Roadmap to a plasma cell: Epigenetic and transcriptional cues that guide B cell differentiation.

Antibody-secreting cells (ASCs) or plasma cells secrete antibodies and form a cornerstone of humoral immunity. B cells that receive activation signals in the presence or absence of T cells initiate a differentiation program that requires epigenetic and transcriptional reprogramming in order to ultimately form ASC. Reprogramming is accomplished through the interplay of transcription factors that initiate gene expression programs and epigenetic mechanisms that maintain these programs and cell fates. An important consideration is that all of these factors are operating in the context of cell division. Recent technical advances now allow mechanistic studies to move beyond genetic studies to identify the promoters and enhancer repertoires that are regulated by epigenetic mechanisms and transcription factors in rare cell types and differentiation stages in vivo. This review will detail efforts to integrate transcriptional and epigenetic changes during B cell differentiation with cell division in vivo. What has emerged is a multiphased differentiation model that requires distinct transcription factors and epigenetic programs at each step. The identification of markers that define each phase will help facilitate the manipulation of B cell differentiation for vaccine development or to treat diseases where antibodies are a component.

High-Throughput Method for Detection of Arbovirus Infection of Saliva in Mosquitoes Aedes aegypti and Ae. albopictus.

Vector competence refers to the ability of a vector to acquire, maintain, and transmit a pathogen. Collecting mosquito saliva in medium-filled capillary tubes has become the standard for approximating arbovirus transmission. However, this method is time-consuming and labor-intensive. Here we compare the capillary tube method to an alternative high-throughput detection method the collection of saliva on paper cards saturated with honey, with (FTA card) and without (filter paper) reagents for the preservation of nucleic acid for Aedes aegypti and Aedes albopictus mosquitoes infected with two emerging genotypes of the chikungunya virus (CHIKV). Model results showed that the Asian genotype CHIKV dissemination in the harvested legs of both Ae. aegypti and Ae. albopictus increased the odds of females having a positive salivary infection and higher salivary viral titers, while for the IOL genotype the same effect was observed only for Ae. aegypti. Of the three tested detection methods, the FTA card was significantly more effective at detecting infected saliva of Ae. aegypti and Ae. albopictus females than the capillary tube and filter paper was as effective as the capillary tube for the Asian genotype. We did not find significant effects of the detection method in detecting higher viral titer for both Asian and IOL genotypes. Our results are discussed in light of the limitations of the different tested detection methods.

Effect of Oral Infection of Mayaro Virus on Fitness Correlates and Expression of Immune Related Genes in Aedes aegypti.

Mayaro virus is a mosquito-borne Alphavirus endemic to forests of tropical South America with a sylvatic cycle involving non-human primates and Haemagogus mosquitoes. Human infection with Mayaro virus causes a febrile illness and long-lasting arthralgia and cases are often associated with exposure to tropical forest habitats. Human movement between tropical forest habitats and urban settings may allow for imported cases and subsequent local transmission by domestic mosquito Aedes aegypti. The relative importance of Ae. aegypti as a vector of Mayaro virus may depend on the pathogenic effects of the virus on fitness correlates, especially those entomological parameters that relate to vectorial capacity. We performed mosquito infection studies and compared adult survival and fecundity of females from Brazilian and Floridian populations of Ae. aegypti following oral ingestion of uninfectious (control) and Mayaro virus infectious blood. Mayaro virus infected and refractory mosquitoes had similar or 30-50% lower fecundity than control (unexposed) mosquitoes, suggesting a reproductive cost to mounting an immune response or phenotypic expression of refractoriness. Survival of adult female mosquitoes and targeted gene expression in the Toll and IMD pathways were not altered by Mayaro virus infection. Adult lifespan and fecundity estimates were independent of measured viral titer in the bodies of mosquitoes. The lack of adverse effects of infection status on female survival suggests that Mayaro virus will not alter vectorial capacity mediated by changes in this parameter.

Patterns of Abundance, Host Use, and Everglades Virus Infection in Culex (Melanoconion) cedecei Mosquitoes, Florida, USA.

