First Viruses Infecting the Marine Diatom Guinardia delicatula

Abstract

The marine diatom Guinardia delicatula is a cosmopolitan species that dominates seasonal blooms in the English Channel and the North Sea. Several eukaryotic parasites are known to induce the mortality of this species. Here, we report the isolation and characterization of the first viruses that infect G. delicatula. Viruses were isolated from the Western English Channel (SOMLIT-Astan station) during the late summer bloom decline of G. delicatula. A combination of laboratory approaches revealed that these lytic viruses (GdelRNAV) are small tailless particles of 35-38 nm in diameter that replicate in the host cytoplasm where both unordered particles and crystalline arrays are formed. GdelRNAV display a linear single-stranded RNA genome of ~9 kb, including two open reading frames encoding for replication and structural polyproteins. Phylogenetic relationships based on the RNA-dependent-RNA-polymerase gene marker showed that GdelRNAV are new members of the Bacillarnavirus, a monophyletic genus belonging to the order Picornavirales. GdelRNAV are specific to several strains of G. delicatula. They were rapidly and largely produced (<12 h, 9.34 × 10⁴ virions per host cell). Our analysis points out the host's variable viral susceptibilities during the early exponential growth phase. Interestingly, we consistently failed to isolate viruses during spring and early summer while G. delicatula developed important blooms. While our study suggests that viruses do contribute to the decline of G. delicatula's late summer bloom, they may not be the primary mortality agents during the remaining blooms at SOMLIT-Astan. Future studies should focus on the relative contribution of the viral and eukaryotic pathogens to the control of Guinardia's blooms to understand the fate of these prominent organisms in marine systems.

Keywords: Western English Channel; diatoms; genomics; host-virus dynamics; single-stranded RNA viruses.

Figure 1

Temporal dynamics of Guinardia delicatula (solid line) and all diatoms (dash line) at SOMLIT-Astan station (Western English Channel) during the September 2015–October 2016 period. All along this period a protocol designed for the isolation of viruses lytic to G. delicatula was applied to seawater samples collected every fortnight. The arrows point to sampling dates on which the virus strains GdelRNAV-01 to 04 were successfully isolated.

Figure 2

Aspect of healthy cultures of G. delicatula RCC3083 (A,C) and infected cultures by GdelRNAV-01 (B,D) that show disintegration of host cells. (A,B) Pictures of the flasks, (C,D) Light microscopy micrographs showing heathy cells with golden brown plastids (C) or cells totally degraded (D). All pictures were taken 14 days post-infection. Scale bars on pictures (C,D) 50 μm.

Figure 3

Ultrathin sections of Guinardia delicatula RCC3083 and negatively stained GdelRNAV-01 particles obtained by TEM. (A) Healthy control. (B–E) G. delicatula infected by GdelRNAV-01 at 72 hpi. (C) Crystalline arrays and dispersed viral particles accumulated in the host cytoplasm. (D) Higher magnification of panel C of GdelRNAV-01 in the host cytoplasm. (E) Negatively stained GdelRNAV-01 particles. Arrows: Crystalline arrays. F, frustule; G, Golgi apparatus; M, mitochondrion; N, nucleus, CH, chloroplast.

Figure 4

Infection kinetic of Guinardia delicatula RCC3083 by GdelRNAV-01. Abundances of diatom hosts in the control culture (black circles) and in infected cultures (open triangles) were obtained using optical microscopy. Viral titers (open hexagons) were estimated using the MPN method. Error bars were estimated based on counts obtained in triplicates of infected cultures. Gray rectangles represent the dark phases. Pictures obtained using transmitted-light and epifluorescence microscopy illustrate the morphology of G. delicatula cells in control and infected cultures at T0 and Tfinal. With epifluorescence microscopy the red natural fluorescence of chloroplasts and green fluorescence of PicoGreen stained nucleic acids are observed. At Tfinal, the green fluorescence is due to the presence of bacteria. Scale bars: 20 μm.

Figure 5

Nucleic acids type of GdelRNAV-01 after extraction. Extracts were treated with DNase treatment, with RNase treatment, or with S1 nuclease treatment.

Figure 6

Schematic genome organization of GdRNAV-01 (9,233 nt). 5′UTR: 5′ untranslated region (1,008 nt), 3′ UTR: 3′ untranslated region (367 nt). The yellow box indicates the replication polyprotein with Hel: Helicase and RdRp: RNA-dependent RNA polymerase. The gray box represents the capsid proteins (CP) with domains corresponding to the Rhv_like superfamily interspaced by the Dicistro_VP4, and the CRPV_capsid superfamily. The green box indicates the possible ORFan. P1 to P5: structural proteins. Segments corresponding to P1 to P5 are not scaled on the genome sequence.

Figure 7

Analysis of the structural proteins of GdelRNAV-01 using SDS-PAGE. Lane Marker: Novex sharp unstained protein standard marker (kDa). Lane GdelRNAV-01: Denatured proteins of purified GdelRNAV-01. P1 to P5 represent the proteins 1 to 5.

Figure 8

Phylogenetic rooted tree based on RdRp sequences of representative viruses from the Picornavirales order. Caliciviridae viruses were taken as outgroup. The star indicates the position of GdelRNAV-01 in the genus Bacillarnavirus. The Maximum Likelihood tree was generated using PhyML 3.0 with 1,000 replications and a LG + G + I + F substitution model according to the SMS analyses. Bootstraps values (%) >80 are shown. Scale bar indicates the number of substitutions per site. Virus abbreviations: ABPV, acute bee paralysis virus NC_002548.1; AiV, Aichi virus, AB010145; AglaRNAV, Asterionellopsis glacialis RNA virus NC_024489; AuRNAV, Aurantiochytrium single-stranded RNA virus), BAE47143; BoCV, Bovine enteric calicivirus, AJ011099; BBW, broad bean wilt virus 1 NC_005289.1; CsfrRNAV, Chaetoceros socialis f. radians RNA virus, AB469874; Csp03RNAV, Chaetoceros sp. number03 RNA virus, AB639040; CtenRNAV type-I, Chaetoceros tenuissimus RNA virus, AB375474; CtenRNAV type-II, AB971661; CtenRNAV_SS10V-39V, AB971662; CtenRNAV_SS10V-45V, AB971663; CRLV, cherry rasp leaf virus, NC_006271.1; CPSMV, cowpea severe mosaic virus, NC_003545; CrPV, cricket paralysis virus, NC_003924; DWV, deformed wing virus, NC_004830; HaRNAV, Heterosigma akashiwo RNA virus, NC_005281; HplV-81, Hubei picorna-like virus 81 strain CJLX25805, KX884540.1; HplV-82, Hubei picorna-like virus 82 KX883688.1; PV, human poliovirus 1 Mahoney, V01149; IFV, infectious flacherie virus NC_003781.1; NV, Norwalk virus, M87661; PYFV, Parsnip yellow fleck virus, D14066; RsRNAV, Rhizosolenia setigera RNA virus, AB243297; RTSV, rice tungro spherical virus, AAA66056; SBV, sacbrood virus, NC_002066; SDV, Satsuma dwarf virus RNA 1 NC_003785.2; SINV-2, Solenopsis invicta virus 2 EF428566.1; TRSV, tobacco ringspot virus RNA 1 NC_005097.1; ToTV, tomato torrado virus RNA 1 NC_009013.1; TrV, triatoma virus, NC_003783.