Littérature scientifique sur le sujet « Emerging viral diseases »

Emerging viral diseases encompass two types, those of new appearance in the population and those that we previously knew about or re-emerging, but that at a certain moment present an exponential increase in incidence or geographic distribution in the form of epidemics or outbreaks. These emerging and re-emerging viruses share a series of characteristics that establish the emerging virus model, such as having an RNA genome, being zoonotic, transmitted by vectors and transmissible to humans, that the virus is able to recognize and provoke a response in receptors. Conserved in several species and inhabiting ecosystems that undergo ecological, demographic or social changes that favor the spread of the virus. There are different factors that contribute to facilitating the emergence of viral infections, although this is made up of three fundamental aspects such as the susceptible population, the virus itself and the environment where both can interact.

Human life is intricately related to it’s surrounding environment which also harbors other animals and some deadly infectious pathogens. Any threat to the environment can thus increase the threat of new and so-called ‘emerging infectious diseases’ (EIDs) especially novel viral infections called ‘emerging viral diseases’. This occurs partly due to changing climate as well as human interference with nature and animal life. An important event in new disease emergence is genetic changes in the pathogen that make it possible to become established in a new host species, productively infect new individuals in the new hosts (typically humans) and create local, regional or worldwide health threats. The world has witnessed some emerging and deadly viral threats in recent past with huge mortality and morbidity. Among them were severe acute respiratory syndrome (SARS), bird flu, swine flu, Middle East respiratory syndrome (MERS), ebola virus disease. Moreover some disease has caused great concern in certain regions including Bangladesh in terms of morbidity, like Nipah virus, Zika virus, Dengue and Chikungunya fever. Here in this article an attempt was made to briefly describe some of these emerging viral infections.Birdem Med J 2017; 7(3): 224-232

The intensive aquaculture remains the world’s fastest growing sector producing food of animal origin. In fact, it is the only animal food-producing sector growing faster than the human population, and provides an acceptable supplement to and substitute for wild fish. A number of cyprinid diseases have emerged globally and their study has become increasingly important. The expansion of aquaculture, which has relied heavily on the movement of animals and farming species new to aquaculture, has been paralleled with disease emergence. In the last few years several emerging or re-emerging fish diseases have been detected in cyprinid fish populations in Serbia. In this paper, the authors overview the major viral threats for cyprinid fishes in Serbia

Viral diseases are an important limiting factor in many crop production systems. Because antiviral products are not available, control strategies rely on genetic resistance or hygienic measures to prevent viral diseases, or on eradication of diseased crops to control such diseases. Increasing international travel and trade of plant materials enhances the risk of introducing new viruses and their vectors into production systems. In addition, changing climate conditions can contribute to a successful spread of newly introduced viruses or their vectors and establishment of these organisms in areas that were previously unfavorable. Tomato is economically the most important vegetable crop worldwide and many viruses infecting tomato have been described, while new viral diseases keep emerging. Pepino mosaic virus is a rapidly emerging virus which has established itself as one of the most important viral diseases in tomato production worldwide over recent years. Begomovirus species and other whitefly-transmitted viruses are invading into new areas, and several recently described new viruses such as Tomato torrado virus and new Tospovirus species are rapidly spreading over large geographic areas. In this article, emerging viruses of tomato crops are discussed.

AbstractEmerging infectious diseases (EIDs) are an increasing threat to public health on a global scale. In recent times, the most prominent outbreaks have constituted RNA viruses, spreading via droplets (COVID-19 and Influenza A H1N1), directly between humans (Ebola and Marburg), via arthropod vectors (Dengue, Zika, West Nile, Chikungunya, Crimean Congo) and zoonotically (Lassa fever, Nipah, Rift Valley fever, Hantaviruses). However, specific approved antiviral therapies and vaccine availability are scarce, and public health measures remain critical. Patients can present with a spectrum of ocular manifestations. Emerging infectious diseases should therefore be considered in the differential diagnosis of ocular inflammatory conditions in patients inhabiting or returning from endemic territories, and more general vigilance is advisable in the context of a global pandemic. Eye specialists are in a position to facilitate swift diagnosis, improve clinical outcomes, and contribute to wider public health efforts during outbreaks. This article reviews those emerging viral diseases associated with reports of ocular manifestations and summarizes details pertinent to practicing eye specialists.

The risks posed by rapidly evolving RNA viruses to human and animal health are well recognized. Epidemics in managed and wildlife populations can lead to considerable economic and biodiversity losses. Yet, we lack understanding of the ecological and evolutionary factors that promote disease emergence. Host-switching viruses may be a particular threat to species important for human welfare, such as pollinating bees. Both honeybees and wild bumblebees have faced sharp declines in the last decades, with high winter mortality seen in honeybees. Infectious and emerging diseases are considered one of the key drivers of declines, acting in synergy with habitat loss and pesticide use. Here I focus on multihost viruses that pose a risk to wild bumblebees. I first identify the risk factors driving viral spillover and emergence from managed honeybees to wild bumblebees, by synthesising current data and literature. Biological factors (i.e. the nature of RNA viruses and ecology of social bees) play a clear role in increasing the risk of disease emergence, but anthropogenic factors (trade and transportation of commercial honeybees and bumblebees) creates the greatest risk of viral spillover to wild bees. Basic knowledge of the pathogenic effect of many common pollinator viruses on hosts other than A. mellifera is currently lacking, yet vital for understanding the wider impacts of infection at a population level. Here, I provide evidence that a common bumblebee virus, Slow bee paralysis virus (SBPV), reduces the longevity of Bombus terrestris under conditions of nutrition stress. The invasion of Varroa destructor as an ectoparasitic viral vector in European honeybees has dramatically altered viral dynamics in honeybees. I test how this specialist honeybee vector affects multi-host pathogens that can infect and be transmitted by both honeybees and wild bumblebees. I sampled across three host species (A. mellifera, B. terrestris and B. pascuorum) from Varroa-free and Varroa-present locations. Using a combination of molecular and phylogenetic techniques I find that this specialist honeybee vector increases the prevalence of four multi-host viruses (deformed wing virus (type A and B), SBPV and black queen cell virus) in sympatric wild bumblebees. Furthermore, wild bumblebees are currently experiencing a DWV epidemic driven by the presence of virus-vectoring Varroa in A. mellifera. Overall this thesis demonstrates that wild bumblebees are at high risk of viral disease emergence. My research adds to the ever-expanding body of evidence indicating that stronger disease controls on commercial bee operations are crucial to protect our wild bumblebees.