Everglades virus (EVEV), subtype II within the Venezuelan equine encephalitis (VEE) virus complex, is a mosquitoborne zoonotic pathogen endemic to south Florida, USA. EVEV infection in humans is considered rare, probably because of the sylvatic nature of the vector, the Culex (Melanoconion) cedecei mosquito. The introduction of Cx. panocossa, a tropical vector mosquito of VEE virus subtypes that inhabits urban areas, may increase human EVEV exposure. Field studies investigating spatial and temporal patterns of abundance, host use, and EVEV infection of Cx. cedecei mosquitoes in Everglades National Park found that vector abundance was dynamic across season and region. Rodents, particularly Sigmodon hispidus rats, were primary vertebrate hosts, constituting 77%-100% of Cx. cedecei blood meals. Humans were fed upon at several locations. We detected EVEV infection in Cx. cedecei mosquitoes in lower and upper regions of Everglades National Park only during the wet season, despite an abundance of Cx. cedecei mosquitoes at other sampling times.

Experimental Vertical Transmission of Chikungunya Virus by Brazilian and Florida Aedes Albopictus Populations.

Chikungunya virus (CHIKV) is a vector-borne alphavirus transmitted by the bites of mosquitoes, specifically infected, female mosquitoes of the invasive Aedes species. In nature, CHIKV can be maintained by vertical transmission, a phenomenon that relates to the transfer of CHIKV from the infected parent to their offspring within the ovary or during oviposition. In the present study, we conducted laboratory experiments to determine vertical transmission with Ae. albopictus populations from Brazil and Florida. Parental Ae. albopictus females were orally infected with the emergent Asian genotype of CHIKV in the first gonotrophic cycle (infectious blood meal) and tested for vertical transmission following the second (non-infectious blood meal) gonotrophic cycle. CHIKV infection and CHIKV viral titer in parental females were significantly related to population origin, with Brazilian Ae. albopictus showing higher viral dissemination and viral titer than the Florida population. Experimental vertical transmission of CHIKV was documented in one pool of female and four pools of male Ae. albopictus from Brazil (minimum infection rate, MIR, of 0.76% and 2.86%, respectively, for females and males). For the Florida population of Ae. albopictus, only one pool of males was positive for CHIKV infection, with an MIR of 1.06%. Our results demonstrate that Ae. albopictus populations from Brazil and Florida show heterogeneous CHIKV dissemination and vertical transmission, which may contribute to the epidemiology of CHIKV and may be particularly relevant to virus survival during inter-epidemic periods.

Optimization of cationic (Q)-paper for detection of arboviruses in infected mosquitoes.

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper's binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated. Sixteen variations of Q-paper were prepared with modifications of the derivatizing reagents and derivatization temperature. The binding capacities of these variations were determined first with 1,3,5-benzenetricarboxylic (BTCA), then viral RNA (purified or in infected mosquito samples) was used for validation. For this, samples with Zika (ZIKV) and chikungunya (CHIKV) RNA or virus-infected Aedes aegypti mosquito bodies were applied to sixteen Q-paper variants. Washing the paper samples with water versus elution with aqueous salt (1 M) gave samples that were analyzed for viral RNA by a PCR-based direct Luminex hybridization assay. The comparison ranked the Q-paper binding capacities from the lowest to the highest. The Q-paper with the highest RNA binding capability was further validated with ZIKV- and CHIKV-infected mosquito saliva.

Chikungunya virus vector competency of Brazilian and Florida mosquito vectors.

Chikungunya virus is a vector-borne alphavirus transmitted by the bites of infected female Ae. aegypti and Ae. albopictus. In Brazil between 2014 and 2016 almost 320 thousand autochthonous human cases were reported and in Florida numerous imported CHIKV viremic cases (> 3,800) demonstrate the potential high risk to establishment of local transmission. In the present study, we carried out a series of experiments to determine the viral dissemination and transmission rates of different Brazilian and Florida populations of Ae. aegypti and Ae. albopictus at 2, 5, and 13 days post-infection for the emergent Asian genotype of CHIKV. Our results show that all tested populations of Ae. aegypti and Ae. albopictus have a high proportion (> 0.80) of individuals with disseminated infection as early as 2 days-post exposure. We found no significant treatment effects of mosquito population origin effects on viral dissemination rates. Transmission rates had a heterogeneous pattern, with US Ae. aegypti and Brazilian Ae. albopictus having the highest proportion of individuals with successful infection (respectively 0.50 and 0.82 as early as 2 days-post infection). Model results found significant effects of population origin, population origin x species, population origin x days post-infection and population origin x species x days post infection.

