Development of a Propidium Monoazide-Based Viability Quantitative PCR Assay for Red Sea Bream Iridovirus Detection

Abstract

Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR

“…To address these points, a sample pre-treatment with DNA intercalants such as PMA and its derivatives (i.e., PMAxx) has been proposed to distinguish between infectious and non-infectious viruses. Recent studies have demonstrated the selective detection of various viruses using PMA, including norovirus, enterovirus, African swine fever viruses, and Red Sea Bream Iridovirus [ 20 , 21 , 22 , 23 ]. Recently, Hong et al [ 18 ] conducted in vitro experiments by inactivating or not inactivating the SARS-CoV-2 with UV light and using sodium dodecyl sulfate as a mild membrane destabilizer to increase the PMA permeability [ 18 ].…”

“…Therefore, only genomes from intact virions will be amplified and detected, while free nucleic acids or those from non-infectious viruses are not considered by a PCR. Initially, this test was primarily used to assess the integrity of bacterial pathogens [ 19 ]; however, it has since been applied to numerous viruses [ 16 , 17 , 18 , 20 , 21 , 22 , 23 ]. With the emergence of SARS-CoV-2, the question has arisen as to whether the use of PMA can aid in identifying viruses capable of infecting and producing new viral progeny from viral remnants or incomplete viruses and free viral genomes, particularly in samples that test positive by molecular assays but have low viral loads and stay positive for prolonged periods.…”

Impact of Red Sea Bream Iridovirus Infection on Rock Bream (Oplegnathus fasciatus) and Other Fish Species: A Study of Horizontal Transmission

“…RSIV has been reported to survive in seawater at 25 °C and 15 °C for approximately 7–10 days [ 42 ]. To investigate the dynamics of RSIV in aquaculture settings, disease progression can be monitored through qPCR or histopathological analysis of fish.…”

Recent Advances in Biochemistry and Molecular Biology of Infectious Diseases

“…Bound viral DNA inhibits qPCR amplification, providing a mechanism to effectively differentiate infectious and inactive viruses. Furthermore, this non-invasive method will aid in establishing a disease prediction system and epidemiological analysis using seawater [9].…”