As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR). In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer. In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential. Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

We will propose a random changepoint model for the analysis of longitudinal CD4 and CD8 T-cell counts, as well as viral RNA loads, for HIV infected subjects following highly active antiretroviral treatment. The data was taken from two studies, one of the Aids Clinical Group Trial 398 and one performed by the Terry Beirn Community Programs for Clinical Research on AIDS. Models were created with the changepoint following both exponential and truncated normal distributions. The estimation of the changepoints was performed in a Bayesian analysis, with implementation in the WinBUGS software using Markov Chain Monte Carlo methods. For model selection, we used the deviance information criterion (DIC), a two term measure of model adequacy and complexity. DIC indicates that the data support a random changepoint model with the changepoint following an exponential distribution. Visual analyses of the posterior densities of the parameters also support these conclusions.

Scope and Contents: Virus specific RNA components of the cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis virus were examined. Studies were carried out both in the presence and absence of defective particle interference. / Thesis / Master of Science (MSc)

Morbilliviruses belong to the family Paramyxoviridae of the Mononegavirale order of viruses. The Mononegavirale order contains viruses which contain negatively-polar, non-segmented and single stranded RNA genomes. This order contains some of most lethal pathogens known to the humankind. Ebola virus and Marburg virus are perhaps the most lethal human pathogens. Rinderpest virus, declared eradicated in 2011, was known to be the most significant cattle killer. Similarly the Canine distemper virus and Rabies virus, two topmost canine pathogens belong to this order. The L protein in the viruses of Morbillivirus genus harbours the viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome and also all the mRNA capping enzymes, viz. RNA 5’ triphosphatase, guanylyltransferase, RNA (guanine-7-)methyltransferase and RNA 5’ cap-dependent (2’-oxo-)methyltransferase. Moreover this protein can act as a protein kinase that can regulate the function of P protein which serves as a switch between transcription and replication. mRNA capping is necessary for the virus for the purpose of exploiting host cellular machinery towards viral protein synthesis. The Rinderpest virus L protein serves as a model to study the capping enzymes of Morbillivirus. RNA triphosphatase (RTPase), the first enzyme of the capping cascade had earlier been located on the L protein. The RTPase minimal domain on the L protein was identified earlier by sequence homology studies done with RTPase proteins of Baculovirus and Vaccinia virus and cloned. The bacterially expressed recombinant domain was shown to possess RTPase activity. The enzymatic activity was characterized and the RTPase was found to be a metal-dependent enzyme which is highly specific to capping viral mRNA. Further characterization of the domain revealed that the domain also possesses nucleotide triphosphatase (NTPase), tripolyphosphatase and pyrophosphatase activities. Two site-directed mutants in motif-A of the domain: E1645A and E1647A were also tested and were found to be essential for the RTPase and NTPase activity. It was also recognized through these mutant studies that the active sites of RTPase and NTPase activities are partially overlapping. Earlier work done with Vesicular stomatitis virus capping enzymes showed that the Rhabdoviridae family of viruses follow unconventional capping pathway utilizing an enzyme polyribonucleotidyltransferase (PRNTase) which transfers GDP to 5’-monophosphated RNA. Characterization of the RTPase activity which converts 5’-triphosphated RNA into 5’-diphosphated RNA is an evidence for the morbilliviruses utilizing the conventional eukaryotic capping cascade. The results show that Paramyxoviridae do not follow unconventional capping pathway for the mRNA capping as has been the paradigm in the past decade.

The prediction of correct secondary structures of large RNAs is one of the unsolved challenges of computational molecular biology. Among the major obstacles is the fact that accurate calculations scale as O(n⁴), so the computational requirements become prohibitive as the length increases. We present a new parallel multicore and scalable program called GTfold, which is one to two orders of magnitude faster than the de facto standard programs mfold and RNAfold for folding large RNA viral sequences and achieves comparable accuracy of prediction. We analyze the algorithm's concurrency and describe the parallelism for a shared memory environment such as a symmetric multiprocessor or multicore chip. We are seeing a paradigm shift to multicore chips and parallelism must be explicitly addressed to continue gaining performance with each new generation of systems. We provide a rigorous proof of correctness of an optimized algorithm for internal loop calculations called internal loop speedup algorithm (ILSA), which reduces the time complexity of internal loop computations from O(n⁴) to O(n³) and show that the exact algorithms such as ILSA are executed with our method in affordable amount of time. The proof gives insight into solving these kinds of combinatorial problems. We have documented detailed pseudocode of the algorithm for predicting minimum free energy secondary structures which provides a base to implement future algorithmic improvements and improved thermodynamic model in GTfold. GTfold is written in C/C++ and freely available as open source from our website.

