Characterization of the transovarial transmission potential, tissue tropisms and genetic determinants of host specificity of single-host flaviviruses

Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts; thus, they are dual-host viruses. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) are single-host viruses because they have host ranges restricted to vertebrates and insects, respectively. Numerous insect-specific flaviviruses (ISFs) including CxFV have been discovered in the last decade and most are widely spread in nature. However, little is known about the mechanism(s) by which ISFs are maintained in nature. In a previous study, CxFV was detected in both female and male mosquitoes collected in the field suggesting that this virus is maintained in nature by vertical transmission. The experiments outlined in chapter 2 were designed to test the hypothesis that efficient transovarial transmission (TOT) of CxFV occurs in the mosquito host. CxFV RNA was detected in 526 of 540 Culex pipiens progeny derived from CxFV-infected females and thus, the filial infection rate was 97.4%. Because all positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that extremely efficient TOT of CxFV occurs in mosquitoes in nature. Tissue tropisms of CxFV were also defined. CxFV RNA was detected in all tissues tested: salivary glands, ovaries, testes, head, fat bodies and midguts. Time course experiments demonstrated that CxFV disseminates to the ovaries as early as 4 days post-inoculation. In chapter 3, the host range and genetic diversity of CxFV was investigated. Previously, a high prevalence of CxFV was reported in Cx. quinquefasciatus in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, five other Culex spp. mosquitoes were tested for evidence of CxFV infection. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have \u3e99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in Yucatan Peninsula of Mexico. In chapter 4, studies were performed to increase our knowledge of the genetic elements that condition the differential host ranges of flaviviruses. Although flaviviruses possess a similar genomic organization, they differ in terms of their host specificity; some flaviviruses infect both vertebrates and arthropods whereas others have a vertebrate-specific or arthropod-specific phenotype. The genetic elements that condition these differential host ranges and transmission cycles have not been identified. Therefore, chimeric viruses were constructed by replacing the capsid (C), premembrane (prM) and envelope (E) genes or the prM-E genes of MODV with the corresponding regions of WNV and CxFV. Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV in a MODV backbone. The virus could infect vertebrate but not mosquito cells, indicating that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype. The three other chimeras did not produce detectable virus. Comparative studies between flaviviruses that possess differential host range profiles will help us understand why some flaviviruses can infect only vertebrate or only invertebrate organisms while other flaviviruses can infect both insect and vertebrate hosts and cause devastating disease in humans and animals

Preparation, structure and properties of gold-based ‘ruby’ sodium silicate glass: A model metal-glass nano-composite

