Molecular and biochemical studies of virus infections in two filamentous brown algae

The filamentous marine brown algae Ectocarpus siliculosus and Feldmannia simplex are infected by host specific DNA-viruses. Under Percoll isolation, Ectocarpus siliculosus-virus (EsV)-particles maintained their infective potential.The EsV has a circular genome of dsDNA with a size of 320 kb. A restriction map has been established. The gene of a major coat protein (gp1) was detected in a genomic library and partly sequenced. Using gpl- sequences for polymerase chain reaction (PCR) amplification analysis, EsV specific sequences could be detected in various symptom-free, clonal cultures of Ectocarpus. The PCR was also used to follow the passage of the virus genome during the meiosis of hosts. A monospecific antibody against recombinant gpl was used for immunostaining and infection experiments.The Feldmannia simplex-virus (FlexV-1) has a circular genome with a size of 220 kb and a 43% G+C content. FsV-DNA contains methylated bases. 5-methylcytosine (5 mC) makes up 12% of the total cytosines.     

PASSAGE OF A MARINE BROWN ALGAL DNA VIRUS FROM ECTOCARPUS FASCICULATUS (ECTOCARPALES,PHAEOPHYCEAE) TO MYRIOTRICHIA CLAVAEFORMIS (DICTYOSIPHONALES,PHAEOPHYCEAE): INFECTION SYMPTOMS AND RECOVERY1

A dsDNA virus (EfasV-1) isolated from Ectocarpus fasciculatus Harvey infected Myriotrichia clavaeformis Harvey, a species belonging to a different brown algal order. The virus did not complete its infection cycle in the foreign host but caused infertility due to malformed reproductive structures. After some time in culture, the host's reproductive capacity was sometimes restored with concomitant loss of at least part of the viral genome. This incidence of interordinal virus transfer is discussed in relation to possibilities for virus-mediated horizontal gene transfer in brown algae.     

Entry of the DNA virus, Ectocarpus fasciculatus virus type 1 (Phycodnaviridae), into host cell cytosol and nucleus

The process by which Ectocarpus fasciculatus virus type 1 (EfasV‐1) infects zoospores of its brown algal host was studied by electron microscopy. Upon virus attachment to the target cell, the integral membrane component of the viral capsid fuses with the host plasma membrane, and the 140‐nm viral DNA‐protein core enters the cytosol. Within 5 min after infection, particles resembling viral cores appeared in the nucleus. The entry mechanism of EfasV‐1 into the host nucleus remains enigmatic.     

A Virus Infection in the Marine Brown Alga Ectocarpus siliculosus (Phaeophyceae)

Laboratory cultures of Ectocarpus siliculosus originating from New Zealand showed a defect in gametangium formation. Nuclear divisions in gametangium initials are not followed by cell wall formation. In the resulting multinucleate cells nuclear DNA increases dramatically, and nuclear membranes disintegrate. Eventually, the entire structure is filled with hexagonal particles of approximately 130 nm diameter. These were isolated and shown by EM to consist of a dense core surrounded by a 3-layered shell. They are released into the culture medium when the host cells burst. Ectocarpus gametes from healthy cultures could be infected by these particles. The resulting partheno-sporophytes developed pathological symptoms, suggesting that the particles are viruses. The expression of the defect is temperature dependent. At 10°C all gametangia are abnormal, while between 15 and 20 °C defective and normal gametangia and gametes are formed on the same plant. Partheno-sporophytes developing from such gametes carry the viral particles expressed in deformed unilocular and plurilocular sporangia.     

Virulence determines beneficial trade‐offs in the response of virus‐infected plants to drought via induction of salicylic acid

It has been hypothesized that plants can get beneficial trade‐offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus‐induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought‐tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus‐infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid‐independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non‐infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.     

Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi‐persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR‐amplified viral DNA fragment, RYNV‐specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus‐like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV‐infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field‐grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV‐infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes.     

