Investigation of Cyprinid Herpesvirus 3 (CyHV-3) Disease Periods and Factors Influencing CyHV-3 Transmission in A Low Stocking Density Infection Trial

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) and important pathogen of aquaculture and wild populations of common carp worldwide. Understanding the relative contributions of direct and indirect transmission of CyHV-3 as well as the factors that drive CyHV-3 transmission can clarify the importance of environmental disease vectors and is valuable for informing disease modeling efforts. To study the mechanisms and factors driving CyHV-3 transmission we conducted infection trials that determined the kinetics of KHVD and the contributions of direct and indirect forms of CyHV-3 transmission, as well as the contributions of contact rate, viral load, pathogenicity and contact type. The incubation period of KHVD was 5.88 + 1.75 days and the symptomatic period was 5.31 + 0.87 days. Direct transmission was determined to be the primary mechanism of CyHV-3 transmission (OR = 25.08, 95%CI = 10.73–99.99, p = 4.29 × 10−18) and transmission primarily occurred during the incubation period of KHVD. Direct transmission decreased in the symptomatic period of disease. Transmissibility of CyHV-3 and indirect transmission increased during the symptomatic period of disease, correlating with increased viral loads. Additionally, potential virulence-transmission tradeoffs and disease avoidance behaviors relevant to CyHV-3 transmission were identified.

Inhibitory properties of crude microalgal extracts on the in vitro replication of cyprinid herpesvirus 3

Microalgae are possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study. An inhibition of the in vitro replication of CyHV-3 could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis crude extracts. At still non-cytotoxic concentrations, viral DNA replication was reduced by over 3 orders of magnitude each compared to the untreated replication controls, while the virus titers were even below the limit of detection (reduction of 4 orders of magnitude). When pre-incubating both cells and virus with C. reinhardtii and H. pluvialis extracts before inoculation, the reduction of viral DNA was even stronger (> 4 orders of magnitude) and no infectious viral particles were detected. Thus, the results of this study indicate that microalgae and cyanobacteria are a promising source of natural bioactive substances against CyHV-3. However, further studies regarding the isolation and identification of the active components of the extracts are needed.

Inhibitory properties of microalgal extracts on the in vitro replication of cyprinid herpesvirus 3

Microalgae often stand out for their high biodiversity as well as their associated large number of potent bioactives. Therefore, they are interesting candidates as possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study for inhibitory effects on viral replication. An inhibition of the in vitro replication of CyHV-3 in common carp brain cells could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis extracts. At still non-cytotoxic concentrations viral DNA replication was reduced by over 3 orders of magnitude (> 99.9 %) each compared to the untreated replication controls, while the virus titers were at or even below the limit of detection. When pre-incubating cells and virus with C. reinhardtii and especially H. pluvialis extracts before inoculation, the reduction of viral DNA and virus titer was even stronger. Based on these results, an intervention in the initial replication steps like viral adsorption or membrane fusion is assumed. Moreover, a protection mechanism preventing the production of viral proteins and the assembly of mature virions is also possible. All in all, the results show that microalgae are a very promising source of natural antiviral substances against CyHV-3.

Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia

Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to 90% of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of 36 RNA libraries representing 10 species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified 18 vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further 27 that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fishes sampled here, suggesting that there is little virus transmission from common carp to native fish species. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.Author SummaryThe ongoing invasion of the common carp in the Murray-Darling Basin, Australia, has wreaked havoc on native freshwater ecosystems. This has stimulated research into the possible biological control of invasive carp through the deliberate release of the virus cyprinid herpesvirus 3 (CyHV-3). However, little is known on the diversity of viruses that naturally circulate in wild freshwater fauna, whether these viruses are transmitted between invasive and native species, nor if CyHV-3 or other cyprinid herpesviruses are already present in the basin. To address these fundamental questions we employed meta-transcriptomic next-generation sequencing to characterise the total assemblage of viruses (i.e. the viromes) in three invasive and seven native fish species cohabiting at 10 sites across 2,200 km of the river system. From this analysis we identified 18 vertebrate-associated viruses across 14 viral families, yet a marked lack of virus transmission between invasive and native species. Importantly, no CyHV-3 was detected. This study shows that freshwater fish harbour a high diversity and abundance of viruses, that viruses have likely been associated with fish for millennia, and that there is likely little direct virus transmission between introduced and native species.