Non-enveloped RNA Research Articles

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

Stability of enveloped and nonenveloped viruses in hydrolyzed gelatin liquid formulation

BackgroundThe thermal stability of viruses in gelatin liquid formulations for medical research and application is poorly understood and this study aimed to examine the thermal stability of 4 enveloped and nonenveloped DNA and RNA viruses in hydrolyzed gelatin liquid formulations.MethodsBovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus, reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period.ResultsThe BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV (log10 reduction value) at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin (60,000 and 160,000) with an LRV above 1. Based on the gelatin type, BHV in alkaline-treated hydrolyzed gelatin samples were unexpectantly more stable than in acid-treated hydrolyzed gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks.ConclusionThe results demonstrated that 5% of 4000 MW hydrolyzed gelatin formulation can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.

Rhinovirus and Cell Death.

Rhinoviruses (RVs) are the etiological agents of upper respiratory tract infections, particularly the common cold. Infections in the lower respiratory tract is shown to cause severe disease and exacerbations in asthma and COPD patients. Viruses being obligate parasites, hijack host cell pathways such as programmed cell death to suppress host antiviral responses and prolong viral replication and propagation. RVs are non-enveloped positive sense RNA viruses with a lifecycle fully contained within the cytoplasm. Despite decades of study, the details of how RVs exit the infected cell are still unclear. There are some diverse studies that suggest a possible role for programmed cell death. In this review, we aimed to consolidate current literature on the impact of RVs on cell death to inform future research on the topic. We searched peer reviewed English language literature in the past 21 years for studies on the interaction with and modulation of cell death pathways by RVs, placing it in the context of the broader knowledge of these interconnected pathways from other systems. Our review strongly suggests a role for necroptosis and/or autophagy in RV release, with the caveat that all the literature is based on RV-A and RV-B strains, with no studies to date examining the interaction of RV-C strains with cell death pathways.

A customized program for the identification of conserved protein sequence motifs.

We searched for viral protein sequences that could be important for tissue tropism. To achieve this goal, human pathogenic viruses were classified according to the tissue they infect (e.g., pulmonary), irrespective of whether they were enveloped or non-enveloped RNA or DNA viruses. Next, we developed an amino acid sequence alignment program and identified the conserved amino acid motif, VAIVLGG, in alphaviruses. The VAIVLGG sequence is located on the structural capsid protein of the chikungunya virus, a mosquito-borne arthrogenic member of the alphaviruses. Capsid protein translocation onto the host cell membrane is a required step for virion budding. Our identified VAIVLGG consensus sequence might potentially be used for developing a pan-vaccine effective against alphaviruses.

Rotavirus-associated seizures and reversible corpus callosum lesion.

Rotavirus is a non-enveloped double-stranded RNA virus that causes severe gastroenteritis in children, but complications are rarely reported. Some reports have shown that rotavirus can induce diverse complications of the central nervous system, such as seizures, encephalopathy with a reversible splenial lesion, encephalitis, cerebral white matter abnormalities, and cerebellitis. Here, we present a 2-year-old patient with seizures, who had an isolated splenial lesion in the corpus callosum on neuroimaging, and the rotavirus antigen detected in faeces.

Canine caliciviruses of four serotypes from military and research dogs recovered in 1963\u22121978 belong to two phylogenetic clades in the Vesivirus genus

BackgroundVesiviruses (family Caliciviridae) had been shown capable of invading a variety of host species, raising concern of their zoonotic potential. Since the 1980’s, several canine caliciviruses (CaCV) isolates have been reported and are phylogenetically related to the vesiviruses with features distinct from both Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) species in phylogeny, serology and cell culture specificities. Etiological studies of canine diseases in dogs used for military services and laboratory studies were conducted in 1963–1978 at the Walter Reed Army Institute of Research. Multiple known and unknown viral pathogens including caliciviruses were recovered.MethodsFour unidentified isolates were recovered in Walter Reed Canine Cells (WRCC) from respiratory, fecal and penile specimens. Physicochemical tests, electron microscopy, viral cultivation in human and animal cells, antibody neutralization assays, and recently the genome sequencing were used to characterize the isolates. Sera from these dogs and their cohorts were tested with the isolates to determine origin and prevalence of the infections.ResultsThe viral isolates were small non-enveloped spherical RNA virions, 27 to 42 nm in diameter with cup-like structures, indicating they are caliciviruses. They propagated in WRCC and MDCK cells, not in either other canine cells or human and other animal cells. Each isolate is antigenically distinct and react with dog sera in respective cohorts. The genomes have nucleotide identities ranging from 70.3% to 90.7% and encode the non-structural polyprotein (1810 amino acids), major capsid protein (691 amino acids) and minor structural protein (134 amino acids). They belong to two different phylogenetic clades in Vesivirus genus with close relation with canine calicivirus (CaCV).ConclusionsThese CaCV isolates have restricted cell tropism, antigenic diversity and genetic variation. Further investigation will shed light on antigenic relation to other vesiviruses, and its pathogenicity for dogs and potential infectivity to other animals. Together with the previously reported CaCV strains provides significant evidence to support the formation of a new CaCV species in the Vesivirus genus.

