Virome capture sequencing finds viruses missed by PCR in flu-like illness cases

Conventional diagnostic tests often come back negative in patients with influenza-like illness, leaving the cause of their respiratory infection undetermined. A new cross-sectional study reports that a technique called Virome Capture Sequencing for Vertebrate Viruses (VCS) was able to detect viruses in nearly half of samples that had already tested negative by multiplex PCR, including respiratory pathogens consistent with the patients’ symptoms.

The research, published in Virology Journal, was led by Sandra Rajme-López and colleagues, with senior authorship including Guillermo M. Ruiz-Palacios and W. Ian Lipkin. The team retrospectively analyzed 440 nasal swab samples from adults enrolled in the ILI002 cohort in Mexico between 2010 and 2014. All of these samples had tested negative for respiratory pathogens using the Respifinder multiplex PCR panel.

Unlike targeted PCR assays, which look for a predefined list of pathogens, VCS is an agnostic approach designed to detect both known and unexpected vertebrate viruses in clinical samples. In the study, total nucleic acids — both DNA and RNA — were extracted from the swabs and sequenced using the VCS method. Bioinformatic analysis was carried out with the Rapid Identification of Microbes (RIM) pipeline, and detected viruses were classified according to their pathogenicity and disease association.

The results showed that 208 of the 440 samples, or 48 percent, yielded viral hits, with a total of 314 viruses identified. A respiratory virus consistent with the patients’ clinical symptoms was found in 17 percent of the samples. Rhinoviruses were the most frequent respiratory viruses identified, underscoring how commonly these pathogens evade standard PCR-based testing.

One of the more notable findings was the detection of influenza C virus (ICV) in five cases, which the authors describe as revealing previously unrecognized circulation of the virus. Phylogenetic analyses of the rhinoviruses showed high diversity among the strains, while genotyping of the ICV strains confirmed lineage conservation.

Beyond respiratory pathogens, the sequencing also revealed a high frequency of non-respiratory viruses, particularly Epstein-Barr virus (EBV) and papillomaviruses. The authors note that this broader view of the viral content of respiratory samples may help advance insights into host-pathogen and microbiota interactions.

According to the study’s conclusions, VCS is able to detect common respiratory pathogens missed by conventional diagnostic methods and can also identify unusual viruses of epidemiological relevance, such as influenza C virus. The authors argue that this capability underscores the technique’s potential value for pandemic preparedness, because an agnostic sequencing approach can flag viruses that targeted panels would never query.

The study was registered as trial NCT01418287 and is published as open access research. As a retrospective cross-sectional analysis of samples from a single cohort collected between 2010 and 2014, the findings describe what viruses were detectable in these PCR-negative samples rather than testing the clinical performance of VCS in routine care.

Subject of Research: Virome capture sequencing to detect viruses in PCR-negative influenza-like illness cases

Article Title: Virome capture sequencing for enhanced detection of respiratory and other viruses in PCR-negative influenza-like illness cases : a cross-sectional study

Article References: Rajme-López, S., P Collins, J., Ramos-Cervantes, P., Ortega-Villa, A. M., Lu, X.-J., de Blas, P. C. G., Shaw-Saliba, K., Lim, C., Wickiser, J. K., Lipkin, W. I., & Ruiz-Palacios, G. M. (2026). Virome capture sequencing for enhanced detection of respiratory and other viruses in PCR-negative influenza-like illness cases : a cross-sectional study. Virology Journal. https://doi.org/10.1186/s12985-026-03311-1

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03311-1

Keywords: virome capture sequencing, influenza-like illness, multiplex PCR, rhinovirus, influenza C virus, Epstein-Barr virus, papillomavirus, respiratory viruses, Mexico, diagnostics, pandemic preparedness, metagenomics