Characterization and Vector Competence Studies of Chikungunya Virus Lacking Repetitive Motifs in the 3′ Untranslated Region of the Genome

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions

Evidence for a Pathophysiological Role of Keratinocyte-Derived Type III Interferon (IFNλ) in Cutaneous Lupus Erythematosus

Type I IFNs (IFNα/β) have been shown to have a central role in the pathophysiology of lupus erythematosus (LE). The recently discovered type III IFNs (IFNλ1/IL29, IFNλ2/IL28a, IFNλ3/IL28b) share several functional similarities with type I IFNs, particularly in antiviral immunity. As IFNλs act primarily on epithelial cells, we investigated whether type III IFNs might also have a role in the pathogenesis of cutaneous LE (CLE). Our investigations demonstrate that IFNλ and the IFNλ receptor were strongly expressed in the epidermis of CLE skin lesions and related autoimmune diseases (lichen planus and dermatomyositis). Significantly enhanced IFNλ1 could be measured in the serum of CLE patients with active skin lesions. Functional analyses revealed that human keratinocytes are able to produce high levels of IFNλ1 but only low amounts of IFNα/β/γ in response to immunostimulatory nuclear acids, suggesting that IFNλ is a major IFN produced by these cells. Exposure of human keratinocytes to IFNλ1 induced the expression of several proinflammatory cytokines, including CXCL9 (CXC-motiv ligand 9), which drive the recruitment of immune cells and are associated with the formation of CLE skin lesions. Our results provide evidence for a role of type III IFNs in not only antiviral immunity but also autoimmune diseases of the skin

Obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses including chikungunya virus, West Nile virus, influenza virus etc. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins was previously determined to be antiviral against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses like chikungunya virus (EC50 = 0.03 μM) and Semliki Forest virus (SFV) (EC50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. Neutral red retention assay revealed that obatoclax induces rapid neutralization of the acidic environment of endolysosomal vesicles and thereby, most likely inhibits viral fusion. Characterization of escape mutants revealed that mutation L369I in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins neither inhibited viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses like Zika virus, West Nile virus and yellow fever virus, which require low pH for fusion, but not of pH-independent picornaviruses like coxasackievirus A9, echovirus 6 and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification preventing viral fusion that could be pursued as a potential broad-spectrum antiviral candidate.</p

Human TLR8 Senses RNA From Plasmodium falciparum-Infected Red Blood Cells Which Is Uniquely Required for the IFN-γ Response in NK Cells

During blood-stage malaria, the innate immune system initiates the production of pro-inflammatory cytokines, including IFN-γ, that are critical to host defense and responsible for severe disease. Nonetheless, the innate immune pathways activated during this process in human malaria remain poorly understood. Here, we identify TLR8 as an essential sensor of Plasmodium falciparum-infected red blood cells (iRBC). In human immune cells, iRBC and RNA purified from iRBC were detected by TLR8 but not TLR7 leading to IFN-γ induction in NK cells. While TLR7 and 9 have been shown to lead to IFN-γ in mice, our data demonstrate that TLR8 was the only TLR capable of inducing IFN-γ release in human immune cells. This unique capacity was mediated by the release of IL-12p70 and bioactive IL-18 from monocytes, the latter via a hitherto undescribed pathway. Altogether, our data are the first reported activation of TLR8 by protozoan RNA and demonstrate both the critical role of TLR8 in human blood-stage malaria and its unique functionality in the human immune system. Moreover, our study offers important evidence that mouse models alone may not be sufficient to describe the human innate immune response to malaria

Evidence for Novel Hepaciviruses in Rodents

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 roden

Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles

Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aα, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK↓T within NS2A. Exchange of serine for lysine at this site (QKT→QST) blocks the production of both NS2Aα and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aα. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aα was replaced with serine. In addition to true reversions, NS2Aα cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aα cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aα site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles