WO2022175356A1 - Control of green algae blooms - Google Patents

Abstract

The present invention relates to ribonucleic acid molecules and pro-polypeptides of defined sequences, and their use as active agents for the biological control of green algae blooms, in particular blooms of green algae belonging to the genus Ulva.

Description

CONTROL OF GREEN ALGAE BLOOMS

FIELD OF INVENTION

The present invention relates to new ribonucleic acid molecules, new pro-polypeptides and new polypeptides, that may be of use in the biological control of green algae blooms, in particular blooms of algae from the genus Ulva.

BACKGROUND OF INVENTION

Contrarily to microalgae, for which economic interest grows each year, macroalgae remain a hazard, in particular to the sea environment and to human’s and animal’s health. Indeed, macroalgae blooms damage marine ecosystems, have a negative impact on local tourism and may result in animal and human deaths. This is notably the case with Ulva lactuca blooms.

Ulva lactuca is a macroalga that belongs to the phylum Chlorophyta, that was first described by Linnaeus in the Baltic Sea in the 18^th century. Ulva lactuca alga is made of a bilayer cell structure, and its thallus has generally a flat bladelike appearance. It is able to grow both with a holdfast such as rocks or free floating. Ulva lactuca algae have the capacity to reproduce with two methods, one being sexual and the other being from fragmentation of the thallus, which is rarely observed in macroalgae. These two methods provide a capacity to rapidly proliferate by covering the water surface, hereby decreasing the biodiversity for other algae species. Ulva lactuca is a polymorphic species regarding the degree of water salinity or symbiosis with bacteria.

Ulva lactuca invades principally beaches and its biodegradation can produce toxic acidic vapors (mainly FhS) that induce death of animals (a horse was reported dead in 2009 on the French Brittany coasts, located west, due to Ulva lactuca biodegradation) and possibly humans.

The first Ulva lactuca bloom to be described was at Belfast (North of Ireland) at the end of the 19^th century. Ulva lactuca blooms were well studied in the Laguna of Venice from the 1930’s with an unexplained decrease observed after the 1990’s. Since the 1980’s, Ulva lactuca blooms have been observed worldwide, from Galicia (Spain) to the Tokyo Bay (Japan), including the coasts from the American continent and Australia. However, the largest events in the world to date remain the green tides observed in the Yellow Sea for ten consecutive years from 2007, which covered 10 % of its surface. In Europe, Brittany north coasts have the biggest Ulva lactuca blooms. It is nowadays acknowledged that Ulva lactuca blooms are mainly the consequences of human activities, noticeably because of increasing amounts of traces of nitrogen and phosphorus in seawaters. In addition, the green tides observed in seas surrounding Belfast and Venice were correlated with increasing rej ection of human wastes.

To date, collection of green algae from seawater or the coastline, e.g, the beach, is the sole solution to cope with Ulva lactuca blooms.

Therefore, there is a need to provide a mean to control and/or eradicate blooms of algae of the genus Ulva, in particular of the species Ulva lactuca, in the seawater or in the land of a coastline contaminated with Ulva.

There is also a need to control Ulva blooms in a safe manner, in particular without emission of toxic acidic vapors, such as, e.g., H2S vapors.

SUMMARY A first aspect of the invention relates to an isolated ribonucleic acid molecule having at least 75% sequence identity with SEQ ID NO: 1, over the entire length.

In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 2, over the entire length, preferably encoding a first pro-polypeptide (pro-polypeptide PI). In certain embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 3, over the entire length, preferably encoding a second pro-polypeptide (pro-polypeptide P2). In some embodiments, the sequence of the ribonucleic acid molecule consists of SEQ ID

NO: 1

Another aspect of the invention relates to an isolated pro-polypeptide PI having at least 75% amino acid identity with SEQ ID NO: 4, over the entire length. In certain embodiments, pro-polypeptide PI is susceptible to be cleaved into two or more viral-like polypeptide(s) selected in the group consisting of a helicase, a 3C or 3C-like protease, and a RNA-dependent RNA polymerase.

One further aspect of the invention relates to an isolated pro-polypeptide P2 having at least 75% amino acid identity with SEQ ID NO: 5, over the entire length. In some embodiments, pro-polypeptide P2 is susceptible to be cleaved into two or more viral capsid-like polypeptide(s).

The invention also pertains to a green alga cell comprising the ribonucleic acid molecule, and/or expressing pro-polypeptide PI, and/or expressing pro-polypeptide P2, as defined herein. In certain embodiments, pro-polypeptide PI is encoded by a ribonucleic acid molecule, and/or pro-polypeptide P2 is encoded by a ribonucleic acid molecule according to the instant invention.

In some embodiments, the green alga cell belongs to the genus Ulva.

A further aspect of the invention relates to an algicide composition comprising at least one ribonucleic acid molecule, and/or at least one pro-polypeptide PI, and/or at least one pro-polypeptide P2 according to the instant invention.

In another aspect, the invention relates to a method for the biological control of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule, and/or a pro-polypeptide PI, and/or a pro-polypeptide P2, and/or an algicide composition according to the instant invention. Another aspect of the invention relates to a method for preventing a bloom of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule, and/or a pro-polypeptide PI, and/or a pro-polypeptide P2, and/or an algicide composition according to the instant invention.

In certain embodiments, the biological control is to be performed in a marine environment.

DEFINITIONS In the present invention, the following terms have the following meanings:

“About” preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

“At least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or more.

“Isolated”, when referred to a subject matter, such as, e.g, a ribonucleic acid, a pro polypeptide or a polypeptide, as defined herein, is intended to mean that said subject matter is no longer within its original and/or natural environment. In some embodiments, the terms “isolated” and “purified” are intended to be synonyms. - “Ribonucleic acid” or “RNA” refers to any poly rib onucl eoti de, which may be unmodified or modified RNA. “Ribonucleic acid” includes, without limitation single- and double- stranded RNA, and RNA that is a mixture of single- and double-stranded regions, positive and negative RNAs that may be single-stranded or double-stranded or a mixture of single- and double-stranded regions. In addition, “Ribonucleic acid” also includes RNAs containing one or more modified bases as well as RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to RNA in the state of the art; thus, “ribonucleic acid” embraces chemically, enzymatically or metabolically modified forms of polyribonucleotides as typically found in nature, as well as the chemical forms of RNA characteristic of microorganisms and eukaryote cells, or from synthetic origin.

Bloom” refers to a rapid and excessive growth of a population. By extension, “algae bloom” refers to a rapid and excessive growth of algae in a given marine environment.

In practice, green algae blooms may be accountable for a “green tide”, which refers to the green coloration of the seawater due the presence of an excessive concentration of green algae in a given perimeter. As used herein, an “algae bloom” is considered as being a pollution matter, because polluted waters, in particular seawaters, and coastline areas, in particular shores and beaches, may become life-threatening to both animal and human, due to the toxic vapors, in particular ThS vapors, that are emitted upon the degradation of the algae.

“Marine environment” refers to the ecosystem from the seawater, including the open sea (or deep sea), the seashore, the estuaries, the coastline. In practice, the coastline encompasses any land or ground surface in direct contact with the sea, e.g, rocks, beaches.

“Control”, “biological control” and “controlling” refer to both the steps, including prophylactic or preventative step, undertaken to prevent or slow down (lessen) a specific deleterious phenomenon, in particular an algae bloom. The environments in need of these steps include those already experiencing said specific deleterious phenomenon, in particular an algae bloom, as well as those prone to experience the specific deleterious phenomenon or those in which the specific deleterious phenomenon is to be prevented. The specific deleterious phenomenon is successfully “controlled” if, after receiving an efficient amount of the ribonucleic acid and/or one or more pro-polypeptide(s) or polypeptide(s) according to the present invention, the environment shows observable and/or measurable reduction in or absence of one or more of the parameters associated with said specific deleterious phenomenon; better quality of the environment. The above parameters for assessing successful control and improvement in the environment are readily measurable by routine procedures familiar to a skilled in the art. In one embodiment, the specific deleterious phenomenon is macroalgae blooms, in particular Ulva lactuca blooms.

“Preventing” refers to keeping from happening, and/or lowering the chance of the occurrence of, at least one parameter of a specific deleterious phenomenon. - “Promoting death” refers to the ability to kill a target. By extension “promoting death of the algae” is intended to refer to the killing or the degradation of the algae. In practice, dead algae are no longer capable of growing, spreading and promote a green tide. In one embodiment, the death of the algae may be accompanied by, or be the consequence of, a whitening, or bleaching, of the tissues of the algae. In practice, the whitening or bleaching of algae’s tissues may be visibly observed by a naked eye.

“Identity”, when used in a relationship between the sequences of two or more nucleic acid sequences or of two or more pro-polypeptides or polypeptides, refers to the degree of sequences relatedness between nucleic acid sequences pro-polypeptides or polypeptides (respectively), as determined by the number of matches between strings of two or more nucleotides or of two or more amino acid residues, respectively.

“Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related nucleic acid sequences or pro-polypeptides or polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence

Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the analyzed sequences. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. N CB/NLM/NIH Bethesda, Md. 20894; Altschul et al, supra). The well-known Smith Waterman algorithm may also be used to determine identity. In one embodiment, the term identity is measured over the entire length of the sequence to which it refers. - “Polypeptide” refers to a linear polymer of at least 50 amino acids linked together by peptide bonds. In some embodiments, a polypeptide refers to a structural polypeptide, i.e., a polypeptide involved in a 2D and/or 3D substructure (assembly of subunits). In some embodiments, the polypeptide refers to a functional polypeptide, i.e., a polypeptide having enzymatic properties. “Pro-polypeptide”, as used herein, refers to a “precursor polypeptide” encoded by one open reading frame (ORF), and susceptible to be cleaved into two or more polypeptides. The term “pro-polypeptide” as used herein can be used interchangeably with the term “polyprotein”.

“Protein” refers to a functional entity formed of one or more peptides or polypeptides, and optionally of non-polypeptides cofactors. In some embodiments, a protein refers to a structural protein, i.e., a protein involved in a 2D and/or 3D substructure (assembly of subunits). In some embodiments, the protein refers to a functional protein, i.e., a protein having enzymatic properties.

As used herein, the expressions “Ulva alga” and “alga of the genus Ulva” are meant to refer to the same subject matter and may substitute to one another. DETAILED DESCRIPTION

The inventors observed that major Ulva lactuca blooms, as those observed in the Yellow Sea or in Brittany, do not happen in Mediterranean Sea, despite the presence of Ulva lactuca, the absence of significant tides (water stagnation) and the presence of abundant sources of nitrogen and phosphorus. The inventors surprisingly observed that Mediterranean seawater samples promote Ulva lactuca death by a bleaching process (whitening of the tissues) and identified a new ribonucleic acid molecule in samples wherein Ulva lactuca bleached. More precisely, the inventors provide herein experimental data showing that the seawater comprise a RNA genome-based microorganism that promotes the death of Ulva lactuca, and hence the control of Ulva lactuca blooms, and that this RNA genome shares low identity with known viral genomes.

A first aspect of the invention relates to an isolated ribonucleic acid molecule having at least 75% sequence identity with SEQ ID NO: 1, over the entire length. As used herein, the term “at least 75% sequence identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence identity.

In practice, the level of identity of 2 ribonucleic acid sequences may be performed by using any one of the known algorithms available from the state of the art. Illustratively, the ribonucleic acid identity percentage may be determined using the CLUSTAL W software (version 1.83) the parameters being set as follows: for slow/accurate alignments: (1) Gap Open Penalty: 15; (2) Gap Extension Penalty: 6.66; (3) Weight matrix: IUB; for fast/approximate alignments: (4) K-tuple (word) size: 2; (5) Gap Penalty: 5; (6) No. of top diagonals: 5; (7) Window size: 4; (8) Scoring Method: PERCENT.

In some embodiments, the isolated ribonucleic acid molecule according to the invention has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 1, over the entire length. In certain embodiments, the isolated ribonucleic acid molecule is represented by a ribonucleic acid sequence consisting of SEQ ID NO: 1. In practice, ribonucleic acid molecule consisting of sequence SEQ ID NO: 1 is a 8,518 bases RNA molecule.

In some embodiments, the ribonucleic acid molecule according to the invention is produced by methods known in the art. In some embodiments, the method for producing the ribonucleic acid molecule of the invention comprises the steps of (i) cultivating a single stranded positive RNA virus whose genomic RNA comprises of consists of the sequence of SEQ ID NO: 1, (ii) extracting the viral genomic RNA comprising of consisting of SEQ ID NO: 1, and optionally (iii) purifying the extracted RNA.

In some embodiments, the method is performed by infecting a culture of algae of the genus Ulva, preferably Ulva lactuca. In one embodiment, the culture is performed by cloning in a bioreactor. In another embodiment, the culture is performed in a culture basin comprising at least 1,000 litter of seawater.

In some embodiments, the isolated ribonucleic acid molecule according to the invention is single stranded or double stranded. In some embodiments, the isolated ribonucleic acid molecule according to the invention is single stranded.

In some embodiments, the isolated ribonucleic acid molecule according to the invention is a positive or negative sense RNA molecule. In some embodiments, the isolated ribonucleic acid molecule according to the invention is a positive sense RNA molecule.

In some embodiments, the isolated ribonucleic acid molecule according to the invention is a single stranded, positive sense RNA molecule.