[ES] Las modificaciones químicas post-transcripcionales implican un nuevo nivel de modulación de la expresión génica. Al comienzo de esta Tesis, algunos componentes del complejo de metilación del nitrógeno en posición 6 de la adenosina (m6A) habían sido caracterizados en plantas. Sin embargo, a diferencia de mamíferos y levadura, ninguno de los 13 homólogos de AlkB (atALKBH1-10B) - potenciales desmetilasas (o erasers) - y las 13 proteínas de la familia YTH (ECT1- 11, AT4G11970 y CPSF30) - potenciales proteínas de reconocimiento de m6A (o readers) - identificadas en el genoma de Arabidopsis se habían caracterizado funcionalmente. Además, varios estudios describen la presencia de m6A en RNAs de virus de mamíferos y las diferentes funciones que desempeña esta modificación en la regulación de esas infecciones. No obstante, no se ha estudiado la posible implicación de este mecanismo molecular en las infecciones virales de plantas. El descubrimiento de la interacción entre la CP del virus del mosaico de la alfalfa (AMV) y una proteína de Arabidopsis (atALKBH9B) con homología a una eraser humana fue el punto de partida de esta Tesis. En este trabajo se confirma esta interacción, y se demuestra que atALKBH9B también puede reconocer los RNAs virales. Los resultados revelan que atALKBH9B tiene la capacidad de desmetilar m6A a partir de moléculas de RNA monocatenario in vitro. Esta proteína se acumula en gránulos citoplasmáticos que se colocalizan con siRNA bodies y se asocian a P-bodies, lo que sugiere que su actividad podría estar relacionada con el silenciamiento y/o degradación de mRNA. Por otro lado, ensayos preliminares muestran que los RNAs del AMV, el virus del mosaico del pepino (CMV), el virus de la arruga del nabo (TCV) y el virus del mosaico de la coliflor (CaMV) se metilan durante la infección en Arabidopsis. Además, para AMV y CMV, los resultados fueron corroborados por UPLC-PDA-Tof-MS y los sitios m6A a lo largo de los RNAs del AMV fueron identificados mediante MeRIP-seq. Los resultados presentados confirman que la relación m6A/A a lo largo de los RNAs virales aumenta en plantas atalkbh9b en comparación con las silvestres, mientras que la traducción y/o replicación se ven afectadas y el movimiento sistémico a los tallos florales está prácticamente bloqueado. A diferencia de la CP de AMV, la de CMV no interacciona con atALKBH9B por Y2H y, como ocurre con el resto de virus analizados (CMV, TCV y CaMV), su ciclo de infección no se ve afectado en plantas atalkbh9b. Además, la secuenciación de mRNA realizada en este trabajo revela que la infección por AMV induce algunos genes de Arabidopsis pertenecientes a la maquinaria m6A, MTA, MTB, VIR y ECT5. De acuerdo con el efecto antiviral dependiente de m6A para el AMV y teniendo en cuenta que ECT2, ECT3 y ECT4 fueron recientemente caracterizadas como readers citoplasmáticas, la supresión del módulo ECT2/ECT3/ECT5 aumenta significativamente los títulos sistémicos de AMV y CMV. El efecto antiviral de ECT2 sobre AMV parece estar modulado por su unión directa a los residuos de m6A presentes en los RNAs virales, ya que un mutante de ECT2 defectuoso en el reconocimiento de m6A pierde la actividad antiviral que sí presenta la proteína original y no es capaz de arrastrar RNAs virales in vivo. Por otro lado, acorde a la localización previamente descrita para ECT2 y ECT4 y la capacidad de ECT2 para experimentar una fase similar al gel in vitro, la expresión transitoria de ECT5 muestra un patrón citoplasmático con formación de agregados. Se propone que, como se ha descrito para las proteínas YTH de mamíferos, la interacción entre las ECTs y el RNA polimetilado (en este caso, RNA viral) promovería la formación de gránulos de estrés y, en consecuencia, reduciría las tasas de traducción y replicación viral. En resumen, en este trabajo se caracteriza la primera m6A eraser de plantas, atALKBH9B, y, por primera vez, se describe la influencia del mecanismo de metilación m6A en las infecciones virales de