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World-wide reports of amphibian population declines have led to increased interest in the reproductive biology of anurans. As a model system, here we present evidence for the effective cryoprotection of sperm from the Puerto Rican frog, Eleutherodactylus coqui, using mixtures of fetal bovine serum (FBS) and dimethylsulfoxide (Me2SO), glycerol or sucrose extenders. Using a fluorescent dye exclusion assay, we found that 53.9 and 50.4% of all sperm with intact membranes prior to freezing maintained membrane integrity after rapid freezing and thawing when protected with either a FBS/glycerol or FBS/sucrose solution, respectively. The methods reported here may be useful for similar work with many of the more than 700 other species in this genus. [Copyright &y& Elsevier]

Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus. [ABSTRACT FROM AUTHOR]

This study investigates ovulation and egg deposition behaviors in the anuran Eleutherodactylus coqui from Puerto Rico in response to stimulation with gonadotropin and gonadotropin releasing hormones. Five hormones were tested by injection over a range of doses, including mammalian LHRH, avian LHRH, fish LHRH, D-Ala6, des-Gly10 ethylamide LHRH and hCG. We report a low level of ovulation and egg deposition in response to all hormones, with the most complete and consistent results from the non-natural D-Ala6, des-Gly10 ethylamide LHRH derivative. To confirm the viability of eggs produced in this manner we performed in vitro fertilization experiments that resulted in the development of normal frogs. Reproductive behaviors in E. coqui are apparently not controlled by a mammalian form of LHRH as reported in other common laboratory anuran species. D-Ala6, des-Gly10 ethylamide LHRH induces ovulation and deposition of mature and fertilizable eggs in E. coqui. [ABSTRACT FROM AUTHOR]

During May 2022-April 2023, dengue virus serotype 3 was identified among 601 travel-associated and 61 locally acquired dengue cases in Florida, USA. All 203 sequenced genomes belonged to the same genotype III lineage and revealed potential transmission chains in which most locally acquired cases occurred shortly after introduction, with little sustained transmission. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor commenting on the report "Allometry of Alarm Calls: Black-Capped Chickadees Encode Information About Predator Size."

Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread. [ABSTRACT FROM AUTHOR]

Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) [1] in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 μM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 μM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 μM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection. [ABSTRACT FROM AUTHOR]

Introduction: Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen and transmitted sexually is a vital question that has, thus far, been inconclusive. Prior studies, with limited numbers, have included men in various stages of infection with most in the recovery phase of the illness. The timing of test results and severity of illness has made recruiting study participants a significant challenge. Our pilot study will examine semen from men with a recent diagnosis of COVID-19 as well as those in the convalescent phase to determine if SARS-CoV-2 can be detected and its relationship, if any, with the severity of the disease. Methods: Eighteen men with a median age of 32 (range, 24-57) who tested positive for COVID-19 by rt-PCR analysis were enrolled and provided a semen sample. The study group demonstrated symptoms of COVID-19 ranging from asymptomatic to moderate and none required hospitalization. Samples were subjected to viral RNA extraction and then processed by real-time RT-PCR using the US Centers for Disease Control and Prevention (CDC, USA) panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes to detect the presence of SARS-CoV-2 RNA. Results: Length of time from diagnosis to providing a specimen ranged from 1 to 28 days (median, 6 days). Fifteen participants were symptomatic and three were asymptomatic, including recovering men, at the time of semen collection. No SARS-CoV-2 was detected in any of the semen samples. Conclusion: Based on these preliminary results and consistent with prior findings, we suggest SARS-CoV-2 is not present in semen during the acute or convalescent phase of COVID-19. [ABSTRACT FROM AUTHOR]

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015 – 2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms. [ABSTRACT FROM AUTHOR]

This study introduces a novel class of imidazolium- and ammonium-based ionic liquids possessing two C12 and C14 tails and thioether linkers designed for lipoplex-mediated DNA delivery. Imidazolium-based ionic liquids displayed efficient gene delivery properties with low toxicity. Thiol–yne click chemistry was employed for the facile and robust synthesis of these thioether-based cationic lipioids with enhanced lipophilicity and low fluidity. [ABSTRACT FROM AUTHOR]

We demonstrate herein a facile synthesis of a series of highly-ordered low-melting protic ionic liquids with linear C16–C20 side chains identical to natural fatty acids, which were produced by the S-alkylation of the 2-mercapto-1-methylimidazole (methimazole) ring. There is considerable structural latitude possible when designing highly lipophilic protic ionic liquids that exhibit low Tm values due to the cumulative disruptive effects of thioether and unsaturation on their packing efficiency. Their bulk thermophysical properties, hydrophobicity and cytotoxicity were also determined. [ABSTRACT FROM AUTHOR]

Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA. [ABSTRACT FROM AUTHOR]

Abstract: Severe dengue virus (DENV) disease symptoms, including dengue hemorrhagic fever and dengue shock syndrome, have been correlated with the presence of pre-existing antibodies that enhance rather than neutralize infections in Fc receptor bearing cells. These antibodies can originate from previous infection with a different serotype of dengue, or from waning antibody titers that occur in infants and young children as they are weaned from breast milk that contains protective dengue-specific antibodies. Despite the apparent importance of this antibody dependent enhancement (ADE) effect, there has been no description of any specific inhibitors of this process. We explored DENV entry inhibitors as a potential strategy to block ADE. Two different peptide entry inhibitors were tested for the ability to block antibody-mediated DENV-2 infection of human, FcRII bearing K562 cells in vitro. Both peptides were able to inhibit ADE, showing that entry inhibitors are possible candidates for the development of specific treatment for severe DENV infection. [ABSTRACT FROM AUTHOR]

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding "pseudoenergies", we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 µM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery. Author Summary: Virus surface proteins mediate interactions with target cells during the initial events in the process of infection. Inhibiting these proteins is therefore a major target for the development of antiviral drugs. However, there are a very large number of different viruses, each with their own distinct surface proteins and, with just a few exceptions, it is not clear how to build novel molecules to inhibit them. Here we applied a computational binding optimization strategy to an atomic resolution structure of dengue virus serotype 2 envelope protein to generate peptide sequences that should interact strongly with this protein. We picked dengue virus as a target because it is the causative agent for the most important mosquito transmitted viral disease. Out of a small number of candidates designed and tested, we identified two different highly inhibitory peptides. To verify our results, we showed that these peptides block virus:cell binding, interfere with a step during viral entry, alter the surface structure of dengue viral particles, and that they interact directly with dengue virus envelope protein. We expect that our approach may be generally applicable to other viral surface proteins where a high resolution structure is available. [ABSTRACT FROM AUTHOR]

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding ''pseudoenergies'', we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 μM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery. [ABSTRACT FROM AUTHOR]

Background: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. Results: Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. Conclusions: HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity. [ABSTRACT FROM AUTHOR]

Abstract: The anti-adhesive compound p-sulfoxy-cinnamic acid, zosteric acid (ZA), is derived from the temperate marine eelgrass, Zostera marina. ZA and five combinatorial chemistries based on ZA were evaluated for their anti-viral properties against dengue virus in a focus forming unit reduction assay. None of the compounds showed evidence of toxicity to the monkey kidney cell line LLCMK-2 over the concentration ranges tested. ZA showed a modest IC50 of approximately 2.3mM against DENV-2. Three other compounds showed IC50 values of 2.5, 2.4, 0.3mM, with a fourth not achieving a 50% inhibitory concentration against DENV-2. The most active compound, CF 238, showed IC50 values of 24, 46, 14 and 47μM against DENV-1, DENV-2, DENV-3 and DENV-4, respectively. CF 238 showed evidence of inhibition at an entry step in the viral life cycle and enhanced virus:cell binding as evidenced by a quantitative RT-PCR assay system. CF 238 may promote inappropriate virus:cell attachments common to all DENV strains that interfere with receptor interactions required for viral entry. These and other related chemistries may be useful as reagents for studying DENV entry, capturing and detecting DENV, and development of pharmaceuticals. [Copyright &y& Elsevier]

Background: The Sykes' monkey and related forms (Cercopithecus mitis) make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk). We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex. Results: The SIVsyk antigen-specific Western blot assay detected more serum samples as positive than either of the cross-reactive ELISA assays. Using this assay, we found that seroprevalence is higher than previously reported, but extremely variable in wild populations (from 0.0 to 90.9%). Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified. Conclusion: We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission. [ABSTRACT FROM AUTHOR]

The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated Boudicca. Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length. ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein, including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that of the gypsy/Ty3 retrotransposons. An additional ORF at the 3' end of the retrotransposon may encode an envelope protein. Phylogenetic comparison based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRnl from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing ≈8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode. [ABSTRACT FROM AUTHOR]

Discusses the origin of the human AIDS viruses human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). How the disease represents cross-species infections; Viruses related to the HIV-1 isolated from the common chimpanzee.

Correction for ‘Biomimetic design of protic lipidic ionic liquids with enhanced fluidity’ by Richard A. O'Brien et al., New J. Chem., 2016, 40, 7795–7803. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about the detection of mosquito-associated dengue virus in Key West, Florida in 2009.