Gold ruby glass has been well-known for its deep red color dating back to Roman times but the role of gold nano-particles (AuNPs) in the creation of color was revealed only in 20th century. Glass alchemists discovered early on that addition of tin promotes the development of color. However, the underlying mechanism, which relies on the availability of electrons for oxidation/reduction and growth of AuNP above a minimum size, remains unclear. Additionally, very little information is available on how the electrical properties of such glasses are affected by the presence of gold nanoparticles or tin doping. In this dissertation, a gold-doped sodium trisilicate glass has been investigated as a model for metal-glass nano-composite in order to unravel the origin of the optical, electrical, and thermal properties of ruby glass in terms of atomistic structure and microstructure. This study also provides an insight on the dynamics of electrons in silicate glasses.We investigated the development of the AuNPs by extended X-ray absorption fine structure (EXAFS), in situ X-ray absorption near edge structure (XANES) analysis, and optical spectroscopy of sodium trisilicate glass doped with ≤ 0.1mol% of gold (as HAuCl4) and varying amounts of SnO 2 (0.005-0.1 mol%). The samples were prepared by the conventional melt-quench technique followed by thermal treatment. XANES was measured at the Au L3 -edge and Sn K-edge while heating the glass up to 550 °C. Development of the ruby color was followed concurrently with in situ optical spectroscopy; the in situ spectroscopy showed a red-shift and a blue-shift of the surface plasmon resonance peak due to AuNPs forming in samples with varying concentration of SnO2 with heat treatment. The XANES and EXAFS indicated transformation of ionic gold to metallic gold, and conversion from Au-O to Au-Au bonds. SnO2 doped samples had lower metallic gold i.e. Au-Au bonds in the as-quenched state, but also had a shorter incubation time and a faster growth of AuNPs during the heat treatment process. The tin remained as Sn4+ ions in the distorted octahedral site surrounded by mainly oxygen throughout i.e. both before and after the heat treatment. The addition of SnO2 helped gold dissolve in the glass matrix during melting, while acting as a nucleating agent at lower temperatures.Based on above results, a new model for the formation of AuNPs controlled by electron (FACE) availability is presented. The model suggests that the electrons involved in the reduction process through Au+1 + e-  Au0 control the growth process and in tin-free glass they mainly come from NBO. To prove this concept, additional electrons were generated by X-ray irradiation. The results showed a faster growth of AuNPs by the availability of these additional electrons, confirming that the reduction of gold ions is the rate limiting process that required availability of electrons. In addition, the X-ray irradiation produced the ruby color more effectively if the sample was irradiated during heating instead of before heating as the irradiation created new defects. However, the effect of the reduction process from X-irradiation diminished when higher SnO2 was present in the glasses. After the reduction stage, the growth of AuNPs, Au0 + Au0  AuNP, was controlled by the diffusion of gold atoms and Ostwald ripening of particles.The major factors that determine the influence of SnO2 on the AuNP formation include: i) Au+1 is more uniformly distributed in glass matrix of the as-quenched sample than in the tin-free sample, ii) the electrons associated with SnO_6^(2-) unit are more mobile than those associated with the NBOs of silicate network, and iii) SnO2 lowers the surface energy as a surfactant so that AuNPs may grow at a faster rate. The experimental results are in excellent qualitative agreement with the proposed FACE model.From scanning transmission electron microscope observations, spherical and non-spherical AuNPs were found in the glass matrix after annealing. In general, the average size of gold nanoparticles increased with heat treatment, as expected from the growth process. A smaller size and narrower size distribution of AuNPs were observed in the sample with higher SnO2 concentration. Different orientations (i.e. multiple grains) were found in several AuNPs, possibly originating from the coalescence of two particles. Some AuNPs appeared to have a core-shell structure but the energy dispersive X-ray spectroscopy did not show higher Sn, Na or Cl concentration in or surrounding the AuNPs.The Open Z-scan technique showed nonlinearity which demonstrated an optical limiting behavior. The samples with higher gold amounts showed a higher nonlinear absorption coefficient (β). The addition of tin to the samples caused the deep ruby red color within the glass but there was a lower β in these samples. Moreover, the tin-doped samples showed a negative or a positive β depending on the intensity of laser. Finally, the addition of both 0.1 mol% gold and 0.1 mol% tin increased the DC electrical conductivity, which originated from the sodium ion transport, as compared to the undoped base glass. The activation energy of DC conductivity decreased when the amount of tin increased with the gold content kept constant. The Na+ ion transport appeared to be different for the two types of dopings: Au doping caused general expansion of molar volume whereas tin doping enhanced the polarizability of the medium

Evidence of Efficient Transovarial Transmission of Culex Flavivirus by Culex pipiens (Diptera: Culicidae)

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F1 adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F1 and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype

Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States

A cross-sectional study was performed to identify operation-level risk factors associated with prevalence of antibody to Bunyamwera (BUN) serogroup viruses in sheep in the United States. Sera were obtained from 5150 sheep in 270 operations located in 22 states (three in the west, nine central states, and 10 in the east) and tested at a dilution of 1:20 by a plaque reduction neutralization test (PRNT) using Cache Valley virus (CVV). Antibodies that neutralized CVV were identified in 1455 (28%) sheep. Animal-level seroprevalence was higher in the east (49%) than the central (17%) and western (10%) states. A convenient subset (n = 509) of sera with antibodies that neutralized CVV was titrated and further analyzed by PRNT using all six BUN serogroup viruses that occur in the United States: CVV, Lokern virus (LOKV), Main Drain virus (MDV), Northway virus (NORV), Potosi virus (POTV), and Tensaw virus (TENV). Antibodies to CVV and LOKV were identified in sheep in all three geographic regions; MDV and POTV activity was detected in the central and eastern states, NORV activity was restricted to the west, and antibodies to TENV were not detected in any sheep. Several management factors were significantly associated with the presence of antibodies to BUN serogroup viruses. For instance, sheep housed during the lambing season inside structures that contained four walls and a roof and a door closed most of the time were more likely to be seropositive than other sheep. In contrast, herded/open-range sheep were less likely to be seropositive than their counterparts. These data can be used by producers to implement strategies to reduce the likelihood of BUN serogroup virus infection and improve the health and management practices of sheep

Characterization of the transovarial transmission potential, tissue tropisms and genetic determinants of host specificity of single-host flaviviruses

Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts; thus, they are dual-host viruses. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) are single-host viruses because they have host ranges restricted to vertebrates and insects, respectively. Numerous insect-specific flaviviruses (ISFs) including CxFV have been discovered in the last decade and most are widely spread in nature. However, little is known about the mechanism(s) by which ISFs are maintained in nature. In a previous study, CxFV was detected in both female and male mosquitoes collected in the field suggesting that this virus is maintained in nature by vertical transmission. The experiments outlined in chapter 2 were designed to test the hypothesis that efficient transovarial transmission (TOT) of CxFV occurs in the mosquito host. CxFV RNA was detected in 526 of 540 Culex pipiens progeny derived from CxFV-infected females and thus, the filial infection rate was 97.4%. Because all positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that extremely efficient TOT of CxFV occurs in mosquitoes in nature. Tissue tropisms of CxFV were also defined. CxFV RNA was detected in all tissues tested: salivary glands, ovaries, testes, head, fat bodies and midguts. Time course experiments demonstrated that CxFV disseminates to the ovaries as early as 4 days post-inoculation. In chapter 3, the host range and genetic diversity of CxFV was investigated. Previously, a high prevalence of CxFV was reported in Cx. quinquefasciatus in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, five other Culex spp. mosquitoes were tested for evidence of CxFV infection. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have >99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in Yucatan Peninsula of Mexico. In chapter 4, studies were performed to increase our knowledge of the genetic elements that condition the differential host ranges of flaviviruses. Although flaviviruses possess a similar genomic organization, they differ in terms of their host specificity; some flaviviruses infect both vertebrates and arthropods whereas others have a vertebrate-specific or arthropod-specific phenotype. The genetic elements that condition these differential host ranges and transmission cycles have not been identified. Therefore, chimeric viruses were constructed by replacing the capsid (C), premembrane (prM) and envelope (E) genes or the prM-E genes of MODV with the corresponding regions of WNV and CxFV. Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV in a MODV backbone. The virus could infect vertebrate but not mosquito cells, indicating that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype. The three other chimeras did not produce detectable virus. Comparative studies between flaviviruses that possess differential host range profiles will help us understand why some flaviviruses can infect only vertebrate or only invertebrate organisms while other flaviviruses can infect both insect and vertebrate hosts and cause devastating disease in humans and animals.</p

Evidence of Efficient Transovarial Transmission of Culex Flavivirus by Culex pipiens (Diptera: Culicidae)

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F1 adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F1 and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature.This article is from Journal of Medical Entomology 48 (2011): 1031–1038, doi:10.1603/ME11043.</p

Minor Elements and Color Causing Role in Spinel: Multi-Analytical Approaches

Natural spinel (MgAl2O4) usually contains some minor and trace elements (e.g., Cr, Co, Fe, V) that may cause various hues. The ratios of these chromophores directly affect the color composition. The red color in spinel is attributed to the combination of significant Cr and V. Magenta and purple to blue and green colors in spinels are affected by the significant Fe concentration, whereas orange color in spinel shows the contribution of significant V content compared to Cr and Fe. After the heating experiment, advanced gemological investigation reveals some noteworthy characteristic features. X-ray absorption spectroscopy (XAS) indicates a greater change in oxidation state, as well as disordering of Fe and V. Broadening of the dominant peak at around 406 cm−1 with occurrences of additional small peaks at around 715–719 cm−1 in Raman spectra, as well as broadening of the 685 nm (R-line) and poorly defined structure of additional peaks (N-lines) in photoluminescence spectra should be significant indicators of spinel undergone heat treatment

Structure and nonlinear optical studies of Au nanoparticles embedded in lead lanthanum borate glass

Metal nanoparticles of gold in various proportions were prepared by melt-quenching technique without any assistance of reducing agent. X-ray absorption near edge structure analysis shows that measurements at the Au L3 edge were made on lanthanum lead borate doped with gold. X-ray absorption near edge structure shows that the oxidation state of Au and the analysis show the presence of gold nanoparticles. Electron microscopy and energy dispersive absorption X-ray spectroscopy confirm the size of Au nanoparticle inside the lead lanthanum borate glass. Nonlinear optical studies of Au doped oxide glass are performed using nanosecond laser pulse excitation. Z-scan experiments with 532 nm wave-length pulses are used to excite glass samples which show that optical limiting effect of these as-prepared glasses doped with Au nanoparticle was also studied. © 2014 Elsevier B.V. All rights reserved