Novel antiviral strategies against emerging viral diseases Doctoral Research School of Biochemistry and Molecular Biology-Cycle XXXII; Tutor: Luisa Bracci; Supervisor: Maurizio Zazzi; Candidate: Adele Boccuto Background Zika virus (ZIKV) Dengue virus (DENV) and West Nile virus (WNV) belong to the Flaviviridae family. The World Health Organization has ranked DENV as the most critical mosquito-borne viral disease and declared ZIKV an international public health emergency. Indeed, WNV is widely established in USA and some European areas and is associated to neuroinvasive disease. Despite intensive work, no specific antiviral therapy is available for ZIKV, DENV or WNV. In addition, increasing co-infections with different flaviviruses or serotypes cocirculating within the same area complicate the clinical outcome and treatment options. The high degree of conservation of some viral enzymes or host factor essential for Flaviviridae replication makes it reasonable to search for “broad-spectrum” antivirals. Materials and Methods The half-maximal cytotoxic concentration (CC50) of candidate inhibitors was calculated by Cell Titer-Glo cell viability assay. We determined the drug concentration inhibiting 50% of viral replication (IC50) of ZIKV, DENV and/or WNV by infection of Huh7 (hepatoma cell line) at a multiplicity of infection (MOI) of 0.005 (DENVZIKV) or 0.0025 (WNV) for 1 h and at the end of incubation treating Huh7 cells with serial dilutions of candidate compound at 37°C and 5% CO2. After 72 h (DENV-ZIKV) or 48h (WNV), the IC50 of compounds interfering with the virus life cycle up to protein production, but not at later steps, was directly measured by immune-detection assay (direct yield reduction assay, DYRA). Otherwise, to determine the impairment of viral replicative capacity exerted at late steps of viral replication, e.g. viral assembly and budding, we performed an additional replicative cycle or by infection of pre-seeded Huh7 (Secondary yield reduction assay, SYRA) with supernatants obtained by first round of infection or by Plaque reduction assay (PRA) as previously described (Vicenti et al., 2018). Selectivity indexes (SI) were calculated as ratio between CC50 and IC50. For in vitro ZIKV resistance selection to sofosbuvir, ZIKV was propagated in the Huh-7 cell line with increasing concentrations of sofosbuvir. Viral clones replicating at each drug increment step were collected and sequenced to detect emergent mutations in the polymerase (NS5) region and sofosbuvir IC50 was measured by DYRA. Results and discussion Evaluation of anti-DENV compounds targeting cellular DEAD-box 3 RNA helicase. Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3 (DDX3) belongs to a family of ATP-dependent RNA helicases and is involved in many aspects of RNA metabolism as well as potentially participating to the antiviral innate immune signaling pathways. On the other hand, many studies highlight DDX3 as an essential host factor for the replication of clinically relevant viruses. As part of the activities planned in the UNAVIR and PANVIR project and in collaboration with First Health Pharmaceuticals, we evaluated the effect of DDX3 helicase inhibitors focusing on antiviral activity against DENV. Overall we tested 51 compounds, 31 showed anti-DENV activity and among these 11 molecules showed promising antiviral activity with median SI 66 (IQR 59-74). Overall, the preliminary data obtained so far support targeting DDX3 protein as a potentially effective strategy for inhibition of DENV replication and feasible broad-spectrum option to minimize the possibilities of generating resistance. The evaluation of the mechanism of action of a DDX3 inhibitor compound are currently under evaluation. Evaluation of candidate antivirals agents targeting viral entry/fusion. Virus entry by enveloped viruses is dependent not only on cellular receptors but also on cellular lipids playing multifaceted roles in viral infections as structural components and as necessary cofactors for viral replication. In this scenario, the possibility to identify broad-spectrum antiviral molecules acting on viral envelopes is of particular interest. We tested derivatives entry inhibitors of the previously identified MAS family (Cagno et al., 2018; Tintori et al., 2018) expected to affect virus-cell membrane fusion during DENV and ZIKV infection. We evaluated the antiviral activity of MAS compounds adapting DYRA and incubating Huh7 cells with serial dilution of MAS molecules for 30 minutes before infection with ZIKV or DENV. Of the ten compounds investigated, five showed antiviral activity both against ZIKV (median SI 16 µM; IQR 12-26) and DENV (median SI 7; IQR 6-14), 3 showed antiviral activity against ZIKV in DYRA (median SI 8; IQR 6.5-17) and anti-DENV activity in SYRA (median SI 11; IQR 9-14), while 2 compounds were completely inactive . Interestingly, the three compound active only against ZIKV were found active also against DENV in SYRA (median SI 11; IQR 9-14). We further evaluated the antiviral activity of MAS9 showing IC50 values of 2.1±1.6 (SI 14) and 2.6±2.2 (SI 12) for DENV and ZIKV, respectively. The evaluation of the mechanism of action confirmed the antiviral activity of MAS9 in viral entry/fusion step of viral replication and through a higher selectivity for viral membranes. These preliminary results support the strategy to target selectively the viral membrane, possibly based on the efficiency of the repair activity of host cell, but not virus, membranes. This class of compounds warrants further investigations to prove and characterize the mechanism of action and the ability to inhibit different emerging enveloped viruses. Evaluation of candidate anti-flaviviral agents targeting the NS3-NS5 interaction. A family of 2,6-diaminopurine derivatives targeting a conserved allosteric pocket on DENV/ZIKV NS5 polymerase, which is required to bind NS3 and generate the functional replication complex, has been recently discovered (Vincettiet al., 2019, 2015). Herein, we analysed a new series of derivatives molecules structurally modified to increase the affinity for the allosteric pocket on NS5 and reduce their cellular toxicity. Of the 21 derivatives compounds investigated against ZIKV and DENV, 5 were cytotoxic, 16 showed anti-ZIKV activity (median SI 9 [IQR 7-26.5]) and 13 showed anti-DENV activity (median SI 11 [IQR 6-13]) in SYRA. The best SI was obtained for compound MR333 with SI of 182 and 77 for ZIKV and DENV, respectively. Moreover, MR333 was found to be active also against WNV with comparable antiviral activity in PRA and SI of 55. This class of compounds appears to be attractive and warrants further evaluation of the mechanism of action and the genetic barrier to the emergence of resistance mutations. Biochemical assays investigating MR333 are underway to confirm and characterize the expected inhibition of multiple viruses based on binding to the highly conserved pocket on the thumb of Flavivirus polymerases. Characterization of in vitro ZIKV resistance to sofosbuvir. Given the high degree of NS5 homology observed among members of the Flaviviridae family (Boldescu et al., 2017; Lim et al., 2013), sofosbuvir has been recently evaluated as an anti-Flavivirus lead candidate. Indeed, the anti-ZIKV activity of sofosbuvir has been showed in vitro with cell-based assay and in animal models. However, sofosbuvir drug genetic barrier has been not yet evaluated with ZIKV. The virus breakthrough time grew with increasing sofosbuvir concentration of 5, 10, 20 and 40 µM (5, 15, 22 and 40±9 days, respectively) with exception of 80 µM (20±2 days). No sequence variations were found up to 20 µM sofosbuvir, while the mutations V360L and V607I were selected in presence of 40 µM sofosbuvir and 80 µM. The mutations C269Y and H289T were individually selected in two experiments with 80 µM sofosbuvir. Globally, majority of mutations observed in Sanger sequencing were confirmed by NGS with addition of N407T, T833I, K834C and W835R mutations which were not associated to sofosbuvir drugpressure. By DYRA we demonstrated the reduced susceptibility to sofosbuvir of viruses carrying the C269Y, V360L and V607I mutations. Moreover, the analysis of NS5 gene showed that the V607I is located near the well-known mutation S282T in SGxxxT consensus sequence of HCV NS5B, within the motif B, and is implicated in drug resistance of HCV toward nucleotide inhibitors (Gane et al., 2016). Interestingly, the V360L mutation is located in a well-known stretch of amino acids termed β-nuclear localization sequence (positions 320-368), whereas, the C269Y is located in the poorly conserved 10-residue linker domain (aminoacidic positions 263 to 272) which is essential for the adequate NS5 conformation. Therefore, the mutations detected are probably related to emergence of ZIKV resistance to sofosbuvir in vitro and, certainly, require further biochemical investigations. References Boldescu, V., Behnam, M.A.M., Vasilakis, N., Klein, C.D., 2017. Broad-spectrum agents for flaviviral infections: Dengue, Zika and beyond. Nat. Rev. Drug Discov. https://doi.org/10.1038/nrd.2017.33Cagno, V., Tintori, C., Civra, A., Cavalli, R., Tiberi, M., Botta, L., Brai, A., Poli, G., Tapparel, C., Lembo, D., Botta, M., 2018. Novel broad spectrum virucidal molecules against enveloped viruses. PLoS One 13, 1– 18. https://doi.org/10.1371/journal.pone.0208333 Gane, E.J., Shiffman, M.L., Etzkorn, K., Morelli, G., Stedman, C., Davis, M.N., Hinestrosa, F., Dvory-Sobol, H., Huang, K.C., Osinusi, A., McNally, J., Brainard, D., McHutchison, J., Thompson, A., Sulkowski, M., 2016. Sofosbuvir/Velpatasvir in Combination with Ribavirin for 24 Weeks is Effective Retreatment for Patients who failed Prior NS5A Containing DAA Regimens: Results of the GS-US-342-1553 Study. J.Hepatol. 64, S147–S148. https://doi.org/10.1016/s0168-8278(16)00037-4 Lim, S.P., Koh, J.H.K., Seh, C.C., Liew, C.W., Davidson, A.D., Chua, L.S., Chandrasekaran, R., Cornvik, T.C., Shi,P.Y., Lescar, J., 2013. A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities. J. Biol. Chem. 288, 31105–31114. https://doi.org/10.1074/jbc.M113.508606 Tintori, C., Iovenitti, G., Ceresola, E.R., Ferrarese, R., Zamperini, C., Brai, A., Poli, G., Dreassi, E., Cagno, V., Lembo, D., Canducci, F., Botta, M., 2018. Rhodanine derivatives as potent anti-HIV and anti-HSV microbicides. PLoS One 13, 1–19. https://doi.org/10.1371/journal.pone.0198478 Vicenti, I., Boccuto, A., Giannini, A., Dragoni, F., Saladini, F., Zazzi, M., 2018. Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro. Virus Res. 244, 64–70. https://doi.org/10.1016/j.virusres.2017.11.003 Vincetti, P., Caporuscio, F., Kaptein, S., Gioiello, A., Mancino, V., Suzuki, Y., Yamamoto, N., Crespan, E., Lossani, A., Maga, G., Rastelli, G., Castagnolo, D., Neyts, J., Leyssen, P., Costantino, G., Radi, M., 2015. Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases. J. Med. Chem. 58, 4964–4975. https://doi.org/10.1021/acs.jmedchem.5b00108 Vincetti, P., Kaptein, S.J.F., Costantino, G., Neyts, J., Radi, M., 2019. Scaffold Morphing Approach to Expand the Toolbox of Broad-Spectrum Antivirals Blocking Dengue/Zika Replication. ACS Med. Chem. Lett. 10, 558–563. https://doi.org/10.1021/acsmedchemlett.8b00583

