of fluid. Lungs full of fluid don't work very well. As a result, sheep infected by BTV, as well as horses infected with African Horse Sickness Virus, will often die by drowning simply as a result of their vasculature leaking fluid into the lungs. I've often considered this reminiscent of what happens with Hantavirus pulmonary syndrome (HPS), caused by new world hantaviruses, including Andes virus and Sin Nombre virus (the latter of which causes sporadic outbreaks across North America). In Eurasia there are related viruses, including Hantaan virus, which cause Hantavirus haemorrhagic fever with renal syndrome (HFRS). Although different, both diseases involve vascular leakage.

A deer mouse: the wild reservoir of hantaviruses in the New World. 

Endothelial cells, those that line the capillaries and other blood vessels, aren't damaged during infection, so how do the vessels become leaky? One suggestion has been that it occurs as a result of the cytokine arm of the immune response, although removing the T cells modulating cytokines appears to have limited impact upon pathology. An alternative hypothesis is that vascular endothelial growth factor (VEGF), which is reportedly elevated during hantavirus infections, degrades vascular endothelium cadherin (VE-cadherin), a molecule with importance for vessel integrity. A recent paper in PLoS Pathogens by Taylor et al contradicts some of this, and suggests a further possibility: activation of the Kallikrein-kinin system (KKS),which leads to the release of bradykinin (BK). BK in turn is an inflammatory molecule that leads to vasodilation and increased vascular permeability.

Although nothing can replicate the real thing, the authors created their own capillaries in a dish and showed that they could be infected. When they looked for a decrease in VE-cadherin (as per hypothesis 2 mentioned above), the levels appeared more or less equal regardless of infection, likewise the amount of VEGF released from the artificial capillaries did not alter significantly as a result of infection.

Home made capillaries: Hantavirus infection (indicated by the presence of nucleocapsid) appears not to alter the expression of vascular endothelial cadherin. Similarly, capillaries infected with ANDV or HTNV still produce VEGF. 

When they looked for BK release as a result of activating the KSS, they found a dramatic increase when the capillaries, or the cells which are used to make the capillaries, were infected with either Hantaan or Andes virus and treated with molecules that would be found in the blood stream of infected patients (FXII, PK and HK). This implies that hantavirus infection induces permeability as a result of a more active KKS.

BK production by cells infected with hantaviruses: Cells infected with HTNV or ANDV produce more BK (and by extension enhanced permeability) relative to mock infected, although less pronounced in the case of pulmonary artery smooth muscle cells (PaSMC).

Clearly, the KKS was working OK, but more so in cells infected with the viruses. An important aspect is the cleavage of HK which ultimately leads to the release of BK. The authors looked at HK cleavage in the presence of FXII and found that cleavage was enhanced in its presence. Going further, when they added an inhibitor of FXIIa (the activated form of FXII), they found the cleavage no longer occurred; FXII is clearly therefore required in the system. 

Measuring resistance: resistance is used as an indication of permeability. Infected cells have enhanced permeability when the KKS factors are applied (A). B-D show the effect on mock, HTNV or ANDV infected cells with the inhibitors CTI (blue) PKSI-527 (red) or HOE 140. 

All of these, and some other, experiments suggest that the KKS and BK liberation may, at least in part, be responsible for the leaky vasculature as a result of hantavirus infection. To look at this the authors used electric cell-substrate impedance sensing to measure the resistance/permeability (leakiness) of confluent layers of endothelial cells infected (or not) with hantaviruses. Cells that were infected, i.e. effectively had an activated KKS and BK present, showed a decrease in cell resistance of up to 50%, compared to a maximum drop of only 10% observed in uninfected cells. The addition of inhibitors against the KSS altered the pattern, reducing the effect observed in infected cells, thus directly implicating alterations in the KKS as being the cause of these changes.

The KKS system and Hantavirus infection: HK and PK are enhanced on the surface of infected cells, leading to cleavage of HK (involving FXII) and subsequent release of BK, and thus enhanced permeability of the endothelial layer. Several drugs are available which target the pathway.

It remains possible, indeed likely, that there are other factors that control the vascular permeability and therefore pathogenesis of hantaviruses. Lungs filling with fluid isn't unique to hantaviruses and there are likely several mechanisms yet to be deciphered. This study does though highlight a new pathway of interest that leads to leaky vessels, and, importantly a pathway for which there are inhibitors: perhaps the leaks can be mended.

Taylor, S.L., Wahl-Jensen, V., Copeland, A.M., Jahrling, P.B., Schmaljohn, C.S. (2013). Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System PLoS Pathogens DOI: 10.1371/journal.ppat.1003470

What's Killing the Koalas?

