High-resolution comparative atomic structures of two Giardiavirus prototypes infecting G. duodenalis parasite.

The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Viruses of protozoan parasites and viral therapy: Is the time now right?

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.

Why we need a European focus on foodborne parasites.

Foodborne parasites (FBP) are recognized as being a neglected pathogen group, often associated with marginalized or disadvantaged populations, especially those living in regions where water supply or sanitation are inadequate. Nevertheless, we are also increasingly recognising that FBP are not just restricted to such places, and even those that do have a circumscribed endemic area may also travel further in our globalised world; FBP are relevant everywhere, including Europe. Against this background, COST Action Euro-FBP (FA1408) was established and ran for a period of 4 years, addressing a number of different questions related to FBP, particularly in the European setting. In this special issue (SI), some of the issues and outputs associated with Euro-FBP are considered in greater depth, as an output also of the final Euro-FBP meeting. As well as more general issues regarding, for example, globalization and climate change, use of economic models, and the value of risk-based surveillance that puts the topic in perspective, individual articles are included that address specific parasites. These include protozoan parasites, such as Cryptosporidium, Giardia, and Toxoplasma, as contaminants of water, shellfish, and fresh produce, fishborne parasites such as Anisakid nematodes, and meatborne parasites, such as Trichinella. Some of the works provide specific data on occurrence or outbreaks, whilst others are concerned with techniques. In addition, implementation of some of the educational and collaborative tools that are unique to COST Actions are described. COST Actions are not generally intended to deliver a scientific endpoint, and Euro-FBP does not do so. However, the articles in this SI, along with other articles published elsewhere during and subsequent to the course of the Action, as direct outputs of the Euro-FBP activities, indicate that FBP are indeed a relevant topic for European scientists.

Why do we need training? - A "Training school on molecular methods used for foodborne parasite diagnostics in different matrices" is a example of knowledge transfer to foster research quality in EU.

Foodborne parasites with zoonotic potential are of particular concern for human health, being responsible for serious and potentially life threatening diseases. In the last decades, the development of molecular biology techniques have been successfully implemented for clinical diagnosis of FBPs in animal or human samples providing cheaper, less labor intensive, reliable and more sensitive tests. It is apparent from recent publications that unsubstantiated molecular methods for parasite detection that have undergone scant evaluation for sensitivity and specificity are becoming increasingly common. The aim of the organized Training Schools was to transfer knowledge on application, optimization and troubleshooting for methods used to extract, amplify, and sequence nucleic acids from contaminated matrices and isolated FBPs. The organized Training Schools fulfilled the trainees' expectations, whom acquired useful knowledge for their research activities.

Comparative evaluation of loop-mediated isothermal amplification (LAMP) vs qPCR for detection of Toxoplasma gondii oocysts DNA in mussels.

The apicomplexan parasite Toxoplasma gondii can infect humans and cause toxoplasmosis. T. gondii has been highly prioritized among the foodborne parasites regarding its global impact on public health. Human infection can occur through multiple routes, including the ingestion of raw or undercooked food contaminated with T. gondii oocysts, such as fresh produce and bivalves. As filter-feeders, bivalves can accumulate and concentrate contaminants, including protozoan (oo)cysts. Although detection of T. gondii in different bivalves by molecular techniques (PCR and qPCR) has been achieved, routine application is currently limited by lack of sensitivity or equipment costs. Here, we describe the assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect T. gondii oocysts in spiked mussels. Detection limit was down to 5 oocysts/g in tissue and 5 oocyst/ml in hemolymph, and, under the experimental conditions tested, LAMP was found to provide a promising alternative to qPCR.

Testing the impact of Whole Genome Amplification on genome comparison using the polyploid flagellated Giardia duodenalis as a model.

The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.

Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready-to-eat salad.

