When VEEV antibodies were present, significantly fewer viral plaques were measured at 72 h p

When VEEV antibodies were present, significantly fewer viral plaques were measured at 72 h p.i. across placental cells and into an embryoid body. The data showed that antibodies to Venezuelan equine encephalitis virus significantly reduced MK-2461 CHIKV viral load in embryoid bodies. The MK-2461 data highlighted the fact that viral pathogenesis can be cell-specific and that exploiting antigenic cross-reactivity could be an avenue for reducing the impact of congenital CHIKV infections. Keywords: chikungunya, congenital infection, antibody cross-reactivity, alphavirus 1. Introduction Chikungunya virus (CHIKV) is an alphavirus vectored by mosquitos and is an enveloped + positive sense single stranded RNA ssRNA virus with a 12 kb genome. In humans, CHIKV can cause a febrile illness punctuated by severe polyarthralgia. Historically, CHIKV infection was thought to be self-limited, but increasing reports are showing that rheumatic and neurological sequelae can linger for years following infection [1,2,3]. When CHIKV emerged in the Western Hemisphere, neuroinvasive disease and congenital infections were reported at rates much MK-2461 higher than the Eastern Hemisphere [4]. CHIKV congenital infections were first reported during the 2005 outbreak on Reunion Island [5,6,7]. After the emergence of CHIKV in the Western Hemisphere, multiple reports of congenital infections were published that documented neurological complications, cardiac defects, respiratory distress, and miscarriage [5,8,9]. Although reports of congenital infection were published in many CHIKV-endemic locations, the vast majority originated from South and Central America [4]. South and Central America, along with the Caribbean, are endemic to several New World alphaviruses including Madariaga virus (MADV), Mayaro virus (MAYV), Eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), and Western equine encephalitis (WEEV). Human exposure prevalence for these viruses can be as high as 80% in some regions [10,11]. VEEV and CHIKV have been found to circulate in the same Central and South American regions [12,13,14,15]. For example, VEEV has been thought to be endemic in southern Mexico for decades, with seroprevalence rates between 18C75%, whereas the seroprevalence of CHIKV in southern Mexico has been determined by some studies to be as high as 85% [13,14]. Additionally, recent studies have indicated that antigenic cross-reactivity, antibody-mediated enhancement, and antibody cross-neutralization of alphaviruses can have a significant impact on the course of infection [16,17,18,19]. The study of congenital infections with these viruses is problematic, as most animal models do not reflect human disease. Although research has shown that non-human primates and sheep can serve as models, these systems can be expensive, labor intensive, and contain small sample sizes [20,21]. As a result, aside from kidney cell line Vero E6 (ATCC CRL-1586) were grown in Dulbeccos modified MK-2461 Eagles medium (DMEM) with 10% FBS, supplemented with penicillin/streptomycin, 1X non-essential amino acids, 1X Glutamax, and 1 mM HEPES. All cell lines were incubated at 37 C/5% CO2. CHIKV (181/25) was obtained from BEI Resources (NR-50345) and expanded once in Vero cells. Polyclonal anti-Venezuelan equine encephalitis virus, TC-83 (subtype IA/B) glycoprotein MK-2461 (antiserum, goat), NR-9404, was obtained through BEI Resources (BEIresources.org) NIAID, NIH. 2.2. Embryoid Body Formation Human-induced pluripotent stem cells (ATCC ACS-1019) were cultured in mTeSR1 media (StemCell Technologies, Vancouver, Canada) on plates coated with vitronectin XF (Stemcell Technologies). ACS-1019 cells were seeded in an AggreWell 400 24-well plate at a density of 2.4 105 cells per well, following the manufacturers directions, in order to initiate EB formation (StemCell Technologies). ACS-1019 were cultured in the AggreWell microwells with AggreWell EB formation media for 72 h at 37 C/5% CO2. After this, the resulting EBs were harvested and divided equally between replicates of each treatment. 2.3. Monolayer Infection and Imaging Monolayers of BeWo and HUVEC cells were infected with 100 infectious units per well. After 48 h, samples were fixed with 4% paraformaldehyde and blocked with 5% lamb serum. Cells were stained with anti-CHIKV monoclonal antibody 3E7b and anti-MAP2 antibody (Novus Biologicals, Littleton, CO, USA). Slides were mounted with ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology, Danvers, MA, USA catalog #8961S) and images were obtained using an Olympus Fluoview 3000 confocal microscope. Images were processed using the Olympus Fluoview FV10-ASW 4.1 software package. 2.4. Trans-Well Co-Culture Corning 12 mm Trans-well-COL collagen-coated 3.0 m pore PTFE membrane insert (Corning, NY, BMP7 USA catalog #3494) were seeded with HUVEC cells on the basolateral side of the insert at a concentration of 1 1.0 105 cells per 200 L, and BeWo cells were seeded on the apical side of the insert at a density of 1 1.5 105 cells.