plantas. / [EN] Post-transcriptional chemical modifications entail a new level of gene expression modulation. At the beginning of this Thesis, some components of the N6-adenosine methylation (m6A) complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) - putative demethylases (or erasers) - and 13 proteins of the YTH family (ECT1-11, AT4G11970 and CPSF30) had been identified in the Arabidopsis genome. However, unlike mammals and yeast, no functional roles had been described for any of these proteins. Besides, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses and the critical roles that this modification plays regulating viral infections. However, the potential relevance of this molecular mechanism on plant viral infections remained fully unexplored. The discovery of the interaction between the AMV CP and an Arabidopsis protein (atALKBH9B) with similarity to a human eraser was the starting point of this Thesis. Here, this interaction is confirmed and it is demonstrated that atALKBH9B can also recognize the viral RNAs. Furthermore, the obtained results prove that atALKBH9B has the capability of demethylating m6A from single-stranded RNA molecules in vitro. This protein was observed to accumulate in cytoplasmic granules that colocalize with siRNA-bodies and associate to P-bodies, suggesting that atALKBH9B activity could be related to mRNA silencing and/or decay processes. On the other hand, preliminary assays show that viral RNAs of AMV, Cucumber mosaic virus (CMV), Turnip crinkle virus (TCV) and Cauliflower mosaic virus (CaMV) become methylated during infection in Arabidopsis. Besides, for AMV and CMV, the results were corroborated by UPLC-PDA-Tof-MS and m6A sites along the RNAs of AMV were identified through MeRIP-seq approach. The results presented here confirm that m6A/A ratio along viral RNAs is increased in atalkbh9b plants compared to wild type, whereas translation and/or replication are impaired and systemic movement to the floral stems is practically blocked. In contrast to AMV, CMV CP does not interact with atALKBH9B by Y2H and, as it occurs with the rest of the assayed viruses (CMV, TCV and CaMV), its infection cycle is not affected in atalkbh9b plants. Furthermore, the mRNA-seq analysis performed in this Thesis reveals that some Arabidopsis factors belonging to the m6A machinery, MTA, MTB, VIR and ECT5 genes, are upregulated upon AMV infection. Consistent with the m6A-dependent antiviral effect for AMV and considering that ECT2, ECT3 and ECT4 were recently characterized as cytoplasmic m6A readers, mutations of ECT2/ECT3/ECT5 Arabidopsis module significantly increase AMV and CMV systemic titers. The antiviral effect of ECT2 on AMV seems to be modulated via its direct binding to the m6A residues presented in the viral RNAs, since an ECT2 mutant defective in m6A recognition loses wild type antiviral activity and is not able to pull down viral RNAs in vivo. On the other hand, according to the previous subcellular localization described for ECT2 and ECT4 and the ability of ECT2 to undergo gel-like phase in vitro, the transitory expression of ECT5 displays a cytoplasmic pattern with the formation of some aggregates. As found for mammal YTH proteins, the interaction between ECTs and poly-methylated RNA (in this case viral RNA) is proposed to promote the formation of stress granules and, consequently, reduce viral translation and replication rates. In summary, in this work, atALKBH9B is reported as the first m6A eraser identified in plants and, for the first time, it is described the influence of m6A methylation mechanism in plant viral infections. / [CA] Les modificacions químiques post-transcripcionals impliquen un nou nivell de modulació de l'expressió gènica. Al començament d'esta Tesi, s'havien caracteritzat alguns components del complex de metilació del nitrogen en posició 6 de la adenosina (m6A) en plantes. No obstant això, a diferència de mamífers i llevat, cap dels 13 homòlegs d'AlkB (atALKBH1-10B) - potencials