Molecular basis of pathogenesis of emerging viruses infecting aquatic animals

Lang Gui,V.Gregory Chinhar,Qiya Zhang

aCollege of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China

bDepartment of Microbiology,University of Mississippi Medical Center,2500 North State Street,Jackson,MS 39216,USA

cState Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China

1.Introduction

Aquatic animals comprise more than 60%of vertebrate species,including extant fish(32,900 species)and amphibians(7302 species)(The World Conservation Union,2014).The aquaculture resources from aquatic animals are regarded as important,highquality protein foods in human diets(Gui&Zhu,2012).As an alternative to beef,aquatic animals are a benefit not only to human health but also to the environment(Cressey,2009).However,viruses can cause devastating diseases in aquatic animals(Nakajima,Inouye,&Sorimachi,1998;Zhang&Gui,2012).For example,ranaviruses,herpesviruses,reoviruses,and rhabdoviruses have several unexpected consequences.These viral pathogens have different characteristics relative to those infecting terrestrial animals with regard to their morphology,genome,symptom,and pathogen-host interaction in aquatic animals(Zhang&Gui,2015).The viral diversity in aquatic animals is due to the diversity of viral hosts and the rapid and cross-species transmission of viruses in different populations.We have previously reported several aquatic animal viral genomes as well as the viral genes associated with.virus replication and assembly,including RGV(Lei,Ou,Zhu,&Zhang,2012),CaHV(Wang,Gui,Chen,&Zhang,2016),SMReV(Ke,He,Pei,&Zhang,2011)and SCRV(Tao,Zhou,Gui,&Zhang,2008).To date,sequencing and investigation of viral gene functions have fostered new insights into the molecular pathogenesis of aquatic animal viruses.Our current understanding of viral pathogens,and the roles of several viral genes in virus replication and virus-host interactions were exhibited.The schematic illustration in Fig.1 briefly describes a framework of symptoms of viral diseases.The molecular basis of virus-host interactions explain viral pathogenesis in aquatic animals.

2.Ranaviruses

The ranaviruses(family Iridoviridae)cause devastating disease and biodiversity decline among farmed and wild aquatic animals around the globe(Chinchar&Waltzek,2014;Jancovich et al.,2011;Stohr et al.,2015),e.g., fish,frogs,and giant salamanders(Fig.1).The features of ranaviruses include low host specificity and infectiousness.Typical symptoms cause by ranaviruses include skin erythema,eye exophthalmos,glass sphere turbidity,retinal hemorrhaging,limb necrosis,and systemic hemorrhaging,especially from the mouth(Chen et al.,2013;Schock,Bollinger,&Collins,2009;ScienceDaily,2014;Zhang,Li,&Gui,1999,2001).

High mortalities occur within days in frogs infected by Rana grylio virus(RGV)and Chinese giant salamanders infected by Andrias davidianus ranavirus(ADRV).

RGV 53R is a ranaviral envelope protein.RGV 53R is essential for viral assembly(Zhao et al.,2008).RGV 53R initially colocalizes with the endoplasmic reticulum(ER)at an early stage of infection,then the ER components are excluded from the virus factories at the stage of virus assembly(Fig.2A).RGV infection has been associated with apoptosis(Huang,Huang,Gui,&Zhang,2007)and dramatic alterations in mitochondrial dynamics,such as mitochondrial fragmentation and crista remodeling.RGV infection induces apoptosis through a mitochondrion-mediated caspase-dependent pathway of cell death(Fig.2B).

3.Herpesviruses

The fish herpesviruses(family Alloherpesviridae in the order Herpesvirales)can induce diseases ranging from latent to fatal infection and cause mass mortality.Recently,disease outbreaks with 100%mortality within one week was caused by Carassius carassius herpesvirus(CaHV)in crucian carp(Fang et al.,2016).The typical signs of this disease included severe necrosis and heavy bleeding of the gills(Fig.1).

G protein-coupled receptors(GPCRs)are seven-transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization.Study of the subcellular localization of CaHV GPCRs may provide valuable information about the precise interactions between herpesvirus and host,and also provide useful targets for antiviral agents in aquaculture.Recently,a series of variants with truncation/deletion/substitution mutations in the GPCR C-terminal of CaHV were constructed(Wang et al.,2016).Different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs.And Lysine-315(K-315)in C-terminal was a key region.When region K-315 or the intact C-terminal was retained,CaHV GPCR formed aggregates at the nuclear side,and colocalized with the Golgi apparatus(Fig.3).

4.Reoviruses

Infection by reoviruses(family Reoviridae)is associated with high mortality in aquatic animals,such as hemorrhagic disease of grass carp,turbot and largemouth bass caused by grass carp reovirus 109(GCRV-109),Scophthal musmaximus reovirus(SMReV)and Micropteru ssalmoides reovirus(MsReV),respectively.The symptoms include obvious hemorrhaging in the muscles of infected fish,eventually leading to death(Fig.1).Recently,the relationship between the genomic structure of reovirus and host fish in saline environments was revealed(Chen,Gao,&Zhang,2015).

NS80,the nonstructural protein from reoviruses,is involved in SMReV replication and assembly by recruiting viral components(Ke,He,&Zhang,2013).After invasion of host cells,NS80 forms a viral factory which then recruits viral proteins.Different regions of NS80 are required for protein interactions.The intercoil region of NS80 is important for self-aggregation,and it is also related to nuclear localization.Other regions of NS80 are required for associations with viral proteins and assembly of new virions that are released,and lead to host cell damage(Fig.4).

