SHIC-Funded Project Improves Next Generation Sequencing for Detection of Emerging Swine Viruses

A Swine Health Information Center-funded study aimed to develop a sensitive and cost-effective NGS method for detecting viral pathogens in swine samples. Led by Dr. Leonardo Cardia Caserta at Cornell University, the project developed an alternative method for enriching viral sequences in swine biological samples when conventional enrichment methods are not feasible. By removing host and bacterial genetic material from samples, there was improved detection sensitivity and percent genome coverage of several viruses. The novel sequencing method detected viruses from 35 genera across 250 swine respiratory samples. Development and validation of diagnostic methods support earlier detection of emerging viruses and strengthens disease surveillance in the swine industry. 

Read the industry summary of study #24-012 here. 

Emerging infectious diseases pose an ongoing risk to the US pork industry, making early detection critical for protecting herd health. While PCR tests are fast and accurate, they only detect known pathogens, leaving new or mutated viruses often undetected. NGS offers broader detection without prior pathogen knowledge, but its routine use is limited by low viral signal in samples and high costs. One of the primary factors contributing to low sensitivity of NGS is the abundance of host and other non-target sequences in clinical samples. 

Therefore, the objectives of the study herein included 1) to develop and optimize host and bacterial ribosomal RNA depletion procedures to increase sensitivity of NGS for viral disease surveillance in swine; and 2) to evaluate and establish cost-effective NGS procedures for emerging viral disease surveillance in swine.  

In this study, a procedure was developed that removes host and bacterial ribosomal RNA from samples before sequencing, allowing viral genetic material to be more easily detected. Researchers evaluated an rRNA depletion–based enrichment method using biotinylated probes and streptavidin-coated beads to improve viral detection in swine respiratory samples, particularly when enzymatic pre-treatments are not feasible due to the use of nucleic acid preservation media.  

A total of 250 samples were tested in this study, including samples known to be positive for porcine astrovirus 4 (PAstV4), porcine parainfluenza virus 1 (PPIV1), porcine hemagglutinating encephalomyelitis virus (PHEV), PCV2, PRRSV, and SVA, as well as samples known to be negative for these emerging pathogens. Application of the new method enabled detection of viruses from 35 genera in the clinical swine respiratory samples. Viruses with single-stranded DNA genomes represented the most viral reads, including parvoviruses and torque teno viruses, which may indicate a bias towards viruses with small DNA genomes. However, RNA viruses such as pestiviruses and pegiviruses were also detected and resulted in high genome coverage, demonstrating the ability of this method to detect RNA viruses. 

Targeted analysis of select viruses, including porcine parvovirus 4 (PPV4), porcine parvovirus 6 (PPV6), porcine circovirus 3 (PCV3), and atypical porcine pestiviruses (APPV), demonstrated improved genome coverage following rRNA depletion. An exogenous mengovirus control confirmed consistent viral recovery across samples. While enzymatic cocktail pretreatment achieved higher sensitivity for some RNA viruses such as PRRSV and SVA, the probe-based depletion method showed strong performance for DNA viruses. This method has potential as a cost-effective approach for unbiased viral surveillance and detection of emerging pathogens in swine. 

The method developed here allows NGS library preparation directly from the extracted nucleic acid, removing the need to re-extract the sample and decreasing hands-on processing time. Nucleic acid that has already been extracted for a PCR test, for example, can be used directly for metagenomic sequencing using the rRNA depletion method developed in this study. The approach improved the detection and genome coverage of several viruses and demonstrated strong performance for DNA viruses. With an estimated cost of $31.54 per sample, this novel and cost-effective method shows promise for improved detection of emerging viruses through NGS.  

Despite the successful sequencing of a wide range of RNA and DNA viral genera, further optimizations are still necessary to increase the detection sensitivity for RNA viruses such as PRRSV and SVA. Additionally, optimization to reduce the cost per sample is needed. Continued development of this protocol will lead to a streamlined workflow and a highly sensitive method for unbiased detection of emerging viruses in swine.

The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at [email protected] or Dr. Lisa Becton at [email protected].