Background Viruses are aetiologic agents of several human infectious diseases, which can be distinguished into acute and chronic. Among the agents causing acute infections, arboviruses, such as Dengue (DENV), West Nile (WNV) and Zika (ZIKV) Viruses, have increasingly spread worldwide. Moreover, the newly discovered Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the COVID-19 disease, is responsible for an incredibly quantity of infections and deaths. Conversely, the Human Immunodeficiency Virus (HIV-1) is a well-known pathogen able to cause chronic latent infection, but it still represents a global public health concern. The main strategy for combating viral infections is a combination of vaccination and antiviral drugs. However, vaccines are available only for a minority of viral pathogens, thus the demand for new antiviral strategies has significantly increased. Anyway, the screening of candidate compounds requires the assessment of their antiviral effects in vitro. Results and discussion An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV, able to distinguish between the inhibitory effect of molecules targeting the early and post-budding phases of viral replication cycle. Considering that the activity of sofosbuvir, a nucleotide analog licensed for HCV infection, has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. We determined for the first time the antiviral activity of sofosbuvir against WNV in both cell-based and enzymatic assays. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern. The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Antiviral activity of molecules was determined in vitro against DENV, WNV, HIV-1 and SARS-CoV-2. Two compounds were able to inhibit viral replication against two different families of viruses (DENV/WNV and SARS-CoV-2), and two compounds were able to inhibit viral replication against viruses with remarkable differences in their replication cycle (DENV/WNV and HIV-1). At the beginning of the pandemic, an Italian network named SCIRE (SARS-CoV-2 Italian Research Enterprise) was created in order to trace SARS-COV-2 evolution. By NGS whole genome sequencing, it was defined that the initial outbreak in Italy was mainly attributable to the SARS-CoV-2 lineage B.1 and to its uncontrolled circulation for an estimated period of 4 weeks. Developing strategies to eradicate chronic HIV-1 infection are a high priority. Recently, it was hypothesized that maraviroc (MVC), may exert a latency reversing effect in addition to its antiviral activity. Thus, the MVC-mediated HIV-1 induction was explored. An increased HIV-1 expression was detected at the highest MVC concentration in two of the three cell line models tested. In ex vivo CD4 T cells, MVC-mediated induction was detected sparsely on individual samples. Such evidences suggest the role of MVC as a weak latency reversing agent of the HIV-1 provirus induction. Conclusions Control of viral infections is a continuous challenge for public health. The development of effective antiviral compounds could be reasonably the key to halt the spread or to mitigate severe clinical manifestations of acute viral diseases. Moreover, antiviral therapy is essential to the maintenance of undetectable viral loads in chronic infection. Furthermore, developing of compounds able to exert a reversal latency effect may be a remarkable benefit in exacerbate chronic viral infections. In conclusion, the key concept of this thesis is to underline the strong need, and partially participate, in enhancing antiviral therapies strategies to counteract the burden of both acute and chronic viral diseases.

Thesis (PhD)-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence.
German Research Foundation (DFG) (project number: KR1293/9-1/13-1)
The Polio Research Foundation and the NHLS Research
Harry Crossley Foundation, the Polio Research Foundation and Stellenbosch University for granting scholarships and bursaries for PhD.

