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