Mouse and nonhuman primate models now serve as useful platforms to study Zika virus (ZIKV) pathogenesis, candidate therapies, and vaccines, but they rely on needle inoculation of virus: the effects of mosquito-borne infection on disease outcome have not been explored in these models. Here we show that infection via mosquito bite delays ZIKV replication to peak viral loads in rhesus macaques. Importantly, in mosquito-infected animals ZIKV tissue distribution was limited to hemolymphatic tissues, female reproductive tract tissues, kidney, and liver, potentially emulating key features of human ZIKV infections, most of which are characterized by mild or asymptomatic disease. Furthermore, deep sequencing analysis reveals that ZIKV populations in mosquito-infected monkeys show greater sequence heterogeneity and lower overall diversity than in needle-inoculated animals. This newly developed system will be valuable for studying ZIKV disease because it more closely mimics human infection by mosquito bite than needle-based inoculations.

Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8 T and NK cell populations Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8 T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8 effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ∼2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common γC and β chain receptors on both CD8 T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8 T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs. Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen.

Zika virus (ZIKV) is a mosquito-transmitted virus that can cause severe defects in an infected fetus. ZIKV is also transmitted by sexual contact, although the relative importance of sexual transmission is unclear. To better understand the role of sexual transmission in ZIKV pathogenesis, a nonhuman primate (NHP) model of vaginal transmission was developed. ZIKV was readily transmitted to mature cycling female rhesus macaque (RM) by vaginal inoculation with 104–106 plaque-forming units (PFU). However, there was variability in susceptibility between the individual RM with 1–>8 vaginal inoculations required to establish infection. After treatment with Depoprovera, a widely used contraceptive progestin, two RM that initially resisted 8 vaginal ZIKV inoculations became infected after one ZIKV inoculation. Thus, Depoprovera seemed to enhance susceptibility to vaginal ZIKV transmission. Unexpectedly, the kinetics of virus replication and dissemination after intravaginal ZIKV inoculation were markedly different from RM infected with ZIKV by subcutaneous (SQ) virus inoculation. Several groups have reported that after SQ ZIKV inoculation vRNA is rapidly detected in blood plasma with vRNA less common in urine and saliva and only rarely detected in female reproductive tract (FRT) secretions. In contrast, in vaginally inoculated RM, plasma vRNA is delayed for several days and ZIKV replication in, and vRNA shedding from, the FRT was found in all 6 animals. Further, after intravaginal transmission ZIKV RNA shedding from FRT secretions was detected before or simultaneously with plasma vRNA, and persisted for at least as long. Thus, ZIKV replication in the FRT was independent of, and often preceded virus replication in the tissues contributing to plasma vRNA. These results support the conclusion that ZIKV preferentially replicates in the FRT after vaginal transmission, but not after SQ transmission, and raise the possibility that there is enhanced fetal infection and pathology after vaginal ZIKV transmission compared to a mosquito transmitted ZIKV.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4-and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIV mac 239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4-or MHC I-downmodulation. Further characterization of this mutant (SIV mac 239 AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIV mac 239 or SIV mac 239 AAA , and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIV mac 239 AAA than in animals infected with wild-type SIV mac 239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo.

Multistep synthesis is a key capstone experience in organic laboratory instruction. Here, a four-step synthesis of avobenzone, an active component in sunscreens, has been developed that can be completed in two 4 h laboratory periods. This synthesis incorporates green principles and includes an aldol condensation, electrophilic addition of bromine to an alkene, an E2 dehalogenation of a dibromide, and hydration of an alkyne. Highlights of this synthesis include the use of coupling constants to identify alkene configuration, NMR analysis to determine preferred tautomeric form, the use of microwave irradiation to reduce reaction times, a solvent-free synthesis of a chalcone, and a three-step reaction that can be completed in a single lab period.

Background: Alzheimer’s disease (AD) is the most common aging-associated neurodegenerative disease; nevertheless, the etiology and progression of the disease is still incompletely understood. We have previously shown that the microbially-derived metabolite trimethylamine N-oxide (TMAO) is elevated in the cerebrospinal fluid (CSF) of individuals with cognitive impairment due to AD and positively correlates with increases in CSF biomarkers for tangle, plaque, and neuronal pathology. Objective: We assessed the direct impact of TMAO on AD progression. Methods: To do so, transgenic 5XFAD mice were supplemented with TMAO for 12 weeks. Neurite density was assessed through quantitative brain microstructure imaging with neurite orientation dispersion and density imaging magnetic resonance imaging (MRI). Label-free, quantitative proteomics was performed on cortex lysates from TMAO-treated and untreated animals. Amyloid-β plaques, astrocytes, and microglia were assessed by fluorescent immunohistochemistry and synaptic protein expression was quantified via western blot. Results: Oral TMAO administration resulted in significantly reduced neurite density in several regions of the brain. Amyloid-β plaque mean intensity was reduced, while plaque count and size remained unaltered. Proteomics analysis revealed that TMAO treatment impacted the expression of 30 proteins (1.5-fold cut-off) in 5XFAD mice, including proteins known to influence neuronal health and amyloid-β precursor protein processing. TMAO treatment did not alter astrocyte and microglial response nor cortical synaptic protein expression. Conclusion: These data suggest that elevated plasma TMAO impacts AD pathology via reductions in neurite density.

Age-related disease may be mediated by low levels of chronic inflammation ("inflammaging"). Recent work suggests that gut microbes may contribute to inflammation via degradation of the intestinal barrier. While aging and age-related diseases including Alzheimer's disease (AD) are linked to altered microbiome composition and higher levels of gut microbial components in systemic circulation, the role of intestinal inflammation and permeability per se remains unclear. To test whether greater gut inflammation is associated with older age and AD pathology, we assessed fecal samples from older adults to measure calprotectin, an established marker of intestinal inflammation which is elevated in diseases of gut barrier integrity. Here we found that calprotectin levels are higher with age, and that higher calprotectin was associated with greater amyloid burden among participants with an amyloid-confirmed AD dementia diagnosis. Calprotectin was also associated with cerebrospinal fluid markers of AD pathology and axonal degeneration, as well as with lower verbal memory function among cognitively unimpaired participants. Together, these findings suggest that intestinal inflammation may play a role in pathology development, and that it may exacerbate the progression toward AD.