In some embodiments, the isolated ribonucleic acid molecule according to the invention is comprised in, or consists of, the genomic RNA of a virus. In some embodiments, the virus is a single stranded, positive sense RNA virus. In some embodiments, the virus belongs the the Picornaviridae family of viruses. In some embodiments, the virus is lytic virus. In some embodiments, the virus is non-integrative. In some embodiments, the virus is non-enveloped. In some embodiments, the virus is not a lysogenic virus. In some embodiments, the virus genomic RNA does not encode an integrase. By the means of bioinformatic tools and algorithms, the inventors found out that the ribonucleic acid molecule of sequence consisting of SEQ ID NO: 1 may encode two open reading frames (ORFs), namely ORF1 encoding a first pro-polypeptide PI, and ORF2 encoding a second pro-polypeptide P2. In some embodiments, the two ORFs are non- overlapping.

In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 2, over the entire length.

In certain embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 2, over the entire length, preferably encoding a first pro-polypeptide (pro-polypeptide PI).

In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity with SEQ ID NO: 2, over the entire length. In certain embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence consisting of SEQ ID NO: 2.

In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 3, over the entire length. In certain embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 3, over the entire length, preferably encoding a second pro-polypeptide (pro-polypeptide P2).

In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity with SEQ ID NO: 3, over the entire length.

In certain embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence consisting of SEQ ID NO: 3. In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 2, over the entire length, and having at least 75% sequence identity with SEQ ID NO: 3, over the entire length. In some embodiments, the ribonucleic acid molecule comprises a ribonucleic acid sequence consisting of SEQ ID NO: 2, and a ribonucleic acid sequence consisting of

SEQ ID NO: 3.

In some embodiments, the sequence of the ribonucleic acid molecule consists of SEQ ID

NO: 1 The present invention further relates to a desoxy rib onucl ei c acid molecule encoding a ribonucleic acid molecule as described hereinabove. In certain embodiments, encoding the ribonucleic acid molecule according to the invention involves at least one step of alternative splicing. In one embodiment, the desoxyribonucleic acid molecule is linear. In another embodiment, the desoxyribonucleic acid molecule is circular. In some embodiments, the de soxy rib onucl ei c acid molecule is in a form selected from the group comprising or consisting of a plasmid, a cosmid, an artificial chromosome and the like.

Another aspect of the invention pertains to an isolated pro-polypeptide PI having at least 75% amino acid identity with SEQ ID NO: 4, over the entire length.

As used herein, the term “at least 75% amino acid identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% amino acid identity.

Illustratively, the amino acid identity percentage may also be determined using the CLUSTAL W software (version 1.83) the parameters being set as follows: for slow/accurate alignments: (1) Gap Open Penalty: 10.00; (2) Gap Extension Penalty: 0.1; (3) Protein weight matrix: BLOSUM; for fast/approximate alignments: (4) Gap penalty: 3; (5) K-tuple (word) size: 1; (6) No. of top diagonals: 5; (7) Window size: 5; (8) Scoring Method: PERCENT.

In certain embodiments, the isolated pro-polypeptide PI has least 80%, preferably at least 90%, more preferably at least 95% sequence identity with SEQ ID NO: 4, over the entire length.

In some embodiments, the isolated pro-polypeptide PI has an amino acid sequence consisting of SEQ ID NO: 4.

In some embodiments, the isolated pro-polypeptide PI is encoded by a ribonucleic acid sequence having at least 75%, preferably at least 85%, more preferably at least 95%, even more preferably at least 99%, sequence identity with SEQ ID NO: 2, over the entire length. In some embodiments, the isolated pro-polypeptide PI is encoded by a ribonucleic acid sequence as set forth in SEQ ID NO: 2.

In certain embodiments, pro-polypeptide PI is susceptible to be cleaved into two or more viral-like polypeptide(s) selected in the group consisting of a helicase, a 3C or 3C-like protease or peptidase, and a RNA-dependent RNA polymerase.

As used herein, the term “viral-like polypeptide” refers to a polypeptide that shares some structural and/or functional identity levels with one or more identified viral polypeptide(s), in particular sequenced polypeptide(s) available from the databases. As used herein, the term “helicase” refers to a protein that moves directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands, using energy from ATP hydrolysis.

As used herein, the term “3C- or 3C-like protease or petidase” refers to cysteine endopeptidases that cleave site-specific peptide bonds of non-terminal sequences. Picornain 3C found in picomaviruses is one example of 3C-protease.

As used herein, the term “RNA-dependent RNA polymerase” refers to an enzymatic protein that catalyzes the replication of RNA from an RNA template. More particularly, RNA-dependent RNA polymerase catalyzes the synthesis of a RNA strand that is complementary to a RNA template.

In some embodiments, pro-polypeptide PI may be cleaved into a helicase, a 3C or SC- like protease, and a RNA-dependent RNA polymerase. In some embodiments, pro-polypeptide PI may not be cleaved into an integrase.

In practice, the inventors observed that the amino acid sequence of pro-polypeptide PI may share some identity with amino acid sequences from databases, including amino acid sequences of viral origin.

In some embodiment, the pro-polypeptide PI according to the invention is post- translationally modified, for example phosphorylated, glycosylated, methylated, acetyl ated, nitrosylated and the like.

A still further aspect of the invention relates to an isolated pro-polypeptide P2 having at least 75% amino acid identity with SEQ ID NO: 5, over the entire length.

In certain embodiments, the isolated pro-polypeptide P2 has least 80%, preferably at least 90%, more preferably at least 95% sequence identity with SEQ ID NO: 5, over the entire length.

In some embodiments, isolated pro-polypeptide P2 has an amino acid sequence consisting of SEQ ID NO: 5.

In some embodiments, the isolated pro-polypeptide P2 is encoded by a ribonucleic acid sequence having at least 75%, preferably at least 85%, more preferably at least 95%, even more preferably at least 99%, sequence identity with SEQ ID NO: 3, over the entire length. In some embodiments, the isolated pro-polypeptide P2 is encoded by a ribonucleic acid sequence as set forth in SEQ ID NO: 3.

In some embodiments, the pro-polypeptide P2 is susceptible to be cleaved into two or more viral capsid-like polypeptide(s). As used herein, the term “viral capsid-like polypeptide” refers to a polypeptide that has some structural and/or functional identity levels with one or more identified viral capsid polypeptide(s). As used herein, the term “capsid” refers to the protein structure of a virus that encloses the viral genetic material. The viral capsid often results from the 3D arrangement of several capsid polypeptides (also referred to as oligomers).

In certain embodiments, the pro-polypeptide P2 may be cleaved into 4 distinct viral capsid-like polypeptides, herein referred to as CPI, CP2, CP3 and CP4. In some embodiments, any one of CPI, CP2, CP3, CP4 and combinations thereof are proteins of the capsid of a virus. In some embodiments, CPI is critical for the cell entry of the virus. In some embodiments, none of CPI, CP2, CP3 and CP4 have an integrase activity.

In practice, the inventors observed that the amino acid sequence of pro-polypeptide P2 may share some identity with amino acid sequences from databases, including amino acid sequences of viral origin.

In some embodiment, the pro-polypeptide P2 according to the invention is post- translationally modified, for example phosphorylated, glycosylated, methylated, acetyl ated, nitrosylated and the like.

In one aspect, the invention relates to a green alga cell comprising the ribonucleic acid molecule as defined herein, and/or expressing pro-polypeptide PI as defined herein, and/or expressing pro-polypeptide P2 as defined in the instant invention. In certain embodiments, the pro-polypeptide PI is encoded by a ribonucleic acid molecule as defined herein, and/or the pro-polypeptide P2 is encoded by a ribonucleic acid molecule according to the instant invention.

In some embodiments, the green alga cell expresses pro-polypeptide PI and pro polypeptide P2, as defined in the instant invention. In certain embodiments, the green alga cell belongs to the genus Ulva. In some embodiments, an alga of the genus Ulva is selected in the group comprising an alga of the species Ulva acanthophora, Ulva anandii, Ulva arasakii, Ulva armoricana, Ulva atroviridis, Ulva beytensis, Ulva bifrons, Ulva brevistipita, Ulva burmanica, Ulva californica, Ulva chae tomorphoides, Ulva clathrate, Ulva compressa, Ulva conglobata, Ulva cornuta, Ulva covelongensis, Ulva crassa, Ulva crassimembrana, Ulva curvata, Ulva denticulate, Ulva diaphana, Ulva elegans, Ulva enteromorpha, Ulva erecta, Ulva expansa Ulva fasciata, Ulva flexuosa, Ulva geminoidea, Ulva gigantea, Ulva grandis, Ulva hookeriana, Ulva hopkirkii, Ulva howensis, Ulva indica, Ulva intestinalis, Ulva intestinaloides, Ulva javanica, Ulva kylinii, Ulva lactuca, Ulva laetevirens, Ulva laingii, Ulva linearis, Ulva linza, Ulva lippii, Ulva litoralis, Ulva littorea, Ulva lobate, Ulva marginata, Ulva micrococca, Ulva mutabilis, Ulva neapolitana, Ulva nematoidea, Ulva ohnoi, Ulva olivascens, Ulva pacifica, Ulva papenfussii, Ulva parva, Ulva paschima, Ulva patengensis, Ulva percursa, Ulva pertusa, Ulva phyllosa, Ulva polyclada, Ulva popenguinensis, Ulva porrifolia, Ulva procera, Ulva profunda, Ulva prolifera, Ulva pseudocurvata, Ulva pseudolinza, Ulva pulchra, Ulva quilonensis, Ulva radiata, Ulva ralfsii, Ulva ranunculata, Ulva reticulata, Ulva rhacodes, Ulva rigida, Ulva rotundata, Ulva saifullahii, Ulva scandinavica, Ulva serrata, Ulva simplex, Ulva sorensenii, Ulva spinulosa, Ulva stenophylla, Ulva sublittoralis, Ulva subulata, Ulva taeniata, Ulva tanneri, Ulva tenera, Ulva torta, Ulva tuberosa, Ulva uncialis, Ulva uncinate, Ulva uncinate, Ulva usneoides, Ulva utricularis, Ulva utriculosa, Ulva uvoides and Ulva ventricosa.

In certain embodiments, the alga of the genus Ulva is selected in a group comprising an alga of the species Ulva armoricana and Ulva lactuca. In one embodiment, the alga of the genus Ulva is an alga of the species Ulva lactuca. Within the scope of the invention an alga of the species Ulva lactuca may also refer to an Enteromorpha alga.

Without wanting to be bound to a theory, the inventors consider that expression of the ribonucleic acid according to the invention, and optionally both pro-polypeptide PI and pro-polypeptide P2, in a green alga cell within a green alga’s tissue may promote the whitening or bleaching of said green alga’s tissue, which results in the death of the green alga’s tissue and hence to the death of the green alga. In addition, the inventors also consider that expression of the ribonucleic acid according to the invention, and optionally both pro-polypeptide PI and pro-polypeptide P2, in a green alga cell may result in the production of viral particles which may be released from whitened or bleached green alga. A further aspect of the invention relates to an algicide composition comprising at least one ribonucleic acid molecule as defined herein, and/or at least one pro-polypeptide PI as defined herein, and/or at least one pro-polypeptide P2 as defined herein.

As used herein, the term “algicide composition” refers to a composition which promotes the control of green alga’s blooms, by delaying, inhibiting or stopping the proliferation of green alga. In some embodiments, the algicide composition according to the invention promotes the death of the green alga, in particular a death mediated by the whitening or bleaching of the green alga’ s tissues. In practice, the bleaching of green alga’ s tissues is monitored or evaluated by the observation of a white coloration of the green alga’ s tissues.

In some embodiments, the algicide composition comprises an effective amount of at least one ribonucleic acid molecule according to the instant invention. In certain embodiments, the algicide composition comprises an effective amount of at least one pro-polypeptide PI, and/or at least one pro-polypeptide P2 according to the instant invention. In some embodiments, the algicide composition comprises an effective amount of at least one pro polypeptide PI and at least one pro-polypeptide P2 according to the instant invention. In some embodiments, the ribonucleic acid molecule according to the invention may be encapsulated, so as to avoid its degradation. In practice, the encapsulation may be obtained naturally, z.e., by the mean of capsid or capsid-like polypeptides, in particular of viral origin. In some embodiments, the capsid or capsid-like polypeptides may be encoded by the pro-polypeptide P2. Alternatively, the encapsulation may be obtained artificially as disclosed in the state of the art.

In some embodiments, the algicide composition further comprises one or more algicide agent. In one embodiment, the one or more algicide agent is a chemical agent. Non- limitative examples of chemical algicide agents include cybutryn, copper (II) sulfate and hexachl orobutadiene . In another embodiment, the one or more algicide agent is a biological agent. Non-limitative examples of biological algicide agents include bacteria of the phylum cyanobacteria and plants of the haloragaceae family.

The invention also relates to the use of a ribonucleic acid molecule as defined herein, and/or a pro-polypeptide PI as defined herein, and/or a pro-polypeptide P2 as defined herein, and/or an algicide composition as defined herein, in the biological control of a green alga, in particular a green alga belonging to the genus Ulva.

In another aspect, the invention further pertains to the use of a ribonucleic acid molecule as defined herein, a pro-polypeptide PI as defined herein, a pro-polypeptide P2 as defined herein, or an algicide composition as defined herein, in preventing a bloom of a green alga, in particular a green alga belonging to the genus Ulva.

Another aspect of the invention relates to a method for the biological control of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule as defined herein, and/or a pro-polypeptide PI as defined herein, and/or a pro-polypeptide P2 as defined herein, and/or an algicide composition as defined herein.

A further aspect of the invention pertains to a method for preventing a bloom of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule as defined herein, a pro-polypeptide PI as defined herein, a pro-polypeptide P2 as defined herein, or an algicide composition as defined herein.

