Identification of a new cell line permissive to porcine reproductive and resporatory syndrome virus replication

Le syndrome reproducteur et respiratoire porcin (SRRP) est une des maladies les plus dévastatrices économiquement pour l'industrie mondiale du porc. L'agent étiologique du SRRP est le virus du SRRP (VSRRP) lequel est connu pour avoir une spécificité d'hôte très restreinte et pour sa transmission par voie aerosol. Les antigènes et les ARN du VSRRP ont été trouvés dans des cellules épithéliales du tractus respiratoire de porcs infectés par le virus. L’interaction entre les macrophages alvéolaires porcins (PAMs) et le VSRRP a été démontrée comme jouant un rôle important dans l’infection causée par le virus. Malgré cela, l’interaction prenant place entre les cellules épithéliales du tractus respiratoire porcin et le virus ne devrait pas être négligée. Jusqu’à présent, la réplication du VSRRP in vitro dans des cellules épithéliales du tractus respiratoire porcin n’a pas été conduite avec succès et les tentatives pour le faire ont échoué. Une nouvelle lignée de cellules épithéliales de poumon de porc (SJPL) est maintenant disponible et sera utilisée dans cette étude afin de déterminer si elle est permissive à la réplication du VSRRP et si elle peut être un modèle approprié pour l’étude de la pathogénèse virale du VSRRP. L’expérimentation a démontré que cette nouvelle lignée cellulaire était permissive à l’infection et à la réplication du VSRRP. Afin de corroborer ces résultats, la cinétique de réplication du virus à été effectuée avec les cellules MARC-145 et SJPL. Aucune différence significative dans la production virale totale n’a été trouvée entre les deux lignées cellulaires. Les cellules SJPL ont permis la réplication de plusieurs souches Nord-Américaines du VSRRP, quoiqu’elles sont légèrement moins efficaces que les cellules MARC-145 pour l’isolement du virus. De plus, les cellules SJPL sont phénotypiquement différentes des cellules MARC-145. Plus précisément, les cellules SJPL sont plus sensibles à l’activation par le VSRRP des pro-caspases 3/7 et plusieurs inducteurs apoptotiques. Elles ont également montré de 8 à 16 fois plus de sensibilité à l’effet antiviral causé par l’IFN-α sur la réplication du virus contrairement aux cellules MARC-145. Ces résultats démontrent que les cellules SJPL pourraient représenter un substitut intéressant aux cellules MARC-145 pour la production d’antigènes pour un vaccin anti-VSRRP. Également, dû à leurs origines (poumon de l’hôte naturel), elles pourraient s’avérer être un modèle in vitro plus approprié pour l’étude de la pathogénèse du VSRRP.

Rôle de la structure du génome viral sur la réplication du virus de l’hépatite C.