desmetilases (o erasers) - i les 13 proteïnes de la família YTH (ECT1- 11, AT4G11970 i CPSF30) - potencials proteïnes de reconeixement de m6A (o readers) - identificades en el genoma d'Arabidopsis s'havien caracteritzat funcionalment. A més, diversos estudis han descrit la presència de residus m6A en RNAs de virus de mamífers i les diferents funcions que exercix esta modificació en la regulació de les infeccions virals. No obstant això, no s'ha estudiat la possible implicació d'este mecanisme molecular en les infeccions virals de plantes. El descobriment de la interacció entre la CP del virus del mosaic de l'alfals (AMV) i una proteïna d'Arabidopsis (atALKBH9B) amb homologia a una eraser humana va ser el punt de partida d'esta Tesi. En este treball es confirma esta interacció, i es demostra que atALKBH9B també pot reconéixer els RNAs virals. Els resultats revelen que atALKBH9B té la capacitat de desmetilar m6A a partir de molècules de RNA monocatenari in vitro. Esta proteïna s'acumula en grànuls citoplasmàtics que es colocalitzen amb siRNA bodies i s'associen a P-bodies, la qual cosa suggerix que l'activitat atALKBH9B podria estar relacionada amb els processos de silenciament i/o degradació de mRNA. D'altra banda, assajos preliminars mostren que els RNAs virals de l'AMV, el virus del mosaic del cogombre (CMV), el virus de l'arruga del nap (TCV) i el virus del mosaic de la coliflor (CaMV) es metilen durant la infecció en Arabidopsis. A més, per AMV i CMV els resultats van ser confirmats per UPLC-PDA-Tof-MS i els llocs m6A al llarg dels RNAs d'AMV s'identificaren mitjançant MeRIP-seq. Els resultats presentats confirmen que la relació m6A/A al llarg dels RNAs virals augmenta en les plantes atalkbh9b en comparació amb les silvestres, mentre que la traducció i/o replicació es veuen afectades i el moviment sistèmic a les tiges florals està pràcticament bloquejat. A diferència de la CP d'AMV, la de CMV no interacciona amb atALKBH9B per Y2H i, com ocorre amb la resta dels virus analitzats (CMV, TCV i CaMV), el seu cicle d'infecció no es veu afectat en plantes atalkbh9b. A més, la seqüenciació de mRNA realitzada en este treball revela que la infecció per AMV indueix alguns gens d'Arabidopsis pertanyents a la maquinària m6A, MTA, MTB, VIR i ECT5. D'acord amb l'efecte antiviral dependent de m6A per a l'AMV i tenint en compte que ECT2, ECT3 i ECT4 van ser recentment caracteritzades com readers citoplasmàtiques, la supressió del mòdul ECT2/ECT3/ECT5 augmenta significativament els títols sistèmics d'AMV i CMV. L'efecte antiviral d'ECT2 sobre AMV sembla estar modulat a través de la seua unió directa als nucleòtids m6A presents en els RNAs virals, ja que un mutant de la proteïna ECT2 defectuós en el reconeixement de m6A perd l'activitat antiviral que sí que presenta la proteïna original i no és capaç d'arrossegar RNAs virals in vivo. D'altra banda, d'acord amb la localització subcel·lular descrita prèviament per a ECT2 i ECT4 i la capacitat d'ECT2 per a experimentar una fase similar al gel in vitro, l'expressió transitòria d'ECT5 mostra un patró citoplasmàtic amb la formació d'agregats. Es proposa que, com s'ha descrit per a les proteïnes YTH de mamífers, la interacció entre les ECTs i el RNA polimetilat (en aquest cas, RNA viral) promouria la formació de grànuls d'estrès i, en conseqüència, reduiria les taxes de traducció i replicació viral. En resum, en este treball es caracteritza la primera m6A eraser de plantes, atALKBH9B, i, per primera vegada, es descriu la influència de l'mecanisme de metilació M6A en les infeccions virals de plantes. / Martínez Pérez, M. (2020). Involvement of the host RNA N6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155976