In some embodiments, the method comprises the steps of: a) contacting the green alga with an effective amount of a ribonucleic acid molecule, and/or a pro-polypeptide PI, and/or a pro-polypeptide P2, and/or an algicide composition according to the instant invention, within a given perimeter; b) observing the whitening or bleaching of the green alga’s tissue in the given perimeter; wherein biological control of a green alga or prevention a bloom of a green alga is operant when substantial amount of green alga’s tissues is whitened or bleached.

As used herein, the term “substantial amount” encompasses at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the green alga’ s tissues from the treated perimeter is whitened or bleached.

In some embodiments, the given perimeter is a marine environment.

In certain embodiments, the biological control is to be performed in a marine environment.

Within the scope of the instant invention, “a marine environment in need thereof’ refers to a seawater ecosystem experiencing or prone to experience Ulva blooms.

In some embodiments, the marine environment may be limited to seawaters, in particular deep sea, seashore, estuaries, and the like.

In some embodiments, the marine environment may be artificial, for example marine aquariums or artificial marine reserves. In practice, assessing whether a marine environment is in need of controlling and/or preventing blooms of an alga of the genus Ulva may be performed by measuring one or more of the following parameters, including the average seawater salinity, the average seawater surface temperature and the average concentration of Ulva in said environment.

Illustratively, measuring the average seawater salinity, i.e. the concentration of salt (in grams) per kg of seawater, may be performed by any method known in the state of the art. Non-limitative examples of methods suitable for measuring seawater salinity includes the measure of electrical conductivity (EC), the measure of total dissolved solids (TDS). In some embodiments, a marine environment in need of controlling and/or preventing blooms of an alga of the genus Ulva may have an average salinity comprised of from about 30 g to about 40 g of salt per kg of seawater. Within the scope of the invention, the expression “from about 30 g to about 40 g of salt per kg of seawater” includes 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g and 40 g of salt per kg of seawater. Illustratively, measuring the average seawater surface temperature may be performed by any method known in the state of the art. Non-limitative examples of methods suitable for measuring the average seawater surface temperature includes satellite microwave radiometers, infrared (IR) radiometers, in situ buoys. In some embodiments, a marine environment in need of controlling and/or preventing blooms of an alga of the genus Ulva may have an average surface temperature comprised of from about 12°C to about 25°C, preferably from about 14°C to about 20°C. Within the scope of the invention, the expression “from about 12°C to about 25°C” includes 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C and 25°C. Illustratively, measuring the average concentration of an alga of the genus Ulva may be performed by any method known in the state of the art. In practice, the biomass of algae in seawater may be assessed by any one of the well-established methods, e.g, methods disclosed in Hambrook Berkman, J.A., and Canova, M.G. (2007, Algal biomass indicators (ver. 1.0): U S. Geological Survey Techniques of Water-Resources Investigations, book 9, chap. A7, section 7.4). Non-limitative examples of methods suitable for measuring the biomass of algae includes the measure of carbon biomass as ash-free dry mass, the measure of the particulate organic carbon (POC), or the quantification of chlorophyll a in a seawater sample.

In some embodiments, Ulva green algae blooms may be controlled in the seawater, in particular prior to running aground on the coastline, in particular on rocks or on beaches.

In some other embodiments, Ulva green algae blooms may be controlled on the coastline, including any land or ground surface ground surface in direct contact with the sea, e.g., rocks, beaches.

In practice, the effective amount of a ribonucleic acid molecule, a pro-polypeptide PI, a pro-polypeptide P2, or an algid de composition according to the instant invention may be contacted with the alga of the genus Ulva that are lying on the coastline. In some embodiments, Ulva algae are killed prior to its natural biodegradation. In practice, the natural biodegradation is initiated when significant amounts of toxic acidic vapors are emitted, in particular H2S vapors. Illustratively, once dead, the green algae may be safely removed and/or stored prior to their final destruction.

In some embodiments, the death of an alga of the genus Ulva may be assessed by the decolorating of the green tissues of algae into white tissues. As used herein, the decolorating of the green tissues of algae into white tissues may also referred to as the “bleaching” of the green tissues of algae. In practice, observation of dead (necrotic) white tissues may be visually assessed or assessed by the mean of naked eye (also referred to unaided eye), or by optical microscopy. In certain embodiments, white tissues may be observed from about 1 day to about 15 days after contacting the effective amount of a ribonucleic acid molecule, a pro-polypeptide PI, a pro-polypeptide P2, or an algicide composition according to the instant invention with the Ulva algae, preferably at day light and/or at a temperature of from about 20°C to about 30°C. Within the scope of the instant invention, the expression “from about 1 day to about 15 days” encompasses 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 days. Within the scope of the instant invention, the expression “from about 20°C to about 30°C” encompasses 20°C, 21°C, 22°C, 23 °C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C and 30°C.

By “effective amount”, it is meant a level or amount of ribonucleic acid, pro- polypeptide^), or of an algicide composition, that is necessary and sufficient for slowing down or stopping the proliferation or the bloom of a green alga, in particular a green alga belonging to the genus Ulva.

In some embodiments, an effective amount of the ribonucleic acid to be used may range from about 1 ^c 10⁵ to about 1 ^c 10¹⁵ copies per kg or L of green alga.

Within the scope of the instant invention, from about lxlO⁵ to about lxlO¹⁵ copies includes lx lO⁵, 2^c10⁵, 3^c10⁵, 4xl0⁵, 5x10^s, 6x l0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2x l0⁶, 3x l0⁶, 4xl0⁶, 5xl0⁶, 6x l0⁶, 7x l0⁶, 8xl0⁶, 9x l0⁶, lxlO⁷, 2^c10⁷, 3^c10⁷, 4^c10⁷, 5xl0⁷, 6x l0⁷, 7xl0⁷, 8xl0⁷, 9^c 10⁷, lx lO⁸, 2^c10⁸, 3^c 10⁸, 4^c10⁸, 5^c10⁸, 6^c10⁸, 7^c10⁸, 8xl0⁸, 9x l0⁸, lx lO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2x l0¹⁰, 3x l0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, 9xl0¹⁰, lxlO¹¹, 2xlO^u, 3xl0^u, 4x lO^u, 5x l0^u, 6xlO^u, 7xlO^u, 8xl0^u, 9xlO^u, lxlO¹², 2xl0¹², 3xl0¹², 4xl0¹², 5xl0¹², 6x l0¹², 7^c 10¹², 8^c10¹², 9^c10¹², 1 ^c 10¹³, 2^c10¹³, 3^c10¹³, 4^c10¹³, 5^c10¹³, 6^c10¹³, 7^c10¹³, 8^c 10¹³, 9^c 10¹³, 1 ^c 10¹⁴, 2^c10¹⁴, 3^c10¹⁴, 4^c10¹⁴, 5^c10¹⁴, 6^c10¹⁴, 7^c10¹⁴, 8^c10¹⁴, 9^c 10¹⁴ and

1 ^c 10¹⁵ copies, per kg or L of green alga.

In certain embodiments, an effective amount of the pro-polypeptide PI and/or pro polypeptide P2 according to the invention may range from about 0.001 mg to about 3,000 mg, per kg or L, preferably from about 0.05 mg to about 1,000 mg, per kg or L.

Within the scope of the instant invention, from about 0.001 mg to about 3,000 mg includes, from about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,100 mg, 1,150 mg, 1,200 mg, 1,250 mg, 1,300 mg, 1,350 mg, 1,400 mg, 1,450 mg, 1,500 mg, 1,550 mg, 1,600 mg, 1,650 mg, 1,700 mg, 1,750 mg, 1,800 mg, 1,850 mg, 1,900 mg, 1,950 mg, 2,000 mg, 2,100 mg, 2,150 mg, 2,200 mg, 2,250 mg, 2,300 mg, 2,350 mg, 2,400 mg, 2,450 mg, 2,500 mg, 2,550 mg, 2,600 mg, 2,650 mg, 2,700 mg, 2,750 mg, 2,800 mg, 2,850 mg, 2,900 mg, 2,950 mg and 3,000 mg per kg or L.

In certain embodiments, the pro-polypeptide PI and/or pro-polypeptide P2 according to the invention is to be used to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of green alga weight. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C are photographs of Enteromorpha algae. Fig. 1A: a green tubular alga formerly called Enteromorpha collected in November 2018 after a bloom in the Trieux fjord (TR) in the north coast of Brittany (48°46’ N, 3°06’W). Fig. IB: the tubular form disappears after an incubation for one month at 20°C and day light exposure with sea water collected in June 2018 in the bay of Marseille (43°18’ N 5°16Έ or spot X (RN) in Figure 2). Fig. 1C: the “Enteromorpha” became atypical Ulva lactuca after three months at 20°C and day light exposure.

Figure 2 is a scheme showing a statistical analysis of in vitro Breton Ulva lactuca proliferation with distinct samples of seawater of the bay of Marseille. Seawater samples collected in June 2018 in three different spots in the bay of Marseille. X (RN) was 43°18’N, 5°16Έ; Y (RS) was 43°15’N, 5°19Έ and Z (PR) was 43°14’N, 5°21Έ. Seawater samples were divided in three groups of tubes (n=36). Breton Ulva lactuca collected in June 2018 at Brehec (north coast of Brittany 48°43’N 2°06’W) were cut in pieces of 1 cm² and put in the three groups of tubes corresponding to spots X, Y and Z in the bay of Marseille collected one day before sampling (Dl). Ulva lactuca proliferation was carried out at 25°C with seawater in 50 ml tubes closed with a tape to induce anoxia. Proliferation was observed in 25 tubes/36 (69 %) in the X spot that corresponds to open sea, while it was 14 tubes/36 (39%) in the Y spots and only 1 tube/36 (2.7 %) in the Z spot, the closest of the shore (see the inserted graph). In tubes where Ulva lactuca could proliferate, confluence was reached after one week and acidity was detected. No acidity was observed in tubes where Ulva lactuca could not grow and Ulva lactuca became white after 5 days. Seawater from the Z spot was kept from D30 to D180 before to be incubated again with Breton Ulva lactuca and proliferation was observed in 12 tubes/36 (33 %) for D30 and in 36 tubes/36 (100 %) for D 180 (see the inserted graph).

Figures 3A-3D are photographs showing the comparison with optical microscopy of Ulva lactuca in three different states. Fig. 3A: Ulva lactuca became white in five days when incubate at 20°C and day light exposure with seawater from Z spot of the bay of Marseille. Fig. 3B: Optical microscopy (10x) of white Ulva lactuca. Ulva lactuca tissue remains unaffected with a regular organization of Ulva cells. Fig. 3C: Optical microscopy (10x) of healthy Ulva lactuca. Fig. 3D: Optical microscopy (10x) of Ulva lactuca after acidic biodegradation. Ulva lactuca tissue is disrupted with release of chlorophytes that remain green in spite of anoxia. Photographed with a camera Nikon D3100 coupled to a Nikon Eclipse Ti L100 microscope (Nikon®, Tokyo, Japan). Figures 4A-4D are photographs and graph showing the fluorescence microscopy after SYBR staining. Fig. 4A-C: sea water from Mediterranean seas inducing bleaching was incubated without Ulva (panel A) and with Ulva (panels B and C). Fig. 4D: shows the virus-like particles amount, expressed as a number of particles/mL. Sea water was filtrated at 0.2 pm.

EXAMPLE 1: Identification of seawater samples that promote death of Ulva lactuca

1) Materials and Methods a) Ulva lactuca polymorphism

Green algae were collected in the Trieux fjord in the North coasts of Brittany (48°46’ N, 3°06’W). Proliferation was carried out in vitro with seawater samples from the bay of

Marseille (Provence, South of France). A green tubular alga formerly called Enteromorpha was collected in November 2018 after a bloom in the Trieux fjord in the north coast of Brittany (48°46’ N, 3°06’W). The incubation was carried out for one month at 20°C and day light exposure with sea water collected in June 2018 in the north bay of Marseille (43°18’ N 5°16Έ; RN). b ) Ulva lactuca proliferation

Seawater samples were collected in surface in springtime (June 2018, 2019 and 2020) in eight different spots, including three different spots at Marseille (Figure 2), 2 spots in Provence, and 3 spots in Brittany (see Table 1). Seawater samples were divided in three groups of tubes (n=36). Breton Ulva lactuca collected in June 2018 at Brehec (north coast of Brittany 48°43’N 2°6O) were cut in pieces of 1 cm² and put in the three groups of falcon tubes (50 ml) corresponding to spots X, Y and Z in the bay of Marseille collected one day before sampling (Dl). Acidity was tested with a Crison pH Meter (Barcelona, Catalonia). The pH meter was calibrated before any measurement. Nitrates was measured with a METRHOM chromato ionic device (Berne, Switzerland) with a Metrosep column A supp 5 150/4 mm with 3.2 mM Na2CCb/L mM NaHCCb as eluant. Sea Water was diluted 1/8 and a standard was use to calibrate the amount of nitrates. c) Optical microscopy

Optical microscopy (10X) was performed on healthy Ulva lactuca before confluence, after acidic biodegradation and on white Ulva lactuca after five days with water sample collected in the Z (PR) spot in the bay of Marseille. Photographs were carried out with a camera Nikon D3100 coupled to a Nikon Eclipse Ti L100 microscope (Nikon®, Tokyo, Japan). d) Diode Array Detection High Performance Liquid Chromatography (DAD HPLC)

Sea water samples were filtered at 0.2 pm and were analyzed on a Beckman® HPLC system gold device with a reverse phase (C8) column using H2O 0.1 % TFA (A) and CH3CN 0.1%TFA (B). The gradient was from 10% to 50% B in 40 min, and then 10 min at 90% B and 10 min at 10% B. Diode Array Detector Beckman® device was coupled after the injector. Flow rate was 0.8 mL/min e) Fluorescence microscopy after SYBR stainins

Mediterranean seas water without and with Ulva lactuca sample was filtrated through 0.22 pm membrane filter (Millex®; cat. n° SLGP033RS) to remove cells and then through 0.02 pm Anodise filters (Whitman®; cat. n° WHA68096002) using a vacuum filtration system to collect viral particles.

