Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel human coronavirus, emerged in late 2019 and rapidly evolved into a pandemic around the world. The coronavirus disease (COVID-19) pandemic has dramatically changed the epidemiology and seasonality of other traditional respiratory viruses, e.g., influenza, respiratory syncytial virus, enterovirus, etc. These traditional respiratory viruses have transmission mode and clinical symptoms similar to SARS-CoV-2 but may differ in clinical outcomes and management. Co-infection between SARS-CoV-2 and one or more traditional respiratory viruses have been reported in the literature but have shown mixed evidence in clinical outcomes. With SARS-CoV-2 evolving into mild Omicron variants, it is believed that SARS-CoV-2 co-circulates with other respiratory viruses, which in turn affect the epidemiology and clinical course of respiratory viral infections. In response to these changes, multiplex molecular tests for SARS-CoV-2 and one or more traditional respiratory viruses are attracting more attention in the field and have been developed into a variety of testing modalities. In this review, we describe the seasonality (i.e., in the Northern Hemisphere), epidemiology, and clinical significance of traditional respiratory viruses and their co-infection with SARS-CoV-2 in the post-COVID era. Furthermore, we review commonly used multiplex molecular tests and their applications for the detection of respiratory viruses and their co-infections. Altogether, this review not only sheds light on the epidemiology and clinical significance of respiratory viral infections and co-infections in the post-COVID era, and but also provides insights into the laboratory-based diagnoses of respiratory viral infections using multiplex molecular testing.

The innate immune response, particularly the interferon-mediated pathway, serves as the first line of defense against viral infections. During virus infection, viral pathogen-associated molecular patterns (PAMPs) are recognized by host pattern recognition receptors (PRRs), triggering downstream signaling pathways. This leads to the activation of transcription factors like IRF3, IRF7, and NF-κB, which translocate to the nucleus and induce the production of type I interferons (IFN-α and IFN-β). Once secreted, type I interferons bind to their receptors (IFNARs) on the surfaces of infected and neighboring cells, activating the JAK-STAT pathway. This results in the formation of the ISGF3 complex (composed of STAT1, STAT2, and IRF9), which translocates to the nucleus and drives the expression of interferon-stimulated genes (ISGs). Some ISGs exert antiviral effects by directly or indirectly blocking infection and replication. Among these ISGs, ISG15 plays a crucial role in the ISGylation process, a ubiquitin-like modification that tags viral and host proteins, regulating immune responses and inhibiting viral replication. However, viruses have evolved counteractive strategies to evade ISG15-mediated immunity and ISGylation. This review first outlines the PAMP-PRR-induced pathways leading to the production of cytokines and ISGs, followed by a summary of ISGylation’s role in antiviral defense and viral evasion mechanisms targeting ISG15 and ISGYlation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel human coronavirus, emerged in late 2019 and rapidly evolved into a pandemic around the world. The COVID-19 pandemic has dramatically changed the epidemiology and seasonality of other traditional res-piratory viruses e.g., influenza, respiratory syncytial virus, enterovirus, etc. These traditional respiratory viruses share similarity in transmission mode and clinical symptoms with SARS-CoV-2 but may differ in clinical outcomes and management. Co-infection between SARS-CoV-2 and one or more traditional respiratory viruses have been reported in the literature but showed mixed evidence in clinical outcomes. With SARS-CoV-2 evolving into mild Omicron variants, it is believed that SARS-CoV-2 co-circulates with other respiratory viruses, which in return affect the epidemiology and clinical course of respiratory viral infections. In response to these changes, multiplex molecular testings for SARS-CoV-2 and one or more traditional respiratory viruses are attracting more attention in the field and have been developed into a variety of testing modalities. In this review, we describe the epidemiology and clinical significance of traditional respiratory viruses and their co-infection with SARS-CoV-2 in the post-COVID era. Further, we review the principle of multiplex molecular testings and their application to the detection of respiratory viruses and their co-infections. Altogether, this review not only sheds light on epidemiology and clinical significance of respiratory viral infections and co-infections in the post-COVID era and but only provides insights on the laboratory-based diagnosis of respiratory viral infections using multiplex molecular testing.