Worldwide occurrence of virus-infections in filamentous marine brown algae

Virus infections were detected inEctocarpus siliculosus andEctocarpus fasciculatus on the coasts of Ireland, California, Peru, southern South America, Australia and New Zealand; in threeFeldmannia species on the coasts of Ireland, continental Chile and Archipelago Juan Fernandez (Chile); and inLeptonematella from Antarctica. Natural populations on the Irish coast contained 3% infected plants inE. fasciculatus, and less than 1% inFeldmannia simplex. On the Californian coast, 15 to 25% ofEctocarpus isolates were infected. Virus symptoms were absent inE. siliculosus from Peru, but appeared after meiosis in laboratory cultures. The virus particles inE. fasciculatus are identical in size and capsid structure to those reported forE. siliculosus, while the virus inF. simplex is smaller and has a different envelope. Our findings suggest that virus infections are a common and worldwide phenomenon in filamentous brown algae.     

Examination of host genome for the presence of integrated fragments of Solenopsis invicta virus 1

A series of oligonucleotide primer pairs covering the entire genome of Solenopsis invicta virus 1 (SINV-1) were used to probe the genome of its host, S. invicta, for integrated fragments of the viral genome. All of the oligonucleotide primer sets yielded amplicons of anticipated size from cDNA created from an RNA template from SINV-1. However, no corresponding amplification was observed when genomic DNA (from 32 colonies of S. invicta) was used as template for the PCR amplifications. Host DNA integrity was verified by amplification of an ant-specific gene, SiGSTS1. The representation of fire ant colonies included both social forms, monogyne and polygyne, and those infected and uninfected with SINV-1. Furthermore, no amplification was observed from genomic DNA from ant samples collected from Argentina or the US. Thus, it appears that SINV-1 genome integration, or a portion therein, has not likely occurred within the S. invicta host genome.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Host-virus interactions in marine brown algae

Ectocarpus-like marine brown algae are frequently parasitized by polyhedric DNA viruses. Infected hosts have been studied in unialgal and axenic cultures, and the present state of knowledge is summarized in regard to stage-specific virus expression, discharge and survival time of virus particles, infection mechanism, association with host's nuclear genome, passage of the virus genome through mitosis and meiosis of the host, suppression of symptoms and spontaneous recovery of infected plants, host specificity and intergeneric transmission, vitality of infected plants, pandemic occurrence of virus infections, molecular data on Ectocarpus and Feldmannia viruses, and algal DNA-viruses as potential vectors for gene transfer. A scheme for the nomenclature of brown algal viruses is proposed.     

Generation of a non‐transmissive Borna disease virus vector lacking both matrix and glycoprotein genes

Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non‐segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non‐cytopathically in the cell nucleus, leading to establishment of long‐lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G ) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non‐transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M ) and G genes in the genome, is reported. rBoDV‐ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG‐GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG‐GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG‐GFP was also demonstrated. These findings indicate that the rBoDV ΔMG‐based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses.

     

Acquisition and Transmission of two Begomoviruses by the B and a non-B Biotype of Bemisia tabaci from Zhejiang, China

Acquisition and transmission was studied of Tomato yellow leaf curl China virus (TYLCCNV) and Tobacco curly shoot virus (TbCSV) by the B and a non‐B biotype (China‐ZHJ‐1) of Bemisia tabaci from Zhejiang, China. The frequency of TYLCCNV and TbCSV detection by PCR in whitefly adults increased with increasing length of feeding on virus‐infected plants. The virus DNA was detected by PCR in 40% of the B biotype adults tested after a period of 30 min access to infected plants and in all adults after a 12‐h period of access. All ZHJ‐1 adults acquired TYLCCNV and TbCSV after a 48‐h period of access to the virus‐infected plants. Viruliferous B and ZHJ‐1 adults retained TYLCCNV DNA for their entire life when placed on healthy cotton plants. Viruliferous ZHJ‐1 adults retained TbCSV DNA for their entire life when placed on healthy cotton plants but the B biotype adults did not. Transmission of TYLCCNV was achieved with one B or ZHJ‐1 adult per plant, and the probability of transmission reached 100% when the number of adults was increased to 10 per plant. The efficiency for TYLCCNV transmission to healthy plants by adults of both B and ZHJ‐1 was much higher than that for TbCSV.     

ENTRY OF A CHLORELLA-VIRUS INTO ITS HOST CELL1

The endosymbiotic Chlorella sp. (Chlorophyceae) of Paramecium bursaria (Ciliata) can be infected by a double-stranded DNA-containing virus (Chlorella-virus) that has a phagelike entry mechanism. Electron micrographs show that soon after attachment of the virus to the algal cell wall, a hole is formed through which the viral DNA enters the alga. Biochemical studies on a European Chlorella-virus system suggest that digestion of the algal cell wall is caused by glycolytic enzymes, one of which was identified as a β-d -glucosidase. Enzymes are bound to the virus capsid and are activated only after or by the attachment of the virus to its cognate alga or to preparations of the algal cell wall. Common features of viral cell wall-digesting enzymes and algal autolysins are discussed.     

Signatures of selection in natural populations adapted to chronic pollution

Background

Ectocarpus siliculosus virus-1 (EsV-1) is a lysogenic dsDNA virus belonging to the super family of nucleocytoplasmic large DNA viruses (NCLDV) that infect Ectocarpus siliculosus, a marine filamentous brown alga. Previous studies indicated that the viral genome is integrated into the host DNA. In order to find the integration sites of the viral genome, a genomic library from EsV-1-infected algae was screened using labelled EsV-1 DNA. Several fragments were isolated and some of them were sequenced and analyzed in detail.

Results

Analysis revealed that the algal genome is split by a copy of viral sequences that have a high identity to EsV-1 DNA sequences. These fragments are interspersed with DNA repeats, pseudogenes and genes coding for products involved in DNA replication, integration and transposition. Some of these gene products are not encoded by EsV-1 but are present in the genome of other members of the NCLDV family. Further analysis suggests that the Ectocarpus algal genome contains traces of the integration of a large dsDNA viral genome; this genome could be the ancestor of the extant NCLDV genomes. Furthermore, several lines of evidence indicate that the EsV-1 genome might have originated in these viral DNA pieces, implying the existence of a complex integration and recombination system. A protein similar to a new class of tyrosine recombinases might be a key enzyme of this system.

Conclusion

Our results support the hypothesis that some dsDNA viruses are monophyletic and evolved principally through genome reduction. Moreover, we hypothesize that phaeoviruses have probably developed an original replication system.     

Ovipositional discrimination by Microplitis rufiventris females between healthy and granulosis virus-infected Spodoptera littoralis larvae

Ovipositional choice tests by Microplitis rufiventris females (Hym., Braconidae) between granulosis virus‐infected (GVI) and non‐infected (NI) Spodoptera littoralis larvae (Lep., Noctuidae), were assessed using discriminatory methods for re‐isolating the NI and virus‐infected hosts after removing the female parasitoid. When M. rufiventris females were given a choice between NI and GVI S. littoralis hosts, the adult females exhibited marked preference (P < 0.01) for the NI (i.e. higher quality) hosts. In this case, M. rufiventris females and S. littoralis GV (SlGV) did not significantly compete for the same type of host larvae and are, generally, compatible. However, when the choice was given between two low qualities of S. littoralis hosts, i.e. virus‐free previously parasitized hosts and viral‐infected hosts a significant preference (P < 0.01) of the parasitoid females for the GVI larvae was observed. In this case, the parasitoid would be at a disadvantage when competing with GV for the same host. However, the parasitoid could be used as an additional tool for the dissemination of biocontrol viruses within different pest populations, i.e. hosts other than S. littoralis. Importantly, the results showed different strategies of parasitoid female in egg‐laying management. When M. rufiventris female was given a choice between healthy and SlGVI hosts, the female deposited more eggs than when she was given a choice between two low qualities of host larvae. The results of the study may have implications in pest management strategies using M. rufiventris and SlGV against S. littoralis larvae.     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.