Then filters were stained with SYBR Gold dye (N',N'-dimethyl-N-[4-[(E)-(3-methyl-l,3- b enzothi azol -2-y li dene)methy 1 ] - 1 -phenylquinolin- 1 -ium-2-yl]-N-propylpropane- 1,3- diamine) that binds reverently to DNA (Invitrogen®; cat. n° SI 1494) at room temperature for 15 min in the dark, and washed three times with 500 pL of sterile 0.02 pm-filtered mQ water. Stained virus-like particles were observed with an epifluorescence Microscope Leica SP2. 2) Results a) Breton Ulva lactuca can STOW in Mediterranean Sea and has different phenotypes regarding salinity

Ulva lactuca is naturally present in the bay of Marseille (Provence, south of France) and appears each year in winter. Ulva grow rapidly from February to March before disappearing rapidly for springtime. Ulva lactuca blooms, as observed in Brittany, were never reported in the bay ofMarseille while this bay has a high concentration in phosphate and nitrogen and shallow beaches. A first hypothesis could be that Breton Ulva lactuca could easily proliferate in Brittany but could not grow in Mediterranean Sea, more particularly, that the nitrate concentration is lower compared to sea water in Brittany. Five spots nearby Marseille were selected and sea water samples were collected and compared to three spots in Brittany (Table 1).

Table 1: Springtime Sea Waters (n=7) from Brittany and Provence

* Statistical analysis of in vitro Breton Ulva bleaching with sea water samples collected for springtime. All experiments (n=8) were carried out with Breton Ulva collected in Trieux fj ord (TR) in the north of Brittany in 2018, 2019 and 2020. Ulva were cut in pieces of 1 cm² and put in sea water (40 mL) in tubes (n=25) closed with a tape and let at day light with an average temperature of 25°C.

The bay ofMarseille is at 20 km of the mouth of the Rhone River and North West winds (Mistral and Tramontane) that are dominant blow regularly from Rhone River to Marseille. Table 1 shows that pH and conductimetry (related mainly to salinity) are lower in RN due probably to the influence of Rhone River. Table 1 shows that the concentration in nitrates in open coastal sea water is equivalent in Brittany (BR and PO) and in Provence (RN, WF, RS). However, nitrate concentration can be much higher in Brittany fjord (TR) or in Mugel calanque (MU) and marina (PR) in Provence.

As shown above, Breton Ulva lactuca can grow rapidly with sea waters from Marseille (Figure 1). Ulva lactuca polymorphism was tested with a green tubular alga formerly called Enteromorpha (Figure 1A) collected in the Trieux fjord nearby the city of Paimpol

(North Brittany) that became a typical Ulva lactuca after three months at 20°C ± 10°C and day light exposure (Figure 1C). This experiment illustrates the importance of salinity in the polymorphism of Ulva lactuca as previously described (Rybak, Ecological Indicators, 2018, 85, 253-261). b ) Breton Ulva lactuca proliferation is different resardins the location and the timing of the water samylins in the bay of Marseille

Natural biodegradation on beaches occurs when Ulva lactuca reach confluence inducing anoxia characterized by production of H2S. For this biodegradation, Ulva can become white due to dehydration. However, this is a different phenomenon that we observed with Breton Ulva lactuca in sea water collected at Marseille. Breton Ulva lactuca were turning white (bleaching) rapidly sometime in one day without dehydration. To simulate this natural process proliferation of Ulva lactuca was carried out with sea water in 50 ml tubes closed with a tape to induce anoxia.

A statistical analysis was carried out with seawater samples collected in three different spots in Brittany and five spots in Provence, including Marseille Bay (Table 1 and Figure

2). Seawater samples were divided in eight groups of tubes (n=36). Breton Ulva lactuca were cut in pieces of 1 cm² and put in the eight groups of tubes corresponding to spots X (RN), Y (RS) and Z (PR) in the bay of Marseille, PR and MU in Provence, TR and BR (North Brittany) and PO (South Brittany), collected one day before sampling (Dl). As shown in Figure IB, the tubular form disappears after an incubation for one month at 20°C and day light exposure with sea water collected in June 2018 in the bay of Marseille (43°18’ N 5°16Έ or spot X (RN)). No bleaching was observed with sea water collected in Brittany for springtime when Ulva proliferation is the highest. Regarding the five different spots in Provence, the number of tubes where proliferation could happen was not the same. Proliferation was observed in 25 tubes/36 (69 %) for the X spot that corresponds to open sea, while it was 14 tubes/36 for the Y spots and only 1 tube/36 for the Z spot, the closest of the shore. In tubes were Ulva lactuca could not grow, Ulva lactuca were becoming white under day light at 20°C in five days with no acidity detected, as shown in Figure 3A. This Ulva lactuca white phenotype was not similar to white dehydrated Ulva lactuca as observed in Brittany, when Ulva lactuca are staying on the shore in low tides. For tubes where Ulva lactuca could proliferate, confluence was reached after one week and acidity was observed according to the regular process of Ulva lactuca biodegradation (Dominguez and Loret, Mar Drugs. 2019 Jun 14; 17(6). Pii: E357). As observed in natural conditions Ulva lactuca remained green under biodegradation. Sea water from Z spot (Figure 2) were kept from D30 to D180 before to be incubated again with Breton Ulva lactuca and the proliferation were observed in 12 tubes/36 for D30 and 36 tubes/36 for D180 (Figure 2). The active principle that promotes the death of Breton Ulva lactuca cells is not a pollutant that would have produced the same effect from D 1 to D 180. Other Breton algae (mainly brown) were not affected with seawaters from Marseille Bay (data not shown). c) Comparison with optical microscopy of Ulva lactuca in three different states shows that tissue is not disrupted in white Ulva lactuca

What happens in Figure 3A showing white Ulva lactuca was studied at a tissue level with optical microscopy. Figure 3B shows that the white tissue of Ulva lactuca remains unaffected with a regular organization of Ulva lactuca cells comparable to healthy Ulva lactuca, which have a thallus composed of tight cells with chlorophytes present in cytoplasm giving a green color to cells (Figure 3C). The white color in Figure 3B indicates that cells are dead but this death is not due to a macro predator or environmental conditions that would have disrupted the tissue organization of the alga tissue as shown in Figure 3C. Interestingly, Ulva lactuca tissues were disrupted, with release of chlorophytes that remain green after acidic biodegradation as shown in Figure 3D. This is not a sporulation that could provide a white color. The main explanation emerging from these preliminary experiments is that a microorganism specific of Ulva lactuca control Ulva lactuca blooms in the Mediterranean Sea. Only a microorganism attack, in particular a viral attack, could explain this rapid death of Ulva lactuca cells without tissue damage. Furthermore, this hypothesis was confirmed. Indeed, when sea water is filtrated at 0.2 pm, Ulva lactuca can still turn white showing that the bleaching activity is not due to planktons, amoeba or bacteria that have a size superior to 0.2 pm. d) Diode Array Detection coupled to High Performance Liquid Chromatography (DAD

HPLC)

Mediterranean seawater inducing bleaching was filtrated at 0.2 pm and then analyzed with a DAD HPLC that makes possible to have a UV spectral analysis of each entity eluting at different times from a hydrophobic C8 column with an acetonitrile gradient. Most of the peaks eluting between 5 to 45 min are characterized by a UV spectral signature with a maximum absorption at 243 nm and correspond to organic macromolecules call colloids. The 3D view of the DAD HPLC run shows that colloids are the major components of sea water filtrated at 0.2 pm. Three peaks have a different UV spectral signature. The peak indicated with a red arrow at 3.5 min might correspond to the presence of viral-like particles and is characterized by a first max. abs. at 266 nm due to nucleic acids and aromatic amino acids. The two other peaks correspond to free nucleic acids at 6 min and free proteins at 45 min characterized respectively by a max. abs. at 260 nm and 280 nm. When Breton Ulva lactuca are added to Mediterranean seawater for five days and when bleaching occurs, the peak corresponding to virus-like particles increases significantly with a maximum absorbance at 266 nm ranging from 7 mAU to 32 mAU. Interestingly, this peak compatible with viral-like particles increases 78 %, while the colloid peaks decrease (due probably to Ulva eating). e) Virus-like particle stain and fluorescence microscopy

Mediterranean seawater without and with Ulva lactuca was filtrated at 0.2 pm and then stained with an aromatic compounds called SYBR Gold dye (for N',N'-dimethyl-N-[4- [(E)-(3 -methyl - 1 , 3 -b enzothi azol -2-y li dene)methy 1 ] - 1 -phenylquinolin- 1 -ium-2-yl]-N- propylpropane- 1 ,3 -diamine) that binds preferentially to DNA. This dye is widely used in virology to stain and visualize virus-like particles (VLPs) present in seawater and other aquatic samples. There are hundreds of published reports using this methodology to count and detected viruses in biological samples (Shibata et al, Aquat Microb Ecol. 2006, 43, 223-231). Figure 4A-C shows that fluorescence microscopy after SYBR staining reveals a high viral production when Ulva lactuca is added to seawater. This high viral-like particles’ production is already significant when Ulva lactuca are still green. However, when Ulva lactuca become white the abundance of viral-like particles reaches 6.5 xlO⁸ VLP/ml, which is an atypical high concentration (Figure 4D). This experiment suggests that virus-like particles may actively be produced and released with higher rates when Ulva lactuca become bleached. f) Conclusion

Altogether, these results demonstrate the correlation between the presence of a nucleic acid molecules, more likely from viral origin and the bleaching, z.e., the death of Ulva lactuca green algae.

EXAMPLE 2: Identification of the nucleic acid molecules from viral-like particles contained in the seawater that correlate with the death of Ulva lactuca

1) Materials and Methods a) Nucleic acids recovering from bleached Ulva lactuca samples About 250 g of bleached leaves of Ulva lactuca were collected and grinded in a blender in about 500 mL of buffer comprising 0.5 M potassium phosphate buffer, pH 7.5. The grinded material was then filtered through gauze. 250 mL of chloroform were added and the mixture was stirred for 10 min at 4°C, and centrifuged to separate the phases at 500xg for 10 min at 4°C. The upper aqueous phase was collected and filtered through Miracloth and further centrifuged at 5,000xg for 10 min at 4°C. The supernatant was filtered through Miracloth, and transferred to an Erlenmeyer flask on ice. b) Metasenomic analysis

Seawaters from tubes displaying bleached Ulva tissues were filtered by the means of a first filter with pore diameter of 0.45 pm and a second filter with pore diameter of 0.22 pm. Filtered samples were stored at -80°C previous to analysis. RNA extraction from bleached Ulva tissues was performed with the MasterPure™ Epicentre® Tissue Samples, according to the manufacturer’s instructions.

TransNGS® Tn5 Index for Illumina® was used for multiplex sample preparation (library) for next generation sequencing. Primers N701, N702 and N505 were used, according to the manufacturer’s instructions. c) Bioinformatic tools

Bioinformatic tools used in this study are the followings: Trimmomatic; SPAdes; BLASTp, BLASTx, BLASTn; HMM search; Prodigal; Genemark; Geneious; Bowtie; Primer-BLAST and primer 3; Gene aligners: MUSCLE, CLUSTALW; i-Tasser; ESyPred3D Web Server 1.0. 21 Results a) Nucleic acid content analysis in bleached Ulva samples

Ulva lactuca samples from Trieuc fjord treated with seawater samples from Spot MU (La Ciotat, France; 43°09’N, 5°36’E; samples SW3) and which have undergone bleaching (presence of white tissue) were analyzed for their RNA content. Table 2 below indicates the raw data stats for 20 samples.

First, bioinformatic analysis confirmed that Ulva genome could be retrieved from the samples, as 18S rRNA gene from Ulva lactuca was recovered (SEQ ID NO. 10). Table 3 below indicates the numbers of contigs obtained in seawater samples SW3.

Abundant SW3 contigs were from microbial origin, mainly Acinetobacter sp., in particular Acinetobacter junii. However, no DNA viral genomes were detected in the samples, excluding the presence of a DNA virus as the microorganism responsible for the death of Ulva lactuca.

Sample SW3 contains however abundant amount of RNA of sequence SEQ ID NO: 1. Bioinformatic analysis of the RNA sequence SEQ ID NO: 1 revealed that it is a single stranded (+) RNA with a poly A, of 8,518 bp. The RNA sequence SEQ ID NO: 1 is dicistronic, z.e., comprises 2 non-overlapping open reading frames (ORFs), encoding pro- polypeptide 1 (SEQ ID NO. 4) and pro-polypeptide 2 (SEQ ID NO. 5). b) Characterization of the identified RNA sequence

Genomic organization of the RNA sequence SEQ ID NO: 1 was found to be parented to the genomic organization of viruses belonging to the family of Picornaviridaea. Pro polypeptide 1 (SEQ ID NO. 4) shares with other picornaviruses the encoding sequence for RNA helicase, a protease (3C-protease) and a RNA dependent RNA polymerase (RdRpl). Pro-polypeptide 2 (SEQ ID NO. 5), on the other hand, shares with other picornaviruses the encoding sequence for a structural capsid polyprotein (CPI, CP2, CP3 and CP4) that after maturation is supposed to form viral structural proteins forming the capsid of the virus. 3D prediction of structural capsid data indicates that is a non- enveloped spherical capsid.

