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BackgroundChikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs).MethodsVRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout.ResultsUpon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection.ConclusionsA VRP based CHIKV neutralization assay using Gluc as readout represents a fast and useful method to determine CHIKV neutralizing antibodies without the need of using infectious CHIKV.

Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research.

Short CommunicationThe alternate triad motif of the poly(ADP-ribose) polymerase-like domain of the human zinc finger antiviral protein is essential for its antiviral activity Institute of Virology, University of Bonn Medical Center, Bonn, GermanyThe human zinc finger antiviral protein (hZAP) gene is spliced to yield a short (hZAP-S) and a long (hZAP-L) isoform. The long isoform possesses a poly(ADP-ribose) polymerase (PARP)-like domain in its C-terminus predicted to be inactive due to alterations in its triad motif compared with bona fide PARPs. Using Sindbis virus as prototype member of alphaviruses we confirmed that hZAP-L is a more potent inhibitor of alphaviruses than hZAP-S. Specific small interfering RNA knockdown of hZAP-L but not hZAP-S demonstrated a role of endogenous hZAP-L in restriction of alphavirus replication. Whilst single amino-acid substitutions in the triad motif of hZAP-L's PARP-like domain reduced the antiviral activity, exchange of all three triad motif residues to alanine or to the amino acids of active PARPs virtually abolished the antiviral effect. Contrary to previous assumptions, these results indicate an essential function of the PARP-like domain in hZAP-L's antiviral activity.

Edited by Henrik G. DohlmanThe triphosphohydrolase SAMHD1 (sterile ␣ motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro. dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication. The lentiviral restriction factor SAMHD12 is a dNTP triphosphohydrolase (1, 2) that inhibits the replication of HIV-1 in myeloid cells (3, 4). The enzyme removes the triphosphate groups of dNTPs, preventing the reverse transcription in newly infected cells (5). In addition, SAMHD1 has been proposed to have ribonuclease activity that degrades the viral genomic RNA upon virus entry (6). HIV-2 and some SIVs counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1 (3, 4, 7). Other SIVs, such as the SIV of African green monkeys, counteract SAMHD1 through the related accessory protein Vpr (8, 9). Vpx delivery into the cell by virions causes a rapid rise in the intracellular dNTP concentration, relieving the block to reverse transcription and allowing productive infection (5, 10). HIV-1 does not encode Vpx and as a result replicates poorly in dendritic and other myeloid cells. The requirement for Vpx for infection of myeloid cells can be partially replaced in culture by the addition of extracellular deoxynucleosides, which are converted into dNTPs by the salvage pathway (5). In addition, HIV-1 can be genetically modified to package Vpx, allowing it to infect myeloid cells (11,12).The catalytic activity of SAMHD1 is reg...