Doctor of Philosophy
Diagnostic Medicine/Pathobiology
Raymond R. R. Rowland
Emerging viral diseases cause significant and widespread economic losses to U.S. swine production. Over the last 25 years, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and porcine epidemic diarrhea virus (PEDV) have emerged or re-emerged, costing the industry billions through increased mortality and clinical or subclinical reductions in growth. Nursery pigs are greatly affected by these viruses due to high susceptibility to primary and secondary infections after weaning. However, clinical disease occurs in only a subpopulation of infected pigs and can vary drastically from sudden death to poor growth performance. This thesis documents a series of 4 studies where nursery pigs were challenged with either PRRSV/PCV2 or PEDV; the associations between clinical outcome and several factors affecting viral pathogenesis were investigated. In the first study, the administration of PRRS modified live virus vaccine prior to co-challenge with PRRSV/PCV2 was shown to protect against PRRS but enhance PCV2 replication and pathogenesis. This study provides insight into the role that PRRS vaccination has in both the control and potentiation of clinical disease. In the second study, microbial populations were compared between pigs with the best and worst clinical outcome following PRRSV/PCV2 co-infection. Increased fecal microbiome diversity was associated with improved clinical outcome; however, worst clinical outcome pigs had prolonged and greater virus replication, highlighting the host response to viral challenge as a primary determinant of clinical outcome. In the third study, 13 clinical phenotypes were compiled for >450 pigs after PRRSV/PCV2 co-infection. Duration of dyspnea and the presence of muscle wasting had the strongest associations with reduced weight gain. This study highlights the opportunity to improve animal welfare and production through improvements in clinical health. In the fourth study, clinical disease was mild to moderate and occurred within the first week after pigs were challenged with PEDV. However, PEDV was detected weeks after clinical disease had resolved and may implicate nursery pigs as an important source of viral carriage and transmission. Overall, the goal of this thesis was to develop models for understanding the impact of emerging and re-emerging viruses to improve recognition and control of disease.

Vegetables play a major role in the livelihoods of the rural poor in Africa. Among major constraints to vegetable production worldwide are diseases caused by a group of viruses belonging to the genus Begomovirus, family Geminiviridae. Begomoviruses are plant-infecting viruses, which are transmitted by the whitefly vector Bemisia tabaci and have been known to cause extreme yield reduction in a number of economically important vegetables around the world. Several begomoviruses have been detected infecting vegetable crops in West and Central Africa (WCA). Small single stranded circular molecules, alphasatellites and betasatellites, which are about half the size of their helper begomovirus genome, have also been detected in plants infected by begomoviruses. In WCA, B. tabaci has been associated with suspected begomovirus infections in many vegetable crops and weed species. Sequencing of viral genomes from crops such as okra resulted in the identification of two previously known begomovirus species (Cotton leaf curl Gezira virus and Okra yellow crinkle virus) as well as a new recombinant begomovirus species (Okra leaf curl Cameroon virus), a betasatellite (Cotton leaf curl Gezira betasatellite) and new alphasatellites. Tomato and pepper plants with leaf curling were shown to contain isolates of new begomoviruses, collectively referred to as West African tomato-infecting begomoviruses (WATIBs), new alphasatellites and betasatellites. To study the potential of weeds serving as begomovirus reservoirs, begomoviruses and satellites in the weed Ageratum conyzoides were characterized. Sequence analyses showed that they were infected by isolates of a new begomovirus (Ageratum leaf curl Cameroon virus) that belong to the WATIBs group, a new betasatellite (Ageratum leaf curl Cameroon betasatellite), an alphasatellite and two types of defective recombinants between a begomovirus and an alphasatellite. Putative recombinations were detected in begomovirus genomes for all four plant species studied, indicating that recombination is an important mechanism for their evolution. A close relationship between the begomoviruses infecting pepper and tomato and A. conyzoides and the detection of the same alphasatellite in them support the idea that weeds are important reservoirs for begomoviruses and their satellites. With this high diversity, recombination potential and transmission by B. tabaci, begomoviruses and ssDNA satellites pose a serious threat to crop production in West and Central Africa.

