Murine norovirus infection of macrophages induces intrinsic apoptosis as the major form of programmed cell death.

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.

Identification of Estrogen Receptor Modulators as Inhibitors of Flavivirus Infection.

Flaviviruses such as Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) are major global pathogens for which safe and effective antiviral therapies are not currently available. To identify antiviral small molecules with well-characterized safety and bioavailability profiles, we screened a library of 2,907 approved drugs and pharmacologically active compounds for inhibitors of ZIKV infection using a high-throughput cell-based immunofluorescence assay. Interestingly, estrogen receptor modulators raloxifene hydrochloride and quinestrol were among 15 compounds that significantly inhibited ZIKV infection in repeat screens. Subsequent validation studies revealed that these drugs effectively inhibit ZIKV, DENV, and WNV (Kunjin strain) infection at low micromolar concentrations with minimal cytotoxicity in Huh-7.5 hepatoma cells and HTR-8 placental trophoblast cells. Since these cells lack detectable expression of estrogen receptors-α and -ß (ER-α and ER-ß) and similar antiviral effects were observed in the context of subgenomic DENV and ZIKV replicons, these compounds appear to inhibit viral RNA replication in a manner that is independent of their known effects on estrogen receptor signaling. Taken together, quinestrol, raloxifene hydrochloride, and structurally related analogues warrant further investigation as potential therapeutics for treatment of flavivirus infections.

Mouse Norovirus Infection Arrests Host Cell Translation Uncoupled from the Stress Granule-PKR-eIF2α Axis.

The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study, we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in the suppression of host translation, and yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signaling cytokines such as beta interferon, interleukin-6, and tumor necrosis factor alpha. Our subsequent analyses revealed that the phosphorylation of eIF2α was mediated via protein kinase R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2α, and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection and that MNV can restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears that MNV utilizes G3BP1 to enhance replication but equally to prevent SG formation, suggesting an anti-MNV property of SGs. Overall, this study highlights MNV manipulation of SGs, PKR, and translational control to regulate cytokine translation and to promote viral replication.IMPORTANCE Viruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate, and cause disease. In retaliation, cells possess various defense mechanisms to detect, destroy, and clear infecting viruses, as well as signal to neighboring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and proinflammatory cytokines. However, virus replication and protein translation still ensue. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SGs), by recruiting the key host protein G3BP1 to the MNV replication complex, a recruitment that is crucial to establishing and maintaining virus replication. Thus, MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.

The Adenosine Analogue NITD008 has Potent Antiviral Activity against Human and Animal Caliciviruses.

The widespread nature of calicivirus infections globally has a substantial impact on the health and well-being of humans and animals alike. Currently, the only vaccines approved against caliciviruses are for feline and rabbit-specific members of this group, and thus there is a growing effort towards the development of broad-spectrum antivirals for calicivirus infections. In this study, we evaluated the antiviral activity of the adenosine analogue NITD008 in vitro using three calicivirus model systems namely; feline calicivirus (FCV), murine norovirus (MNV), and the human norovirus replicon. We show that the nucleoside analogue (NA), NITD008, has limited toxicity and inhibits calicivirus replication in all three model systems with EC50 values of 0.94 µM, 0.91 µM, and 0.21 µM for MNV, FCV, and the Norwalk replicon, respectively. NITD008 has a similar level of potency to the most well-studied NA 2'-C-methylcytidine in vitro. Significantly, we also show that continual NITD008 treatment effectively cleared the Norwalk replicon from cells and treatment with 5 µM NITD008 was sufficient to completely prevent rebound. Given the potency displayed by NITD008 against several caliciviruses, we propose that this compound should be interrogated further to assess its effectiveness in vivo. In summary, we have added a potent NA to the current suite of antiviral compounds and provide a NA scaffold that could be further modified for therapeutic use against calicivirus infections.

Antiviral Candidates for Treating Hepatitis E Virus Infection.

Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC50], 0.03 µM; half-maximal cytotoxic concentration [CC50], >100 µM) and GPC-N114 (EC50, 1.07 µM, CC50, >100 µM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 µM GPC-N114 or 2.50 µM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC50, 1.97 µM; CC50, >100 µM) and reduced replicon RNA levels (47.2% reduction at 10 µM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.

The Microtubule-Associated Innate Immune Sensor GEF-H1 Does Not Influence Mouse Norovirus Replication in Murine Macrophages.

Norovirus is an acute infection of the gastrointestinal tract causing rapid induction of vomiting and diarrhoea. The infection is sensed and controlled by the innate immune system, particularly by the RNA helicase MDA-5 and type I and III interferons (IFNs). We have observed that intracellular replication of murine norovirus (MNV) occurs in membranous clusters proximal to the microtubule organising centre, a localisation dependent on intact microtubules. Recently, it was shown that the host protein guanine nucleotide exchange factor-H1 (GEF-H1) is a microtubule-associated innate immune sensor that activates interferon Regulatory Factor 3 to induce the production of type I IFNs. Thus, we interrogated the potential role of GEF-H1 in controlling MNV infections. We observed that GEF-H1 was recruited to the MNV replication complex; however RNAi-mediated suppression of GEF-H1 did not outwardly affect replication. We furthered our studies to investigate the impact of GEF-H1 on MNV innate detection and observed that GEF-H1 did not contribute to type I IFN induction during MNV infection or influenza virus infection but did result in a small reduction of interferonâ»ß (IFNß) during West Nile virus infection. Intriguingly, we discovered an interaction of GEF-H1 with the viral MNV non-structural protein 3 (NS3), an interaction that altered the location of GEF-H1 within the cell and prevented the formation of GEF-H1-induced microtubule fibres. Thus, our results indicate that GEF-H1 does not contribute significantly to the innate immune sensing of MNV, although its function may be modulated via interaction with the viral NS3 protein.

Potential Therapeutic Agents for Feline Calicivirus Infection.

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 µM and 2.7 µM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2'-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 µM and 2.5 µM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.

Mouse Norovirus Infection Reduces the Surface Expression of Major Histocompatibility Complex Class I Proteins and Inhibits CD8+ T Cell Recognition and Activation.

Human noroviruses are highly infectious single-stranded RNA (ssRNA) viruses and the major cause of nonbacterial gastroenteritis worldwide. With the discovery of murine norovirus (MNV) and the introduction of an effective model for norovirus infection and replication, knowledge about infection mechanisms and their impact on the host immune response has progressed. A major player in the immune response against viral infections is the group of major histocompatibility complex (MHC) class I proteins, which present viral antigen to immune cells. We have observed that MNV interferes with the antigen presentation pathway in infected cells by reducing the surface expression of MHC class I proteins. We have shown that MNV-infected dendritic cells or macrophages have lower levels of surface expression of MHC class I proteins than uninfected and bystander cells. Transcriptional analysis revealed that this defect is not due to a decreased amount of mRNA but is reflected at the protein level. We have determined that this defect is mediated via the MNV NS3 protein. Significantly, treatment of MNV-infected cells with the endocytic recycling inhibitor dynasore completely restored the surface expression of MHC class I proteins, whereas treatment with the proteasome inhibitor MG132 partly restored such expression. These observations indicate a role for endocytic recycling and proteasome-mediated degradation of these proteins. Importantly, we show that due to the reduced surface expression of MHC class I proteins, antigen presentation is inhibited, resulting in the inability of CD8+ T cells to become activated in the presence of MNV-infected cells.IMPORTANCE Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis worldwide and impose a great burden on patients and health systems every year. So far, no antiviral treatment or vaccine is available. We show that MNV evades the host immune response by reducing the amount of MHC class I proteins displayed on the cell surface. This reduction leads to a decrease in viral antigen presentation and interferes with the CD8+ T cell response. CD8+ T cells respond to foreign antigen by activating cytotoxic pathways and inducing immune memory to the infection. By evading this immune response, MNV is able to replicate efficiently in the host, and the ability of cells to respond to consecutive infections is impaired. These findings have a major impact on our understanding of the ways in which noroviruses interact with the host immune response and manipulate immune memory.

Membrane alterations induced by nonstructural proteins of human norovirus.

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.

Ancient recombination events and the origins of hepatitis E virus.

BACKGROUND: Hepatitis E virus (HEV) is an enteric, single-stranded, positive sense RNA virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. Tracing the evolutionary ancestry of HEV has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry. RESULTS: To reveal close protein relatives of the Hepeviridae family, similarity searching of the GenBank database was carried out using a complete Orthohepevirus A, HEV genotype I (GI) ORF1 protein sequence and individual proteins. The closest non-Hepeviridae homologues to the HEV ORF1 encoded polyprotein were found to be those from the lepidopteran-infecting Alphatetraviridae family members. A consistent relationship to this was found using a phylogenetic approach; the Hepeviridae RdRp clustered with those of the Alphatetraviridae and Benyviridae families. This puts the Hepeviridae ORF1 region within the "Alpha-like" super-group of viruses. In marked contrast, the HEV GI capsid was found to be most closely related to the chicken astrovirus capsid, with phylogenetic trees clustering the Hepeviridae capsid together with those from the Astroviridae family, and surprisingly within the "Picorna-like" supergroup. These results indicate an ancient recombination event has occurred at the junction of the non-structural and structure encoding regions, which led to the emergence of the entire Hepeviridae family. The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses, whilst its non-structural region is related to viruses of the Potyviridae; a large family of plant-infecting viruses with a flexible filamentous rod-shaped virion. Thus, we identified a separate inter-viral family recombination event, again at the non-structural/structural junction, which likely led to the creation of the Astroviridae. CONCLUSIONS: In summary, we have shown that new viral families have been created though recombination at the junction of the genome that encodes non-structural and structural proteins, and such recombination events are implicated in the genesis of important human pathogens; HEV, astrovirus and rubella virus.

A Multi-Site Study of Norovirus Molecular Epidemiology in Australia and New Zealand, 2013-2014.

BACKGROUND: Norovirus (NoV) is the major cause of acute gastroenteritis across all age groups. In particular, variants of genogroup II, genotype 4 (GII.4) have been associated with epidemics globally, occurring approximately every three years. The pandemic GII.4 variant, Sydney 2012, was first reported in early 2012 and soon became the predominant circulating NoV strain globally. Despite its broad impact, both clinically and economically, our understanding of the fundamental diversity and mechanisms by which new NoV strains emerge remains limited. In this study, we describe the molecular epidemiological trends of NoV-associated acute gastroenteritis in Australia and New Zealand between January 2013 and June 2014. METHODOLOGY: Overall, 647 NoV-positive clinical faecal samples from 409 outbreaks and 238 unlinked cases of acute gastroenteritis were examined by RT-PCR and sequencing. Phylogenetic analysis was then performed to identify NoV capsid genotypes and to establish the temporal dominance of circulating pandemic GII.4 variants. Recombinant viruses were also identified based on analysis of the ORF1/2 overlapping region. FINDINGS: Peaks in NoV activity were observed, however the timing of these epidemics varied between different regions. Overall, GII.4 NoVs were the dominant cause of both outbreaks and cases of NoV-associated acute gastroenteritis (63.1%, n = 408/647), with Sydney 2012 being the most common GII.4 variant identified (98.8%, n = 403/408). Of the 409 reported NoV outbreaks, aged-care facilities were the most common setting in both Western Australia (87%, n = 20/23) and New Zealand (58.1%, n = 200/344) while most of the NoV outbreaks were reported from hospitals (38%, n = 16/42) in New South Wales, Australia. An analysis of a subset of non-GII.4 viruses from all locations (125/239) showed the majority (56.8%, n = 71/125) were inter-genotype recombinants. These recombinants were surprisingly diverse and could be classified into 18 distinct recombinant types, with GII.P16/GII.13 (24% of recombinants) the most common. CONCLUSION: This study revealed that following its emergence in 2012, GII.4 Sydney 2012 variant continued to be the predominant cause of NoV-associated acute gastroenteritis in Australia and New Zealand between 2013 and 2014.

