@article{hernandez_qu_volk_showell_hoyo_ellison-barnes_johnson_2024, title={Address correspondence to Raquel G. Hernandez, MD, MPH, (e-mail: Raquel.hernandez@jhmi.edu).}, volume={24}, ISSN={["1876-2867"]}, DOI={10.1016/j.acap.2024.05.002}, abstractNote={To describe the Coronovirus 19 (COVID-19) pandemic impact among mothers of young children (0-8 years) and assess prepandemic factors associated with greater pandemic impact and psychosocial distress.}, number={7}, journal={ACADEMIC PEDIATRICS}, author={Hernandez, Raquel G. and Qu, Xueqi and Volk, Heather and Showell, Nakiya N. and Hoyo, Cathrine and Ellison-Barnes, Alejandra and Johnson, Sara B.}, year={2024}, pages={1076–1085} }
 @article{sosnowski_rojo-wissar_peng_parade_sharkey_hoyo_murphy_hernandez_johnson_2024, title={Maternal childhood adversity and infant epigenetic aging: Moderation by restless sleep during pregnancy}, volume={66}, ISSN={["1098-2302"]}, DOI={10.1002/dev.22464}, abstractNote={AbstractMaternal exposure to childhood adversity is associated with detrimental health outcomes throughout the life span and may have implications for offspring. Evidence links maternal adverse childhood experiences (ACEs) to detrimental birth outcomes, yet the impact on the infant's epigenome is unclear. Moreover, maternal sleep habits during pregnancy may influence this association. Here, we explore whether restless sleep during pregnancy moderates the association between exposure to maternal childhood adversity and infant epigenetic age acceleration in 332 mother–infant dyads (56% female; 39% Black; 25% Hispanic). During the second trimester, mothers self‐reported childhood adversity and past‐week restless sleep; DNA methylation from umbilical vein endothelial cells was used to estimate five epigenetic clocks. Multivariable linear regression was used to test study hypotheses. Despite no evidence of main effects, there was evidence of an interaction between maternal ACEs and restless sleep in predicting infant epigenetic age acceleration using the EPIC gestational age clock. Only infants whose mothers reported exposure to both ACEs and restless sleep demonstrated accelerated epigenetic aging. Results provide preliminary evidence that maternal childhood adversity and sleep may influence the infant epigenome.}, number={2}, journal={DEVELOPMENTAL PSYCHOBIOLOGY}, author={Sosnowski, David W. and Rojo-Wissar, Darlynn M. and Peng, Gang and Parade, Stephanie H. and Sharkey, Katherine and Hoyo, Cathrine and Murphy, Susan K. and Hernandez, Raquel G. and Johnson, Sara B.}, year={2024}, month={Feb} }
 @article{azevedo_cardoso_caldas_vieira werneck_cohen_yamamoto_silva meira_meneses_erthal_sa_et al._2020, title={The impact of Zika virus infection on human semen: a case study}, volume={15}, ISSN={["1746-0808"]}, DOI={10.2217/fvl-2019-0159}, abstractNote={ The outbreak of Zika virus (ZIKV) in Brazil and in many countries was marked by the occurrences of fetal malformations and neurological effects upon infection of pregnant women. Like other members of the Flaviviridae family, ZIKV in semen is infectious and sexually transmitted. Viral persistence in the male genital tract is unresolved and still needs further understanding. A regular semen donor was followed from the acute phase of ZIKV infection, up to 8 months after ZIKV in his body fluids became undetected. Immunofluorescence assay was performed in semen samples before and after the infection. A significant decrease in sperm concentration and in the percentage of progressive motility of sperm was observed. The methodology adopted for clearance of sexually transmitted viruses decreased virus load, but the complete clearance of ZIKV from semen was not achieved. }, number={1}, journal={FUTURE VIROLOGY}, author={Azevedo, Renata Campos and Cardoso, Maria Cecilia A. and Caldas, Lucio Ayres and Vieira Werneck, Caio Luis and Cohen, Cristiane Napoleao and Yamamoto, Kristie Aimi and Silva Meira, Guilherme Louzada and Meneses, Marcelo Damiao and Erthal, Maria Cecilia and Sa, Paulo Gallo and et al.}, year={2020}, month={Jan}, pages={5–12} }
 @article{hernandez_glaros_rizzo_ferreira_2019, title={Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells}, volume={9}, ISSN={["2331-8325"]}, DOI={10.21769/BioProtoc.3239}, abstractNote={Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.}, number={10}, journal={BIO-PROTOCOL}, author={Hernandez, Raquel and Glaros, Trevor and Rizzo, Gabrielle and Ferreira, Davis F.}, year={2019}, month={May} }
 @article{schuchman_kilianski_piper_vancini_ribeiro_sprague_nasar_boyd_hernandez_glaros_2018, title={Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication}, volume={92}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00694-18}, abstractNote={ABSTRACT
          Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus, to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts, and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro, and Chikungunya viruses. The most significant finding was that in addition to the host proteins, SINV nonstructural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics.
          
            IMPORTANCE
            Pathogenic alphaviruses, such as Chikungunya and Mayaro viruses, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens, such as dengue fever, West Nile, and Yellow fever viruses. With few exceptions, there are no vaccines or prophylactics for these agents, leaving one-third of the world population at risk of infection. Identifying effective antivirals has been a long-term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.