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a foodborne zoonosis with a global distribution and estimated to cause up to 20% of the total foodborne disease burden in Europe. Association between T. gondii infection and the consumption of unwashed raw fruits and vegetables contaminated with oocysts has been reported and the increasing habit to eat pre-washed ready-to-eat salads poses a new potential risk for consumers. It is therefore important to trace the occurrence of potential contamination with this parasite to guarantee the safety of ready-to-eat vegetables. Detection of T. gondii in vegetables by molecular techniques has been achieved but low sensitivity (PCR) or expensive equipments (qPCR) limit routine applicability. Here, we describe the development and validation of a sensitive and robust method relying on a LAMP assay, targeting the 529 bp locus, to detect T. gondii oocysts down to 25 oocysts/50 g in ready-to-eat baby lettuce. The LAMP has been also adapted for a faster visualization of the result by a lateral flow dipstick chromatographic detection method.

Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions.

Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies.

Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.

Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

Giardia duodenalis 14-3-3 protein is polyglycylated by a tubulin tyrosine ligase-like member and deglycylated by two metallocarboxypeptidases.

The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis.

Dematin, a component of the erythrocyte membrane skeleton, is internalized by the malaria parasite and associates with Plasmodium 14-3-3.

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.

The Nitrobenzoxadiazole Derivative NBDHEX Behaves as Plasmodium falciparum Gametocyte Selective Inhibitor with Malaria Parasite Transmission Blocking Activity.

This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.

Contamination of Soil, Water, Fresh Produce, and Bivalve Mollusks with Toxoplasma gondii Oocysts: A Systematic Review.

Toxoplasma gondii is a major foodborne pathogen capable of infecting all warm-blooded animals, including humans. Although oocyst-associated toxoplasmosis outbreaks have been documented, the relevance of the environmental transmission route remains poorly investigated. Thus, we carried out an extensive systematic review on T. gondii oocyst contamination of soil, water, fresh produce, and mollusk bivalves, following the PRISMA guidelines. Studies published up to the end of 2020 were searched for in public databases and screened. The reference sections of the selected articles were examined to identify additional studies. A total of 102 out of 3201 articles were selected: 34 articles focused on soil, 40 focused on water, 23 focused on fresh produce (vegetables/fruits), and 21 focused on bivalve mollusks. Toxoplasma gondii oocysts were found in all matrices worldwide, with detection rates ranging from 0.09% (1/1109) to 100% (8/8) using bioassay or PCR-based detection methods. There was a high heterogeneity (I2 = 98.9%), which was influenced by both the sampling strategy (e.g., sampling site and sample type, sample composition, sample origin, season, number of samples, cat presence) and methodology (recovery and detection methods). Harmonized approaches are needed for the detection of T. gondii in different environmental matrices in order to obtain robust and comparable results.

MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms.

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: organization of the protein domains affects the protein secretion pathway.

Cpa135 is a multidomain antigenic protein secreted at the sporozoite stage of the Apicomplexa protozoan Cryptosporidium parvum. Previous studies have shown that the protozoan flagellate parasite Giardia duodenalis is a suitable system for the heterologous expression of secreted proteins of Apicomplexa. Here, we designed three different Cpa135 variants fused to a C-terminal HA tag in order to test their expression in G. duodenalis under the control of the inducible promoter of the cyst wall protein 1 gene (cwp1). The three Cpa135 chimeras encompassed different portions of the protein; CpaG encodes the entire polypeptide of 1574 amino acids (aa); CpaGΔC includes the first 826 aa at the N-terminus; and CpaGΔN consists in of the final 833 aa at the C-terminus. Immunoblot experiments showed that CpaG and CpaGΔN maintained the epitopes recognized by anti-C. parvum-specific human serum. The intracellular localization and transport of the three Cpa135 variants were studied by immunofluorescence in combination with G. duodenalis-specific antibodies. CpaGΔC was mainly accumulated in the endoplasmic reticulum and the intact form was also excreted in the medium. Differently, the Cpa135 chimeras possessing an intact C-terminus (CpaG and CpaGΔN) were transported towards the forming cyst wall possibly and were not detected in the medium. Furthermore, the full-length CpaG was incorporated into the cyst wall. The data presented suggest that the C-terminus of Cpa135, which includes a cysteine reach domain, could influence the secretion of the chimeric proteins.

Molecular Methods for the Detection of Toxoplasma gondii Oocysts in Fresh Produce: An Extensive Review.

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.

Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates.

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/-2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.