Table 4 below indicates the percentage similarity of pro-polypeptide 1 (SEQ ID NO. 4) with existing sequenced pro-polypeptides.

Therefore, bioinformatic analysis of pro-polypeptide 1 provide similarity with sequencec pro-polypeptide 1 from picomaviruses.

Table 5 below indicates the percentage similarity of pro-polypeptide 2 (SEQ ID NO. 5) with existing sequenced pro-polypeptides.

Therefore, bioinformatic analysis of pro-polypeptide 2 provide similarity with sequenced pro-polypeptide 2 from picomaviruses.

Table 6 below indicates the percentage similarity of capsid-like polypeptides CPI, CP2, CP3 and CP4 encoded by polypeptide 2 with existing sequenced polypeptides.

In addition, this RNA molecule is polyadenylated at the 3’ terminus (n=25 bp). c) Evidences that the RNA molecule actively undergoes transcription and is able to promote infection

Upon viral RNAs purification, it was observed that the seawater samples comprise high levels of the virus-like single strand RNA, as well as a double stranded RNA (“ds RNA”), that would be indicative evidence that the RNA molecule replicates. In addition, because the RNA molecule possesses several binding sites for RNA-dependent RNA polymerase (“RDRP”), this would explain why there is a smear of intermediates replicative forms.

Consequently, the newly identified RNA molecule is able to replicate in the presence of Ulva lactuca. d) Abundance of the RNA molecule in the sample

1,912,720 out of the 36,616.914 reads were assembled to produce contigs, meaning that approximately 5.22% of the sequence reads recruited against the newly identified RNA molecule of sequence SEQ ID NO: 1. In addition, the RNA molecule of sequence SEQ ID NO: 1 represented 100 orders of magnitude higher as compared to the 18S rRNA gene of Ulva lactuca. e) Mode of action

In silico bioinformatic analysis demonstrated that the RNA molecule lacks the sequences encoding integrases that are typical and absolutely necessary to follow a lysogenic cycle. Thus, the RNA molecule belongs to a strict lytic virus, that does lead to killing the host cell. This agrees with the observations that putatively infected tissue of U lactuca loses the typical green pigmentation in addition to signs of degeneration ( e.g . less consistency of tissue) (Loret et al., 2020). SEQUENCES USED HEREIN

SEQ ID NO: 1 (Virus-like particle’s RNA genome)