Le virus de l'hépatite C (VHC) touche 3% de la population mondiale et environ 30% des patients chroniquement infectés développeront une fibrose hépatique. Son génome est un ARN simple brin de polarité positive qui possède un cadre ouvert de lecture flanqué de deux régions non traduites hautement conservées. Différents facteurs peuvent influencer le cycle de réplication du VHC. Deux d’entre eux ont été étudiés dans cette thèse. Tout d'abord, nous nous sommes intéressés à l'effet des structures secondaires et tertiaires du génome sur la réplication du VHC. Les extrémités 5' et 3' du génome contiennent des structures ARN qui régulent la traduction et la réplication du VHC. Le 3'UTR est un élément structural très important pour la réplication virale. Cette région est constituée d’une région variable, d’une séquence poly(U/C) et d’un domaine hautement conservé appelé région X. Des études in vitro ont montré que le 3'UTR possède plusieurs structures ARN double brin. Cependant, les structures ARN telles qu'elles existent dans le 3'UTR dans un contexte de génome entier et dans des conditions biologiques étaient inconnues. Pour élucider cette question, nous avons développé une méthode in situ pour localiser les régions ARN simple brin et double brin dans le 3'UTR du génome du VHC. Comme prédit par les études antérieures, nous avons observé qu’in situ la région X du 3’UTR du génome présente des éléments ARN double brin. Étonnamment, lorsque la séquence poly (U/UC) est dans un contexte de génome entier, cette région forme une structure ARN double brin avec une séquence située en dehors du 3'UTR, suggérant une interaction ARN-ARN distale. Certaines études ont démontré que des structures ARN présentes aux extrémités 5’ et 3' du génome du VHC régulent à la fois la traduction et la réplication du VHC. Cela suggère qu'il y aurait une interaction entre les extrémités du génome qui permettrait de moduler ces deux processus. Dans ce contexte, nous avons démontré l'existence d'une interaction distale ARN-ARN, impliquant le domaine II du 5'UTR et la séquence codante de NS5B du génome du VHC. En outre, nous avons démontré que cette interaction joue un rôle dans la réplication de l'ARN viral. Parallèlement, nous avons étudié l'impact d'une molécule immuno-modulatrice sur la réplication du VHC. La fibrose hépatique est une manifestation majeure de l’infection par le VHC. Hors, il a été montré qu'une molécule immuno-modulatrice appelée thalidomide atténuait la fibrose chez les patients infectés par le VHC. Cependant, son impact sur la réplication virale était inconnu. Ainsi, nous avons étudié l'effet de cette molécule sur la réplication du VHC in vitro et nous avons démontré que la thalidomide active la réplication du virus en inhibant la voie de signalisation de NF-kB. Ces résultats soulignent l’importance de la voie de signalisation NF-kB dans le contrôle de la réplication du VHC, et sont à prendre en considération dans l’établissement d’un traitement contre la fibrose hépatique.

Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from MARC-145 cell line

Background Airborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM) and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro. Results The SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFNα antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFNβ mRNA and no up regulation of IFNα mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFNγ and TNF-α mRNAs only in infected MARC-145 cells. Conclusions In conclusion, the SJPL cells are permissive to PRRSV. In addition, they are phenotypically different from MARC-145 cells and are an additional tool that could be used to study PRRSV pathogenesis mechanisms in vitro.

The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.

The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Replication of hepatitis B in HBsAg-positive siblings

Persistent hepatitis B virus (HBV) replication is important for progression of chronic liver diseases. To understand whether there is a trend of HBV replication in siblings or not, 1850 relatives of patients with hepatocellular carcinoma (HCC) were examined prospectively for liver function test, viral markers and HBV DNA. The prevalence of HBsAg in the parents', siblings', children's and grandchildren's generations were 43.4%, 57.2%, 35.5% and 32.1%, respectively. The prevalence of hepatitis B e antigen (HBeAg) in sibling's generation (mean age 44.4 years) was 19%, which is higher than that of asymptomatic HBsAg carriers. For siblings in the children's generation, the prevalence of HBeAg in hepatitis B surface antigen (HBsAg) carriers declined from 40% in the eldest siblings to 19% in the youngest siblings. In 75 families clustered with three or more HBsAg carrier siblings, the mean age for seven families of which all siblings remained HBeAg + was younger, whereas the mean age for 35 families of which all siblings had cleared HBeAg was older. For the remaining 33 families, in only 10 families had the older siblings cleared the HBeAg earlier than the younger siblings. Twenty families showed that younger siblings cleared the HBeAg earlier than the older or middle siblings. We concluded that HBV replication in HCC relatives cannot be explained by familial tendency alone. A significant number of younger siblings appeared to have a shorter HBV replication phase than their older siblings. The possible role of this in maternal–fetal transmission is discussed.

Ribosome inactivating proteins (RIPs) from momordica charantia for anti viral therapy

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.

