F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters

Abstract

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated. This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.

F-Specific RNA Bacteriophages Model the Behavior of Human Noroviruses during Purification of Oysters: the Main Mechanism Is Probably Inactivation Rather than Release

“…Again, this difference could be explained by a higher growth rate for FRNAPH-I compared to FRNAPH-II, as previously reported (22). For example, for the same initial concentration of 0.5 PFU for both genogroups, Hartard et al (25) demonstrated that 57% of ICC-RT-qPCR detected FRNAPH-I against only 14% for FRNAPH-II. Because of the high rate of bacteriophage multiplication induced by this method, we recommend performing the analysis on four samples at a time to limit contamination risk.…”

“…When focusing only on purification conditions applied to oysters coming from polluted areas, many studies have shown not only the incidence of physicochemical conditions (i.e., water temperature, salinity, and dissolved oxygen) on shellfish physiology but also the effect of the initial microorganism load (i.e., natural or artificial contamination) (10,(35)(36)(37). For these reasons, we decided to work with naturally NoV-contaminated oysters that were also positive for FRNAPH, because of the high correlation previously demonstrated (25). The initial batch had been collected in winter and the high E. coli count observed (1,200 MPN/100 g of FIL) indicate a fecal contamination.…”

“…However, relaying is a less common practice than depuration, which may have potentially contributed to the relative absence of relay-associated outbreaks. Conversely, another study found no NoV genome reduction differences between relaying and depuration after 30 days; indeed, a similar and slight decrease in NoV genome levels was observed in both cases (25). However, because of the limited number of studies on this topic, there are discrepancies in the effectiveness of purification in NoV genome removal.…”

“…Among them, genogroups II (FRNAPH-II) and III (FRNAPH-III) are mainly associated with human fecal pollution (24), while genogroups I (FRNAPH-I) and IV (FRNAPH-IV) are mainly related to animal fecal pollution. A positive correlation has recently been demonstrated by Hartard et al between the genomes of FRNAPH-II and NoV in oysters during purification processes (22), showing that FRNAPH-II could be proposed as a potential indicator to evaluate NoV risk in shellfish (25). On the other hand, it has been demonstrated that FRNAPH are excreted by only a part of the population (26%) (26).…”

Metagenomic Analysis of Infectious F-Specific RNA Bacteriophage Strains in Wastewater Treatment and Disinfection Processes

“…The sizes, shape structures, and genomes of FRNAPHs are similar to those of noroviruses [1,2], which have caused numerous outbreaks of gastroenteritis in multiple countries [3]. Furthermore, FRNAPH behavior, abundance, and survival in the environment including during water treatment are also similar to those of human enteric viruses [1,2,[4][5][6]. Thus, they serve as potential indicators of water contamination and the fates of viruses in aquatic environments and wastewater treatment plants (WWTPs) [4][5][6].…”