Defining the chemokine basis for leukocyte recruitment during viral encephalitis.

UNLABELLED: The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain. IMPORTANCE: Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely, CXCR3 and CCR2, does not alter viral titers within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis.

Assessment of a novel real-time pan-flavivirus RT-polymerase chain reaction.

Outbreaks of West Nile virus (WNV) have occurred intermittently in regions around the Mediterranean coast, and the virus may have become established in Northern Italy and Romania, with reported intermittent outbreaks in Spain, Hungary, and France. WNV has also spread rapidly throughout the Americas since its introduction into New York in 1999. This capacity to emerge in new geographical locations and to spread rapidly together with the current increase in incidence of other flaviviruses such as tick-borne encephalitis virus, dengue virus, and Usutu virus has prompted us to design a novel pan-flavivirus RT-polymerase chain reaction for the purpose of surveillance for a range of flaviviruses. The assay utilizes degenerate primers targeting the flavivirus NS5 gene (RNA-dependent RNA polymerase) and detects a range of flaviviruses, including WNV. A small panel of WNV bird samples obtained from the United States has been shown to be detected using this assay. The amplicon generated is of sufficient size to provide sequence data to confirm the identity of the virus detected and undertake limited phylogenetic analysis. Testing using this assay has shown its ability to detect a range of tick-borne flaviviruses, particularly louping ill virus that is endemic in areas of the United Kingdom. The assay has been used to survey 160 bird samples and 1000 mosquito samples from the United Kingdom and found no evidence for WNV.