UIJUIJIJIJIJIJIJIJIJIJIJUIJIJIJIJIJIJIJUUUUGGAAUUACGAGCUACGAGAGCAUC

GCAUGAAAAUGUUCGAACUAGAGAGGGAAAUUUCCACCCAGCGUCACCGC

CGGGUUUAAAACCUCUAAAGUUCUAAUAACUUUAUGCGAUGACACAGAG

GAGAACCUCUCGUCAAUCUAUUACACUGUAAAAUAGACCACAUGGCUCGA

CCAUGUGUGGGAAAGCCCACACAUGAUCGUGUGAACGCUUUCCUUAGAG

AGUGACAAUAUUUACGUCACUUGGUAUGCGAGCAGGAGCCCCUAUGAAA

A AG A AU AGGGA A A A AU CUU CU GCU GCU GC AGU GU A A A AUU GU A AC C A AU

GAUAUUGAUUCGCAGUUUCGCGAUAAUCAAAGGAUACAGUUUGGCUACC

UUGAUUCAAUCCUGUAUAAGGAGGAACCUCGGUUUGUCCUGGUUCAUUA

CAUUCAGCAUAUCUUAAUGCUGUUUGAAAAGGGAGUUCAUACUCCAAAC

CAGGUUGAGAAGCCACGCCAGUUAAAGCUGUUCCAGCUGCACUGUCUUGG

CAAUUGUCCGCACGCCAUUUUUCGCGGACAACUCUUGACAAACUGUAUGA

ACCGUUAUCGUUAAUAAUAUUAACUUGCGAUUCGUUUGACAAGUACUCG

GGAUUUCUACCUACCGAUAAAGGUUGUAAUUGGUUAUUUGUGCCACAAU

AUAGCACUUUCCAACGACAUCCUCCUCGAAAGCCAACAUAUGCCUGCAUG

CAAUAACGCAUCAUGGUCAUAGGUAACAAAUUAUAAUUUAUUGGGCCUG

C GAU AGGGGUC AUU GC AGA AGC A AU ACU GGAU C C AU A AGAU GGAC C AGG

ACCCUGAGGGUAGAUAAAAGUUCGGUACAUAACUGUGUACCAAUUGUUU

ACAUUAAGAGAUUCUAAAGGCUCUAAAGUCCUAAAGUAAUUAUAUCUCU

UAAGUAAAGAUCUGAAGGAAACAACAGCCUCACCGUAAUAUACGUGAGA

UUGUUCCUUACAGAAUUCCGUGUAUUCACCAUUAAGAACAUAAGUAGUA

UCUUGUUCUGGCAAAUUUUCGUCCAUGGUCAUACCUUCUGUAGCCAUUU

GUGCUAAGGUAGGUGGACCAACAUCAGUCGUAGAGUUUGCAUAAGCAAC

AUCUUGACCUAAUGUUCCACGUGGAUUUCUCACUUCGUAAGAAUCACCAC

CGGAAAUGAAGACAUUCACGGAAACGCUGCUAUCCGUAAUAGGAGCAGC

AAGUGCAUUAAGUACAUAUACAUUAAGACGACCAUUGCAAUUAGCAACA

UCAUCAGCAUCUGUACCUAUAGCACCAGUAGCACCCUCCCAAGCAGAAAG

UUUUU G AGCU CU A A AG AU AU C A AU CUUU CU AU A AGCUU CUUU CU GGGU C

CAGUUGAUUUCAAAAGUAACAUCGCGUUCUUCUGAAAUAUCAACAAUAU GAGAGUAACGGUCAUUAGUAUCUGUCACAGUACCCGCCGUUGAUAACGU GGGUUCAUAAACAAACAAUAAUCGUCCUCUAUGAUAUUGUGAAGCCACA AUCUGAAAUCUAUAUCUUAGACUUCCAGACCAGUUAUUGAAAGGAUAAG AAACAAAACUCAAAGGAGUUUGACAUCUUACCACUGUAUCAUUAGAACC AGUUUGAUAGAUUGGUGCUAUCAUUGGAUGUACUUGAAUAGAAUACAAG AGCCCAGUGUUCUGGGCUACAAUUGUACUCCAAUUGAAGUAAUCAAUGA A AGCUU C AC GCUU GACC A A AU AUCC A A A AGAC AUUU GAU CUU CUU C AC CU A ACCC A ACGGU GGU GGGGUC A AU GGU GAGCU CUU GCUU GGGAU C A AG AG UUAAUUUGAAGAUAGGAUCUGCGCCUGAUGUAUUGGCUAAAUUGCCAAU AGGCUGCGAUUUAUAAAACGCAGUAUCUGUUAGAACUGCAGGUCGUGAA AAUCCAAAAAGACGCGCAAUGCUGCUAACAGCACCACUCGCUAUCUGGGU UGAUUUUGCAAAUUUACCAAUAUAUGGUAUUUCUGUAAAAUAGCCAGCA AAGUCGGCCAAUAAAGCUGCAGGAGCGGACACUACACCAUCAGGUUUGU GUUCAUCUUCUCCAGAAGUAUUGGUAAACGUUGGUUUAGCUUUCUUCUU U GGUUUU CU CUU CUU GGU CUU CUU ACU GUUUU GA AU CUU AU C A AC AGC C AU CU GAGCU GUU GC AGGAGC AGC AGCU GU A AGAC C AGC GA AUU CU AC AU CUUCC AUCC AC GC A A A A AUU GA A AUUU C A ACUU C AU C AGU GGC AC CGUU G GAAUGUUGUAGUUGGGUUAAUUCCCAAAUUUCCAAUACACCCAUACGAU CAAUAGUUUCUUGAUCAGUUAAAUCAAUCCAAUUGGUGGCCGAAAAGAA AGGC C AC GAG AUUU GUU GU GGUU G AUUU GUU GAU GG AU C A AU A A AU AC A UGUGGGCGUUGCGAGUAUAAGCAAGCCAUGUUAUUCAUUGUCUUAUUAC UACCAGCUGCAUUUUCAUCCGUAUAGGAUAAACUAACACCUGGAGUUAC AGGACCAUCAGUAAGUGUAUUGUUGUCGAAUUUUGAAGGUCUAAGACCA ACAAACAUUCGACCAUAAUGAAAAGGUGAACCAUUAACCAAAAUCUUGA GUUUAAGAGUUCCAUGAAGUAAUUUGAAAGUUUCUAUUUUGUUAGCAAC UUUAGAAUCACUAAGGAAUAAAUCCCAAGGAUUAAUAGCAUUCACAUAU UGUGGAGACUCAUUUACUUCCCAAAUCUUAGAAAAAAUUCUAAUAGGAC GGGACAUAAACGUAGUCAAAUCAACAGCUUGUGAAUUAGCCUUUUCGUA AGUCGGAUCUGACAAAGGUUUACCGACCAAAAGUUGAUAUUGUUCAACU U GGU C AGA A A A AUU A AC AUUU GU CU C GACUU GA AGACC AGA AU CUU C AU GUUCUACUUUUGCUUCAACUGCACUUUGUACAGUAAUAUCUGAAUCAUU AGU AGU AG AUU CU GGUU GUUU GGUUU CUU C AU GU AG AGU AGG A AG AG A A UAACCUAAGGCUCUCUCAUAUUCUAUCAUAUCAUGAAGUUUGCAAGUUU GACACUUGCACCUGACAUGGUCCACCUUAGGUGUACAACAUGUGCUAGGC AUAUGGGUUGGCAUUUUAAAAUUGAAGUUCAUAAUGCUUUAAAUUUAAG GGACGUACCCUAAAGAGGCUAUCUGCGCGUGCCUGUGCGUGCUAUAAGG UUGCAAACCAUAAAUUAUCGAGCUCCCAAUUAAGGGCGACGUAACUAUU UUUUGUGAAGGAAAUAAUUGUAAAACCUAUAUAAAAUUACAACUCUCCA AACCUUUCAAUAUAAAGUUCAUCAAAAGUUUUAAGAGCACAAUUGGGUA A AU A AC AU CU C A A AUCGA AUU C AU CU A AU A AUUCGUU GAGA A AU GU AUU UUCCCGUACAAAUUUCUUCUUACCAUAUUGGAAAUAUUCCGUAUUGGCG GAAGAAAUAAUUUGAGCACACUGUUCUUUAAAAGUAAUAGUCUUAGAUC UAGUGCAAACACUAAGACUUUUAAGAAUAGAUCCUUCAUCUAAAGGAGC UU G A AUU AC AC CUU GA AC AU A A AC AGAU GGAGU A A AUUUU CU CUU A AGA AAAUCAACAUCUUUAACGUUAAUAAAAGGUACAGAUUCAGACUCUUUAU CAGCCAUUGUAUAAGUAACUCCUCUUUUAGAAAGAGCAGUACUAAUGGU GGUAUGAUUUAACCAAGGAAUACUAGAAGAAGCACAAUUAUCAUCGCCA UAAGUUAAAAUGGCAAGUGAAUCUUGAAAUCUAGCAUAAUCUAUUUCUU GAACACCUCUUUCUUCUUCAAUAUCCAUGGCAGCUAGCAUCAUAUACAUA AUGUUGCACAUGCCAUUAAUUGGAGUAGUAAGAGGAUGACCACUAGGGU UACUACCAAACAUACCUACCACGGUUCCAAAAACAUUGGAUAGUGGAUA GCAAAUAUCAGUAGCUAGACCGCGCAUAAUAGUUAAGUCUUCAUCAGAC CAACCAUUGUCACGGCAAAUUCUAAUGAGAACAUUAAAACUAGCAGUCA UAAGCUCAGGAGGCAUAUUCUUAUCGAAUGCUUUAUAAUCGCCCGCAAU CAUAUUAUUCUUUCCAUGUUGGGUAAUAUAUUCAUAAAUUGCAGUCCAA UCAUCUCCAUGAGCAUUGGCACCGAUGGCCAUACCGAAUUCAGUUCUCUU UUUACCAGAAACUAAUGGAAUAAUCCAAAGGUAGUACAUGCGGACCAUA AUCAUAAAAUGAAGUGGUCCACUGUUAAAAAUUCUACAUUUCUGUUUCA AG AGUUUU G ACU C GG A A AC GGGCU C AU CUUU A A A AUU G A AGU C C C A A AC AAUAUCGCCGCGUUCACCUCUCAAGUAUUUUUCUUUGAGGCGAUUGAAU UCUAGC AUAAU AUU AUC AUU AAGUUCAU AUUUU ACGGUGUGUCCUGGUU CAGGAUCACCUAAAUGU AGAU AUUUGGAUUUAGCUCCUUUAUGAGC AAA ACCACCUGAUGUAGAUGCAGGAAUUCUGUCAAUAUAUGACGCUCCGUCA UAUCCAUUGACACCUACGUCGAUAUCGUAGGGCUCACAGGGAAUAAAGA AUUUAUGUUCUUCGAUCUUCCUUGUGAACCAAGCAUAUAACGCGUCUUC UGCGCGUUGAAUAUUCUUGGCUGGAAAACUAGGUUUUACAAACAUAGGG GCAACAUUUUGUUUACACGCCAAAGAGGAUUUAAUACCUUUUGGUGAAA AAUGUGUAAAAUCUAAUCUACCAUAAUGCUGUAAAAUACGUUCUGCCAU AAUAGUAUGACAAACCAUAGUUUUCAUUUUACGUCUAUGAGUAUUAAUA GAACCAAUCAUCAUCAUUGAACCUCCAUCAGUUAGUCUGAUAGGACAUU UCUUGUCUUGGGUUUGAGUAAUUGAUAGAUCUUCUACUGUCUCAUAGUG UUCGUUUAAAUCAACGCCAUUAUAUGAUAAUGGAGUAAAUUUGGAAUCA UCCAAAUCUGGGUUGAUUAAACAGGCUGCAAAGACUUUAUAUCUACCAC CAAAGGUAGCUUUCUUUGCACCAACAUGGAAACCAGCGAUAAAACACCCA UUAGGGGUCUUAACGAUGUAUGGUGCUCCACAUUCGCCUAUAAUAGUUG GCACUUUGGUGUAUGCCAUAUAACCAUUAUAAGCAUAUGAAAAAUAUUU AUCAUUAUAAGAGAUGGGACUAGUUUGCAUAGCUUCAAAAUCUUUAUAA GCCAUAAUACCGUCUCUUUGACGAAUAACCAUUGUACCAGAUGUUUUCCC UUUAAUCAUUCCAGGUAAUAGGAACUUACGCACAUCGCGAAAAGUUCCU AAAGCUGAAUGUCUAAAGAAUAUAACAUCACUUGGUCCAUAGCGUUCAU CAAAUUCAAAACAUGAUUCAUCAACUAAGACCUUAAAUCUGCUAGGUCC UCCUUGUUUGGUAAUAUCGUGUCUAAUGAUAUCCAUUCUAAGAGGAAAU ACAUCUUUAAGAGAAUCAAAGAAAUGUUUAGGCAAAGCCAUAACAUUUC CGUACAGUCCGAGACCACAUAUCCUAUGAUAUUGUCGCUCGGCGAGAUUU UCU GCU AUC AGAU GAC AGC AGUUU AGCU C A AC ACU AUU GACU A ACU GGU CAAAAGUUACUGUACUAGGAGGUCCACUCAAUCGUGUAAUAUUUUCAUA UU G AGCU GAC C A AU AGUU GG AGU C AG AUU G AUUUU C AU C A AC CUCU GGU UUU GAUU GU GGUU GA AU AGCU GAGU AC A A AU GU ACU AC AGU AU AC A AGC ACCCUAAAAGUGCAACAGUACCACCAACCGAAUACUUGUUAUAAUAUUU AGAU AAGUCAUGUCUAAUAGACUGAACUAAAAU ACC AUGCCCAUAGGCA CGUCUACAAGCGUUCGUAGCCCAAUCUUGCCAAGUGUCACAAAGACACCU AGUAGCAAGAAACAUAGGCAAAUAGGUAAACCACCAAGGCACUCCAAACC UCGAAGGUAAUUUUCUUAUACAAAUAACGACCUUUUUUGUAAAAUAAUU AGGUUCAUUAUUUCUAUAAGCUUUUGCGCAUACUUUAUGAAGUAGCCAA U A A AU GGCU A ACU G A A A A AUU GU AC AC A AU AC A AU GAGU G AGCU G AU AU UUUCAACCACAGUCAUAGUACAAUUAUAAGCUUUCAUAAUGUUGUCAAC ACU C AUUU GAGGACGAGU GC A AUC AC A A AGC GCU GAU GGA AC GU GAC AU GUUUUACAAAAUUCACUAUCCAUAAAAUGCUCAACACUCUUCUUGGCAU GUUCGCCACGAAAGUGAUGAGGCUUUUGUACAUGUUCUAGUAAAAAUCU AGAUAAUUGAGCGAAAGUCAUAGUAGGACAGCGUGUUUUGCUAUCAUAC CAUUGUUUAUCUUCGUCAUUCCAGUAAACUGGGGUAGAUUUAUUUGCUA GUACUUUGUACUUACGGACAUGAAAUACAUGAAGUUCAUGAUUUUUAUA UCCGUCUUCGGUGAGAUCUCCUUGGAGCUGUUGUUCACCAGGUUUCUUA UACUCUUCCCGAACCAUGGUCUCAAUAAAGAGAUCACGUCUAUAUGCACC ACCACCAGGUCGAAAGACCUUGGAAAUACCAGCAUCAUAAGAGUUGGUA GUCUUCAUAACAUACUUACAUGUAAAAGGAAUCAUACCUUUAUCUUCUA AGUUU GCUU G AUU GGU A AC AU AU GC A AC AGU AUUU GC A A AUU GG AU GC A CUUUGAAUUAGCGCCUCCUUUCUUUUGCAAAUUAAUAUCAUCAUUGAAU UGAUCAAUAUCAUCAAUAAUACAAACUUCAUGAGAAGCUUUAAAUUCUG ACAUAUAUUCAUCAUCUUCAUUAAAGACAUAUUUUAGUUUGGGAUCAAA UUUAGUACUUGAUACACCAAGAUAUCUAUCAUUUUCGUAUAAGCAAUGU AAUAGCUUAUCAGUGAGUGUAGAUUUACCCACACCUGGAGCACUGUACA AAACAAUAGAAAUUGGAGCUUUACGCUCGAUUUUAAUACAAAGUUUAUC AU C A ACU CU GU AUUU GU A AC GAG AU A A AGC AG AUU GUU GCUUUUU G AGG AUAGCUGCCUUAUACUUAUCCGCGGAAUAGAAACUAAGCAAUUUCUUUC CAGUUUCCAGAGUCUCAUCGACUUUACGUCGAUAAUCAUCCAAAGACAUU UCAUGAUUCGCUAGCAAAGCGAAUUUAUCACUAUAAUAAGUGAGAAAGU CAAAAUCCUUCUCAUAUUCGAUGAUUUCUUUAUCAUCAAUAUAUAUAGC GUUAUAAUUACCAGUUUCAAGAUAUAAAAUAACCUUGUCGGUAAUAAAA GUUGCACCAUCAAUAAUAUGAAGUGCAAUAUUUACAGCAUGAGUAUUGU UAUACUCCUUUUGUAAAUUCUUAGCGUGUAACUCGGAAAAUCCGAGCCA CGUGGAAUCAACUCCACUUUUAACAAAAAGUGGGGUACAAACAAGAAAA GAACAAAAAGUAGCAAAUUUCUUAGCGAAAUCACUAUUUUUAAUUUGUU CAGUUUUAGACAAACACUGUCUAAUAUCUUGUAAAAUCUUCUUGAAGGA CACCUUAUCUAUCGCACCUUGCGCAUAGGUAAGAUUUCCACCUUCUUUCU UAGGGCGUCUAUACAAAGACACACCAAGACACAAGAAAAUAAAUGAUAA AACAUAUUUUUCAUAUUUUCUUGUAAAUUGAGAAUACGUCAAGCCAAAC AUGUCUCGCAAAUACGAAUGUAUUGCGGUAAUGAAUGACAUAGGAGAAG UACACUCGGAUAACCGAAUAAAGAAAUUCGUAUGGAUAUACAAAUAUUC UU C A AU GUU AGUU G AUUU CUUU C C A A A A ACUUU GG A AG A A AU AGUUU C G UUUUUAGAGAUAACGGAAUCUAGCAUUUUAAUGUCUUGCUGAGACAAAA AUUU AAAAUAAUCCAUAAUUGAAGUAUUAUAUCGGGCAUUACCCGU AAA AACAGCCUUCAGCGGCUUAAACUGACUUAAGUUGUCACCUAUAAAUGUU CUCACUUAAGAGAAACUAAGGUACUUUUAUGGCAACUAGUACGAAACCC UUUGUUUUACUAAACAAAGAAAAGUUCAUCCUACAUAUUUAUAAUUAAA UUAAUAAUUAAUUGUCUAUAGGGUCGACAAAAUAAAAAAGGGGACCUGU UUCUCCUCUUUAUUUAUACAUAAAAAAUUUAAUCGUUUCAAAAAUGCUC GUCGUGAGAGCAGUUAGCGUUAAGGGGGGAACAGAGCCUAAUCCGCGAG AAUAAGUUCUGGAGCCAAUCUACCUGUAACUUACGUGUUUACAACACCAC AGUCGAUUUUCUCUGAACACCUCUAAUACUAAAAAGUUCACGAAUCUAC AUUAAUUCUGUCGUCUAAAUUUCCCUGCAAAAUAAAGCGGGACGCCAAG UUGGGCGCAGAUAAAAUUCUAUACAGCUCAUAAAGAGCUGUACAGUGUC CGACUCCCAGAACGGCCAGUUCGUGGUUAUGGAUUCCCACAGACUGUUGA GC GGGG ACU C AC C GU G AU GGGC CUC C C AGGUU C CU GGCU G AU CUUU C GGU GCCCGCUGUCCCCAAUC

SEQ ID NO: 2 (RNA sequence of ORF1 encoding pro-polypeptide 1)