Alteration of the proline at position 7 of the HIV-1 spacer peptide p1 suppresses viral infectivity in a strain dependent manner

The HIV-1 spacer peptide p1 is located in the C-terminus of the Gag polyprotein and separates the nucleocapsid (NC) and p6(Gag). Research centered on p1 has been limited and as yet no function has been ascribed to this spacer peptide. We have previously found that the conserved p1 proline residues (position 7 and 13) are critical for replication in the HIV-1 strain HXB2-BH10. In this study we have focused on the proline rich p1-p6(Gag) C-terminus of HIV-1. We individually examined the role of p1 proline's in multiple strains of HIV-1 and investigated the role of three proline residues in p6(Gag) (P24, P25 and P30). Assessment of the HXB2-BH10 based mutants revealed that Gag-Pol incorporation relative to Gag decreased in the p1 mutant virions, with the double proline mutant the most impaired. Mutating both p1 proline residues was found to abolish infectivity in multiple strains of HIV-1. Independent mutation of the p1 proline at position 7 resulted in a strain-dependent suppression of viral infectivity. This defect correlates with the presence of a tyrosine residue at position 9 of p1 and occurs in the early phase of the HIV-1 replication cycle. The p1 proline residues were found to be functionally distinct from P24, P25 and P30 in p6(Gag). This work affords novel insights into our understanding of the role of p1 in HIV-1 replication.

Lipid rafts and HIV-1 : from viral entry to assembly of progeny virions

Background: Lipid rafts are currently an intensely investigated topic of cell biology. In addition to a demonstrated role in signal transduction of the host cell, lipid rafts serve as entry and exit sites for microbial pathogens and toxins, such as FimH-expressing enterobacteria, influenza virus, measles virus and cholera toxin. Furthermore, caveolae, a specialised form of lipid raft, are required for the conversion of the non-pathogenic prion protein to the pathogenic scrapie isoform.

Objectives: A number of reports have shown, directly or indirectly, that lipid rafts are important at various stages of the human immunodeficiency virus type-1 (HIV-1) replication cycle. The purpose of this paper is to provide a brief overview of the role of membrane-associated lipid rafts in cell biology, and to evaluate how HIV-1 has hijacked this cellular component to support HIV-1 replication. Special sections are devoted to discussing the role of lipid rafts in (1) the entry of HIV-1, (2) signal transduction regulation in HIV-1-infected cells, (3) the trafficking of HIV-1 proteins via lipid rafts during HIV-1 assembly; and a further section discusses the role of cholesterol in mature HIV-1.

Summary: Like a number of other pathogens, HIV-1 has evolved to rely on the host cell lipid rafts to support its propagation during multiple stages of the HIV-1 replication cycle. This review has highlighted the importance of lipid rafts in HIV-1 replication.

Maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity

Production of the human immunodeficiency virus type 1 (HIV_-1) Gag-Pol precursor protein results from a −1 ribosomal frameshifting event. In infected cells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20:1, a ratio that is conserved among retroviruses. To examine the impact of this ratio on HIV_-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus-producing cells by cotransfecting HIV_-1 proviral DNA with an HIV_-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other contains an inactive protease [PR(−)]. In an attempt to produce viral particles with Gag/Gag-Pol ratios ranging from 20:21 to 20:1 (wild type), 293T cells were cotransfected with various ratios of wild-type proviral DNA and proviral DNA from either Gag-Pol expression vector. Viral particles derived from cells with altered Gag/Gag-Pol ratios via overexpression of PR(−) Gag-Pol showed a ratio-dependent defect in their virion protein profiles. However, the defects in virion infectivity were independent of the nature of the Gag-Pol expression vector, i.e., PR(+) or PR(−). Based on equivalent input of reverse transcriptase activity, we estimated that HIV_-1 infectivity was reduced 250- to 1,000-fold when the Gag/Gag-Pol ratio in the virion-producing cells was altered from 20:1 to 20:21. Although virion RNA packaging was not affected by altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to 20:21 progressively reduced virion RNA dimer stability. The impact of the Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-Pol PR(−) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(−) in a 20:21 ratio contained mainly monomeric RNA. Our observations provide the first direct evidence that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol proteins is also important for RNA dimerization and that stable RNA dimers are not required for encapsidation of genomic RNA in HIV_-1.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process.

Inhibition of HIV-1 replication by balsamin, a ribosome inactivating protein of Momordica balsamina

Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4(+) T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reverse-transcription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections.

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.