Enveloped RNA viruses like human immunodeficiency virus type-1 (HIV-1) and SARS-CoV-2 enter host cells through fusion with the plasma membrane, a process facilitated by specific viral envelope proteins that recognize and bind to receptors expressed on the host cell surface. These receptors can diverge based on the type of cell and virus. For HIV-1, the primary receptors on myeloid cells are CD4 and CCR5 or CXCR4. For SARS-CoV-2, although the primary receptor is ACE2, other myeloid-cell specific sialic acid binding lectins can also facilitate entry. Following cellular invasion, different viral RNA species can be detected by distinct host nucleic acid sensors, resulting in type I interferons and pro-inflammatory cytokine induction. While these innate immune responses are essential for controlling viral infections, overactivation can lead to chronic inflammation, tissue damage, and disease pathogenesis. Herein, I examine the contribution of HIV-1 and SARS-CoV-2 de-novo RNA expression and the molecular mechanisms that contribute to innate immune activation in myeloid cells. Despite advancements in combination antiretroviral therapy (ART) in suppressing systemic viral replication in individuals infected with HIV, residual viral RNA expression in tissue reservoirs remains a significant hindrance to curative efforts. I hypothesized that persistent expression of viral RNAs in myeloid cells triggers dysregulated innate immune activation, and inflammasomes activation. This study centers on the long-lived tissue-resident innate immune cells - macrophages and microglia, which, owing to their self-renewing nature, operate as reservoirs of viral RNA production, and are thought to lead to chronic immune activation even in the absence of productive replication. Our previous studies suggest that de novo expression of unspliced intron-containing HIV-1 RNA (herein referred to as icRNA) triggers activation of pro-inflammatory cytokines in myeloid cells. Here, I demonstrate that cytosolic expression of HIV-1 icRNA, but not multiply-spliced viral RNAs induces inflammasome activation, LDH release and IL-1β secretion in productively infected monocyte-derived macrophages (MDM) and induced pluripotent stem cell (iPSC)-derived microglia. Interestingly, knockdown of RLRs, RIG-I and MDA5 or endosomal TLRs failed to abrogate HIV-1 icRNA-induced IL-1β secretion. Rather, knockdown of NLRP1, but not NLRP3, inflammasome resulted in a significant reduction in IL-1β secretion, underscoring NLRP1's pivotal role in the HIV-1 icRNA-induced IL-1β secretion. Furthermore, Rev-Crm1-dependent nucleocytoplasmic export of HIV-1 icRNA was required for NLRP1-mediated Caspase-1 activation, IL-1β secretion, LDH release and cell death. Similarly, SARS-CoV-2, while not establishing productive infection in macrophages, can activate these cells, contributing to a hyper-inflammatory response marked by the heightened expression of pro-inflammatory cytokines, which is understood to be a principal driver of COVID-19 pathology. SARS-CoV-2 established an abortive infection in macrophages. CD169, a macrophage-specific sialic-acid binding lectin, mediated ACE2-independent SARS-CoV-2 entry in human macrophages and establishment of restricted infection. Interestingly, CD169-mediated SARS-CoV-2 entry in macrophages led to the expression of viral genomic and subgenomic RNAs, with negligible viral protein expression and no release of infectious virus particles, implying a post-entry restriction to SARS-CoV-2 replication in macrophages that was curbed by exogenous ACE2 expression. Despite restricted viral RNA expression, cytoplasmic RLRs, RIG-I and MDA5, sensed abortive viral transcripts, and induced pro-inflammatory responses in a MAVS dependent manner. This dissertation reveals striking parallels between the role of viral RNAs in driving pro-inflammatory responses in HIV-1 and SARS-CoV-2 infections. These findings collectively underscore the central role of cytoplasmic sensing of viral RNAs and their contribution to chronic inflammation in virus-infected myeloid cells. Elucidating these molecular mechanisms further may pave the way for novel therapeutic interventions to mitigate the persistent innate immune activation and immunopathology detected in HIV-1 and SARS-CoV-2 infected individuals.

