8/12/2023 0 Comments Virus spike definition![]() ![]() The post-mortem analysis represents a pivotal tool in understanding the biological characteristics and the pathogenesis of SARS-CoV-2. The rapid spread and the limited knowledge of the virus require continuous updating by the experts improving particularly the investigation on post-mortem biomaterials. The RT-PCR assays currently available target E, N, S, and ORF1b (including RNA-dependent RNA polymerase-RdRp) genes. To date, the RT-PCR is the gold standard to identifying the SARS-CoV-2 infection according to the WHO recommendations. The SARS-CoV-2 diagnosis is currently based on the clinical manifestations associated with the detection of virus RNA through real-time reverse transcription polymerase chain reaction (RT-PCR), primarily in the nasopharyngeal and oropharyngeal swabs. SARS-CoV-2 shares with other SARS-CoV more than 90% amino acid identity of the structural proteins, excluding the S gene, which diverges. The S1 domain of SARS-CoV-2 showed only 40% of homology with other coronaviruses and great variability in amino acids resulting in a high affinity for binding to the human receptor angiotensin-converting enzyme 2 (ACE-2). The S protein is composed of two subunits, the S1 domain responsible for the receptor-binding and the S2 domain associated with the envelope. The N protein forms the helical capsid to accommodate the genome, the M and E are needed for the virus assembly, and the S protein mediates the host cell recognition and the entry of the virus. The replicase gene ORF1ab encodes a large polyprotein (pp1ab), which is proteolytically cleaved into 16 non-structural proteins (NSPs) that are involved in the transcription and replication of the virus. The genome of SARS-CoV-2 is larger compared to other RNA viruses and it is organized as follows, 5′ to 3′: two flanking untranslated regions (UTRs), a single long open reading frame (ORF1ab), a non-structural polyprotein, four structural proteins-spike (S), envelope (E), membrane (M), nucleocapsid (N)-and five accessory proteins-ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10. The viral structures and genome of SARS-CoV-2 showed a unique feature compared to all other coronaviruses. Phylogenetically, the SARS-CoV-2 genome is closely related to two bat coronaviruses, bat-SLCoVZC45 and bat-SL-CoVZXC21 (89–96.3% sequence homology), while it has less sequence similarity (79–82%) with SARS-CoV and MERS-CoV. Before the infection caused by SARS-CoV-2, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are the best-known examples of large-scale epidemic coronavirus-associated involvement in severe acute respiratory syndromes. SARS-CoV-2 belongs to the family of the coronaviruses (CoVs) that are enveloped, positive-sense, single-stranded RNA viruses. The World Health Organization (WHO) has officially declared the SARS-CoV-2 disease a pandemic with a public health emergency of international concern. In January 2020, a severe acute respiratory syndrome, caused by the Novel Coronavirus 2019 SARS-CoV-2, was firstly identified in China and quickly spread throughout the world. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR however, it is able to localize the virus in the cellular context since it preserves the morphology. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. ![]() All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. ![]() The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2 thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. ![]()
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