Accurate and reliable surface-enhanced Raman spectroscopy assay for early detection of SARS-CoV-2 RNA with exceptional sensitivity.

This research proposes a highly sensitive and simple surface-enhanced Raman spectroscopy (SERS) assay for the detection of SARS-CoV-2 RNA using suitably designed probes specific for RdRp and N viral genes attached to a Raman marker. The sensitivity of the assay was optimized through precise adjustments to the conditions of immobilization and hybridization processes of the target RNA, including modifications to factors such as time and temperature. The assay achieved a remarkable sensitivity down to 58.39 copies/mL, comparable to or lower than the sensitivities reported for commercial fluorescent polymerase chain reaction (PCR) based methods. It has good selectivity in discriminating SARS-CoV-2 RNA against other respiratory viruses, respiratory syncytial virus (RSV), and influenza A virus. The reliability of the assay was validated by testing 24 clinical samples, including 12 positive samples with varying cycle threshold (Ct) values and 12 negative samples previously tested using real-time PCR. The assay consistently predicted true results that were in line with the PCR results for all samples. Furthermore, the assay demonstrated a notable limit of detection (LOD) of Ct (38 for RdRp gene and 37.5 for N-gene), indicating its capability to detect low concentrations of the target analyte and potentially facilitating early detection of the pathogen.

Crimean-Congo hemorrhagic fever virus in ticks collected from imported camels in Egypt.

Crimean-Congo hemorrhagic fever (CCHF) is one of the utmost broadly distributed tick-borne viruses, with an infection resulting in a fatality rate of up to 30%. During this study period, 25,000 hard adult ticks of Hyalomma species were collected from freshly slaughtered imported camels to determine the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) and genetic lineage of the virus. Ticks were pooled and analyzed for the existence of CCHFV using nested RT- PCR and real-time reverse transcription PCR; the genome was detected in 18 (1.44%) tick pools. Partial genome sequences reveal an adjacent relationship with strains from South Africa to Namibia, Nigeria, Sudan, Senegal, and Mauritania, corresponding to the Africa I and III genotypes. This study indicates the presence of CCHFV in Egypt and illustrates the potential for tick-borne dissemination of the virus. Further studies focused on not only tick samples, but also human samples are epidemiologically valuable to obtain exact data in the region.

Stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus causing a highly fatal respiratory disease in humans. Confirmation of MERS-CoV infection and molecular study on the virus may require transportation of samples to specialized laboratories. While freezing at -80 °C is the gold standard method for RNA preservation, maintaining the integrity of viral RNA during transport will require additional precautions and, as a result, increase transport costs. We aimed at testing the stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature for 16 weeks. Respiratory samples spiked with stock culture of MERS-CoV were extracted and loaded on QIAamp Viral RNA Mini Kit spin columns and preserved at room temperature. Amount of viral RNA was evaluated periodically by real-time quantitative reverse-transcription polymerase chain reaction. Minimal changes in cycle threshold values over the study period were noted, suggesting stability of viral RNA by this preservation method.

Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is highly critical, because the SARS-CoV receptor influenced pathology, and its localization in the deep respiratory tract is thought to have restricted the transmissibility of SARS. Our data show that hCoV-EMC does not need the SARS-CoV receptor to infect human cells. Moreover, the virus is capable of infecting human, pig, and bat cells. This is remarkable, as human CoVs normally cannot replicate in bat cells as a consequence of host adaptation. Our results implicate that the new virus might use a receptor that is conserved between bats, pigs and humans suggesting a low barrier against cross-host transmission.

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.

A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.

Alkhurma haemorrhagic fever--a viral haemorrhagic disease unique to the Arabian Peninsula.

The emergence and re-emergence of human and animal pathogens on a global scale continues unabated. One such pathogen is the arbovirus that causes Alkhurma haemorrhagic fever, which emerged in the Kingdom of Saudi Arabia in the mid 1990s. It has since re-emerged in other regions of the country and threatens to widen its area of endemicity beyond the peninsula. Human and animal movements, especially those associated with the annual mass gathering event of Hajj (pilgrimage) may facilitate introduction into other continental masses, where it must be differentiated from dengue and other similar arboviral haemorrhagic fevers. In addition to dengue and Kadam viruses, which are known to be endemic in Saudi Arabia, it is thought that other flaviviruses exist in the region, though undetected. Collectively, these viruses present diagnostic challenges that may confound the recognition of clinical cases of Alkhurma haemorrhagic fever. The Saudi Ministry of Health is making concerted efforts to expand the evidence base in order to enhance the diagnostic and preventive protocols used to address the challenge of Alkhurma haemorrhagic fever.