Hosts have two main strategies for coping with infections: resistance and tolerance. Resistance is aimed at preventing or eliminating parasites, whereas the goal of tolerance is to maintain performance regardless of parasite burden. The balance between resistance and tolerance within a host may mediate host competence, or the propensity of a host to infect other hosts or vectors. Hosts with high tolerance and low resistance to an infection, for instance, may be highly competent and possess the greatest potential to act as superspreaders. These superspreading hosts will contribute disproportionately to transmission, thus posing the greatest risk to other hosts within a population or community. Understanding the drivers of heterogeneity in host competence therefore has broad implications for the management of infectious diseases in nature. Host tolerance is typically quantified as the slope of the relationship between host performance and parasite burden. The majority of host tolerance studies have been conducted at the level of genotypes, populations and species. Individual hosts often exhibit variation in competence, with some individuals contributing more or less to transmission than the population/species average. Despite the clear importance of understanding tolerance at the individual-level, such studies are rare and may be particularly challenging in field contexts due to the need for repeated performance-burden measurements. I used the house sparrow (HOSP) – West Nile virus (WNV) system to investigate differences among two alternative approaches to estimating individual tolerance: the scope and position methods. The scope method estimates tolerance traditionally as the slope of multiple performance-burden measurements over time within an individual; alternatively, the position method required only one measurement for each individual, thus characterizing tolerance via among-individual variation in host defense. We found strong relationships between scope and position estimates of individual tolerance, suggesting that the position method may be an appropriate proxy to use in field studies. I also compared tolerance estimates derived from different metrics of performance. There were weak correlations among these estimates of tolerance, implying that tolerance estimated by measuring a single trait may not be indicative of tolerance at the level of the whole individual or their contribution to disease processes. Understanding the physiological mediators of host competence may help to pinpoint at-risk and risky individuals (or genotypes, populations and species) within natural communities, thus facilitating the development of more targeted disease management strategies. Cytokines and glucocorticoids have been identified as potent mediators of host defense. Pro-inflammatory cytokines may act to promote resistance, whereas anti-inflammatory cytokines and glucocorticoids tend to mediate host tolerance. I investigated the dynamics of pro-inflammatory cytokine IFN-γ, anti-inflammatory cytokine IL-10, and the major avian glucocorticoid, corticosterone (CORT), following WNV exposure in HOSP. I then assessed the influence of these three mediators on resistance and tolerance to WNV infection. I found unusual dynamics for the three mediators across the infection period: IFN-γ expression was not induced by WNV exposure, IL-10 expression was dampened by WNV exposure, and CORT levels were higher in unexposed individuals. Despite the unique response of HOSP to WNV exposure seen here, we did find that constitutive expression of IFN-γ and IL-10 mediate resistance and tolerance to WNV, respectively. Unexpectedly, we also found evidence for protective (pro-resistance) effects of CORT, which contrasts with previous evidence for the role of CORT in mediating WNV infections. Combined, the results of this study suggest that hosts with constitutively high IL-10 and low IFN-γ expression may have high potential to act as superspreaders of disease, thus becoming critical targets in designing WNV-control strategies in passerines. The methods by which we quantify host tolerance may greatly affect the conclusions we are able to draw from such studies. To date, a variety of definitions and techniques have been used to study tolerance in animals. In chapter three, I briefly summarize past plant and animal tolerance research, highlighting discrepancies among researchers in their motivations, definitions and techniques for studying tolerance. For instance, I discuss biases in the literature regarding the use of range versus point tolerance, vigor, and laboratory versus field studies. In particular, I expound upon the nature of the performance metrics used in the majority of tolerance estimations in the literature, and discuss the ecological implications of these metrics. To conclude, I offer suggestions for overcoming the challenges associated with studying tolerance and encourage a unified way forward in the field, emphasizing the selection of system-specific and ecologically relevant tolerance metrics. My thesis research has employed physiological and behavioral methods in an ecological context to better understand the heterogeneities that exist in host competence. By combining empirical data in the HOSP-WNV system with conceptual and methodological strategies for assessing host defenses, this research has broadened our knowledge of host responses in the WNV system in a manner that may be applicable to understanding and managing disease dynamics in diverse natural communities.

Ranaviruses are large dsDNA viruses that are considered emerging pathogens, and they are known to cause mortality events in amphibian and fish populations. This research utilizes experimental and genomic data to elucidate the mechanisms driving the evolution and spread of ranaviruses, with a focus on host switching within the genus. In Chapter 1, we utilize virus challenge assays to examine potential transfer of ranaviruses between cultured juvenile largemouth bass (M. salmoides) and bullfrog tadpoles (Rana catesbeiana). Additionally, a commonly used antiparasitic treatment containing malachite green and formalin (MGF) was utilized to suppress the immune system of largemouth bass to assess the susceptibility of immunocompromised fish to ranaviruses. The results indicate that tadpoles are not susceptible to Largemouth Bass Virus (LMBV), but that bass are susceptible to ranaviruses isolated from amphibians. Furthermore, immunocompromised fish were more susceptible to both LMBV and FV3 infections than immunocompetent fish. In Chapter 2, we used eight sequenced ranavirus genomes and two selection-detection methods (site-based and branch-based) to identify genes that exhibited signatures of positive selection, potentially due to the selective pressures at play during host switching. We found evidence of positive selection acting on four genes via the site-based method, three of which are newly-acquired genes unique to ranavirus genomes. Our results suggest that the group of newly acquired genes in the ranavirus genome may have undergone recent adaptive changes that have facilitated interspecies and interclass host switching. In Chapter 3, we annotated and analyzed the nearly complete genomic sequence of LMBV to determine its taxonomic classification. The available genomic content and phylogenetic evidence suggests that LMBV is more closely related to amphibian-like ranaviruses (ALRVs) than grouper ranaviruses, and this is further supported by greater genomic collinearity between LMBV and ALRVs. This data suggests that the classification of LMBV as a ranavirus is warranted. The results presented here will help to clarify the taxonomic relationships of ranaviruses, and will also be useful in developing management strategies to limit interspecific and intraspecific viral spread. The information garnered from this research will have far-reaching implications in studies of amphibian conservation, disease evolution, and virology.
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The COVID-19 pandemic has made a huge impact on various aspects of life around the globe. An important step in tackling issues caused by COVID-19 and other thoracic pathologies is to find approaches that will automate the detection of such diseases from medical images. Medical images such as chest x-rays are widely available around the globe. Traditional medical image classification techniques required extensive work for finding distinctive features in medical images and using them for building accurate models for classification. The modern approaches are highly focused on using models based on machine learning for similar tasks. Convolutional Neural Networks (CNNs) are a type of artificial neural network based upon the mathematical linear operation called convolution. CNNs have a few different layers and are widely used for computer vision tasks. A major advantage of CNNs is that they could extract spatial features from images automatically. In this work, various convolutional neural networks are implemented and tested for the classification of chest x-ray images from four classes of normal, viral pneumonia, lung opacity, and Covid-19. The most efficient model achieves an accuracy of 90.69% and a recall and precision of 91.25% and 92.03% across all four classes.