Abstract Feline immunodeficiency virus (FIV) is an important infectious agent of domestic cats worldwide. It has been classified into the Lentivirus genus of the Retroviridae family, together with human immunodeficiency virus (HIV). Five FIV subtypes (A, B, C, D and E) have been described based on sequence variation of the V3-V5 region of the envelope (env) gene. There is considerable sequence diversity within and between subtypes, which has been a major obstacle in the development of a successful vaccine. However, an FIV vaccine that incorporates inactivated whole viruses from subtypes A and D is now commercially available. Although the vaccine has been shown to be efficacious in protecting against challenge with homologous and a heterologous (subtype B) subtypes, its effectiveness against other viral variants is unknown. Therefore, identifying the type and diversity of FIV strains in different regions is important to establish the potential efficacy of the vaccine in areas where vaccination is to be implemented. The proviral DNA sequence of the V3-V5 region of the env gene was determined for 102 FIV-infected cats from locations in Australia, New Zealand and South Africa. Subtype A was the predominant subtype in Australia and South Africa, although subtype B and C were also identified in each of these countries, respectively. Both subtypes A and C were also present in New Zealand. Of interest, there were some samples in New Zealand and South Africa that demonstrated subtype assignment discrepancies when different regions of the genome were analysed, suggesting co-infection and/or recombination. Cats infected with FIV exhibit varying degrees of immunological impairment. Currently, prognosis for an FIV-infected cat is based on clinical signs alone, which is a relatively subjective measure. In HIV-infected patients it is recognised that viral RNA load correlates with disease stage and prognosis. This PhD research tested whether viral RNA load may be a useful prognostic marker in FIV infection. A real-time PCR assay was developed to quantify plasma viral RNA load in 42 FIV-infected cats at three different clinical stages (1:healthy, 2:unwell without signs of immunodeficiency, 3:unwell with signs of immunodeficiency). In cats older than 5 years of age, log-transformed viral RNA loads were significantly higher in cats in category 3 compared to cats in category 1. There were no significant differences in the viral RNA load of older cats in category 2 compared to category 1. There were no cats younger than 5 years of age in category 3 and there was no significant difference in viral RNA load between young cats in categories 1 and 2. Of the 15 cats for which follow-up data was available, eight showed no change in clinical signs, and seven showed a worsening of clinical signs with six of these showing a progression of clinical category including death. One of the cats in category 2 that progressed clinically had one of the highest viral RNA loads of cats in that category. Three of four cats from category 3 that were followed had either died or been euthanised. Two of these cats had among the highest viral RNA loads in the whole study, while the remaining cat (for which the definitive cause of death was not confirmed) had a relatively low viral RNA load. In summary, measurement of viral RNA load was found to be a potentially useful clinical and prognostic marker but further work is required to better assess its usefulness to veterinarians. Serum acute phase proteins were investigated as possible candidate markers of FIV disease with the aim of developing a more simplified assay that could be used as a prognostic marker for FIV infection. Blood samples from 43 FIV-infected and 25 FIV-negative cats were assayed for the concentration of four acute phase proteins. Both healthy and sick cats were included in the study. Compared to healthy cats, sick cats had significantly higher concentrations of serum amyloid A (P<0.05). Alpha 1-acid glycoprotein and haptoglobin were also found to be in higher concentrations in sick cats (P<0.1). Other variables such as age and gender were also associated with acute phase protein concentrations. With respect to FIV infection, it was found that in sick cats, serum amyloid A, in combination with the age of the cat, was the best predictor of FIV viral RNA load. Alpha 1-acid glycoprotein and haptoglobin were not significantly associated with FIV viral RNA load. Although health status did not influence albumin levels, they were found to be significantly lower in FIV-positive cats in comparison to FIV-negative cats (P<0.05). The frequent monitoring of viral RNA loads and CD4+ lymphocyte counts that is performed on HIV-infected patients is cost prohibitive in veterinary patients. This study showed that there is potential for the use of acute phase protein concentrations (in particular serum amyloid A) as alternative prognostic tools in FIV-infected cats. Further work, particularly longitudinal studies, is required to more definitively define changes in viral RNA load and acute phase protein concentrations throughout the course of FIV infection.