AUGGAUUAUUUUAAAUUUUUGUCUCAGCAAGACAUUAAAAUGCUAGAUU C C GUU AU CUCU A A A A AC G A A ACU AUUU CUU C C A A AGUUUUU GG A A AG A A AUCAACUAACAUUGAAGAAUAUUUGUAUAUCCAUACGAAUUUCUUUAUU CGGUUAUCCGAGUGUACUUCUCCUAUGUCAUUCAUUACCGCAAUACAUUC GUAUUUGCGAGACAUGUUUGGCUUGACGUAUUCUCAAUUUACAAGAAAA U AU G A A A A AU AU GUUUU AU C AUUU AUUUU CUU GU GU CUU GGU GU GU CUU UGUAUAGACGCCCUAAGAAAGAAGGUGGAAAUCUUACCUAUGCGCAAGG UGCGAUAGAUAAGGUGUCCUUCAAGAAGAUUUUACAAGAUAUUAGACAG U GUUU GU CU A A A ACU G A AC A A AUU A A A A AU AGU G AUUU C GCU A AG A A AU UUGCUACUUUUUGUUCUUUUCUUGUUUGUACCCCACUUUUUGUUAAAAG UGGAGUUGAUUCCACGUGGCUCGGAUUUUCCGAGUUACACGCUAAGAAU UUACAAAAGGAGUAUAACAAUACUCAUGCUGUAAAUAUUGCACUUCAUA UUAUUGAUGGUGCAACUUUU AUU ACCGACAAGGUU AUUUU AUAUCUUGA AACUGGUAAUUAUAACGCUAUAUAUAUUGAUGAUAAAGAAAUCAUCGAA U AU G AG A AGG AUUUU G ACUUU CU C ACUU AUU AU AGU G AU A A AUU C GCUU UGCUAGCGAAUCAUGAAAUGUCUUUGGAUGAUUAUCGACGUAAAGUCGA U G AG ACU CU GG A A ACU GG A A AG A A AUU GCUU AGUUU CU AUU C C GC GG AU A AGU AU A AGGC AGCU AU C CU C A A A A AGC A AC A AU CU GCUUU AU CU C GUU ACAAAUACAGAGUUGAUGAUAAACUUUGUAUUAAAAUCGAGCGUAAAGC UCCAAUUUCUAUUGUUUUGUACAGUGCUCCAGGUGUGGGUAAAUCUACA CUCACUGAUAAGCUAUUACAUUGCUUAUACGAAAAUGAUAGAUAUCUUG GUGUAUCAAGUACUAAAUUUGAUCCCAAACUAAAAUAUGUCUUUAAUGA AG AU G AU G A AU AU AU GU C AG A AUUU A A AGCUU CU C AU G A AGUUU GU AUU AUU G AU G AU AUU G AU C A AUU C A AU G AU G AU AUU A AUUU GCA A A AG A A AG GAGGCGCUAAUUCAAAGUGCAUCCAAUUUGCAAAUACUGUUGCAUAUGU UACCAAUCAAGCAAACUUAGAAGAUAAAGGUAUGAUUCCUUUUACAUGU AAGUAUGUUAUGAAGACUACCAACUCUUAUGAUGCUGGUAUUUCCAAGG UCUUUCGACCUGGUGGUGGUGCAUAUAGACGUGAUCUCUUUAUUGAGAC CAUGGUUCGGGAAGAGUAUAAGAAACCUGGUGAACAACAGCUCCAAGGA GAU CU C AC CGA AGAC GGAU AU A A A A AU C AU GA ACUU C AU GU AUUU C AU G UCCGU AAGUACAAAGUACU AGC AAAUAAAUCUACCCC AGUUU ACUGGAA UGACGAAGAUAAACAAUGGUAUGAUAGCAAAACACGCUGUCCUACUAUG ACUUUCGCUCAAUUAUCUAGAUUUUUACUAGAACAUGUACAAAAGCCUC AU C ACUUU C GU GGC G A AC AU GC C A AG A AG AGU GUU G AGC AUUUU AU GG A UAGUGAAUUUUGUAAAACAUGUCACGUUCCAUCAGCGCUUUGUGAUUGC ACUCGUCCUCAAAUGAGUGUUGACAACAUUAUGAAAGCUUAUAAUUGUA CUAUGACUGUGGUUGAAAAUAUCAGCUCACUCAUUGUAUUGUGUACAAU UUUUCAGUUAGCCAUUUAUUGGCUACUUCAUAAAGUAUGCGCAAAAGCU UAUAGAAAUAAUGAACCUAAUU AUUUU AC AAAAAAGGUCGUUAUUUGUA UAAGAAAAUUACCUUCGAGGUUUGGAGUGCCUUGGUGGUUUACCUAUUU GCCUAUGUUUCUUGCUACUAGGUGUCUUUGUGACACUUGGCAAGAUUGG GCUACGAACGCUUGU AGACGUGCCUAUGGGCAUGGU AUUUU AGUUCAGU CUAUUAGACAUGACUUAUCUAAAUAUUAUAACAAGUAUUCGGUUGGUGG UACUGUUGCACUUUUAGGGUGCUUGUAUACUGUAGUACAUUUGUACUCA GCU AUU C A AC C AC A AU C A A A AC C AGAGGUU GAU GA A A AUC A AU CU GACU CCAACUAUUGGUCAGCUCAAUAUGAAAAUAUUACACGAUUGAGUGGACC UCCUAGUACAGUAACUUUUGACCAGUUAGUCAAUAGUGUUGAGCUAAAC UGCUGUCAUCUGAUAGCAGAAAAUCUCGCCGAGCGACAAUAUCAUAGGA UAUGUGGUCUCGGACUGUACGGAAAUGUUAUGGCUUUGCCUAAACAUUU CUUUGAUUCUCUUAAAGAUGUAUUUCCUCUUAGAAUGGAUAUCAUUAGA CACGAUAUUACCAAACAAGGAGGACCUAGCAGAUUUAAGGUCUUAGUUG AU G A AU C AU GUUUU G A AUUU G AU G A AC GCU AU GG AC C A AGU G AU GUU AU AUUCUUUAGACAUUCAGCUUUAGGAACUUUUCGCGAUGUGCGUAAGUUC CUAUUACCUGGAAUGAUUAAAGGGAAAACAUCUGGUACAAUGGUUAUUC GUCAAAGAGACGGUAUUAUGGCUUAUAAAGAUUUUGAAGCUAUGCAAAC UAGUCCCAUCUCUUAUAAUGAUAAAUAUUUUUCAUAUGCUUAUAAUGGU UAUAUGGCAUACACCAAAGUGCCAACUAUUAUAGGCGAAUGUGGAGCAC CAUACAUCGUUAAGACCCCUAAUGGGUGUUUUAUCGCUGGUUUCCAUGU UGGUGCAAAGAAAGCUACCUUUGGUGGUAGAUAUAAAGUCUUUGCAGCC UGUUUAAUCAACCCAGAUUUGGAUGAUUCCAAAUUUACUCCAUUAUCAU AUAAUGGCGUUGAUUUAAACGAACACUAUGAGACAGUAGAAGAUCUAUC AAUUACUCAAACCCAAGACAAGAAAUGUCCUAUCAGACUAACUGAUGGA GGUUCAAUGAUGAUGAUUGGUUCUAUUAAUACUCAUAGACGUAAAAUGA AAACUAUGGUUUGUCAUACUAUUAUGGCAGAACGUAUUUUACAGCAUUA UGGUAGAUUAGAUUUUACACAUUUUUCACCAAAAGGUAUUAAAUCCUCU UUGGCGUGUAAACAAAAUGUUGCCCCUAUGUUUGUAAAACCUAGUUUUC CAGCCAAGAAUAUUCAACGCGCAGAAGACGCGUUAUAUGCUUGGUUCAC AAGGAAGAUCGAAGAACAUAAAUUCUUUAUUCCCUGUGAGCCCUACGAU AUCGACGUAGGUGUCAAUGGAUAUGACGGAGCGUCAUAUAUUGACAGAA UU C CU GC AU CU AC AU C AGGU GGUUUU GCU C AU A A AGG AGCU A A AU C C A A AUAUCUACAUUUAGGUGAUCCUGAACCAGGACACACCGUAAAAUAUGAA CUUAAUGAUAAUAUUAUGCUAGAAUUCAAUCGCCUCAAAGAAAAAUACU U GAG AGGU G A AC GC GGC G AU AUU GUUU GGG ACUU C A AUUUU A A AG AU G A GCCCGUUUCCGAGUCAAAACUCUUGAAACAGAAAUGUAGAAUUUUUAAC AGUGGACCACUUCAUUUUAUGAUUAUGGUCCGCAUGUACUACCUUUGGA UUAUUCCAUUAGUUUCUGGUAAAAAGAGAACUGAAUUCGGUAUGGCCAU CGGUGCCAAUGCUCAUGGAGAUGAUUGGACUGCAAUUUAUGAAUAUAUU ACCCAACAUGGAAAGAAUAAUAUGAUUGCGGGCGAUUAUAAAGCAUUCG AUAAGAAUAUGCCUCCUGAGCUUAUGACUGCUAGUUUUAAUGUUCUCAU UAGAAUUUGCCGUGACAAUGGUUGGUCUGAUGAAGACUUAACUAUUAUG CGCGGUCUAGCUACUGAUAUUUGCUAUCCACUAUCCAAUGUUUUUGGAA CCGUGGUAGGUAUGUUUGGUAGUAACCCUAGUGGUCAUCCUCUUACUAC UCCAAUUAAUGGCAUGUGCAACAUUAUGUAUAUGAUGCUAGCUGCCAUG G AU AUU G A AG A AG A A AG AGGU GUU C A AG A A AU AG AUU AU GCU AG AUUU C AAGAUUCACUUGCCAUUUUAACUUAUGGCGAUGAUAAUUGUGCUUCUUC UAGUAUUCCUUGGUUAAAUCAUACCACCAUUAGUACUGCUCUUUCUAAA AGAGGAGUUACUUAUACAAUGGCUGAUAAAGAGUCUGAAUCUGUACCUU UUAUUAACGUUAAAGAUGUUGAUUUUCUUAAGAGAAAAUUUACUCCAUC UGUUUAUGUUCAAGGUGUAAUUCAAGCUCCUUUAGAUGAAGGAUCUAUU CUU AAAAGUCUUAGUGUUUGCACUAGAUCUAAGACU AUU ACUUUUAAAG AACAGUGUGCUCAAAUUAUUUCUUCCGCCAAUACGGAAUAUUUCCAAUA UGGUAAGAAGAAAUUUGUACGGGAAAAUACAUUUCUCAACGAAUUAUUA GAUGAAUUCGAUUUGAGAUGUUAUUUACCCAAUUGUGCUCUUAAAACUU UU G AU G A ACUUU AU AUU G A A AGGUUU GG AG AGUU GU A A

SEQ ID NO: 3 (RNA sequence of ORF2 encoding pro-polypeptide 2)

AUGAUAGAAUAUGAGAGAGCCUUAGGUUAUUCUCUUCCUACUCUACAUG AAGAAACCAAACAACCAGAAUCUACUACUAAUGAUUCAGAUAUUACUGU AC A A AGU GC AGUU GA AGC A A A AGU AGA AC AU GA AGAUU CU GGU CUU C A A GU C GAG AC A A AU GUU A AUUUUU CU G AC C A AGUU G A AC A AU AU C A ACUUU UGGUCGGUAAACCUUUGUCAGAUCCGACUUACGAAAAGGCUAAUUCACA AGCUGUUGAUUUGACUACGUUUAUGUCCCGUCCUAUUAGAAUUUUUUCU A AG AUUU GGG A AGU A A AU G AGU CUC C AC A AU AU GU G A AU GCU AUU A AU C CUUGGGAUUUAUUCCUUAGUGAUUCUAAAGUUGCUAACAAAAUAGAAAC UUU C A A AUU ACUU C AU GG A ACU CUU A A ACU C A AG AUUUU GGUU A AU GGU UCACCUUUUCAUUAUGGUCGAAUGUUUGUUGGUCUUAGACCUUCAAAAU UCGACAACAAUACACUUACUGAUGGUCCUGUAACUCCAGGUGUUAGUUU AUCCUAUACGGAUGAAAAUGCAGCUGGUAGUAAUAAGACAAUGAAUAAC AUGGCUUGCUUAUACUCGCAACGCCCACAUGUAUUUAUUGAUCCAUCAAC AAAUCAACCACAACAAAUCUCGUGGCCUUUCUUUUCGGCCACCAAUUGGA UUGAUUUAACUGAUCAAGAAACUAUUGAUCGUAUGGGUGUAUUGGAAAU UUGGGAAUUAACCCAACUACAACAUUCCAACGGUGCCACUGAUGAAGUU G A A AUUU C A AUUUUU GC GU GG AU GG A AG AU GU AG A AUU C GCU GGU CUU A CAGCUGCUGCUCCUGCAACAGCUCAGAUGGCUGUUGAUAAGAUUCAAAAC AGUAAGAAGACCAAGAAGAGAAAACCAAAGAAGAAAGCUAAACCAACGU UUACCAAUACUUCUGGAGAAGAUGAACACAAACCUGAUGGUGUAGUGUC CGCUCCUGCAGCUUUAUUGGCCGACUUUGCUGGCUAUUUUACAGAAAUAC CAUAUAUUGGUAAAUUUGCAAAAUCAACCCAGAUAGCGAGUGGUGCUGU UAGCAGCAUUGCGCGUCUUUUUGGAUUUUCACGACCUGCAGUUCUAACA GAUACUGCGUUUUAUAAAUCGCAGCCUAUUGGCAAUUUAGCCAAUACAU CAGGCGCAGAUCCUAUCUUCAAAUUAACUCUUGAUCCCAAGCAAGAGCUC ACCAUUGACCCCACCACCGUUGGGUUAGGUGAAGAAGAUCAAAUGUCUU UUGGAUAUUUGGUCAAGCGUGAAGCUUUCAUUGAUUACUUCAAUUGGAG UACAAUUGUAGCCCAGAACACUGGGCUCUUGUAUUCUAUUCAAGUACAU CCAAUGAUAGCACCAAUCUAUCAAACUGGUUCUAAUGAUACAGUGGUAA GAUGUCAAACUCCUUUGAGUUUUGUUUCUUAUCCUUUCAAUAACUGGUC UGGAAGUCUAAGAUAUAGAUUUCAGAUUGUGGCUUCACAAUAUCAUAGA GGACGAUUAUUGUUUGUUUAUGAACCCACGUUAUCAACGGCGGGUACUG UGACAGAUACUAAUGACCGUUACUCUCAUAUUGUUGAUAUUUCAGAAGA ACGCGAUGUUACUUUUGAAAUCAACUGGACCCAGAAAGAAGCUUAUAGA A AG AUU G AU AU CUUU AG AGCU C A A A A ACUUU CU GCUU GGG AGGGU GCU A CUGGUGCUAUAGGUACAGAUGCUGAUGAUGUUGCUAAUUGCAAUGGUCG UCUUAAUGUAUAUGUACUUAAUGCACUUGCUGCUCCUAUUACGGAUAGC AGCGUUUCCGUGAAUGUCUUCAUUUCCGGUGGUGAUUCUUACGAAGUGA GA A AUCC AC GU GGA AC AUU AGGU C A AGAU GUU GCUU AU GC A A ACU CU AC GACUGAUGUUGGUCCACCUACCUUAGCACAAAUGGCUACAGAAGGUAUG ACCAUGGACGAAAAUUUGCCAGAACAAGAUACUACUUAUGUUCUUAAUG GUGAAUACACGGAAUUCUGUAAGGAACAAUCUCACGUAUAUUACGGUGA GGCUGUUGUUUCCUUCAGAUCUUU ACUUAAGAGAUAUAAUU ACUUU AGG ACUUUAGAGCCUUUAGAAUCUCUUAAUGUAAACAAUUGGUACACAGUUA UGUACCGAACUUUUAUCUACCCUCAGGGUCCUGGUCCAUCUUAUGGAUCC AGUAUUGCUUCUGCAAUGACCCCUAUCGCAGGCCCAAUAAAUUAUAAUU UGUUACCUAUGACCAUGAUGCGUUAUUGCAUGCAGGCAUAUGUUGGCUU U C G AGG AGG AU GU C GUU GG A A AGU GCU AU AUU GU GGC AC A A AU A AC C A A UUACAACCUUUAUCGGUAGGUAGAAAUCCCGAGUACUUGUCAAACGAAU CGCAAGUUAAUAUUAUUAACGAUAACGGUUCAUACAGUUUGUCAAGAGU UGUCCGCGAAAAAUGGCGUGCGGACAAUUGCCAAGACAGUGCAGCUGGA ACAGCUUUAACUGGCGUGGCUUCUCAACCUGGUUUGGAGUAUGAACUCCC UUUU C A A AC AGC AUU A AGAU AU GCU G A AU GU A AU GA AC C AGG AC A A ACC GAGGUUCCUCCUUAUACAGGAUUGAAUCAAGGUAGCCAAACUGUAUCCU UUGAUUAUCGCGAAACUGCGAAUCAAUAUCAUUGGUUACAAUUUUACAC UGCAGCAGCAGAAGAUUUUUCCCUAUUCUUUUUCAUAGGGGCUCCUGCUC GCAUACCAAGUGACGUAAAUAUUGUCACUCUCUAA

SEQ ID NO: 4 (Amino acid sequence of pro-polypeptide PI)