How can a doctor diagnose new diseases with little historical knowledge, which are emerging over time? Active learning is a promising way to address the problem by querying the most informative samples. Since the diagnosed cases for new disease are very limited, gleaning knowledge from other domains (classical prescriptions) to prevent the bias of active leaning would be vital for accurate diagnosis. In this paper, a framework that attempts to glean knowledge from multiple domains for active learning by querying the most uncertain and representative samples from the target domain and calculating the importance weights for re-weighting the source data in a single unified formulation is proposed. The weights are optimized by both a supervised classifier and distribution matching between the source domain and target domain with maximum mean discrepancy. Besides, a multiple domains active learning method is designed based on the proposed framework as an example. The proposed method is verified with newsgroups and handwritten digits data recognition tasks, where it outperforms the state-of-the-art methods.

Background and aims: In 2020 a global pandemic was declared caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). The pandemic is still ongoing and continues to cause considerable mortality and morbidity world-wide and new variants of the virus are emerging. Rapid development and rollout of vaccines for SARS-CoV-2 is in progress to counter the pandemic but has been tempered by the emergence of new SARS-CoV-2 variants, many of which exhibit reduced vaccine effectiveness. To date there is no approved antiviral treatment for coronavirus disease 2019 (COVID-19). Several studies have shown that Manuka honey has virucidal/antiviral effect. Methylglyoxal (MG), a bioactive component in Manuka honey, has antiviral activity in vitro. MG may modify arginine residues in the functional domains of viral spike and nucleocapsid proteins, resulting in loss of charge, protein misfolding and inactivation. The aim of this study was to characterize the antiviral activity of Manuka honey against SARS-CoV-2 in vitro Materials and methods: Wild-type SARS-CoV-2 with titers of multiplicities of infection (MOI) 0.1 and 0.05 were incubated with 2-fold serial dilutions of 250+ Manuka honey (equivalent to 250 to 31 µM) in infection medium (Dulbecco's Modified Eagle Medium + 2% fetal bovine serum + 100 units/ml penicillin + 100 µg/ml streptomycin) for 3 h. Manuka honey treated and untreated control SARS-CoV-2 was incubated with confluent cultures of Vero cells in vitro for 1 h, cultures washed with phosphate-buffered saline and incubated in fresh infection medium at 37°C for 4 - 5 days until 70% of virus control cells displayed cytopathic effect. We also studied the effect of scavenging MG in Manuka Honey with aminoguanidine (AG; 500 µM) on virucidal activity. The antiviral activity of MG was judged by median tissue culture infectious dose (TCID50) assays. Data analysis was by logistic regression. TCID50 (mean ± SD) was deduced by interpolation. Results: Diluted Manuka honey inhibited SARS-CoV-2 replication in Vero cells. SARS-CoV-2 was incubated in diluted Manuka honey in medium at 37°C for 3 h before adding to Vero cells. Manuka honey dilutions down to 125 µM MG equivalents completely inhibited cytopathic effect of SARS-CoV-2 whereas 31.25 µM and 62.5 µM MG equivalents had limited effect. Logistic regression and interpolation of the cytopathic effect indicated that the TCID50 = 72 ± 2 µM MG equivalents for MOI of 0.1. Prior scavenging of MG by addition of AG resulted in virus replication levels equivalent to those seen in the virus control without AG. Conclusion: Manuka honey has antiviral activity against SARS-CoV-2 when incubated with the virus in cell-free media at no greater than ca. 40-fold dilutions of 250+ grade. Anti-viral activity was inhibited by AG, consistent with the anti-viral effect being mediated by MG. Manuka honey dilutions in MG equivalents had similar antiviral effect compared to authentic MG, also consistent with MG content of Manuka honey mediating the antiviral effect. Whilst Manuka honey may inactivate SARS-CoV-2 in cell-free culture medium, its antiviral activity in vivo for other than topical application may be limited because of the rapid metabolism of MG by the glyoxalase system and limited bioavailability of oral MG.

