Detection and identification of coronaviruses in human tissues using electron microscopy.

The identification of viral particles within a tissue specimen requires specific knowledge of viral ultrastructure and replication, as well as a thorough familiarity with normal subcellular organelles. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored how challenging the task of identifying coronavirus by electron microscopy (EM) can be. Numerous articles have been published mischaracterizing common subcellular structures, including clathrin- or coatomer- coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles in SARS-CoV-2 positive patient tissue specimens. To counter these misinterpretations, we describe the morphological features of coronaviruses that should be used to differentiate coronavirus particles from subcellular structures. Further, as many of the misidentifications of coronavirus particles have stemmed from attempts to attribute tissue damage to direct infection by SARS-CoV-2, we review articles describing ultrastructural changes observed in specimens from SARS-CoV-2-infected individuals that do not necessarily provide EM evidence of direct viral infection. Ultrastructural changes have been observed in respiratory, cardiac, kidney, and intestinal tissues, highlighting the widespread effects that SARS-CoV-2 infection may have on the body, whether through direct viral infection or mediated by SARS-CoV-2 infection-induced inflammatory and immune processes. HIGHLIGHTS: The identification of coronavirus particles in SARS-CoV-2 positive tissues continues to be a challenging task. This review provides examples of coronavirus ultrastructure to aid in the differentiation of the virus from common cellular structures.

Detection of SARS-CoV-2 in Neonatal Autopsy Tissues and Placenta.

Severe coronavirus disease in neonates is rare. We analyzed clinical, laboratory, and autopsy findings from a neonate in the United States who was delivered at 25 weeks of gestation and died 4 days after birth; the mother had asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and preeclampsia. We observed severe diffuse alveolar damage and localized SARS-CoV-2 by immunohistochemistry, in situ hybridization, and electron microscopy of the lungs of the neonate. We localized SARS-CoV-2 RNA in neonatal heart and liver vascular endothelium by using in situ hybridization and detected SARS-CoV-2 RNA in neonatal and placental tissues by using reverse transcription PCR. Subgenomic reverse transcription PCR suggested viral replication in lung/airway, heart, and liver. These findings indicate that in utero SARS-CoV-2 transmission contributed to this neonatal death.

Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus.

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

Best practices for correctly identifying coronavirus by transmission electron microscopy.

This guidance provides clear, concise strategies for identifying coronaviruses by transmission electron microscopy of ultrathin sections of tissues or infected tissue cultures. These include a description of virus morphology as well as cell organelles that can resemble viruses. Biochemical testing and caveats are discussed. Numerous references provide information for documentation and further study.