ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping.

OBJECTIVES: ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS: Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS: Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS: Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.

Full genome sequences and preliminary molecular characterization of three tick-borne encephalitis virus strains isolated from ticks and a bank vole in Slovak Republic.

The full genome sequences of three tick-borne encephalitis virus strains, two isolated from Ixodes ricinus ticks and one from the brain of a bank vole, Myodes glareolus, originating from the Slovak Republic were determined. Nucleotide sequences were found to be very similar (>99.5 % nt-identity) with only one distinct amino acid (aa) difference to each other. They all shared 30 aa-changes when compared to type strain Neudoerfl, isolated in neighboring Austria. An internal poly(A)-stretch similar to that of strain Neudoerfl was found only in TBEV strain 114 from a tick. Despite this heterogeneity in the 3'-NCR, the high level of sequence identity was striking, indicating a low rate of nucleotide substitutions of TBEV strains in Slovakia and no obvious relation to the host species.

Complete Genome Sequence of Tick-Borne Encephalitis Virus Strain A104 Isolated from a Yellow-Necked Mouse (Apodemus flavicollis) in Austria.

Tick-borne encephalitis virus (TBEV) strain A104 was isolated from the brain of a yellow-necked mouse in Austria in 1990. The complete genome sequence was 11,097 nucleotides long. Comparison with TBEV prototype strain Neudoerfl showed 32 amino acid exchanges and the absence of an internal poly(A) stretch within the 3' noncoding region.

Clinical and virological characterization of imported cases of Chikungunya fever.

A Chikungunya virus (CHIKV) epidemic emerged in the Indian Ocean islands of the Comores, Reunion, Mayotte, Mauritius, the Seychelles and Madagascar in 2005 resulting in the infection of about 250.000 inhabitants and travellers in only one year. Beginning in March 2006 increasing numbers of CHIKV-like febrile illnesses were reported from various parts of India. We investigated 70 consecutive German travellers returning from the affected areas and presenting with arthralgia and/or fever suggestive of CHIKV infection. Eleven patients had serological evidence of CHIKV infection. Real-time RT-PCR for CHIKV was positive in two cases, one who returned from Mauritius and the other who came back from Rajasthan, Northern India. In both cases CHIKV was isolated and sequencing of the entire viral genome was performed. The nucleotide sequence data obtained for both CHIKV strains revealed a high level of identity to CHIKV isolates from the ongoing epidemic. In detail, we found only 18 nucleotide exchanges between the isolates from Mauritius and Rajasthan, resulting in only six amino acid changes (nsP1 T128K, T376M, nsP3 S472N, capsid P23S, V27I and E1-protein A226V). Although the excessive dimension of the 2005/2006 outbreak in the Indian Ocean islands was at least in part accounted to the naïve population affected, our results of the Rajasthan isolate support that the emergence of this CHIKV subtype may rather be a result of a better viral fitness. This has been previously accounted to a A226V change in the E1 protein of the new CHIKV variant when compared to other CHIKV data available. This mutation, supposedly resulting in high-titred viremia in humans and/or an enhanced adaptation to the vector population resulting in increased transmission rates, was also found in our CHIKV isolate from Mauritius. The spread of an African CHIKV to Asia further demonstrates how fast viruses can emerge and establish in places where competent vectors are prevalent.