Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli

Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.     

Blood‐feeding and immunogenic Aedes aegypti saliva proteins

Mosquito‐transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar‐fed and blood‐fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar‐ or blood‐based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar‐fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up‐regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2‐DE profiles of immunogenic Ae. aegypti SG BF‐related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.     

In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection

The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8⁺ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4⁺ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a⁺ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.     

Relationships between IL-17+ Subsets,Tregs and pDCs That Distinguish among SIV Infected Elite Controllers,Low, Medium and High Viral Load Rhesus Macaques

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4⁺ Tregs, CD8⁺ Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4⁺ Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8⁺ Tregs was also noted with a moderate increase in ECs but further increases of CD8⁺ Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4⁺ Tregs and increases in the levels of CD8⁺ Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17⁺ subsets in GIT as CD3^−/CD8⁺/NKG2a⁻ and CD3⁺/CD8⁺/NKG2a⁺ subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design.     

Prospective field study of transovarial dengue‐virus transmission by two different forms of Aedes aegypti in an urban area of Bangkok,Thailand

A prospective field study was conducted to determine transovarial dengue‐virus transmission in two forms of Aedes aegypti mosquitoes in an urban district of Bangkok, Thailand. Immature Aedes mosquitoes were collected monthly for one year and reared continuously until adulthood in the laboratory. Mosquitoes assayed for dengue virus were processed in pools and their dengue virus infection status was determined by one‐step RT‐PCR and nested‐PCR methods. Of a total 15,457 newly emerged adult Ae. aegypti, 98.2% were dark and 1.8% of the pale form. The results showed that the minimum infection rate (MIR) by transovarial transmission (TOT) of dengue virus during the one‐year study ranged between 0 to 24.4/1,000 mosquitoes. Dengue virus TOT increased gradually during the hot summer months, reaching a peak in April‐June, while dengue cases peaked in September, a rainy month near the end of the rainy season. Therefore, mosquito infections due to TOT were prevalent four months before a high incidence of human infections. TOT dengue virus infections occurred in both forms of Ae. aegypti. All four dengue serotypes were detected, with DEN‐4 predominant, followed by DEN‐3, DEN‐1, and DEN‐2, respectively.     

In Vivo Administration of a JAK3 Inhibitor to Chronically SIV Infected Rhesus Macaques Leads to NK Cell Depletion Associated with Transient Modest Increase in Viral Loads

Innate immune responses are reasoned to play an important role during both acute and chronic SIV infection and play a deterministic role during the acute stages on the rate of infection and disease progression. NK cells are an integral part of the innate immune system but their role in influencing the course of SIV infection has been a subject of debate. As a means to delineate the effect of NK cells on SIV infection, use was made of a Janus kinase 3 (JAK3) inhibitor that has previously been shown to be effective in the depletion of NK cells in vivo in nonhuman primates (NHP). Extensive safety and in vitro/in vivo PK studies were conducted and an optimal dose that depletes NK cells and NK cell function in vivo identified. Six chronically SIV infected rhesus macaques, 3 with undetectable/low plasma viral loads and 3 with high plasma viral loads were administered a daily oral dose of 10 mg/kg for 35 days. Data obtained showed that, at the dose tested, the major cell lineage affected both in the blood and the GI tissues were the NK cells. Such depletion appeared to be associated with a transient increase in plasma and GI tissue viral loads. Whereas the number of NK cells returned to baseline values in the blood, the GI tissues remained depleted of NK cells for a prolonged period of time. Recent findings show that the JAK3 inhibitor utilized in the studies reported herein has a broader activity than previously reported with dose dependent effects on both JAK2 and JAK1 suggests that it is likely that multiple pathways are affected with the administration of this drug that needs to be taken into account. The findings reported herein are the first studies on the use of a JAK3 inhibitor in lentivirus infected NHP.     

Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following Infection with Dengue Virus

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.     

Preparation of squid skin collagen hydrolysate as an antihyaluronidase,antityrosinase, and antioxidant agent

A collagen was isolated from squid skin, a processing waste product. The biofunctional activities of enzymatic squid skin collagen hydrolysates were determined to produce a value-added material. Five low-molecular-mass hydrolysate fractions, F1 (>30 kD), F2 (10–30 kD), F3 (3–10 kD), F4 (1–3 kD), and F5 (<1 kD), were manufactured from its enzymatic hydrolysate by ultrafiltration. Fraction F3 had the strongest antihyaluronidase inhibitory activity. Gly, Val, and Pro were major amino acids in F3, while Met, Tyr, and His were minor ones. The molecular mass of F3 was in the range of 3.4 to 10 kD. F3 exhibited copper chelating ability in a concentration-dependent manner. The ferrous chelating ability of F3 was almost 50% at 200 µg/mL. F3 also inhibited tyrosinase activity by 39.65% at 1 mg/mL. Furthermore, F3 had stronger hydroxyl radical scavenging activity (IC₅₀ = 149.94 µg/mL) than ascorbic acid (IC₅₀ = 212.94 µg/mL). Therefore, the squid collagen hydrolysate can be utilized as a nutraceutical or cosmeceutical agent.