Search

Currently guidelines for disinfection of water with free chlorine, while primarily developed for potable water, are often used for virus disinfection of nitrified recycled water of >1 NTU (Nephelometric Turbidity Unit). More information is needed on the disinfection efficacy of free chlorine for viruses in waters of varying turbidity and pH due to significant reuse of treated wastewater of varying quality. In this study, disinfection efficacy in nitrified/denitrified activated sludge treated wastewater was investigated for coxsackievirus B5 (CB5), an enterovirus known to be highly resistant to free chlorine. The required chlorine contact times (CT) values (mg.min/L) for inactivation of CB5 were established in treated wastewater at 10 °C and of varying turbidity (0.2, 2, 5 and 20 NTU) and pH (7, 8 and 9). CTs were calculated to achieve 1 to 4 log10 inactivation. Robust data is presented in support of the chlorine CT values required to inactivate a chlorine-resistant virus in a range of turbidities and pHs in treated wastewaters. The testing method used a conservative approach and the data presented have been used to develop the free chlorine virus inactivation guildelines for recycled water in Victoria and South Australia, Australia.

Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

Background: Extraction of viral nucleic acids from serum samples is widely used in diagnostic pathology tests. However, the heterogeneous nature of non-serum samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. Objectives: Six different methods were compared for optimal extraction of viral DNA or RNA from four types of non-serum specimens. Study design: The DNA viruses used were herpes simplex virus and cytomegalovirus. The RNA viruses were poliovirus, rotavirus and small round structured virus. The specimens used were from respiratory, genital, faecal and peripheral blood mononuclear cell samples. The extracted nucleic acids were amplified by PCR and detected in an enzyme immunoassay using digoxygenin-labelled amplicons. Results and conclusions: For extraction of viral DNA, the phenol-chloroform method yielded the highest amount of DNA as judged by endpoint titration. The three methods compared for extraction of viral RNA used guanidine isothiocyanate and the QiaRNA kit was shown to yield the highest amount of viral RNA.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein., Author Summary Viperin is a virally induced host protein that has been previously shown to have antiviral activity against a variety of viruses. Here we have demonstrated that viperin is also anti-viral against the medically significant arbovirus, dengue virus. Viperin was able to inhibit dengue virus at the level of viral replication, and cell lines unable to produce normal levels of viperin grew the virus to higher titres. These anti-dengue effects of viperin were mediated by amino acid residues in its C-terminus, and did not require structural domains of the N-terminal region as has been previously shown by us and others for the related virus, hepatitis C virus. Viperin was also demonstrated to co-localise and interact with the dengue capsid protein on the surface of lipid droplets, as well as with the NS3 protein and viral RNA. Viperin's association with NS3 was further demonstrated to be involved in its anti-dengue activities. The anti-viral activities of viperin presented in this manuscript show both similarities and contrasts with other described anti-viral mechanisms for the protein and highlight the diverse nature of this unique anti-viral host protein.

Tumor necrosis factor alpha (TNF-alpha) is believed to play a significant role in the pathogenesis of dengue virus (DV) infection, with elevated levels of TNF-alpha in the sera of DV-infected patients paralleling the severity of disease and TNF-alpha release being coincident with the peak of DV production from infected monocyte-derived macrophages (MDM) in vitro. Since macrophages are a primary cell target in vivo for DV infection, we investigated the potential antiviral role of TNF-alpha in regulating DV replication in MDM. While pretreatment of MDM with TNF-alpha had a minor inhibitory effect, addition of TNF-alpha to MDM with established DV infection had no effect on DV replication as measured by DV RNA levels or progeny virus production. Blocking endogenous TNF-alpha using short interfering RNA or inhibitory TNF-alpha antibodies also had no effect on infectious DV production or viral RNA synthesis. Together, these results demonstrate that DV replication in MDM is not affected by TNF-alpha. Additionally, normal cellular TNF-alpha signaling, measured by quantitation of TNF-alpha-induced stimulation of transcription from an NF-kappaB-responsive reporter plasmid or NF-kappaB protein nuclear translocation, was blocked in DV-infected MDM and Huh7 cells. Thus, DV replication in MDM is not affected by TNF-alpha, and infected cells do not respond normally to TNF-alpha stimulation. It is therefore unlikely that the increased production of TNF-alpha seen in DV infection directly effects DV clearance by reducing DV replication, and the ability of DV to alter TNF-alpha responsiveness highlights another example of viral subversion of cellular functions.

Summary Aims To ascertain whether isolates of Neisseria meningitidis in South Australia (SA) have become less susceptible to antimicrobial agents. The patients studied were children and adults in SA with either meningococcal bacteraemia or meningitis or both. Methods The susceptibility of meningococci to 11 antimicrobial agents, including sulphonamides, penicillin and rifampicin, was tested by agar dilution, and in the case of six of the drugs, by E test also. Results Resistance to folate antagonists emerged in 1979 and became very common. Resistance peaked in 1995 at 76% of strains. Relative insusceptibility to penicillin was first encountered amongst strains isolated in 1985, and, while the incidence of such strains increased slightly, the overall incidence was low at 10 (5.2%) of 190 strains tested. Meningococci relatively insusceptible to rifampicin were encountered as early at 1971 and did not become more common. The incidence of such strains at 26 (13.7%) of 189 strains tested was higher than that for penicillin. For the 11-year period 1989-1999 of ≥84 strains tested all were susceptible to ceftriaxone, chloramphenicol and ciprofloxacin; 98% were susceptible to azithromycin and 97% were susceptible to minocycline. Shifts in MIC values for these drugs were not detected. Conclusions Resistance was common to sulphonamides and co-trimoxazole, however ≥95% meningococci tested were susceptible to drugs commonly used in the treatment of meningococcal disease, including penicillin and ceftriaxone. Relative insusceptibility to rifampicin was more common but did not increase during the 29-year period. For all drugs tested, except rifampicin, there was good agreement between agar dilution and E test results.