Activation of CXCL-8 Transcription by Hepatitis E Virus ORF-1 via AP-1.

Hepatitis E virus (HEV) is a small nonenveloped single-stranded positive-sense RNA virus and is one of the major causes for acute hepatitis worldwide. CXCL-8 is a small multifunctional proinflammatory chemokine. It was reported recently that HEV infection significantly upregulates CXCL-8 gene expression. In this study, we investigated the mechanism of HEV-induced CXCL-8 transcriptional activation. Using CXCL-8 promoter reporters of different lengths ranging from −1400 to −173, we showed that −173 promoter has the highest promoter activity in the presence of HEV genomic RNA, indicating that the −173 promoter contains sequences responsible for CXCL-8 activation by HEV. Ectopic expression of the ORF-1 protein can upregulate the −173 CXCL-8 promoter activity. In contrast, expression of the ORF-2 protein suppresses the CXCL-8 promoter activity and expression of the ORF-3 protein has no effect on the CXCL-8 promoter activity. We further showed that AP-1 is required for CXCL-8 activation because neither HEV genomic RNA nor the ORF-1 protein can upregulate the −173 CXCL-8 promoter in the absence of the AP-1 binding sequence. Taken together, our results showed that HEV and HEV ORF-1 protein activate the CXCL-8 promoter via AP-1. This novel function of HEV ORF-1 protein should contribute to our understanding of HEV-host interactions and HEV-associated pathogenesis.

Antiviral activity of a composition of Gentiana lutea L., Primula veris L., Sambucus nigra L., Rumex spec. andVerbena officinalis L. (Sinupret®) against viruses causing respiratory infections

Sinupret ® , an aqueous-ethanolic extract from five plants, Gentiana lutea L., Primula veris L., Sambucus nigra L., Rumex spec. and Verbena officinalis L., is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about basic mechanisms of its potential antiviral activity. Therefore experiments have been performed for the detection of its antiviral activity against a broad panel of human pathogenic enveloped and non-enveloped RNA and DNA viruses causing infections of the upper respiratory tract: influenza A virus, parainfluenza virus, human rhinovirus B, coxsackievirus, adenovirus C, and respiratory syncytial virus. Determination of virus production was performed after treatment of the infected cells with non-toxic concentrations of the commercially available product using plaque-reduction assays, analyses of cytopathogenic effects and ELISAs for viral proteins. Antiviral activity of Sinupret ® could be detected independent of the type of the viruses in RNA as well as in DNA virus infected cell cultures and also against coated and uncoated viruses. A very strong inhibitory activity was obvious against adenovirus and respiratory syncytial virus infections. These results demonstrate that Sinupret ® showed a broad antiviral activity which basic mechanisms are still unclear. This has to be elucidated in further studies.

In vitro evaluation of the antiviral effects of the homeopathic preparation Gripp-Heel on selected respiratory virusesThis article is one of a selection of papers published in this special issue (part 2 of 2) on the Safety and Efficacy of Natural Health Products.

