Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case

Abstract

Background: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection.

Objectives: We aimed to determine and characterise the etiological agent responsible for the poor outcome.

Methods: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection.

Findings: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR.

Main conclusions: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Fig. 1:. summarised timeline of the clinical evolution of patient 6439.

Fig. 2:. detection of viral coinfection of patient 6439. (A) Plaque assay of the nasopharyngeal swab sample from patient 6439 (6439_SW) showing the indicated sample dilutions in duplicate wells. (B) The two different plaque phenotypes observed in this sample are highlighted in the inset of the 1:30 dilution duplicates (dashed panel). Red arrowheads correspond to the larger and turbid plaques with undefined borders, whereas blue arrowheads indicate the smaller and clearer plaques with well-defined circular borders. Depth of coverage diagram across the reference genome for Herpes simplex virus 1 (HSV-1) (C) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (D) in kilobase (Kb) obtained through virome analysis. The basic structure of both genomes is indicated below the diagrams. SARS-CoV-2: non-structural coding region (red) indicated by ORF1a&b (the overlapping open reading frames 1a and 1b); and structural and accessory coding regions (blue and green) highlighting the S (spike) and N (nucleocapsid) genes. HSV-1: UL and US (blue) stand for Unique Long and Unique Short regions, respectively; and TR(L/S) and IR(L/S) (red) stand for Terminal and Internal - inverted - Repeats flanking the UL and the US regions, respectively.

Fig. 3:. characterisation of the two plaque phenotypes. Plaque reduction neutralisation test (PRNT) using double-negative, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), anti-Herpes simplex virus 1 (HSV-1) sera. Columns correspond to fixed Vero cell monolayers subjected to the following conditions from left to right: uninfected cells (cell control), 6439_CM - inoculated cells without serum (virus control), and cells inoculated with 6439_CM pre-incubated with the indicated dilutions of the corresponding serum.