Objectives: (1) Evaluate Immunocompetence-OTL-containing Chromosomal Regions (ICRs), marked by microsatellites or candidate genes, for magnitude of direct effect and for contribution to relationships among multiple immunocompetence, disease-resistance, and growth traits, in order to estimate epistatic and pleiotropic effects and to predict the potential breeding applications of such markers. (2) Evaluate the interaction of the ICRs with genetic backgrounds from multiple sources and of multiple levels of genetic variation, in order to predict the general applicability of molecular genetic markers across widely varied populations. Background: Diseases cause substantial economic losses to animal producers. Emerging pathogens, vaccine failures and intense management systems increase the impact of diseases on animal production. Moreover, zoonotic pathogens are a threat to human food safety when microbiological contamination of animal products occurs. Consumers are increasingly concerned about drug residues and antibiotic- resistant pathogens derived from animal products. The project used contemporary scientific technologies to investigate the genetics of chicken resistance to infectious disease. Genetic enhancement of the innate resistance of chicken populations provides a sustainable and ecologically sound approach to reduce microbial loads in agricultural populations. In turn, animals will be produced more efficiently with less need for drug treatment and will pose less of a potential food-safety hazard. Major achievements, conclusions and implications:. The PI and co-PIs had developed a refined research plan, aiming at the original but more focused objectives, that could be well-accomplished with the reduced awarded support. The successful conduct of that research over the past four years has yielded substantial new information about the genes and genetic markers that are associated with response to two important poultry pathogens, Salmonella enteritidis (SE) and Escherichia coli (EC), about variation of immunocompetence genes in poultry, about relationships of traits of immune response and production, and about interaction of genes with environment and with other genes and genetic background. The current BARD work has generated a base of knowledge and expertise regarding the genetic variation underlying the traits of immunocompetence and disease resistance. In addition, unique genetic resource populations of chickens have been established in the course of the current project, and they are essential for continued projects. The US laboratory has made considerable progress in studies of the genetics of resistance to SE. Microsatellite-marked chromosomal regions and several specific genes were linked to SE vaccine response or bacterial burden and the important phenomenon of gene interaction was identified in this system. In total, these studies demonstrate the role of genetics in SE response, the utility of the existing resource population, and the expertise of the research group in conducting such experiments. The Israeli laboratories had showed that the lines developed by selection for high or low level of antibody (Ab) response to EC differ similarly in Ab response to several other viral and bacterial pathogens, indicating the existence of a genetic control of general capacity of Ab response in young broilers. It was also found that the 10w-Ab line has developed, possibly via compensatory "natural" selection, higher cellular immune response. At the DNA levels, markers supposedly linked to immune response were identified, as well as SNP in the MHC, a candidate gene responsible for genetic differences in immunocompetence of chickens.

Original objectives and revisions-The proposed research included these original objectives: field validation of diagnostic tests (PCR), the development and evaluation of new sensitive tools (LC-PCR/TaqManPCR, antibody detection by ELISA) including their use to study the ecology and the epidemiology of KHV (virus distribution in the environment and native cyprinids) and the carrier status of fish exposed experimentally or naturally to KHV (sites of virus replication and potential persistence or latency). In the course of the study we completed the genome sequence of KHV and developed a DNA array to study the expression of KHV genes in different conditions. Background to the topics-Mass mortality of koi or common carp has been observed in Israel, USA, Europe and Asia. These outbreaks have reduced exports of koi from Israel and have created fear about production, import, and movements of koi and have raised concerns about potential impacts on native cyprinid populations in the U.S.A. Major conclusions-A suite of new diagnostic tools was developed that included 3 PCR assays for detection of KHV DNA in cell culture and fish tissues and an ELISA assay capable of detecting anti-KHV antibodies in the serum of koi and common carp. The TKPCR assay developed during the grant has become an internationally accepted gold standard for detection of viral DNA. Additionally, the ELISA developed for detecting serum anti-KHV antibodies is now in wide use as a major nonlethal screening tool for evaluating virus status of koi and common carp populations. Real time PCR assays have been able to detect viral DNA in the internal organs of survivors of natural and wild type vaccine exposures at 1 and 10³ genome equivalents at 7 months after exposure. In addition, vaccinated fish were able to transmit the virus to naive fish. Potential control utilizing hybrids of goldfish and common carp for production demonstrated they were considerably more resistant than pure common carp or koi to both KHV (CyHV-3). There was no evidence that goldfish or other tested endemic cyprinids species were susceptible to KHV. The complete genomic sequencing of 3 strains from Japan, the USA, and Israel revealed a 295 kbp genome containing a 22 kbp terminal direct repeat encoding clear gene homologs to other fish herpesviruses in the family Herpesviridae. The genome encodes156 unique protein-coding genes, eight of which are duplicated in the terminal repeat. Four to seven genes are fragmented and the loss of these genes may be associated with the high virulence of the virus. Viral gene expression was studies by a newly developed chip which has allowed verification of transcription of most all hypothetical genes (ORFs) as well as their kinetics. Implications, both scientific and agricultural- The results from this study have immediate application for the control and management of KHV. The proposal provides elements key to disease management with improved diagnostic tools. Studies on the ecology of the virus also provide insights into management of the virus at the farms that farmers will be able to apply immediately to reduce risks of infections. Lastly, critical issues that surround present procedures used to create “resistant fish” must be be resolved (e.g. carriers, risks, etc.). Currently stamping out may be effective in eradicating the disease. The emerging disease caused by KHV continues to spread. With the economic importance of koi and carp and the vast international movements of koi for the hobby, this disease has the potential for even further spread. The results from our studies form a critical component of a comprehensive program to curtail this emerging pathogen at the local, regional and international levels.