5.Rhabdoviruses

Fish rhabdoviruses(family Rhabdoviridae)are classified into three genera,Novirhabdovirus,Perhabdovirus and Vesiculovirus.The genomes of more than 100 fish rhabdovirus strains have been sequenced,and serious rhabdoviral pathogens have been isolated and characterized from diseased aquatic animals in China,such as mandarin fish (Siniper cachuatsi rhabdovirus,SCRV),turbot(Scophthal musmaximus rhabdovirus,SMRV), flounder(Paralichthys olivaceus rhabdovirus,PORV),and farmed rice field eels(Monopterus albus rhabdovirus,MoARV)(Ou,Zhu,Chen,&Zhang,2013;Zhu&Zhang,2014).Symptoms include anaemia,bulging eyes,ascites,and gangrenous ulcers(Fig.1)and sick fish gasp at the water surface,and their movements become agitated.Prior to death,abnormal clinical signs include loss of equilibrium,disorganized swimming, accumulation of skin mucus, skin hemorrhages,and excessive internal blood clotting.Accordingly,fish rhabdovirus infections frequently cause high mortality.

Fig.2.53R is essential for RGV assembly and mitochondrion-mediated apoptosis.The viral proteins:Rana grylio virus 53R(RGV-53R).(A)The role of 53R in assembly:The cell compartment collapses to give rise to precursors of the viral inner envelope,and these precursors are rapidly recruited inside the assembly sites.The capsids are then gradually assembled on one side of the inner envelope.As the assembled viral particles start to close,the viral genome is engulfed.Finally,the mature intracellular virions accumulate in the cytoplasm of infected cells within large paracrystalline arrays or are released by budding from the plasma membrane.(B)Mitochondrion-mediated apoptosis induced by RGV:Virus induced mitochondrial fragmentation,increased intracellular Ca2+,activated caspase-3 and caspase-9,dissipated the mitochondrial membrane potential(Δψm),and activated transcription factor AP-1 and nuclear factor NF-κB.Chromatin condensation and DNA fragmentation were also observed and finally,apoptosis led to death.

Fig.3.GPCR C-terminal mutants affect the subcellular localization of CaHV.Carassius carassius herpesvirus G-protein-coupled receptor(CaHV-GPCR)is 349 amino acids(aa),and the localization of GPCR was affected by the C-terminus(aa 315-349).Lysine-315(K)was the key region of the C-terminus.CaHV GPCR aggregated at the nuclear site,and CaHV GPCR colocalizated with Golgi apparatus is represented by yellow.The arrows indicate CaHV GPCR and associated proteins might intracellular transport to peripheral cytoplasm by Golgi apparatus.(For interpretation of the references to colour in this figure legend,the reader is referred to the Web version of this article.)

Fig.4.NS80's crucial role in SMReV assembly by recruiting viral components.Scophthal musmaximus reovirus NS80(SMReV-NS80)viral protein has an important role in SMReV assembly by recruiting viral components VP1,VP2,VP3,VP4,VP5,VP6,NS38,and RNA.In brief,aa 1-55 of NS80 and aa 20-39 of NS38 interact with each other and aa 56-85 of NS80 interacts with VP3,and aa 550-742 of NS80 associates with VP1,VP2,and VP4.The interaction of SMReV-NS80(NS80 regions required)ensures self-aggregation and selective recruitment of viral proteins to viral factories for SMReV assembly.

The genomes of rhabdoviruses generally contain five structural genes,namely the nucleocapsid protein(N)gene,phosphoprotein(P)gene,matrix protein(M)gene,glycoprotein(G)gene,and RNA-dependent RNA polymerase(L)gene.The G and N genes in DNA vaccine plasmids(G-vaccine and N-vaccine)against SCRV were constructed,and the antiviral immune response elicited in mandarin fish was determined(Chen,Lei,&Zhang,2012).A type I IFN antiviral immune response was rapidly triggered by the G-vaccine in fish.Vaccination with G-vaccine decreased histological lesions and suppressed viral replication in fish organs.The expression levels of IFN system genes in vaccinated fish were strongly upregulated after injection and the immune response was rapidly triggered and a significant antiviral defense was observed(Fig.5).

6.Conclusion

Despite the recent progress in understanding aquatic animal viruses,many critical questions remain unanswered.Including:(1)Why the emergence and resurgence of viruses occur frequently in aquatic animal?(2)How does the viruses cross aquatic animal barriers and be transferred between different aquatic animals?(3)How can we identificate viruses from other families that are emerging?(4)What is the molecular basis of viral cross-species transmission?(5)How can viral gene expression be prevented or inhibited to block the invasion and replication of aquatic animal viruses?Answering such questions through investigation will lead to important breakthroughs in the control and prevention of aquatic animal viral diseases.

Fig.5.Antiviral defense in fish induced by G protein DNA vaccine against SCRV.A plasmid containing the SCRV-G gene was injected in mandarin fish and the antiviral immune response was determined.The expression levels of type I IFN system genes,including the interferon regulation factor-7 gene,the myxovirus resistance gene,and the virus inhibitory protein gene were strongly up-regulated in mandarin fish after injection with SCRV-G gene.The results showed a protective effect against SCRV challenge with a relative percentage survival(RPS)of 77.5%with G-vaccine.

Acknowledgements

This work was partially supported by the following grants:the National Natural Science Foundation of China(31430091,31302214,31772890),the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA08030202),and the Project of State Key Laboratory of Freshwater Ecology and Biotechnology(2016FBZ01).

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