Coronaviruses

Introduction. The cycle threshold (Ct) value in real-time PCR (RT-PCR) is where a target-specific amplification signal becomes detectable and can infer viral load, risk of transmission and recovery. Use of Ct values in routine practice is uncommon.

Gap Statement. There is a lack of routine use of Ct values when reporting RT-PCR results in routine practice.

Aim. To automatically insert Ct values and interpretive comments when reporting SARS-CoV-2 RT-PCR to improve patient management.

Methodology. Routine Ct values across three different RT-PCR platforms were reviewed for concordance at presentation and clearance in patients with COVID-19. An indicative threshold (IT) linked to viral clearance kinetics was defined at Ct30 to categorize Ct values as low and high, reflecting high and low viral loads respectively.

Results. The different gene targets of each platform showed high correlation and kappa score agreement (P<0.001). Average Ct values were automatically generated with values ≤Ct30 reported as positive and >Ct30 as reactive; interpretive comments were added to all reports. The new reporting algorithm impacted on: physician interpretation of SARS-CoV-2 results; patient management and transfer; staff surveillance; length of stay in quarantine; and redefinition of patient recovery.

Conclusion. Incorporation of Ct values into routine practice is possible across different RT-PCR platforms and adds useful information for patient management. The use of an IT with interpretive comments improves clinical interpretation and could be a model for reporting other respiratory infections. Withholding Ct values wastes useful clinical data and should be reviewed by the profession, accreditation bodies and regulators.

Given the importance of disinfecting showerheads from Legionella species and the lack of instructions as to how to successfully achieve this, the aim of this study was to examine the ability of domestic steam disinfection to successfully disinfect showerheads from Legionella species. Steam disinfection of Legionella pneumophila [n=3; L. pneumophila serogroup 2–15 (wildtype environmental water isolate); L. pneumophila serogroup 1 NCTC11192 (reference strain); L. pneumophila serogroup 1 (wildtype environmental water isolate)], L. erythra (wildtype environmental water isolate) and L. bozemanii CRM11368M (reference strain) were examined in this study. Steam disinfection employing a baby bottle steam disinfector device eradicated all Legionella organisms tested. Steam disinfection, when performed properly under the manufacturer’s instructions, offers a relatively inexpensive, simple, versatile and widely available technology for the elimination of Legionella species from contaminated showerheads. We therefore advocate the employment of such devices to regularly disinfect showerheads and shower tubing in hairdressing salons, barber shops and gyms, as a critical control in the elimination of these organisms from these sources, thereby enhancing customer/client/staff safety.

There have been 2040 laboratory-confirmed cases of Middle East respiratory syndrome coronavirus (MERS-CoV) in 27 countries, with a mortality rate of 34.9 %. There is no specific therapy. The current therapies have mainly been adapted from severe acute respiratory syndrome (SARS-CoV) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. The development of specific therapies and vaccines is therefore urgently required. We examine existing and potential therapies and vaccines from a molecular perspective. These include viral S protein targeting; inhibitors of host proteases, including TMPRSS2, cathepsin L and furin protease, and of viral M(pro) and the PL(pro) proteases; convalescent plasma; and vaccine candidates. The Medline database was searched using combinations and variations of terms, including ‘Middle East respiratory syndrome coronavirus’, ‘MERS-CoV’, ‘SARS’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘S protein’, ‘DPP4’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. There are many options for the development of MERS-CoV-specific therapies. Currently, MERS-CoV is not considered to have pandemic potential. However, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. Well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies.

Coronaviruses are enveloped RNA viruses that have evolved complex relationships with their host cells, and modulate their lipid composition, lipid synthesis and signalling. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes in viral infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to lipid rafts. Because cholesterol-rich microdomains appear to be a general feature of the entry mechanism of non-eneveloped viruses and of several coronaviruses, the purpose of this study was to analyse the contribution of lipids to the infectivity of canine coronavirus (CCoV). The CCoV life cycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-β-cyclodextrin (MβCD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from the cell membrane resulted in a dose-dependent reduction, but not abolishment, of virus infectivity, and at a concentration of 15 mM, the reduction in the infection rate was about 68 %. MβCD treatment was used to verify if cholesterol in the envelope was required for CCoV infection. This resulted in a dose-dependent inhibitory effect, and at a concentration of 9 mM MβCD, infectivity was reduced by about 73 %. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cell membranes, or interfering with lipid metabolism, may have strong antiviral potential. It will be interesting in the future to analyse the membrane microdomains in the CCoV envelope.

Like severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus (HCoV)-NL63 employs angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. SARS-CoV infection causes robust downregulation of cellular ACE2 expression levels and it has been suggested that the SARS-CoV effect on ACE2 is involved in the severity of disease. We investigated whether cellular ACE2 downregulation occurs at optimal replication conditions of HCoV-NL63 infection. The expression of the homologue of ACE2, the ACE protein not used as a receptor by HCoV-NL63, was measured as a control. A specific decrease for ACE2 protein level was observed when HCoV-NL63 was cultured at 34 °C. Culturing the virus at the suboptimal temperature of 37 °C resulted in low replication of the virus and the effect on ACE2 expression was lost. We conclude that the decline of ACE2 expression is dependent on the efficiency of HCoV-NL63 replication, and that HCoV-NL63 and SARS-CoV both affect cellular ACE2 expression during infection.

A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-α displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.

In order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5′ end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).

Several strains of the enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) have been shown to replicate in alveolar macrophages maintained in vitro. A distinct cytopathic effect was observed at a multiplicity of infection ≥0.1. Infected cells released infectious virus. The extent of both virus production and cell destruction was highly dependent upon the virus input. At low input, cell viability was affected only slightly, and a delayed and persistent virus production could be observed. TGEV infection of macrophages also led to a marked synthesis of type I interferon. Thus, the possibility that alveolar macrophages act as an extra-intestinal target for TGEV must be considered.

Evidence for the replication of human coronavirus strain 229E (HCV 229E) in macrophages is presented. Virus antigen was detected in macrophages by an immunofluorescent technique 24 h after infection and virus particles were observed in the cisternae of the endoplasmic reticulum by electron microscopy. Giant cells were observed by light and scanning electron microscopy, and large multinucleate cells were seen by thin-section electron microscopy, suggesting that HCV 229E can induce syncytial formation in cultured human macrophages. Furthermore, the production of infectious virus by macrophages was demonstrated by an infectious centre assay.