          }, number={14}, journal={JOURNAL OF VIROLOGY}, author={Schuchman, Ryan and Kilianski, Andy and Piper, Amanda and Vancini, Ricardo and Ribeiro, Jose M. C. and Sprague, Thomas R. and Nasar, Farooq and Boyd, Gabrielle and Hernandez, Raquel and Glaros, Trevor}, year={2018}, month={Jul} }
 @article{schuchman_vancini_piper_breuer_ribeiro_ferreira_magliocca_emmerich_hernandez_brown_2018, title={Role of the vacuolar ATPase in the Alphavirus replication cycle}, volume={4}, ISSN={2405-8440}, url={http://dx.doi.org/10.1016/J.HELIYON.2018.E00701}, DOI={10.1016/J.HELIYON.2018.E00701}, abstractNote={We have shown that Alphaviruses can enter cells by direct penetration at the plasma membrane (R. Vancini, G. Wang, D. Ferreira, R. Hernandez, and D. Brown, J Virol, 87:4352-4359, 2013). Direct penetration removes the requirement for receptor-mediated endocytosis exposure to low pH and membrane fusion in the process of RNA entry. Endosomal pH as well as the pH of the cell cytoplasm is maintained by the activity of the vacuolar ATPase (V-ATPase). Bafilomycin is a specific inhibitor of V-ATPase. To characterize the roll of the V-ATPase in viral replication we generated a Bafilomycin A1(BAF) resistant mutant of Sindbis virus (BRSV). BRSV produced mature virus and virus RNA in greater amounts than parent virus in BAF-treated cells. Sequence analysis revealed mutations in the E2 glycoprotein, T15I/Y18H, were responsible for the phenotype. These results show that a functional V-ATPase is required for efficient virus RNA synthesis and virus maturation in Alphavirus infection.}, number={7}, journal={Heliyon}, publisher={Elsevier BV}, author={Schuchman, Ryan M. and Vancini, Ricardo and Piper, Amanda and Breuer, Denitra and Ribeiro, Mariana and Ferreira, Davis and Magliocca, Joseph and Emmerich, Veronica and Hernandez, Raquel and Brown, Dennis T.}, year={2018}, month={Jul}, pages={e00701} }
 @article{magliocca_vancini_hernandez_brown_2016, title={Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly}, volume={90}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00948-16}, abstractNote={ABSTRACT
          
            A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface.
            35
            S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer.
          
          
            IMPORTANCE
            Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.
          }, number={18}, journal={JOURNAL OF VIROLOGY}, author={Magliocca, Joseph and Vancini, Ricardo and Hernandez, Raquel and Brown, Dennis T.}, year={2016}, month={Sep}, pages={8372–8380} }
 @article{schuchman_vancini_piper_breuer_ribeiro_farreira_magliocca_emmerich_hernandez_brown_2016, title={The role of the vacuolar ATPase in alphavirus replication.}, volume={27}, journal={Molecular Biology of the Cell}, author={Schuchman, R. and Vancini, R. and Piper, A. and Breuer, D. and Ribeiro, M. and Farreira, D. and Magliocca, J. and Emmerich, V. and Hernandez, R. and Brown, D.}, year={2016} }
 @misc{vancini_hernandez_brown_2015, title={Alphavirus Entry into Host Cells}, volume={129}, journal={Molecular basis of viral infection}, author={Vancini, R. and Hernandez, R. and Brown, D.}, year={2015}, pages={33–62} }
 @article{briggs_smith_piper_huitt_spears_quiles_ribeiro_thomas_brown_hernandez_2014, title={Live Attenuated Tetravalent Dengue Virus Host Range Vaccine Is Immunogenic in African Green Monkeys following a Single Vaccination}, volume={88}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00541-14}, abstractNote={ABSTRACT
          
            The causative agent of dengue fever, dengue virus (DENV), is transmitted by mosquitoes, and as distribution of these insects has expanded, so has dengue-related disease. DENV is a member of the
            Flaviviridae
            family and has 4 distinct serotypes (DENV-1, -2, -3, and -4). No lasting cross protection is afforded to heterologous serotypes following infection by any one of the individual serotypes. The presence of nonneutralizing antibodies to one serotype can facilitate the occurrence of more-severe dengue hemorrhagic fever through immune enhancement upon infection with a second serotype. For this reason, the development of a safe, tetravalent vaccine to produce a balanced immune response to all four serotypes is critical. We have developed a novel approach to produce safe and effective live-attenuated vaccines for DENV and other insect-borne viruses. Host range (HR) mutants of each DENV serotype were created by truncating transmembrane domain 1 of the E protein and selecting for strains of DENV that replicated well in insect cells but not mammalian cells. These vaccine strains were tested for immunogenicity in African green monkeys (AGMs). No vaccine-related adverse events occurred. The vaccine strains were confirmed to be attenuated
            in vivo
            by infectious center assay (ICA). Analysis by 50% plaque reduction neutralization test (PRNT
            50
            ) established that by day 62 postvaccination, 100% of animals seroconverted to DENV-1, -2, -3, and -4. Additionally, the DENV HR tetravalent vaccine (HR-Tet) showed a tetravalent anamnestic immune response in 100% (16/16) of AGMs after challenge with wild-type (WT) DENV strains.
          
          
            IMPORTANCE
            We have generated a live attenuated viral (LAV) vaccine capable of eliciting a strong immune response in African green monkeys (AGMs) in a single dose. This vaccine is delivered by injecting one of four attenuated serotypes into each limb of the animal. 100% of animals given the vaccine generated antibodies against all 4 serotypes, and this response was found to be balanced in nature. This is also one of the first studies of dengue in AGMs, and our study suggests that viremia and antibody response in AGMs may be similar to those seen in DENV infection in humans.
          }, number={12}, journal={JOURNAL OF VIROLOGY}, author={Briggs, Caitlin M. and Smith, Katherine M. and Piper, Amanda and Huitt, Emerson and Spears, Carla J. and Quiles, Michelle and Ribeiro, Mariana and Thomas, Malcolm E. and Brown, Dennis T. and Hernandez, Raquel}, year={2014}, month={Jun}, pages={6729–6742} }
 @article{piper_ribeiro_smith_briggs_huitt_nanda_spears_quiles_cullen_thomas_et al._2013, title={Chikungunya Virus Host Range E2 Transmembrane Deletion Mutants Induce Protective Immunity against Challenge in C57BL/6J Mice}, volume={87}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.03357-12}, abstractNote={ABSTRACT
          
            A vaccine against Chikungunya virus (ChikV), a reemerging pathogenic arbovirus, has been made by attenuating wild-type (WT) virus via truncation of the transmembrane domain (TMD) of E2 and selecting for host range (HR) mutants. Mice are a standard model system for ChikV disease and display the same symptoms of the disease seen in humans. Groups of mice were inoculated with one of three ChikV HR mutants to determine the ability of each mutant strain to elicit neutralizing antibody and protective immunity upon virus challenge. One mutant, ChikV TM17-2, fulfilled the criteria for a good vaccine candidate. It displayed no reactogenicity at the site of injection, no tissue disease in the foot/ankle and quadriceps, and no evidence of viral persistence in foot/ankle tissues 21 days after infection. Upon challenge with a highly pathogenic strain of ChikV, the mutant blocked viral replication in all tissues tested. This study identified a ChikV HR mutant that grows to high levels in insect cells but was restricted in the ability to assemble virus in mammalian cells
            in vitro
            . The study demonstrates that these HR strains are attenuated in the mammalian host and warrant further development as live-attenuated vaccine strains.
          }, number={12}, journal={JOURNAL OF VIROLOGY}, author={Piper, Amanda and Ribeiro, Mariana and Smith, Katherine M. and Briggs, Caitlin M. and Huitt, Emerson and Nanda, Kavita and Spears, Carla J. and Quiles, Michelle and Cullen, John and Thomas, Malcolm E. and et al.}, year={2013}, month={Jun}, pages={6748–6757} }
 @article{vancini_kramer_ribeiro_hernandez_brown_2013, title={Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane}, volume={435}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2012.10.013}, abstractNote={Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.}, number={2}, journal={VIROLOGY}, author={Vancini, Ricardo and Kramer, Laura D. and Ribeiro, Mariana and Hernandez, Raquel and Brown, Dennis}, year={2013}, month={Jan}, pages={406–414} }
 @article{brown_hernandez_2012, title={Infection of cells by alphaviruses}, volume={726}, journal={Viral molecular machines}, author={Brown, D. T. and Hernandez, R.}, year={2012}, pages={181–199} }
 @article{smith_nanda_mccarl_spears_piper_ribeiro_quiles_briggs_thomas_thomas_et al._2012, title={Testing of Novel Dengue Virus 2 Vaccines in African Green Monkeys: Safety, Immunogenicity, and Efficacy}, volume={87}, ISSN={["1476-1645"]}, DOI={10.4269/ajtmh.2012.12-0004}, abstractNote={The immunogenicity and safety of three novel host-range vaccines containing deletions in the transmembrane domain of dengue virus serotype 2 (DV2) E glycoprotein were evaluated in African green monkeys. The shorter transmembrane domains are capable of functionally spanning an insect but not a mammalian cell membrane, resulting in production of viral mutants that have reduced infectivity in mammalian hosts but efficient growth in insect cells. Groups of four monkeys received one dose each of test vaccine candidate with no booster immunization. After immunization, levels of viremia produced by each vaccine were determined by infectious center assay. Vaccine recipient immune response to wild-type DV2 challenge was measured on Day 57 by enzyme-linked immunosorbent assay and plaque reduction neutralization test. Two vaccines, DV2ΔGVII and DV2G460P, generated neutralizing antibody in the range of 700–900 50% plaque reduction neutralization test units. All three vaccine strains decreased the length of viremia by at least two days. No safety concerns were identified.}, number={4}, journal={AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE}, author={Smith, Katherine M. and Nanda, Kavita and McCarl, Victoria and Spears, Carla J. and Piper, Amanda and Ribeiro, Mariana and Quiles, Michelle and Briggs, Caitlin M. and Thomas, Gwynneth S. and Thomas, Malcolm E. and et al.}, year={2012}, month={Oct}, pages={743–753} }
 @article{he_piper_meilleur_hernandez_heller_brown_2012, title={Conformational Changes in Sindbis Virus Induced by Decreased pH Are Revealed by Small-Angle Neutron Scattering}, volume={86}, ISSN={["0022-538X"]}, url={http://europepmc.org/abstract/med/22156534}, DOI={10.1128/jvi.06569-11}, abstractNote={ABSTRACT
          Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.}, number={4}, journal={JOURNAL OF VIROLOGY}, author={He, Lilin and Piper, Amanda and Meilleur, Flora and Hernandez, Raquel and Heller, William T. and Brown, Dennis T.}, year={2012}, month={Feb}, pages={1982–1987} }
 @article{hunt_hernandez_brown_2011, title={Role of the Vacuolar-ATPase in Sindbis Virus Infection}, volume={85}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.01864-10}, abstractNote={ABSTRACT
          
            Bafilomycin A
            1
            is a specific inhibitor of the vacuolar-ATPase (V-ATPase), which is responsible for pH homeostasis of the cell and for the acidification of endosomes. Bafilomycin A
            1
            has been commonly used as a method of inhibition of infection by viruses known or suspected to follow the path of receptor-mediated endocytosis and low-pH-mediated membrane fusion. The exact method of entry for Sindbis virus, the prototype alphavirus, remains undetermined. To further investigate the role of the V-ATPase in Sindbis virus infection, the effects of bafilomycin A
            1
            on the infection of BHK and insect cells by Sindbis virus were studied. Bafilomycin A
            1
            was found to block the expression of a virus-encoded reporter gene in both infection and transfection of BHK cells. The inhibitory effects of bafilomycin A
            1
            were found to be reversible. The results suggest that in BHK cells in the presence of bafilomycin A
            1
            , virus RNA enters the cell and is translated, but replication and proper folding of the product proteins requires the function of the V-ATPase. Bafilomycin A
            1
            had no significant effect on the outcome of infection in insect cells.
          }, number={3}, journal={JOURNAL OF VIROLOGY}, author={Hunt, Sabrina R. and Hernandez, Raquel and Brown, Dennis T.}, year={2011}, month={Feb}, pages={1257–1266} }
 @article{smith_nanda_spears_ribeiro_vancini_piper_thomas_thomas_brown_hernandez_2011, title={Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype}, volume={8}, ISSN={["1743-422X"]}, DOI={10.1186/1743-422x-8-289}, abstractNote={There are over 700 known arboviruses and at least 80 immunologically distinct types that cause disease in humans. Arboviruses are transmitted among vertebrates by biting insects, chiefly mosquitoes and ticks. These viruses are widely distributed throughout the world, depending on the presence of appropriate hosts (birds, horses, domestic animals, humans) and vectors. Mosquito-borne arboviruses present some of the most important examples of emerging and resurgent diseases of global significance. A strategy has been developed by which host-range mutants of Dengue virus can be constructed by generating deletions in the transmembrane domain (TMD) of the E glycoprotein. The host-range mutants produced and selected favored growth in the insect hosts. Mouse trials were conducted to determine if these mutants could initiate an immune response in an in vivo system. The DV2 E protein TMD defined as amino acids 452SWTMKILIGVIITWIG467 was found to contain specific residues which were required for the production of this host-range phenotype. Deletion mutants were found to be stable in vitro for 4 sequential passages in both host cell lines. The host-range mutants elicited neutralizing antibody above that seen for wild-type virus in mice and warrant further testing in primates as potential vaccine candidates. Novel host-range mutants of DV2 were created that have preferential growth in insect cells and impaired infectivity in mammalian cells. This method for creating live, attenuated viral mutants that generate safe and effective immunity may be applied to many other insect-borne viral diseases for which no current effective therapies exist.}, journal={VIROLOGY JOURNAL}, author={Smith, Katherine M. and Nanda, Kavita and Spears, Carla J. and Ribeiro, Mariana and Vancini, Ricardo and Piper, Amanda and Thomas, Gwynneth S. and Thomas, Malcolm E. and Brown, Dennis T. and Hernandez, Raquel}, year={2011}, month={Jun} }
 @article{hernandez_brown_2010, title={Growth and Maintenance of Baby Hamster Kidney (BHK) Cells}, volume={17}, ISSN={1934-8525}, url={http://dx.doi.org/10.1002/9780471729259.MCA04HS17}, DOI={10.1002/9780471729259.MCA04HS17}, abstractNote={AbstractThe BHK21 cell line was established in 1961 from the kidneys of 5 Syrian hamsters from litter number 21. Since this time, this cell line has been a laboratory standard for the growth of countless viruses and the study of many biological processes. The specific use for the growth of Sindbis virus is described, although it may apply to many types of viruses. Curr. Protoc. Microbiol. 17:A.4H.1‐A.4H.7. © 2010 by John Wiley & Sons, Inc.}, number={1}, journal={Current Protocols in Microbiology}, publisher={Wiley}, author={Hernandez, Raquel and Brown, Dennis T.}, year={2010}, month={May}, pages={A.4H.1-A.4H.7} }
 @article{hernandez_brown_2010, title={Growth and Maintenance of Chick Embryo Fibroblasts (CEF)}, volume={17}, ISSN={1934-8525}, url={http://dx.doi.org/10.1002/9780471729259.MCA04IS17}, DOI={10.1002/9780471729259.MCA04IS17}, abstractNote={AbstractPrimary cultures of chick embryo fibroblasts (CEF) are widely used for the cultivation of viruses. Protocols for the growth and maintenance of CEF cells in the laboratory are provided. Curr. Protoc. Microbiol. 17:A.4I.1‐A.4I.8. © 2010 by John Wiley & Sons, Inc.}, number={1}, journal={Current Protocols in Microbiology}, publisher={Wiley}, author={Hernandez, Raquel and Brown, Dennis T.}, year={2010}, month={May}, pages={A.4I.1-A.4I.8} }
 @article{hernandez_brown_2010, title={Growth and Maintenance of Mosquito Cell Lines}, volume={17}, ISSN={1934-8525}, url={http://dx.doi.org/10.1002/9780471729259.MCA04JS17}, DOI={10.1002/9780471729259.MCA04JS17}, abstractNote={AbstractMosquito cells (Aedes albopictus) are among the most common insect cells emerging as new sources of cell cultures to use in basic research and in the pharmaceutical industry. They adapt well to growth in suspension; can be used in bioreactors for the production of expressed proteins, virus, and virus‐like particles; can be used in studies requiring lower growth temperatures than mammalian cells (28°C or below); and (because they are cholesterol auxotrophs) can be adapted to grow in dilipidated or serum‐free medium for experiments requiring these conditions. Procedures applicable to the laboratory maintenance of mosquito cell lines are described. Curr. Protoc. Microbiol. 17:A.4J.1‐A.4J.8. © 2010 by John Wiley & Sons, Inc.}, number={1}, journal={Current Protocols in Microbiology}, publisher={Wiley}, author={Hernandez, Raquel and Brown, Dennis T.}, year={2010}, month={May}, pages={A.4J.1-A.4J.8} }
 @article{mudiganti_hernandez_brown_2010, title={Insect response to alphavirus infection-Establishment of alphavirus persistence in insect cells involves inhibition of viral polyprotein cleavage}, volume={150}, ISSN={["1872-7492"]}, DOI={10.1016/j.virusres.2010.02.016}, abstractNote={Alphavirus persistence in the insect vector is an essential element in the vector–host transmission cycle of the virus and provides a model to study the biochemical and molecular basis for virus–vector coexistence. The prototype alphavirus Sindbis (SV) establishes persistent infections in invertebrate cell cultures which are characterized by low levels of virus production. We hypothesized that antiviral factors may be involved in decreasing the virus levels as virus persistence is established in invertebrate cells. Transcription profiles in Drosophila S2 cells at 5 days post-infection with SV identified families of gene products that code for factors that can explain previous observations seen in insect cells infected with alphaviruses. Genomic array analysis identified up-regulation of gene products involved in intracellular membrane vesicle formation, cell growth rate changes and immune-related functions in S2 cells infected with SV. Transcripts coding for factors involved in different aspects of the Notch signaling pathway had increased in expression. Increased expression of ankyrin, plap, syx13, unc-13, csp, rab1 and rab8 may aid in formation of virus containing vesicles and in intracellular transport of viral structural proteins. Possible functions of these gene products and relevant hypotheses are discussed. We confirmed the up-regulation of a wide-spectrum protease inhibitor, Thiol-ester containing Protein (TEP) II. We report inhibition of the viral polyprotein cleavage at 5 days post-infection (dpi) and after superinfection of SV-infected cells at 5 dpi. We propose that inefficient cleavage of the polyprotein may, at least in part, lead to reduced levels of virus seen as persistence is established.}, number={1-2}, journal={VIRUS RESEARCH}, author={Mudiganti, Usharani and Hernandez, Raquel and Brown, Dennis T.}, year={2010}, month={Jun}, pages={73–84} }
 @article{he_piper_meilleur_myles_hernandez_brown_heller_2010, title={The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering}, volume={84}, ISSN={["1098-5514"]}, url={http://europepmc.org/abstract/med/20219936}, DOI={10.1128/jvi.00044-10}, abstractNote={ABSTRACT
          The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.}, number={10}, journal={JOURNAL OF VIROLOGY}, author={He, Lilin and Piper, Amanda and Meilleur, Flora and Myles, Dean A. A. and Hernandez, Raquel and Brown, Dennis T. and Heller, William T.}, year={2010}, month={May}, pages={5270–5276} }
 @article{nanda_vancini_ribeiro_brown_hernandez_2009, title={A high capacity Alphavirus heterologous gene delivery system}, volume={390}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2009.05.026}, abstractNote={A novel replication competent Sindbis virus based gene delivery vector has been developed for the introduction of genetic cargo into cell lines in vitro and potentially, animal models in vivo. This delivery system expands the previous uses of Sindbis virus as a gene delivery system in that no replicons are required and the resulting cargo containing virus particles are infectious. The heterologous vector is based on a morphological mutant in C, Ser180/Gly183 which produces larger than the normal size T = 4 virus particles of 70 nm in size. This mutant produced particles up to 205 nm in size equal to a triangulation number of 36. It was postulated that because the Ser180/Gly183 mutant was capable of assembling such large particles, that increasing the size of the RNA genome incorporated into this mutant capsid protein would favor the assembly of larger than T = 4 wild type sized virions. The first generation prototype larger vehicle, described here, carries a ~ 18 kb cDNA insert, however it is conceivable that RNA as large as 32 kb could be transcribed and packaged. The large variant produces a high virus titer of ~ 109 pfu/ml from either mammalian or insect cells in culture. Multiple passages of the virus show no loss of the inserted genetic material.}, number={2}, journal={VIROLOGY}, author={Nanda, Kavita and Vancini, Ricardo and Ribeiro, Mariana and Brown, Dennis T. and Hernandez, Raquel}, year={2009}, month={Aug}, pages={368–373} }
 @article{hafer_whittlesey_brown_hernandez_2009, title={Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells}, volume={83}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.00755-09}, abstractNote={ABSTRACT
          
            Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (
            Aedes albopictus
            ) cell lines; one other insect cell line, Sf21 from
            Spodoptera frugiperda
            ; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.
          }, number={18}, journal={JOURNAL OF VIROLOGY}, author={Hafer, Amanda and Whittlesey, Rebecca and Brown, Dennis T. and Hernandez, Raquel}, year={2009}, month={Sep}, pages={9113–9121} }
 @article{kononchik_nelson_hernandez_brown_2009, title={Helical virus particles formed from morphological subunits of a membrane containing icosahedral virus}, volume={385}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2008.12.015}, abstractNote={The classic publication by Caspar and Klug in 1962 [Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27:1–24.] has formed the basis of much research on virus assembly. Caspar and Klug predicted that a single virus morphological unit could form a two dimensional lattice composed of 6-fold arrays (primitive plane), a family of icosahedra of increasing triangulation numbers (T) and helical arrays of varying length. We have shown that icosahedral viruses of varying T numbers can be produced using Sindbis virus [Ferreira, D. F. et al. 2003. Morphological variants of Sindbis virus produced by a mutation in the capsid protein. Virology 307:54–66]. Other studies have shown that Sindbis glycoproteins can also form a 2-dimensional lattice confirming Caspar and Klug's prediction of the primitive plane as a biologically relevant structure [VonBonsdorff, C. H., and S. C. Harrison. 1978. Sindbis virus glycoproteins form a regular icosahedral surface lattice. J. Virol. 28:578]. In this study we have used mutations in the glycoproteins of membrane containing Sindbis virus to create helical-virus-like particles from the morphological subunits of a virus of icosahedral geometry. The resulting virus particles were examined for subunit organization and were determined to be constructed of only 6-fold rotational arrays of the virus glycoproteins. A model of the tubular virus particles created from the 6-fold rotational arrays of Sindbis virus confirmed the observed structure. These experiments show that a common morphological unit (the Sindbis E1–E2 heterodimer) can produce three different morphological entities of varying dimensions in a membrane-containing virus system.}, number={2}, journal={VIROLOGY}, author={Kononchik, Joseph R., Jr. and Nelson, Steevenson and Hernandez, Raquel and Brown, Dennis I.}, year={2009}, month={Mar}, pages={285–293} }
 @misc{hernandez_paredes_2009, title={Sindbis virus as a model for studies of conformational changes in a metastable virus and the role of conformational changes in in vitro antibody neutralisation}, volume={19}, ISSN={["1099-1654"]}, DOI={10.1002/rmv.619}, abstractNote={AbstractSindbis virus has long been the prototypical alphavirus used in the study of assembly of icosahedral viruses which contain membranes. Much is known about the assembly pathway and molecular architecture of this virus, and models of mechanisms of infection and release of the viral RNA have been proposed. This review will focus on the structural aspects of in vitro antibody neutralisation of a metastable alphavirus, Sindbis virus, which results from antibody induced conformational changes. For the alphaviruses, structures of in vitro antibody induced conformational neutralisation for Sindbis virus, and receptor occlusion for Sindbis and Ross River virus (RRV) have been reported. A model is presented which could extend the mechanism of antibody induced conformational neutralisation to any metastable virus structure. Copyright © 2009 John Wiley & Sons, Ltd.}, number={5}, journal={REVIEWS IN MEDICAL VIROLOGY}, author={Hernandez, Raquel and Paredes, Angel}, year={2009}, month={Sep}, pages={257–272} }
 @article{smith_nanda_slominski_hernandez_brown_thomas_2008, title={Construction and characterization of mutant Dengue2 virus vaccine candidates displaying a host-range phenotype}, journal={Vaccine}, author={Smith, K. M. and Nanda, K. and Slominski, C. J. and Hernandez, R. and Brown, D. T. and Thomas, M. E.}, year={2008} }
 @article{hernandez_paredes_brown_2008, title={Sindbis virus conformational changes induced by a neutralizing anti-E1 monoclonal antibody}, volume={82}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.02673-07}, abstractNote={ABSTRACT
          A rare Sindbis virus anti-E1 neutralizing monoclonal antibody, Sin-33, was investigated to determine the mechanism of in vitro neutralization. A cryoelectron microscopic reconstruction of Sindbis virus (SVHR) neutralized with FAb from Sin-33 (FAb-33) revealed conformational changes on the surface of the virion at a resolution of 24 Å. FAb-33 was found to bind E1 in less than 1:1 molar ratios, as shown by the absence of FAb density in the reconstruction and stoichiometric measurements using radiolabeled FAb-33, which determined that about 60 molecules of FAb-33 bound to the 240 possible sites in a single virus particle. FAb-33-neutralized virus particles became sensitive to digestion by endoproteinase Glu-C, providing further evidence of antibody-induced structural changes within the virus particle. The treatment of FAb-33-neutralized or Sin-33-neutralized SVHR with low pH did not induce the conformational rearrangements required for virus membrane-cell membrane fusion. Exposure to low pH, however, increased the amount of Sin-33 or FAb-33 that bound to the virus particles, indicating the exposure of additional epitopes. The neutralization of SVHR infection by FAb-33 or Sin-33 did not prevent the association of virus with host cells. These data are in agreement with the results of previous studies that demonstrated that specific antibodies can inactivate the infectious state of a metastable virus in vitro by the induction of conformational changes to produce an inactive structure. A model is proposed which postulates that the induction of conformational changes in the infectious state of a metastable enveloped virus may be a general mechanism of antibody inactivation of virus infectivity.}, number={12}, journal={JOURNAL OF VIROLOGY}, author={Hernandez, Raquel and Paredes, Angel and Brown, Dennis T.}, year={2008}, month={Jun}, pages={5750–5760} }
 @article{wang_hernandez_keith_brown_2007, title={Infection of cells by Sindbis virus at low temperature}, volume={362}, DOI={10.1016/j.virol.2006.12.036}, abstractNote={Sindbis virus, which belongs to the family Togaviridae genus Alphavirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for Alphaviruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This virus–host membrane fusion model, well established by influenza virus, has been applied to other unrelated membrane-containing viruses including Alphaviruses. The other mechanism proposes direct penetration of the cell plasma membrane by the virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373–386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311–3320] studies. We have examined the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis virus infects cells in a temperature- and time-dependent fashion.}, number={2}, journal={Virology}, author={Wang, G. B. and Hernandez, R. and keith and Brown, D. T.}, year={2007}, pages={461–467} }
 @article{whitehurst_soderblom_west_hernandez_goshe_brown_2007, title={Location and role of free cysteinyl residues in the Sindbis virus E1 and E2 glycoproteins}, volume={81}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.02859-06}, abstractNote={ABSTRACT
          Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.}, number={12}, journal={JOURNAL OF VIROLOGY}, author={Whitehurst, Christopher B. and Soderblom, Erik J. and West, Michelle L. and Hernandez, Raquel and Goshe, Michael B. and Brown, Dennis T.}, year={2007}, month={Jun}, pages={6231–6240} }
 @article{heldt_hernandez_mudiganti_gurgel_brown_carbonell_2006, title={A  colorimetric assay for viral agents that produce cytopathic effects}, volume={135}, DOI={10.1016/j.j.viromet.2006.01.022}, number={1}, journal={Journal of Virological Methods}, author={Heldt, C. L. and Hernandez, R. and Mudiganti, U. and Gurgel, P. V. and Brown, D. T. and Carbonell, R. G.}, year={2006}, pages={56–65} }
 @article{heldt_hernandez_mudiganti_gurgel_brown_carbonell_2006, title={A colorimetric assay for viral agents that produce cytopathic effects}, volume={135}, ISSN={0166-0934}, url={http://dx.doi.org/10.1016/j.jviromet.2006.01.022}, DOI={10.1016/j.jviromet.2006.01.022}, abstractNote={Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.}, number={1}, journal={Journal of Virological Methods}, publisher={Elsevier BV}, author={Heldt, Caryn L. and Hernandez, Raquel and Mudiganti, Usharani and Gurgel, Patrick V. and Brown, Dennis T. and Carbonell, Ruben G.}, year={2006}, month={Jul}, pages={56–65} }
 @article{west_hernandez_ferreira_brown_2006, title={Mutations in the endodomain of Sindbis virus glycoprotein E2 define sequences critical for virus assembly}, volume={80}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.80.9.4458-4468.2006}, abstractNote={ABSTRACT
          Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (Δ406-407, Δ409-411, and Δ414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.}, number={9}, journal={JOURNAL OF VIROLOGY}, author={West, J and Hernandez, R and Ferreira, D and Brown, DT}, year={2006}, month={May}, pages={4458–4468} }
 @article{mudiganti_hernandez_ferreira_brown_2006, title={Sindbis virus infection of two model insect cell systems - A comparative study}, volume={122}, DOI={10.1016/j.virusres.2006.06.007}, abstractNote={Sindbis, the prototype of the Alphaviruses causes mosquito-borne diseases in mammals and replicates in a wide variety of vertebrate and invertebrate cell cultures. This characteristic can be exploited to use the vast array of Drosophila genetic information available for investigations of the interaction of Sindbis virus with an alternate invertebrate host. For this purpose, a comparative study of Sindbis virus infection of Schnieder-2 Drosophila (S2) cells to cells of the mosquito Aedes albopictus (clone U4.4) was undertaken. After infection, vertebrate cells die within 24–48 h, while invertebrate cell cultures survive an acute phase of infection and become persistently infected. In this study, infection of a model Drosophila system, S2 cells, was compared to U4.4 cells. Virus production, the time course of the establishment of persistence and changes in growth properties of the S2 cells upon infection, were studied in comparison to those of the U4.4 cells. S2 cells survived acute Sindbis infection without any significant cytopathology and continued to produce low levels of virus characteristic of persistently infected cells. S2 cells produced 10 PFU/cell on day 1 post-infection, which falls to 2 PFU/cell on day 2. This result is in contrast to U4.4 cells, which produce peak virus titer on day 2 post-infection and establish persistence by day 5. Onset of the persistent phase of infection of either U4.4 or S2 cells did not result in any change in morphology or growth characteristics. This study establishes S2 cells as an additional invertebrate model system to study the interactions of an invertebrate host with Sindbis virus.}, number={1-2}, journal={Virus Research}, author={Mudiganti, U. and Hernandez, R. and Ferreira, D. and Brown, D. T.}, year={2006}, pages={28–34} }
 @article{whitehurst_willis_sinodis_hernandez_brown_2006, title={Single and multiple deletions in the transmembrane domain of the Sindbis virus E2 glycoprotein identify a region critical for normal virus growth}, volume={347}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2005.11.029}, abstractNote={Sindbis virus is composed of two nested T = 4 icosahedral protein shells containing 240 copies each of three structural proteins: E1, E2, and Capsid in a 1:1:1 stoichiometric ratio. E2 is a 423 amino acid glycoprotein with a membrane spanning domain 26 amino acids in length and a 33 amino acid cytoplasmic endodomain. The interaction of the endodomain with the nucleocapsid is an essential step in virus maturation and directs the formation of the outer protein shell as envelopment occurs. A previous study had determined that deletions in the transmembrane domain could affect virus assembly and infectivity (Hernandez et al., 2003. J. Virol. 77 (23), 12710–12719). Unexpectedly, a single deletion mutant (from 26 to 25 amino acids) resulted in a 1000-fold decrease in infectious virus production while another deletion of eight amino acids had no affect on infectious virus production. To further investigate the importance of these mutants, other single deletion mutants and another eight amino acid deletion mutant were constructed. We found that deletions located closer to the cytoplasmic (inner leaflet) of the membrane bilayer had a more detrimental effect on virus assembly and infectivity than those located closer to the luminal (outer leaflet) of the membrane bilayer. We also found that selective pressure can restore single amino acid deletions in the transmembrane domain but not necessarily to the wild type sequence. The partial restoration of an eight amino acid deletion (from 18 to 22 amino acids) also partially restored infectious virus production. The amount of infectious virus produced by this revertant was equivalent to that produced for the four amino acid deletion produced by site directed mutagenesis. These results suggest that the position of the deletion and the length of the C terminal region of the E2 transmembrane domain is vital for normal virus production. Deletion mutants resulting in decreased infectivity produce particles that appear to be processed and transported correctly suggesting a role involved in virus entry.}, number={1}, journal={VIROLOGY}, author={Whitehurst, CB and Willis, JH and Sinodis, CN and Hernandez, R and Brown, DT}, year={2006}, month={Mar}, pages={199–207} }
 @article{nelson_hernandez_ferreira_brown_2005, title={In vivo processing and isolation of furin protease-sensitive alphavirus glycoproteins: a new technique for producing mutations in virus assembly}, volume={332}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2004.12.013}, abstractNote={Sindbis virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding, and maturation.}, number={2}, journal={VIROLOGY}, author={Nelson, S and Hernandez, R and Ferreira, D and Brown, DT}, year={2005}, month={Feb}, pages={629–639} }
 @article{hernandez_ferreira_sinodis_litton_brown_2005, title={Single amino acid insertions at the junction of the Sindbis virus e2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity}, volume={79}, DOI={10.1128/JVI.79.7682-7697.2005}, number={12}, journal={Journal of Virology}, author={Hernandez, R. and Ferreira, D. and Sinodis, C. and Litton, K. and Brown, D. T.}, year={2005}, pages={7682–7697} }
 @article{paredes_ferreira_horton_saad_tsuruta_johnston_klimstra_ryman_hernandez_chiu_et al._2004, title={Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion}, volume={324}, DOI={10.1016/j.virus.2004.03.046}, number={2}, journal={Virology}, author={Paredes, A. M. and Ferreira, D. and Horton, M. and Saad, A. and Tsuruta, H. and Johnston, R. and Klimstra, W. and Ryman, K. and Hernandez, R. and Chiu, W. and et al.}, year={2004}, pages={373–386} }
 @article{paredes_ferreira_horton_saad_tsuruta_johnston_klimstra_ryman_hernandez_chiu_et al._2004, title={Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion}, volume={324}, ISSN={0042-6822}, url={http://dx.doi.org/10.1016/j.virol.2004.03.046}, DOI={10.1016/j.virol.2004.03.046}, abstractNote={Alphaviruses have the ability to induce cell–cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell–cell fusion and cell–virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane–cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus–cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.}, number={2}, journal={Virology}, publisher={Elsevier BV}, author={Paredes, Angel M and Ferreira, Davis and Horton, Michelle and Saad, Ali and Tsuruta, Hiro and Johnston, Robert and Klimstra, William and Ryman, Kate and Hernandez, Raquel and Chiu, Wah and et al.}, year={2004}, month={Jul}, pages={373–386} }
 @article{hernandez_rusa_rusa_lopez_mijangos_tonelli_2004, title={Controlling PVA hydrogels with gamma-cyclodextrin}, volume={37}, ISSN={["0024-9297"]}, DOI={10.1021/ma048375i}, abstractNote={We report on the preparation and characterization of poly(vinyl alcohol) (PVA) hydrogels formed during freeze−thaw (F−T) cycles of their aqueous solutions containing γ-cyclodextrin (γ-CD). Crystalline inclusion compound (IC) formation was observed between PVA and γ-CD in these gels at low concentrations of γ-CD (γ-CD:PVA molar ratios < 1:25). Confirmation of the existence of the channel structure for γ-CD was achieved by characterizing the dried PVA/γ-CD hydrogels with solid-state DSC, TGA, WAXD, and 13C NMR. Some aspects regarding the mechanism and structure of PVA gels obtained via F−T cycles in the presence/absence of γ-CD are presented based on UV−vis, swelling, solution 1H NMR, and rheological observations. It was observed that the swelling and rheological responses of the aqueous PVA gels formed during F−T cycles in the presence of γ-CD can be controlled by adjustment of the PVA:γ-CD molar ratio employed during their gelation.}, number={25}, journal={MACROMOLECULES}, author={Hernandez, R and Rusa, M and Rusa, CC and Lopez, D and Mijangos, C and Tonelli, AE}, year={2004}, month={Dec}, pages={9620–9625} }
 @article{hernandez_nelson_salm_brown_alpert_2004, title={Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column}, volume={120}, DOI={10.1016/j.viromet.2004.04.013}, number={2}, journal={Journal of Virological Methods}, author={Hernandez, R. and Nelson, S. and Salm, J. R. and Brown, D. T. and Alpert, A. J.}, year={2004}, pages={141–149} }
 @article{hernandez_nelson_salm_brown_alpert_2004, title={Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column}, volume={120}, ISSN={0166-0934}, url={http://dx.doi.org/10.1016/j.jviromet.2004.04.013}, DOI={10.1016/j.jviromet.2004.04.013}, abstractNote={Analysis and purification of specific PCR products from PCR reactions can be problematic due to several issues relating to amplification and low product yield. The use of HPLC as a preparative tool in PCR product analysis is common but has not replaced traditional electrophoretic techniques for purifying DNA to be used in subsequent experiments. Gel purification of PCR products can result in a net loss greater than 50% of the starting DNA amount. Thus, this method of recovery can become the limiting factor in the overall cloning protocol. This paper describes a simple and relatively inexpensive micro-preparative HPLC method to purify and analyze nM quantities of DNA. A microbore polyethyleneimine-based anion-exchange column fractionates PCR mixtures in less than 40 min with a recovery of the purified specific product as high as 80%, thus eliminating the need for gel purification. Using this method, nested PCR products from Sindbis virus differing by 18 bp in some cases and a 277 bp fragment from West Nile virus were resolved and quantified. This method differs from existing methodologies because separation is based on size and charge as well as the overall G + C content of the PCR product.}, number={2}, journal={Journal of Virological Methods}, publisher={Elsevier BV}, author={Hernandez, Raquel and Nelson, Steevenson and Salm, Jeffery R and Brown, Dennis T and Alpert, Andrew J}, year={2004}, month={Sep}, pages={141–149} }
 @article{hernandez_sinodis_horton_ferreira_yang_brown_2003, title={Deletions in the transmembrane domain of a Sindbis virus glycoprotein alter virus infectivity, stability, and host range}, volume={77}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.77.23.12710-12719.2003}, abstractNote={ABSTRACT
          The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and protein-membrane interactions during virus assembly.}, number={23}, journal={JOURNAL OF VIROLOGY}, author={Hernandez, R and Sinodis, C and Horton, M and Ferreira, D and Yang, CN and Brown, DT}, year={2003}, month={Dec}, pages={12710–12719} }
 @article{ferreira_hernandez_horton_brown_2003, title={Morphological variants of Sindbis virus produced by a mutation in the capsid protein}, volume={307}, ISSN={["0042-6822"]}, DOI={10.1016/S0042-6822(02)00034-X}, abstractNote={Sindbis virus is a complex aggregate of RNA, protein and lipid. The virus is organized as two nested T = 4 icosahedral protein shells between which is sandwiched a lipid bilayer. The virus RNA resides within the inner protein shell. The inner protein shell is attached to the outer protein shell through contacts to proteins in the outer shell, which penetrate the lipid bilayer. The data presented in the following manuscript show that mutations in the capsid protein can result in the assembly of the virus structural proteins into icosahedra of different triangulation numbers. The triangulation numbers calculated, for these morphological variants, follow the sequence T = 4, 9, 16, 25 and 36. All fall into the class P = 1 of icosadeltahedra as was predicted by Caspar and Klug (1962). The data support their hypothesis that families of icosahedra would be developed by altering the distance between the points of insertion of the five-fold axis. This capsid protein defect also results in the incorporation of much of the capsid protein, into large cytoplasmic aggregates of protein and RNA. These observations support models suggesting that the geometry of a pre-formed nucleocapsid organizes the assembly of the virus membrane proteins into a structure of identical configuration and argues against models suggesting that assembly of the membrane glycoproteins directs the assembly of the nucleocapsid.}, number={1}, journal={VIROLOGY}, author={Ferreira, D and Hernandez, R and Horton, M and Brown, DT}, year={2003}, month={Mar}, pages={54–66} }
 @article{hernandez_luo_brown_2001, title={Exposure to low pH is not required for penetration of mosquito cells by Sindbis virus}, volume={75}, ISSN={["0022-538X"]}, DOI={10.1128/jvi.75.4.2010-2013.2001}, abstractNote={ABSTRACT
          
            It is widely held that the penetration of cells by alphaviruses is dependent on exposure to the acid environment of an endosome. The alphavirus Sindbis virus replicates in both vertebrate and invertebrate cell cultures. We have found that exposure to an acid environment may not be required for infection of cells of the insect host. In this work, we investigated the effects of two agents (NH
            4
            Cl and chloroquine), which raise the pH of intracellular compartments (lysosomotropic weak bases) on the infection and replication of Sindbis virus in cells of the insect host
            Aedes albopictus
            . The results show that both of these agents increase the pH of endosomes, as indicated by protection against diphtheria toxin intoxication. NH
            4
            Cl blocked the production of infectious virus and blocked virus RNA synthesis when added prior to infection. Chloroquine, in contrast to its effect on vertebrate cells, had no inhibitory effect on infectious virus production in mosquito cells even when added prior to infection. Treatment with NH
            4
            Cl did not prevent the penetration of virus RNA into the cell cytoplasm or translation of the RNA to produce a precursor to virus nonstructural proteins. These data suggest that while these two drugs raise the pH of endosomes, they do not block insect cell penetration. These data support previous results published by our laboratory suggesting that exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses.
          }, number={4}, journal={JOURNAL OF VIROLOGY}, author={Hernandez, R and Luo, TC and Brown, DT}, year={2001}, month={Feb}, pages={2010–2013} }
 @article{pletnev_zhang_mukhopadhyay_fisher_hernandez_brown_baker_rossmann_kuhn_2001, title={Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold}, volume={105}, ISSN={["0092-8674"]}, DOI={10.1016/S0092-8674(01)00302-6}, abstractNote={There are 80 spikes on the surface of Sindbis virus arranged as an icosahedral surface lattice. Each spike consists of three copies of each of the glycoproteins E1 and E2. There are two glycosylation sites on E1 and two on E2. These four sites have been located by removal of the glycosylation recognition motifs using site-specific mutagenesis, followed by cryoelectron microscopy. The positions of these sites have demonstrated that E2 forms the protruding spikes and that E1 must be long and narrow, lying flat on the viral surface, forming an icosahedral scaffold analogous to the arrangement of the E glycoprotein in flaviviruses. This arrangement of E1 leads to both dimeric and trimeric intermolecular contacts, consistent with the observed structural changes that occur on fusion with host cell membranes, suggesting a similar fusion mechanism for alpha- and flaviviruses.}, number={1}, journal={CELL}, author={Pletnev, SV and Zhang, W and Mukhopadhyay, S and Fisher, BR and Hernandez, R and Brown, DT and Baker, TS and Rossmann, MG and Kuhn, RJ}, year={2001}, month={Apr}, pages={127–136} }
 @article{hernandez_lee_nelson_brown_2000, title={A single deletion in the membrane-proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly}, volume={74}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.74.9.4220-4228.2000}, abstractNote={ABSTRACT
          The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.}, number={9}, journal={JOURNAL OF VIROLOGY}, author={Hernandez, R and Lee, H and Nelson, C and Brown, DT}, year={2000}, month={May}, pages={4220–4228} }