Polioviruses, Coxsackieviruses, and Echoviruses belonging to the Picornaviridae family of positive-stranded, non-enveloped viruses, are highly infectious and associated with a range of illnesses in children from minor febrile illness to severe, potentially fatal conditions (eg, aseptic meningitis, encephalitis, paralysis and myocarditis). The viruses encodes 11 viral proteins along with the transient set of intermediates unique to viral propagation. 3A, one of the non-structural proteins, plays a crucial role in viral replication by anchoring the replication complex to the membrane vesicle and by recruiting essential cellular factors to the site of replication. It is an 89 amino-acid longprotein, and consists of a soluble N-terminal region and a hydrophobicC-terminal region. The soluble region contains two amphipathic alphahelices that form a hairpin, which are flanked by unstructured regions.Since, Enteroviruses have limited coding capacity,viral protein interactions with cellular proteins and lipids are essentialin viral replication, translation, polyprotein processing andpathogenesis. Understanding these interactions is essential inunderstanding the molecular mechanisms associated pathogenesis, andidentifying drug targets. Our studies are aimed at identifying hostfactors interacting with 3A protein and their functional significance invirus replication.We have identified thepotential 3A-interacting cellular candidate proteins using pull-down followed by liquid chromatography associated mass spectrometry. Gene ontology analysis revealed asignificant enrichment in cellular pathways, functions, and proteindomains in comparison with the control. Further studies were focused on Sec12 (guanine nucleotideexchange factor), ACBD3 (acyl-CoA binding domain containing 3) andPhosphatidylinositol 4-kinase beta (PI4KIIIß) interactions with the 3Aprotein, and their significance in viral replication. Sec12 (GEF) initiates the COPII vesicle-mediated ER-to-Golgi membrane trafficking by recruiting and activating the small GTP binding protein Sar1A to the membrane, which further recruits Sec23/24, cargo and Sec13/31 coat proteins to form functional COPII vesicles.We demonstrated that Sec12 and 3A interact directly in the ER through their C-terminal hydrophobic regions in oligomerization independent manner, leading toreduced the level of recruitment of individual COPII components such as Sar1A, Sec24A, and Sec31A to the membranes, thereby inhibiting virus replication. But in infected cells, other viral proteins such as 2B and 2BC likely stabilize the membrane-recruited Sar1A to support the viral replication. The viral proteins, ACBD3, PI4KIIIß interacted and co-localized with the Echovirus 3A protein.Knockdown of Sec12 or PI4KIIIß and expression of 3A or DN-Sar1A inhibited Echovirus replication, unlike proteins which support the COPII vesicle mediated ER-to-Golgi trafficking.Our results collectively indicate Sec12 is a crucial component in the anterograde membrane trafficking and is a novel host factor in Echovirus replication.

Les huit ARN viraux (ARNv) génomiques des influenzavirus de type A sont sélectivement empaquetés dans les virions, vraisemblablement sous la forme d’un complexe supramoléculaire maintenu par des appariements ARN/ARN entre signaux d’empaquetage. Afin d’améliorer la compréhension des règles qui gouvernent ce mécanisme, nous avons déterminé le réseau d’interactions formé entre les ARNv de la souche modèle de laboratoire A/Puerto Rico/8/34 (H1N1). Nous avons ensuite défini, au nucléotide près et par deux approches in vitro, les séquences des ARNv impliquées dans la formation de certaines interactions. Le rôle fonctionnel de deux d’entre elles a été testé en contexte infectieux. Nous avons également poursuivi l’étude d’une interaction identifiée au laboratoire entre deux ARNv de la souche A/Moscou/10/99 (H3N2) circulante. Enfin, nous avons collaboré avec le Dr L. Brown (Australie) sur l’étude du rôle d’une interaction entre deux ARNv de la souche A/Udorn/307/72 (H3N2). / The genome of influenza A viruses comprises eight viral RNAs (vRNAs) likely to be selectively packaged into progeny virions as organized supramolecular complexes where vRNAs are held together by base pairing within the packaging signals. To better understand the rules governing this mechanism, we investigated the vRNA/vRNA interaction network of the A/Puerto Rico/8/34 (H1N1) strain. We then identified, at the nucleotide level, using two in vitro approaches, the vRNA sequences involved in several of the interactions. Two of them were functionally tested in an infectious context. We also studied an interaction previously identified in the laboratory between two vRNAs belonging to the circulating A/Moscow/10/99 (H3N2) strain. Finally, we collaborated with Dr L. Brown (Australia) in order to assess the role of an interaction between two vRNAs of the A/Udorn/307/72 (H3N2) strain.