Extrinsic incubation temperature impacts on Zika virus evolution and vector competence during systemic Aedes infection

Includes bibliographical references.2020 Summer.Arthropod-borne viruses (arboviruses) are distinctive in that they are required to constantly replicate in different hosts and in a wide range of temperatures for their perpetuation in nature. Vertebrate hosts tend to maintain temperatures of approximately 37°C - 40°C, but arthropods hosts are poikilotherms and subject to ambient temperatures which can have a daily temperature fluctuation of > 10°C. Invertebrate host genus, species, and strain in combination with arbovirus strain and preparation methods are known to have large impacts on vector competence and vectorial capacity. Seemingly small differences in host geographic isolation, virus strain, and preparation methods can have significant impacts on vector competence studies. The role of temperature on the ability of an arthropod vector to acquire, maintain, and transmit a pathogen has been investigated for numerous arboviruses. Changing the extrinsic incubation temperature between distinct constant temperatures has been shown to alter arbovirus vector competence, extrinsic incubation period, and mosquito survival, in which moderate temperatures of 28°C-32°C are optimal and temperatures higher and lower have deleterious effects. The mean and range of daily temperature fluctuations (diurnal temperature) have likewise been shown to influence arbovirus perpetuation and vector competence, in which large daily temperature fluctuations negatively affect mosquito development, survival, and vector competence. However, little is known as to how temperature alters arbovirus genetic diversity during systemic mosquito infection or how differences in arbovirus hosts and viral strains impact arbovirus genetic diversity in relationship to temperature. Therefore in the study completed in chapter two, we characterized the impact that constant temperatures of 25°C, 28°C, 32°C, and 35°C, and the diurnal fluctuation from 25°C to 35°C during extrinsic incubation periods have on the Puerto Rican isolate of Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti (Poza Rica) and Aedes albopictus (Florida) mosquitoes. To characterize the impact that temperature has on ZIKV population diversity in different host species and viral isolates, in the study completed in chapter three, we used a Tapachula, Mexico Aedes aegypti line and a Chiapas, Mexico ZIKV isolate to assess ZIKV population dynamics during 20°C, 24°C, 28°C, 32°C, 34°C, and 36°C constant extrinsic incubation temperatures. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species, while transmission peaked at 10 days post-infection for Aedes aegypti and 14 days post-infection in Aedes albopictus. The diurnal temperature group is not predicted by the constant temperature distribution. Instead, when using the mean daily temperature of the diurnal group as a predicter, its VC lies between the moderate (28°C and 32°C) and extreme (25°C and 35°C) temperature group VCs. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the ZIKV selective environment during infection. During mosquito infection, constant temperatures more often elicited positive selection whereas diurnal temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes and has distinct effects on the selective environment within mosquitoes. Additionally, the selective pressures induced by temperature are consistent across host species and viral strain and have similar impacts on shaping the viral population structure. However, input viral populations are still a driving factor of diversity and expansion during systemic mosquito infection. While our findings and those of others suggest that vector competence is impacted unimodally regardless of temperature, this is only applicable for constant temperatures. Future work assessing daily temperature fluctuation range and mean are needed to have a clear understanding of the role extrinsic incubation temperature plays on vector competence

Impact of Extrinsic Incubation Temperature on Natural Selection During Zika Virus Infection of Aedes Aegypti and Aedes Albopictus

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of \u3e 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes

Impact of Simultaneous Exposure to Arboviruses on Infection and Transmission by Aedes aegypti Mosquitoes

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection

Mosquitoes Transmit Unique West Nile Virus Populations During Each Feeding Episode

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level

Modulation of apoptosis by V protein mumps virus

<p>Abstract</p> <p>Background</p> <p>The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway.</p> <p>Findings</p> <p>We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process.</p> <p>Conclusions</p> <p>The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.</p

Variation in Competence for ZIKV Transmission by Aedes aegypti and Aedes albopictus in Mexico

BACKGROUND: ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59-Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. CONCLUSIONS/SIGNIFICANCE: Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8-51% in Ae. aegypti and from 2-26% in Ae. albopictus

Taraxacum officinale and Urtica dioica extracts inhibit dengue virus serotype 2 replication in vitro

Abstract Background Urtica dioica, Taraxacum officinale, Calea integrifolia and Caesalpinia pulcherrima are widely used all over the world for treatment of different illnesses. In Mexico, these plants are traditionally used to alleviate or counteract rheumatism and inflammatory muscle diseases. In the present study we evaluated the activity of aqueous and methanolic extracts of these four plants, on the replication of dengue virus serotype 2 (DENV2). Methods Extraction process was carried out in a Soxtherm® system at 60, 85 and 120 °C; a chemical fractionation in silica gel chromatography was performed and compounds present in the active fractions were identified by HPLC-DAD-ESI/MSn. The cytotoxic concentration and the inhibitory effect of extracts or fractions on the DENV2 replication were analyzed in the BHK-21 cell line (plaque forming assay). The half maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated for the extracts and fractions. Results The methanolic extracts at 60 °C of T. officinale and U. dioica showed the higher inhibitory effects on DENV2 replication. After the chemical fractionation, the higher activity fraction was found for U. dioica and T. officinale, presenting IC50 values of 165.7 ± 3.85 and 126.1 ± 2.80 μg/ml, respectively; SI values were 5.59 and 6.01 for each fraction. The compounds present in T. officinale, were luteolin and caffeoylquinic acids derivatives and quercertin diclycosides. The compounds in the active fraction of U. dioica, were, chlorogenic acid, quercertin derivatives and flavonol glycosides (quercetin and kaempferol). Conclusions Two fractions from U. dioica and T. officinale methanolic extracts with anti-dengue activity were found. The compounds present in both fractions were identified, several recognized molecules have demonstrated activity against other viral species. Subsequent biological analysis of the molecules, alone or in combination, contained in the extracts will be carried out to develop therapeutics against DENV2

Effect of gallium as an additive in hydrodesulfurization WS2/gamma-Al2O3 catalysts

Diaz de Leon, J. N. Picquart, M. Villarroel, M. Vrinat, M. Gil Llambias, F. J. Murrieta, F. de los Reyes, J. A.The effect of gallium in W/Ga(x)-gamma-Al2O3 catalysts was investigated in the hydrodesulfurization of dibenzothiophene. The gamma-Al2O3 carrier was modified by the gallium addition prior to W impregnation and further calcination at 723 K. An electrophoretic study was carried out on the calcined Ga samples at 0, 0.29, 0.55, 0.84, 1.09, 1.43, 1.71 and 2.22 wt.% of Ga and it indicated that Ga addition affected significantly the surface of alumina. The presence of at least two gallium species was proposed, at low gallium content GaAl2O4 species decreased the isoelectric point (IEP) and probably the formation of Ga2O3 at higher contents increased again the IEP. Raman spectroscopy showed that the gallium incorporation had a strong effect in the formation of WOx species at the surface of catalysts. The highest amount of irregular WOx particles calculated by the Raman (O=W=O+W=O)/W-O-W band intensities ratio was found on the W/Ga(1.09)-gamma-Al2O3 catalyst. The presence of gallium induced higher activity on the dibenzothiophene hydrodesulfurization reaction in all catalysts tested. The highest Raman ratio and the highest activity were found for the W/Ga(1.09)-gamma-Al2O3 catalyst, suggesting that better dispersed W species could be responsible for the highest HDS activities. (C) 2010 Elsevier B.V. All rights reserved

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation