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[Truncate abstract] Flaviviruses are small, positive-stranded RNA viruses belonging to the family Flaviviridae. Flavivirus infection in humans could cause diseases ranging from febrile illnesses to fatal encephalitis. Mice provide a useful small animal model to study flavivirus-induced encephalitis in humans since mice also develop encephalitis during flavivirus infection. Some strains of mice have been shown to be resistant to flavivirus challenge and this resistance is conferred by a single autosomal dominant gene, designated as Flvr. Recently, OAS1b gene has been identified to be a gene candidate for Flvr. Several congenic resistant mouse strains have been developed by introducing resistance genes from outbred or wild mice onto the genetic background of susceptible C3H mice. These new resistant strains that carry different allelic variants at the Flv locus include C3H/PRI-Flvr (RV), C3H.MOLD-Flvmr (MOLD) and C3H.M.domesticus-Flvr-like (DUB), the latter two being developed in the same laboratory in which the work described in this thesis was accomplished. Preliminary studies in this laboratory found that flavivirus resistant mice are vulnerable to certain flavivirus infections, particularly when challenged by intracerebral (i.c.) route. Intracerebral (i.c.) challenge with flaviviruses such as West Nile virus (WNV) Sarafend strain and Kunjin virus (KUNV) MRM16 strain were found to induce high mortality in flavivirus resistant mice while infection with Murray Valley encephalitis virus (MVEV) OR2 strain did not cause any apparent disease in the same mice. ... Thus, it can be concluded that CD8+ T cells exerted harmful effect to resistant DUB mice during KUNV i.c. infection by producing excessive IFN[gamma] that could be toxic, causing functional loss of the CNS cells. It was shown from in vitro studies that WNV had the highest tropism for macrophages and dendritic cells, followed by KUNV. MVEV however did not replicate well in these cells. This combined with the data from the in vivo studies indicates that macrophages might be involved in the pathogenesis of intraperitoneal (i.p.) infection of WNV but not KUNV and MVEV. The reason for this could be that the production of KUNV in macrophages may not be high enough to induce viraemia and subsequent fatal encephalitis in mice. In contrast, MVEV appears to use different mechanism or cells for virus dissemination. Although macrophages may not be involved in KUNV pathogenesis after i.p. infection, the fact that macrophages support KUNV replication in vitro may indicate the possibility that blood-borne macrophages were recruited to the brain where they can get infected with KUNV during i.c. infection and therefore could participate in KUNV pathogenesis in DUB mice. This study provides evidence for the first time on the detrimental effect of host antiviral immunity and inflammatory mediators during flavivirus i.c. infection in resistant mice. However, it also launches a new question on the selective cell tropism of KUNV versus MVEV responsible for inducing different pattern of immune responses and consequently leading to different outcomes of infection in resistant mice.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importance. Since its introduction into the United States in 1999, WNV has spread throughout most of the country and has caused major epidemics of neuroinvasive disease (Hayes and Gubler, 2005). SLEV is endemic to the United States and is maintained in an enzootic transmission cycle in Florida.The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in central Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medical alerts to warn the population. Current serological detection methods for sentinel chickens include hemagglutination inhibition antibody test (HAI), IgM antibody capture enzyme-linkedimmunosorbent assay (MAC-ELISA), and Plaque Reduction Neutralization Test (PRNT).These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological methods would be of benefit. Microsphere-based immunoassays (MIAs) are a more rapid serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodies to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicken sera from 2005 were assayed using the MIA for WNV and 424 sera from multiple years were assayed for SLEV. The capability of the MIA to multiplex allowed for simultaneous detection of antibodies to WNV and SLEV in sentinel chicken sera. The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sensitive and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection and improvement to the current surveillance system.

Thesis (Ph. D.)--Georgia State University, 2007. / Title from file title page. Margo Brinton, committee chair; W. David Wilson, Teryl Frey, committee members. Electronic text (120 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Nov. 20, 2008. Includes bibliographical references.

<p dir="ltr">It is with great pleasure that I present the culmination of my exploration into the process of flavivirus assembly, with particular emphasis on the envelope glycoproteins and C protein of ZIKV and DENV2, within the subsequent four chapters of this dissertation.</p><p dir="ltr">Beginning in Chapter 2, we describe findings from a structure-function study of the ZIKV prM and E transmembrane helices (TMHs) and their role in virus assembly. Using a mutagenesis approach in a ZIKV reporter virus particle (RVP) system to increase throughput and discovery, substantial information was obtained showcasing a novel function for specific residues located within a short (4 residue) connecting region between the two TMHs of prM protein – denoted as the prM TMH “turn” residues. During translation of the prM and E proteins, these TMH “turn” residues face towards the cytosolic side of the ER membrane. This orientation has been hypothesized to possibly play a role during viral assembly interactions between the envelope glycoproteins and the nucleocapsid core of flaviviruses. However, no information to date has supported or refuted this theory. Overall, a single amino acid change within the prM TMH “turn” residues was found to be highly detrimental to viral assembly, ultimately leading to the loss of capsid integration into released sub viral particles and the alteration of the lipid membrane architecture. We surmised that lipid interactions around the region of the mutation were perturbed, leading to a loss of assembly capabilities but interestingly maintaining the budding mechanisms. The work of Chapter 2 will be submitted for publication to a peer reviewed journal shortly after the submission of this dissertation.</p><p dir="ltr">Chapter 3 expands on the ZIKV RVP results described in Chapter 2 by detailing a series of mutagenesis experiments into the role of the prM and E TMHs in the fully infectious ZIKV and DENV2 systems. Mutations within the prM TMH “turn” residues of DENV2 were found to also perturb virus infectivity, with two mutations within prM completely eliminating infectivity. The two mutants were found to be capable of producing NS5 and intracellular E protein that had been glycosylated, indicating that translation was intact and that E protein trafficking into the trans-Golgi network still occurred. However, unlike the results discussed in Chapter 2, the DENV2 mutants did not release any detectable E protein into their supernatants. This suggested that while the mutants could generate viral proteins and somehow undergo protein trafficking into the Golgi (signifying potential particle maturation), no particles were released. The DENV2 results were supported by reciprocal mutations in the prM proteins of ZIKV using fully infectious cDNA clones. The ZIKV prM mutants also eliminated virus infectivity and prevented the release of the E protein into the supernatant, indicating no release of viral particles, infectious or otherwise. Overall, the mutations in the fully infectious DNEV2 and ZIKV systems add further support for a novel role of the prM TMHs in flavivirus assembly.</p><p dir="ltr">Chapter 4 describes our efforts to reconstitute the flavivirus envelope glycoproteins into natively derived lipid nanoparticles for in vitro assembly analysis. Styrene-maleic acid copolymers (SMAs) were utilized for this study due to their ability to self-polymerize into highly hydrophobic chains in aqueous solutions. These hydrophobic chains can imbed themselves into lipid membranes to escape the aqueous environment, and in doing so “cut out” ~10nm diameter “patches” of native lipid membranes, along with any integrated membrane proteins. This “lipid/protein patch” is referred to as a styrene-maleic acid lipid nanoparticle (SMALP). Initially, attempts were made to generate SMALPs using purified Kunjin virus (KUNV) particles as the source of membrane lipids and glycoproteins due to their rapid growth rate and homogenous particle population. Unfortunately, attempts to generate SMALPs using purified KUNV were unsuccessful. It is hypothesized that the membrane curvature of purified KUNV particles generated a sterically and energetically unfavorable environment for SMALP generation, leading to the complete destruction of the particles during SMA mixing. To circumvent this issue, cells transfected with either WT or mutant ZIKV RVP cDNA were fractionated and purified ER membrane samples were mixed with SMAs to generate SMALPs. Western blot analysis suggested that the SMALP generation was successful. However, further experimentation is warranted to confirm this outcome and the structural integrity of the envelope glycoproteins within the SMALP.</p><p dir="ltr">Chapter 5 describes collaborative work on the identification of a novel compound inhibitor against flavivirus assembly, specifically targeting C protein’s interactions with RNA. This work was done in conjunction with a visiting scholar from the Indian Institute of Technology Mandi – Dr. Prateek Kumar – during his time at Purdue University from August 2022-May 2023. Much of the foundational computation work was done by Prateek prior to his arrival at Purdue University. As such, while the full context and results for the entirety of the study will be discussed, this chapter will primarily focus on the in vitro experimental results that were gathered directly by me, or results that were produced by Prateek and myself equally. This chapter demonstrates that a novel small molecule inhibitor against ZIKV C protein can, in fact, diminish ZIKV assembly by impeding C protein’s binding to RNA, prevent efficient RNA replication through binding and disruption of NS2B/3 protease, and perturb virus binding and entry prior to infection by also binding to E protein. Moreover, the novel molecule was also found to disrupt DENV2 infection as well, albeit to a lesser degree than ZIKV. This multifaceted molecule was recommended for further study in animal systems to continue testing its safety and efficacy for treatment of ZIKV and DENV2 in humans. A co-authorship manuscript has been completed on the work from this chapter and is currently awaiting submission to a peer reviewed journal.</p><p dir="ltr">Finally, Chapter 6 will combine the conclusions from the above chapters and discuss, in detail, aspects pertaining to the future of studies aiming to better understand the assembly of flaviviruses. This chapter will focus on how the link between viral assembly and membrane lipid architecture fits with previously established literature and what future directions could be employed to answer the questions proposed within.</p>

As Neotropical migratory birds, Prothonotary Warblers are exposed to parasites in both tropical and temperate regions and may act as dispersal agents between geographic areas. This study identifies the prevalence of Haemosporidia, West Nile Virus (WNV), and St. Louis Encephalitis virus (SLEV) in this species. A total of 71.6% of captured Prothonotary Warblers were infected with Haemosporidia during the 2008 breeding season, and infection prevalence increased throughout the season. This temporal change in prevalence is likely due to infection relapse and transmission of new infections. No correlations between reproductive effort and infection status were observed, nor were any associations between infection prevalence and nest box location identified. WNV and SLEV were present in 37.5% and 6.3% of sampled Prothonotary Warblers, respectively. These results warrant more detailed analyses of pathogen transmission dynamics in this population, physiological mechanisms that affect infection susceptibility, and spatial and temporal trends in infection that may exist.

Rondônia apresenta área com rica diversidade de artrópodes, porém pouco se conhece sobre a transmissão de arbovírus por estas espécies. O presente trabalho visou detectar arbovírus, por meio da RT-PCR e da Duplex RT-PCR, nas espécies de dipteros coletados no Estado, bem como caracterizá-los pela reação de sequenciamento. A RT-PCR e a Duplex RT-PCR detectaram as suspensões dos vírus Mayaro e Oropouche até 104 e 101 TCID50/mL, respectivamente, porém o vírus Dengue 2 em pools contendo menos de três mosquitos infectados foi negativa. O controle endógeno foi detectado em 66,8 % das amostras, sendo que, em pools contendo entre um e três mosquitos, a detecção foi aproximadamente metade da detecção nos pools contendo entre 11 e 15. Em 0,66 % dos pools foi encontrado o vírus Oropouche e em outros 0,66 %, o vírus Cacipacoré. O vírus Oropouche foi detectado em Coquillettidia sp. e Deinocerites sp., enquanto o vírus Cacipacoré foi encontrado em Anopheles sp. e Culex sp. As técnicas possibilitaram a detecção dos arbovírus pesquisados nos pools coletados em Rondônia. / The Rondônia state has an area with rich arthropods diversity although the knowledge about the arboviruses transmition for these species is poor. The present work aimed to detect arboviruses through RT-PRC and RT-PCR Duplex in the diptera species collected in the region as well as their characterization through the sequence reaction. The RT-PRC and RT-PCR Duplex detected the Mayaro and Oropouche virus suspensions until 104 e 101 TCID50/mL respectively, although it was negative for the Dengue 2 virus in pools containing less than three infected mosquitoes. The endogenous control was detected in 66,8 % of samples and from pools containing from one to three mosquitoes the detection rate was approximately half from that obtained from pools with 11 to 15 mosquitoes. Oropouche virus was found in 0,66 % of pools and Cacipacore virus also in 0,66 % of pools. Oropouche virus was detected in Coquillettidia sp. and Deinocerites sp. while Cacipacoré virus was found in Anopheles sp. and Culex sp. The techniques allowed the detection of examined arboviruses in the pools collected from Rondonia.

Rondônia apresenta área com rica diversidade de artrópodes, porém pouco se conhece sobre a transmissão de arbovírus por estas espécies. O presente trabalho visou detectar arbovírus, por meio da RT-PCR e da Duplex RT-PCR, nas espécies de dipteros coletados no Estado, bem como caracterizá-los pela reação de sequenciamento. A RT-PCR e a Duplex RT-PCR detectaram as suspensões dos vírus Mayaro e Oropouche até 104 e 101 TCID50/mL, respectivamente, porém o vírus Dengue 2 em pools contendo menos de três mosquitos infectados foi negativa. O controle endógeno foi detectado em 66,8 % das amostras, sendo que, em pools contendo entre um e três mosquitos, a detecção foi aproximadamente metade da detecção nos pools contendo entre 11 e 15. Em 0,66 % dos pools foi encontrado o vírus Oropouche e em outros 0,66 %, o vírus Cacipacoré. O vírus Oropouche foi detectado em Coquillettidia sp. e Deinocerites sp., enquanto o vírus Cacipacoré foi encontrado em Anopheles sp. e Culex sp. As técnicas possibilitaram a detecção dos arbovírus pesquisados nos pools coletados em Rondônia. / The Rondônia state has an area with rich arthropods diversity although the knowledge about the arboviruses transmition for these species is poor. The present work aimed to detect arboviruses through RT-PRC and RT-PCR Duplex in the diptera species collected in the region as well as their characterization through the sequence reaction. The RT-PRC and RT-PCR Duplex detected the Mayaro and Oropouche virus suspensions until 104 e 101 TCID50/mL respectively, although it was negative for the Dengue 2 virus in pools containing less than three infected mosquitoes. The endogenous control was detected in 66,8 % of samples and from pools containing from one to three mosquitoes the detection rate was approximately half from that obtained from pools with 11 to 15 mosquitoes. Oropouche virus was found in 0,66 % of pools and Cacipacore virus also in 0,66 % of pools. Oropouche virus was detected in Coquillettidia sp. and Deinocerites sp. while Cacipacoré virus was found in Anopheles sp. and Culex sp. The techniques allowed the detection of examined arboviruses in the pools collected from Rondonia.