Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax.

Currently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.

Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

Nuclear factor of activated T cells (NFAT) comprises a family of transcription factors that regulate T cell development, activation and differentiation. NFAT signalling can also mediate granulocyte and dendritic cell (DC) activation, but it is unknown whether NFAT influences their development from progenitors. Here, we report a novel role for calcineurin/NFAT signalling as a negative regulator of myeloid haematopoiesis. Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment. Culturing bone marrow cells in media supplemented with Flt3-L in the presence of the calcineurin/NFAT inhibitor Cyclosporin A increased numbers of differentiated DC. Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis. In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development. The finding that inhibition of NFAT enhances myeloid development provides a novel insight into understanding how the treatment with drugs targeting calcineurin/NFAT signalling influence the homeostasis of the innate immune system.

Viperin restricts chikungunya virus replication and pathology.

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Highly conserved syntenic blocks at the vertebrate Hox loci and conserved regulatory elements within and outside Hox gene clusters.

Hox genes in vertebrates are clustered, and the organization of the clusters has been highly conserved during evolution. The conservation of Hox clusters has been attributed to enhancers located within and outside the Hox clusters that are essential for the coordinated "temporal" and "spatial" expression patterns of Hox genes in developing embryos. To identify evolutionarily conserved regulatory elements within and outside the Hox clusters, we obtained contiguous sequences for the conserved syntenic blocks from the seven Hox loci in fugu and carried out a systematic search for conserved noncoding sequences (CNS) in the human, mouse, and fugu Hox loci. Our analysis has uncovered unusually large conserved syntenic blocks at the HoxA and HoxD loci. The conserved syntenic blocks at the human and mouse HoxA and HoxD loci span 5.4 Mb and 4 Mb and contain 21 and 19 genes, respectively. The corresponding regions in fugu are 16- and 12-fold smaller. A large number of CNS was identified within the Hox clusters and outside the Hox clusters spread over large regions. The CNS include previously characterized enhancers and overlap with the 5' global control regions of HoxA and HoxD clusters. Most of the CNS are likely to be control regions involved in the regulation of Hox and other genes in these loci. We propose that the regulatory elements spread across large regions on either side of Hox clusters are a major evolutionary constraint that has maintained the exceptionally long syntenic blocks at the HoxA and HoxD loci.

Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes.

With about 24,000 extant species, teleosts are the largest group of vertebrates. They constitute more than 99% of the ray-finned fishes (Actinopterygii) that diverged from the lobe-finned fish lineage (Sarcopterygii) about 450 MYA. Although the role of genome duplication in the evolution of vertebrates is now established, its role in structuring the teleost genomes has been controversial. At least two hypotheses have been proposed: a whole-genome duplication in an ancient ray-finned fish and independent gene duplications in different lineages. These hypotheses are, however, based on small data sets and lack adequate statistical and phylogenetic support. In this study, we have made a systematic comparison of the draft genome sequences of Fugu and humans to identify paralogous chromosomal regions ("paralogons") in the Fugu that arose in the ray-finned fish lineage ("fish-specific"). We identified duplicate genes in the Fugu by phylogenetic analyses of the Fugu, human, and invertebrate sequences. Our analyses provide evidence for 425 fish-specific duplicate genes in the Fugu and show that at least 6.6% of the genome is represented by fish-specific paralogons. We estimated the ages of Fugu duplicate genes and paralogons using the molecular clock. Remarkably, the ages of duplicate genes and paralogons are clustered, with a peak around 350 MYA. These data strongly suggest a whole-genome duplication event early during the evolution of ray-finned fishes, probably before the origin of teleosts.

Hox gene clusters in the Indonesian coelacanth, Latimeria menadoensis.

The Hox genes encode transcription factors that play a key role in specifying body plans of metazoans. They are organized into clusters that contain up to 13 paralogue group members. The complex morphology of vertebrates has been attributed to the duplication of Hox clusters during vertebrate evolution. In contrast to the single Hox cluster in the amphioxus (Branchiostoma floridae), an invertebrate-chordate, mammals have four clusters containing 39 Hox genes. Ray-finned fishes (Actinopterygii) such as zebrafish and fugu possess more than four Hox clusters. The coelacanth occupies a basal phylogenetic position among lobe-finned fishes (Sarcopterygii), which gave rise to the tetrapod lineage. The lobe fins of sarcopterygians are considered to be the evolutionary precursors of tetrapod limbs. Thus, the characterization of Hox genes in the coelacanth should provide insights into the origin of tetrapod limbs. We have cloned the complete second exon of 33 Hox genes from the Indonesian coelacanth, Latimeria menadoensis, by extensive PCR survey and genome walking. Phylogenetic analysis shows that 32 of these genes have orthologs in the four mammalian HOX clusters, including three genes (HoxA6, D1, and D8) that are absent in ray-finned fishes. The remaining coelacanth gene is an ortholog of hoxc1 found in zebrafish but absent in mammals. Our results suggest that coelacanths have four Hox clusters bearing a gene complement more similar to mammals than to ray-finned fishes, but with an additional gene, HoxC1, which has been lost during the evolution of mammals from lobe-finned fishes.