Gripp-Heel is a homeopathic preparation frequently used in the treatment of respiratory viral infections such as various types of influenza and the common cold. The antiviral activity of Gripp-Heel was studied in vitro on human pathogenic enveloped and nonenveloped RNA and DNA viruses. Before the antiviral assays, in vitro cytotoxicity of Gripp-Heel was determined with cells used for the infection experiments (HeLa, HEp-2, MDCK, BGM) as well as with mitogen-stimulated peripheral blood mononuclear leukocytes. A concentration of 0.5 of the commercially available product slightly reduced cell viability and proliferative capacity, and experiments on antiviral activity were determined starting with a dilution of 0.2 of the commercially available product. The antiviral activity was determined against a broad panel of enveloped and nonenveloped DNA and RNA viruses with plaque reduction assay, cytopathogenic assays, virus titrations, analysis of the viral proteins in virus-specific enzyme immunoassays, and haemagglutination tests. Control substances were acyclovir (10 microg/mL), ribavirin (6 microg/mL), and amantadine hydrochloride (5 microg/mL), depending on the virus type. Gripp-Heel demonstrated dose-dependent in vitro activity (significant reductions of infectivity by 20% to 40%) against Human herpesvirus 1, Human adenovirus C serotype 5, Influenza A virus, Human respiratory syncytial virus, Human parainfluenza virus 3, Human rhinovirus B serotype 14, and Human coxsackievirus serotype A9. The mechanisms of this antiviral activity are still unclear, but type I interferon induction might be a possible explanation. Further research on this homeopathic preparation seems warranted.

Molecular biology of astroviruses: selected highlights.

Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5-6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates that viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length cDNA clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORF1a. These studies demonstrate that the full-length ORF1a product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

Viral induced demyelination.

Viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell‐cell interactions and mechanisms of myelin destruction. They illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral‐induced tissue damage. These studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (CNS). Although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (MS). This notion is supported by the persistent view that a viral infection acquired during adolescence might initiate MS after a long period of quiescence.Demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non‐enveloped RNA and DNA viruses (Table 1). The mechanisms that ultimately result in the loss of CNS myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. Although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. This suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus‐specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. Virus‐induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own T cell compartment. Alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive T cells, i.e. molecular mimicry. Although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms.

Hepatitis E: an overview and recent advances in clinical and laboratory research.

Hepatitis E virus (HEV) is a non-enveloped RNA (7.5 kb) virus that is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in southeast and central Asia, the Middle East, parts of Africa and Mexico. Hepatitis E virus infection spreads by the faecal-oral route (usually through contaminated water) and presents after an incubation period of 8-10 weeks with a clinical illness resembling other forms of acute viral hepatitis. Clinical attack rates are the highest among young adults. Asymptomatic and anicteric infections are known to occur. Chronic HEV infection is not observed. Although the mortality rate is usually low (0.07-0.6%), the illness may be particularly severe among pregnant women, with mortality rates reaching as high as 25%. Recent isolation of a swine virus resembling human HEV has opened the possibility of zoonotic HEV infection. Studies of pathogenetic events in humans and experimental animals reveal that viral excretion begins approximately 1 week prior to the onset of illness and persists for nearly 2 weks; viraemia can be detected during the late phase of the incubation period. Immunoglobulin M antibody to HEV (anti-HEV) appears early during clinical illness but disappears rapidly over a few months. Immunoglobulin G anti-HEV appears a few days later and persists for at least a few years. There is no specific treatment available for hepatitis E virus infection. Ensuring a clean drinking water supply remains the best preventive strategy. Recombinant vaccines are being developed that may be particularly useful for travellers to disease-endemic areas and for pregnant women.

In vitro Studies of the Antiviral Range of Cicloxolone sodium and Identification of Cell Lines Tolerant to the Drug

To identify suitable host cells permissive for virus growth but resistant to the triterpenoid compound cicloxolone sodium (CCX), 18 cell lines were studied for tolerance to treatment for 48 h with increasing drug concentrations. IC50 values and replication indices of the IC50, at 300 μm CCX and in the absence of drug, were determined. Eight cell lines proved resistant, six sensitive and four intermediate in sensitivity to CCX. Four of the resistant cell lines were then selected as appropriate hosts for investigating the antiviral effect of CCX. Infectious yield dose–response experiments were carried out with viruses from eight different families comprising enveloped and non-enveloped DNA and RNA viruses. The RNA viruses included some with genomes that were segmented or non-segmented, positive-stranded, negative-stranded or double-stranded. No consistent relationship between sensitivity to CCX and any of these characteristics was found. Three classes of response were obtained: class 1 [VSV (Indiana), Influenza A, EHV-1 and BHV-1] showed a consistent, progressive CCX dose-dependent 100→100000-fold reduction in infectious virus yield; class 2 (Bunyamwera and Germiston, Polio-1, Reovirus-3 and Adenovirus-5) exhibited initial sensitivity (10–100-fold) but increasing the dose had minimal further effect on yield, with plateau values reached between 100 and 200 μm CCX; class 3 (SFV) appeared to be unaffected by CCX treatment. Plaque reduction assays revealed that HCMV and VZV are also highly sensitive to CCX inhibition.