Bradley, B. T. | Search bioRxiv

bioRxiv2022

Characterization of the cytopathic effect of monkeypox virus isolated from clinical specimens and differentiation from common viral exanthems

While the practice of viral culture has largely been replaced by nucleic acid amplification tests, circumstances still exist in which the availability of viral culture will allow for the diagnosis of infections not included in a providers differential diagnosis. Here, we examine the cytopathic effect (CPE) and clinical data associated with eighteen cases of monkeypox virus (MPXV) isolated from nineteen clinical samples submitted for viral culture. During the study period a total of 3,468 viral cultures were performed with herpes simplex virus most commonly isolated (646/3,468; 18.6%), followed by monkeypox virus (19/3,468; 0.6%) and varicella zoster virus (12/3,468; 0.4%). Most MPXV-positive samples were obtained from males (14/19) and taken from genital (7/19) or rectal lesions (5/19). Cycle threshold values of tested samples ranged from 15.3 to 29.0. Growth of MPXV in cell culture was rapid, yielding detectable CPE at a median of 2 days (range: 1-4) often with >50% of the monolayer affected in RMK, BGM, A549, and MRC-5 cell lines. As clinical features of MPXV, HSV, and VZV lesions may overlap, CPE patterns were comparted between viruses. HSV CPE developed in a similar time frame (median: 2 days, range: 1-7) but was more often negative in RMK cells relative to MPXV. VZV grew more slowly (median: 9 days, range: 5-11) and demonstrated CPE affecting [≤]25% of cell monolayers when positive. Viral culture remains an important tool for the detection of rare or emerging viral pathogens, particularly when high viral load specimens are easily obtained.

microbiology↗

bioRxiv2021

In vivo generation of BK and JC polyomavirus defective viral genomes in human urine samples associated with higher viral loads

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory ddPCR of urine samples taken from immunosuppressed individuals, we show clinical BKPyV and JCPyV strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were universally derived from type I subtype BKPyV. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology. IMPORTANCEDefective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens -- no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes with the majority affecting genes required for viral replication. Longitudinal analysis showed these DVGs can persist during an infection, but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests these parasitic sequences exist across the many classes of viruses capable of causing human disease.

microbiology↗