VRTFIGDNLSQFKPLKAVFTGNARYNTSIMDYFKFLSQQDIKMLDSVISKNETIS SKVFGKKSTNIEEYLYIHTNFFIRLSECTSPMSFITAIHSYLRDMFGLTYSQFTRK YEKYVLSFIFLCLGVSLYRRPKKEGGNLTYAQGAIDKVSFKKILQDIRQCLSKTE QIKN SDF AKKF ATF C SFL VCTPLF VK S GVD S TWLGF SELHAKNLQKEYNNTH A VNIALHIIDGATFITDKVILYLETGNYNAIYIDDKEIIEYEKDFDFLTYYSDKFALL ANHEMSLDD YRRKVDETLET GKKLL SF Y S ADKYK AAILKKQQ S ALSRYKYRV DDKLCIKIERKAPISIVL Y S APGVGKSTLTDKLLHCLYENDRYLGV S STKFDPKL KYVFNEDDEYMSEFKASHEVCIIDDIDQFNDDINLQKKGGANSKCIQFANTVAY VTN Q ANLEDKGMIPF T CK YVMKTTN S YD AGI SK VFRPGGGA YRRDLFIETM VR EEYKKPGEQQLQGDLTEDGYKNHELHVFHVRKYKVLANKSTPVYWNDEDKQ W YD SKTRCPTMTF AQL SRFLLEH V QKPHHFRGEHAKK S VEHFMD SEF CKT CH VPSALCDCTRPQMSVDNIMKAYNCTMTVVENISSLIVLCTIFQLAIYWLLHKVC AK AYRNNEPNYFTKK VVICIRKLP SRF GVPWWFT YLPMFL ATRCLCDTW QDW ATNACRRAY GHGIL V Q SIRHDL SK YYNK Y S VGGT VALLGCL YT VVHL Y S AIQP Q SKPE VDEN Q SD SNYW S AQ YENITRL S GPP S T VTFDQL VN S VELNCCHLI AENL AERQYHRICGLGLYGNVMALPKHFFDSLKDVFPLRMDIIRHDITKQGGPSRFKV LVDESCFEFDERYGPSDVIFFRHSALGTFRDVRKFLLPGMIKGKTSGTMVIRQRD GIMAYKDFEAMQTSPISYNDKYFSYAYNGYMAYTKVPTIIGECGAPYIVKTPNG CFI AGFH V GAKK ATF GGRYK VF A ACLINPDLDD SKF TPL S YNGVDLNEHYET V EDLSITQTQDKKCPIRLTDGGSMMMIGSINTHRRKMKTMVCHTIMAERILQHY GRLDF THF SPKGIK S SL ACKQN V APMF VKP SFP AKNIQRAED AL Y AWF TRKIEE HKFFIPCEPYDIDVGVNGYDGASYIDRIPASTSGGFAHKGAKSKYLHLGDPEPG HTVKYELNDNIMLEFNRLKEKYLRGERGDIVWDFNFKDEPVSESKLLKQKCRIF N S GPLHFMIMVRM YYLWIIPL V S GKKRTEF GM AIGANAHGDD WT AIYE YIT QH GKNNMIAGDYKAFDKNMPPELMTASFNVLIRICRDNGWSDEDLTIMRGLATDI C YPLSNVF GTVV GMF GSNPSGHPLTTPINGMCNIMYMML AAMDIEEERGVQEI D Y ARF QD SL AILT Y GDDNC AS S SIPWLNHTTIST AL SKRGVT YTM ADKESES VP FINVKDVDFLKRKFTPSVYVQGVIQAPLDEGSILKSLSVCTRSKTITFKEQCAQII S S ANTEYF Q Y GKKKFVRENTFLNELLDEFDLRCYLPNC ALKTFDEL YIERF GEL SEQ ID NO: 5 (Amino acid sequence of pro-polypeptide P2)

MIE YERALGY SLPTLHEETKQPE S TTND SDIT V Q S AVEAK VEHED S GLQ VETN V

NF SDQVEQ Y QLLVGKPLSDPTYEKANSQ AVDLTTFMSRPIRIF SKIWEVNESPQ YVN AINP WDLFL SD SK V ANKIETFKLLHGTLKLKIL VNGSPFH Y GRMF V GLRP S KFDNNTLTDGPVTPGVSLSYTDENAAGSNKTMNNMACLYSQRPHVFIDPSTNQ PQQISWPFFSATNWIDLTDQETIDRMGVLEIWELTQLQHSNGATDEVEISIFAW MED VEF AGLT A A AP AT AQM A VDKIQN SKKTKKRKPKKK AKPTF TNT S GEDEH KPDGVV S AP AALLADF AGYFTEIP YIGKF AKSTQIASGAV S SIARLFGF SRPAVLT DTAFYKSQPIGNLANTSGADPIFKLTLDPKQELTIDPTTVGLGEEDQMSFGYLVK REAFIDYFNWSTIVAQNTGLLYSIQVHPMIAPIYQTGSNDTVVRCQTPLSFVSYP FNNWSGSLRYRFQIVASQYHRGRLLFVYEPTLSTAGTVTDTNDRYSHIVDISEE RD VTFEINWTQKE A YRKIDIFRAQKL S A WEGAT GAIGTD ADD V AN CN GRLN V Y VLNAL AAPITDSSVSVNVFISGGDSYEVRNPRGTLGQDVAYANSTTDVGPPTLA QM ATEGMTMDENLPEQDTT YVLNGEYTEF CKEQ SHVYY GE AVV SFRSLLKRY NYFRTLEPLESLNVNNWYTVMYRTFIYPQGPGPSYGSSIASAMTPIAGPINYNLL PMTMMRY CMQ A Y V GFRGGCRWK VL Y CGTNN QLQPL S V GRNPEYL SNES Q VN IINDNGS Y SL SRVVREKWRADNCQD S AAGT ALT GVASQPGLEYELPF QT ALRY AECNEPGQTEVPPYTGLNQGSQTVSFDYRETANQYHWLQFYTAAAEDFSLFFFI GAP ARIP SD VNI VTL SEQ ID NO: 6 (Amino acid sequence of capsid-like polypeptide CPI)

MIE YERALGY SLPTLHEETKQPE S TTND SDIT V Q S AVEAK VEHED S GLQ VETN V NF SDQVEQ Y QLLVGKPLSDPTYEKANSQ AVDLTTFMSRPIRIF SKIWEVNESPQ YVN AINP WDLFL SD SK V ANKIETFKLLHGTLKLKIL VNGSPFH Y GRMF V GLRP S KFDNNTLTDGPVTPGVSLSYTDENAAGSNKTMNNMACLYSQRPHVFIDPSTNQ PQQISWPFF SATNWIDLTDQETIDRMGVLEIWELTQLQHSNGATDEVEISIFAW MED VEF AGLT A A AP AT AQM A VDKIQN SKKTKKRKPKKK AKPTF TNT S SEQ ID NO: 7 (Amino acid sequence of capsid-like polypeptide CP2)

SRPAVLTDTAFYKSQPIGNLANTSGADPIFKLTLDPKQELTIDPTTVGLGEEDQM

SFGYLVKREAFIDYFNWSTIVAQNTGLLYSIQVHPMIAPIYQTGSNDTVVRCQTP

LSFVSYPFNNWSGSLRYRFQIVASQYHRGRLLFVYEPTLSTAGTVTDTNDRYSH I VDI SEERD VTFEINWT QKE A YRKIDIFRAQKL S AWEGAT GAIGTD ADD V AN CN GRLNVYVLNALAAPITDSSVSVNVFISGGDSYEVRNPRGTLGQDVAYANSTTD VGPPTLAQMATE

SEQ ID NO: 8 (Amino acid sequence of capsid-like polypeptide CP3)

GMTMDENLPEQDTT YVLNGEYTEF CKEQ SHVYY GE AVV SFRSLLKRYNYFRT LEPLESLNVNNWYTVMYRTFIYPQGPGPSYGS SIASAMTPIAGPINYNLLPMTM MRY CMQ A Y V GFRGGCRWK VL Y CGTNN QLQPL S VGRNPE YL SNE S Q VNIINDN GS Y SL SRVVREKWRADNCQD S AAGT ALT GVASQPGLEYELPFQT ALRY AECNE PGQTEVPPYTGLNQGSQTVSFDYRETANQYHWLQFYTAAAEDFSLFFFIGAPAR IPSDVNIVTL SEQ ID NO: 9 (Amino acid sequence of capsid-like polypeptide CP4)

GEDEHKPDGVV S AP AALL ADF AGYFTEIPYIGKF AKSTQIASGAV S SIARLF GF

SEQ ID NO: 10 (DNA nucleic acid encoding 18sRNA of Viva lactuca)

ATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAA GTATAAACAGTTTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTAGA GTTTATTTGATGGTACCACACTACTCGGATAACCGTAGTAAAGCTACAGCTA ATACGTGCGTAACTCCCGACTTACGAAGGGACGTATTTATTAGATTCAAGGC CGACCGTGCTTGCACGTCTTTGGTGAATCATGGTAACTTCACGAATCGCAGG GTCTATCCCGGCGATGTTTCATTCAACTTTCTGCCCTATCAACTTTCGACGGT AGT AT AGAGGACT ACCGTGGTGGT AACGGGT GACGGAGGATT AGGGTTCGA TTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCA GGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACAATAAATATC AATTCTGGGCCACATGGTCCGGTAATTGGAATGAGTACAATGTAAACGCCT TAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTC C AGCTCC AAT AGCGT AT ATTT AAGTTGTT GC AGTT AAAAAGCTCGT AGTTGG ATTTCGGGTGGGACGCAGCGGTCTCGCATTGCGTTTGTACTGCTGCAGCCCT CCTTCTTGCCGGGGGCGGCCCTTCGGACTTCACTGTCTGGGGGTCAGAATCG GCGATGTTACTTTGAGTAAATTAGAGTGTTCAAAGCAAGCTTACGCTCTGAA TATAATAGCATGGGATAACACGACAGGACTCTGGCCTATCGTGTTGGTCTAT AGGACCAGAGTAATGATTAAGAGGGACAGTCGGGGGCATTCGTATTCCGTT GT C AGAGGTGAAATTCTT GGATTT ACGGAAGACGAAC ATCTGCGAA AGC AT TTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTTGGGGGCTCGAAGAC GATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGATTGG CGGGTGTTTTTTTGATGACCCCGCCAGCACCTCATGAGAAATCAAAGTTTTT GGGTTCCGGGGGGAGT AT GGTCGC AAGGCTGAAACTT AAAGGAATTGACGG AAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGA AAACTTACCAGGTCCAGACATAGGAAGGATTGACAGATTGAGAGCTCTTTC TTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTG TCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTGACGT CTGCTGCGGCAGTCGCGCGCTTCTTAGAGGGACTGTTGGCGTCTAGCCAATG GAAGTATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGC ACGCGCGCTACACTGATACGTTCAACGAGCTCCTTGACCGAGAGGCCCGGG TAATCTTTGAAACCGTATCGTGATGGGGATAGAACATTGCAATTATTGTTCT TCAACGAGGAATGCCTAGTAAGCGCGAGTCATCATCTCGCGTTGATTACGTC CCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAACGTGCTGGTG AAGCGTTAGGACTGGAACTTTGGGCCGGTCTCCTGCTCATTGTTTCGGGAAT TTCGTTGAACCCTCCCGTTTAGAGGAAGGAGAAGTCGTAACAAGGTCTCCG TAGGTGAACCTG

Claims

1. An isolated ribonucleic acid molecule having at least 75% sequence identity with SEQ ID NO: 1, over the entire length.

2. The isolated ribonucleic acid molecule according to claim 1, wherein the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least 75% sequence identity with SEQ ID NO: 2, over the entire length, preferably encoding a first pro-polypeptide (pro-polypeptide PI).

3. The isolated ribonucleic acid molecule according to claim 1 or 2, wherein the ribonucleic acid molecule comprises a ribonucleic acid sequence having at least

75% sequence identity with SEQ ID NO: 3, over the entire length, preferably encoding a second pro-polypeptide (pro-polypeptide P2).

4. The isolated ribonucleic acid molecule according to any one of claims 1 to 3, wherein the sequence of the ribonucleic acid molecule consists of SEQ ID NO: 1.

5. An isolated pro-polypeptide PI having at least 75% amino acid identity with SEQ

ID NO: 4, over the entire length.

6. The isolated pro-polypeptide PI according to claim 5, wherein pro-polypeptide PI is susceptible to be cleaved into two or more viral-like polypeptide(s) selected in the group consisting of a helicase, a 3C or 3C-like protease, and a RNA-dependent RNA polymerase.

7. An isolated pro-polypeptide P2 having at least 75% amino acid identity with SEQ ID NO: 5, over the entire length.

8. The isolated pro-polypeptide P2 according to claim 7, wherein pro-polypeptide P2 is susceptible to be cleaved into two or more viral capsid-like polypeptide(s).

9. A green alga cell comprising the ribonucleic acid molecule as defined in any one of claims 1 to 4, and/or expressing pro-polypeptide PI, as defined in claim 5 or 6, and/or expressing pro-polypeptide P2, as defined in claim 7 or 8.

10. The green alga cell according to claim 9, wherein pro-polypeptide PI is encoded by a ribonucleic acid molecule according to claim 2, and/or pro-polypeptide P2 is encoded by a ribonucleic acid molecule according to claim 3.

11. The green alga cell according to claim 9 or 10, wherein the green alga cell belongs to the genus Ulva.

12. An algicide composition comprising at least one ribonucleic acid molecule according to any one of claims 1 to 4, and/or at least one pro-polypeptide PI according to claim 5 or 6, and/or at least one pro-polypeptide P2 according to claim 7 or 8.

13. A method for the biological control of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule according to any one of claims

1 to 4, and/or a pro-polypeptide PI according to claim 5 or 6, and/or a pro polypeptide P2 according to claim 7 or 8, and/or an algicide composition according to claim 12.

14. A method for preventing a bloom of a green alga, in particular a green alga belonging to the genus Ulva, comprising the step of contacting the green alga with an effective amount of a ribonucleic acid molecule according to any one of claims 1 to 4, and/or a pro-polypeptide PI according to claim 5 or 6, and/or a pro polypeptide P2 according to claim 7 or 8, and/or an algicide composition according to claim 12.

15. The method according to claim 13 or 14, wherein the biological control is to be performed in a marine environment.