GenBank accession numbers for the individual heavy and light chain nucleotide sequences of the recombinantly expressed IgG molecules are JF806449-JF806467. The protein was expressed and purified as described in Material and Methods. (B) Kinetic data for binding of SM5-1 to gB and Dom II-GST. SM5-1 was used as Fab-fragment generated by papain digestion. Kinetic experiments were performed at 25uC using a Biacore T100 (GE Healthcare, Germany). gB and Dom II-GST proteins were captured on the Series S Sensor Chip CM5 (GE Healthcare, Germany) using N-hydroxysuccinimide-N-ethyl-N-dimethylamino-propyl-carboimide chemistry. PBS, 0.05% P20 was used as the running buffer. Approximately 300 RU of Dom II-GST and GST as reference surface were captured with a contact time of 400 sec and a flow rate of 10 ml/min. Different concentrations of SM5-1 (33.33, 11.11 and 3.7 nM) were injected with a contact time of 90 sec, dissociation time of 600 sec and a flow rate of 30 ml/min. The sensor surface was regenerated between each binding reaction with 10 mM glycine (pH 2.0) with a contact time of 20 sec and a flow rate of 30 ml/min. The kinetics were fitted to a 1:1 binding model. k a , apparent association rate constant; k d , apparent dissociation rate constant; K D , apparent dissociation equilibrium. (C) Circular dichroism (CD) measurement of Dom II protein demonstrating structural folding. PreScission Protease (GE Healthcare, Germany) was used to cleave the GST-Tag. Dom II protein at a concentration of 7.8 mM was dialyzed against 20 mM sodium phosphate buffer (pH 7.0) and filtrated. CD measurement was performed at 20uC using a Jasco J-815 CD Spectrometer (Jasco, Japan) and a cuvette with 0.1 cm path length. Spectrum was registered from 185 to 260 nm and was corrected for the contribution of phosphate buffer. Spectrum was accumulated eight times with a band width of 1.0 nm and a sensitivity of 100 mdeg. The scan speed was 20 nm/min, the time response 1 sec and the data pitch 0.1 nm. (TIF) Figure S4 Quality controls of Dom II-specific polyclonal antibody preparation. (A) The affinity purified polyclonal antibody fraction is specific for Dom II of gB. ELISA plates were coated with gB, AD-1, AD-2 and Dom II, respectively, and tested with various antibodies. Dom II poly: affinity purified IgG fraction, Serum pool pre: Serum pool before affinity purification, Serum post: Serum pool after affinity purification, AD-2-specific mab: C23, anti-AD-1-specific mab: 89-104, anti-GST: murine mab specific for GST. (B) The affinity purified polyclonal antibody fraction does not contain detectable antibodies against additional envelope glycoproteins of HCMV. Cos7 cells were transfected with the plasmids indicated in the top row. 48 h later the cells were fixed and incubated with the affinity purified IgG fraction (upper panel) and control antibodies (lower panel). Binding of the primary antibody was detected by incubation with appropriate FITCconjugated secondary antibody. Anti-HA: mouse mab specific for HA, C23: anti-AD-2 human mab, 14-16A: mouse mab specific for the gM/gN complex, SA4: mouse mab specific for gH. Antibody purification was performed twice with similar results. (TIF) Figure S5 Sequence alignment of HCMV strains and clinical isolates. Full length HCMV protein sequences from Genbank and EMBL databases (accession numbers on the left) were aligned to the HCMV TB40 strain. The regions for AD-2, domain I, domain II as well as AD-1 are depicted. Two regions of hyper-variability lie close to the N-terminus and C-terminal from domain II in a linker region. The protein alignment was performed with the Geneious software v4. 8  Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease. The Chikungunya virus (CHIKV), an arthropod-borne virus (arbovirus), is a single-stranded positive-sense RNA virus with three genotypes. The virus is of the Alphavirus genus in the Togaviridae family [1, 2] . CHIKV has been shown to infect and be transmitted by Ae. aegyptii and Ae. albopictus mosquitoes. It was identified in East Africa in the early 1950s and since then has caused epidemics in continental Africa, the Indian Ocean region, and countries of Southeast Asia such as India, where since 2006 suspected cases have been estimated to be 1.39 million, and Singapore [3] [4] [5] [6] . The only reported outbreak outside these areas was in Italy in the Emilia Romagna region in 2007. Small nonepidemic imported cases have been reported in other regions such as North America, France and Japan, which were caused by travelers returning from affected areas [7] [8] [9] .