My current employer made his name working on a retrovirus: Jaagsiekte sheep retrovirus (JSRV). JSRV is a betaretrovirus whose greatest claim to fame is killing Dolly the sheep, but it has revealled many aspects of sheep and retrovirus biology. One of the attributes most associated with viruses is that they're obligate intracellular parasites: without a cell to replicate in, viruses are often little more than a bunch of molecules. Retroviruses take this to the extreme and insert into a cells genomic DNA in order to replicate. At it's simplest this involves the virus like any other infecting a somatic cell, intergrating into the cellular DNA, replicating and exiting. This is the 'exogenous' form. 

Alternatively, if the cell happens to be a germ cell, from which sperm or eggs are produced as a precursor to another individual, the retroviral DNA will be inherited by Mendelian inheritance as for any other gene. When this happens, the virus is now regarded as being 'endogenous'.

In the last few years a type-C retrovirus, Koala retrovirus (KoRV), has been linked to the development of Koala immunodeficiency syndrome (KIDS). As the term suggests, KIDS results in a depleted immune system, resulting in enhanced vulnerability to infectious diseases and cancers. KIDS has become a prominent killer of koalas, particularly those in captivity, where the majority of studies have been performed. KoRV's closest relative appears to be gibbon ape leukemia virus (GALV) which, like KoRV, causes lymphomas and leukemia.

KoRV represents an example of a very young endogenisation event, with the intergation event thought to be only around 100 years ago, and the integrated copies are able to generate infectious viruses. A recent paper in PNAS describes the isolation of a variant of KoRV in San Diego and Los Angeles zoos (Xu et al 2013). All of the koalas at the zoos contained the endogenised form of KoRV. However, in six koalas at Los Angeles zoo, including 3 that died, they found an additional, slightly different, KoRV sequence (KoRV-B, as opposed to the original KoRV-A). The majority of the changes were in the U3 region of the long terminal repeats (LTR). Particularly striking was that parts of the virus responsible for binding to the cell receptor looked more like those of an exogenous virus as opposed to the more endogenous form possessed by KoRV-A. Indeed, when they looked at receptor usage, KoRV-B used a different receptor to KoRV-A and GALV.

Mother to Joey transmission: Joeys only become infected with KoRV-B when the dam is infected, even if the sire is positive, implying the virus is transmissible rather than inherited. (Xu et al 2013)

Further evidence of the infectious nature of KoRV-B came from the observation of infected (or not) joeys born to infected (or not) parents in a family at Los Angeles zoo. A positive joey was only born when the mother was positive; it was possible for a joey to be born negative for KoRV-B even if the father was positive for KoRV-B (as long as the mother is negative).

Koalas would appear in a bad way. However, endogenous retroviruses aren't necessarily harmful. On the contrary, some may be beneficial. A prime example is the JSRVs. The presence of endogenous JSRVs results in so-called 'late restriction'. Interference of exogenous JSRV replication by the presence of endogenous JSRVs is ultimately beneficial for the sheep.

Endogenous JSRVs: a variety of genomic arrangements of JSRVs found in the sheep genome. (Arnaud et al 2007)

Inevitably, as the sheep genome is targeted in further rounds of infection by exogenous JSRVs, a tug of war develops such that a balance exits between the late restriction imparted by endogenous JSRV(s) and the exogenous JSRV. The sheep genome has been invaded multiple times by JSRV, to the extent that the domestication of sheep can be traced based upon which endogenous JSRVs are present in the genome of a particular sheep breed (Chessa et al 2009).
Whether it's too late for something similar with the koalas, time will tell.

Wenqin Xu, Cynthia K. Stadler, Kristen Gorman, Nathaniel Jensen, David Kim, HaoQiang Zheng, Shaohua Tang,, & William M. Switzer, Geoffrey W. Pye, Maribeth V. Eiden (2013). An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1304704110

Arnaud F, Caporale M, Varela M, Biek R, Chessa B, Alberti A, Golder M, Mura M, Zhang YP, Yu L, Pereira F, Demartini JC, Leymaster K, Spencer TE, & Palmarini M (2007). A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses. PLoS pathogens, 3 (11) PMID: 17997604

Chessa B, Pereira F, Arnaud F, Amorim A, Goyache F, Mainland I, Kao RR, Pemberton JM, Beraldi D, Stear MJ, Alberti A, Pittau M, Iannuzzi L, Banabazi MH, Kazwala RR, Zhang YP, Arranz JJ, Ali BA, Wang Z, Uzun M, Dione MM, Olsaker I, Holm LE, Saarma U, Ahmad S, Marzanov N, Eythorsdottir E, Holland MJ, Ajmone-Marsan P, Bruford MW, Kantanen J, Spencer TE, & Palmarini M (2009). Revealing the history of sheep domestication using retrovirus integrations. Science (New York, N.Y.), 324 (5926), 532-6 PMID: 19390051