Longitudinal Sequence and Functional Evolution within Glycoprotein E2 in Hepatitis C Virus Genotype 3a Infection.

The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384-661). Within E2 are three variable regions located at the N-terminus (HVR1; 384-411), and internally at 460-480 (HVR2) and 570-580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411-428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.

Nonnucleoside inhibitors of norovirus RNA polymerase: scaffolds for rational drug design.

Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide, causing over 200,000 deaths a year. NoV is nonenveloped, with a single-stranded RNA genome, and is primarily transmitted person to person. The viral RNA-dependent RNA polymerase (RdRp) is critical for the production of genomic and subgenomic RNA and is therefore a prime target for antiviral therapies. Using high-throughput screening, nearly 20,000 "lead-like" compounds were tested for inhibitory activity against the NoV genogroup II, genotype 4 (GII.4) RdRp. The four most potent hits demonstrated half-maximal inhibitory concentrations (IC50s) between 5.0 µM and 9.8 µM against the target RdRp. Compounds NIC02 and NIC04 revealed a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 RdRp inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious GV.1 murine norovirus (MNV). NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with 50% effective concentrations (EC50s) of 30.1 µM and 71.1 µM, respectively, while NIC10 and NIC12 had no observable effect on the NoV GI.1 replicon. In the MNV model, NIC02 reduced plaque numbers, size, and viral RNA levels in a dose-dependent manner (EC50s between 2.3 µM and 4.8 µM). The remaining three compounds also reduced MNV replication, although with higher EC50s, ranging from 32 µM to 38 µM. In summary, we have identified novel nonnucleoside inhibitor scaffolds that will provide a starting framework for the development and future optimization of targeted antivirals against NoV.

Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis.

The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

Norovirus RNA-dependent RNA polymerase is phosphorylated by an important survival kinase, Akt.

Viruses commonly use host cell survival mechanisms to their own advantage. We show that Akt, an important signaling kinase involved in cell survival, phosphorylates the RNA-dependent RNA polymerase (RdRp) from norovirus, the major cause of gastroenteritis outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the more prevalent norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs at a residue (Thr33) located at the interface where the RdRp finger and thumb domains interact and decreases de novo activity of the polymerase. This finding provides fresh insights into virus-host cell interactions.

Multiplex PCR testing detection of higher-than-expected rates of cervical mycoplasma, ureaplasma, and trichomonas and viral agent infections in sexually active australian women.

Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction.

Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.

The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection.

BACKGROUND/AIMS: The role of cytotoxic T lymphocytes (CTL) in limiting viral replication and producing hepatocellular injury in patients with chronic hepatitis C virus (HCV) infection is controversial. METHODS: Intrahepatic and peripheral blood HCV-specific CTL activity against the entire HCV polyprotein was assessed in 26 patients. CTL responses were assessed after effector lymphocytes were re-stimulated for 6 days in vitro using HCV-vaccinia virus-infected autologous cells expressing HCV antigens. Serum and hepatic viral loads were measured and immunohistochemistry for CD3 and CD8 was performed to localise and enumerate effector cells in liver. RESULTS: A positive CTL response was detected in 39/52 (75%) of assays conducted with intrahepatic mononuclear cells and 21/52 (40%) of peripheral blood assays (P<0.001). The presence of an intrahepatic CTL response was associated with low hepatic viral load (P=0.004). Hepatic lobular infiltration by CD8(+)T cells correlated weakly with serum alanine aminotransferase levels (r=0.42, P=0.04) and no relationship was demonstrated between CTL activity and histological evidence of liver damage. CONCLUSIONS: HCV-specific CTL activity is found more commonly in liver than in blood. An inverse relationship between CTL responses and viral load supports the hypothesis that HCV-specific CTL limit viral replication in patients with chronic HCV infection.