Laboratory Evaluation of Commercially Available Platforms to Detect West Nile and Zika Viruses From Honey Cards.

Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV and ZIKV, Pro-Lab Diagnostics ProAmpRT WNV loop-mediated amplification (LAMP) and ZIKV LAMP assays, and the Rapid Analyte Measurement Platform (RAMP) WNV assay. Real-time RT-PCR was the most sensitive assay and the most robust to viral RNA degradation over time. To maximize the detection of virus, honey cards should be left in the traps ≤1 d if using LAMP assays and ≤3 d if using real-time RT-PCR to detect viruses from field samples. The WNV RAMP assay, although effective for pool screening, lacks sensitivity required for honey card surveillance. Future studies may determine the minimum number of infectious mosquitoes required to feed on a honey card that would be reliably detected by the LAMP or RAMP assays.

Diurnal Temperature Range and Chikungunya Virus Infection in Invasive Mosquito Vectors.

Climate strongly influences the geographic distribution and timing of mosquito-borne disease outbreaks. Environmental temperature affects phenotypic traits of mosquitoes including vector competence for arboviruses mediated by changes in infection, extrinsic incubation period and in rates of transmission. Most experiments, however, are done at constant temperatures. In nature, mosquitoes are more likely to experience daily fluctuations in temperature. Here we compare disseminated infection (leg infection) and saliva infection of Aedes aegypti (L.) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) from Florida following oral exposure to an Asian genotype of chikungunya virus emergent in the Americas. We evaluated experimentally the effect of variable temperature regimens on disseminated infection and saliva infection of these Aedes species. Each of three temperature regimes had approximately the same average temperature (27-28°C), but differed in the magnitude of the diurnal temperature range (DTR). The large DTR was 8.0°C (range 23-31°C) and the small DTR was 4.0°C (range 26-30°C) which approximate ranges in different locations of Florida during July-October when risk of transmission is highest. The constant temperature was set at 27°C. Testing three geographic populations of each mosquito species, significant effects on disseminated infection were detected for an interaction between temperature regime and geographic population for both Ae. aegypti and Ae. albopictus. There were no significant treatment effects of temperature, geographic population, or temperature by geographic population interaction on saliva infection for either mosquito species. Constant temperature resulted in a higher viral load in the saliva of Ae. albopictus, but not Ae. aegypti, compared to conditions where the temperature fluctuated.

Transmission risk of two chikungunya lineages by invasive mosquito vectors from Florida and the Dominican Republic.

Between 2014 and 2016 more than 3,800 imported human cases of chikungunya fever in Florida highlight the high risk for local transmission. To examine the potential for sustained local transmission of chikungunya virus (CHIKV) in Florida we tested whether local populations of Aedes aegypti and Aedes albopictus show differences in susceptibility to infection and transmission to two emergent lineages of CHIKV, Indian Ocean (IOC) and Asian genotypes (AC) in laboratory experiments. All examined populations of Ae. aegypti and Ae. albopictus mosquitoes displayed susceptibility to infection, rapid viral dissemination into the hemocoel, and transmission for both emergent lineages of CHIKV. Aedes albopictus had higher disseminated infection and transmission of IOC sooner after ingesting CHIKV infected blood than Ae. aegypti. Aedes aegypti had higher disseminated infection and transmission later during infection with AC than Ae. albopictus. Viral dissemination and transmission of AC declined during the extrinsic incubation period, suggesting that transmission risk declines with length of infection. Interestingly, the reduction in transmission of AC was less in Ae. aegypti than Ae. albopictus, suggesting that older Ae. aegypti females are relatively more competent vectors than similar aged Ae. albopictus females. Aedes aegypti originating from the Dominican Republic had viral dissemination and transmission rates for IOC and AC strains that were lower than for Florida vectors. We identified small-scale geographic variation in vector competence among Ae. aegypti and Ae. albopictus that may contribute to regional differences in risk of CHIKV transmission in Florida.