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Bioprocesses converting carbon dioxide with molecular hydrogen to methane (CH4) are currently being developed to enable a transition to a renewable energy production system. In this study, we present a comprehensive physiological and biotechnological examination of 80 methanogenic archaea (methanogens) quantifying growth and CH4 production kinetics at hyperbaric pressures up to 50 bar with regard to media, macro-, and micro-nutrient supply, specific genomic features, and cell envelope architecture. Our analysis aimed to systematically prioritize high-pressure and high-performance methanogens. We found that the hyperthermophilic methanococci Methanotorris igneus and Methanocaldococcoccus jannaschii are high-pressure CH4 cell factories. Furthermore, our analysis revealed that high-performance methanogens are covered with an S-layer, and that they harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase. Thus, high-pressure biological CH4 production in pure culture could provide a purposeful route for the transition to a carbon-neutral bioenergy sector., Mauerhofer et al. examine 80 species of methanogenic archaea at high pressures and evaluate growth and methane production, identifying Methanotorris igneus and Methanocaldococcoccus jannaschii as high-pressure methane cell factories. They find that high-performance methanogens are covered with an S-layer and harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1–HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Geminiviruses cause severe damage to agriculture worldwide. The replication (Rep) protein is the indispensable viral protein for viral replication. Although various functional domains of Rep protein in Geminivirus spp. have been characterized, the most carboxyl terminus of Rep protein was not available. We have reported the first cassava-infecting geminivirus, Sri Lankan cassava mosaic virus (SLCMV-HN7 strain), in China. In this study, we reported the second Chinese SLCMV strain, SLCMV-Col, and conducted comparative genomic analysis between these two SLCMV strains. The virulence of SLCMV-Col is much stronger than SLCMV-HN7, indicated by the higher virus titer, more severe symptoms, and more extent host defense. We functionally characterized that Rep protein, a 7-amino-acid motif at the most carboxyl terminus, is essential for Rep protein accumulation and virulence of SLCMV. We also provided evidence suggesting that the motif could also enhance triggering of salicylic acid (SA) defense against SLCMV infection in Nicotiana benthamiana. The significance of the balance between virulence and host SA defense responses in expanding invasions of SLCMV is also discussed.

DNA methylation and DNA methyltransferases (MTases) – the enzymes that introduce the methylation mark into the DNA – have been studied for almost 70 years. In this chapter, we review key developments in the field that led to our current understanding of the structures and mechanisms of DNA MTases and the essential biological role of DNA methylation, including the discovery of DNA methylation and DNA MTases, the cloning and sequence analysis of bacterial and eukaryotic MTases, and the elucidation of their structure, mechanism, and regulation. We describe genetic studies that contributed greatly to the evolving views on the role of DNA methylation in human development and diseases, the invention of methods for the genome-wide analysis of DNA methylation, and the biochemical identification of DNA MTases and the family of TET enzymes, which are involved in DNA demethylation. We finish by highlighting critical questions for the next years of research in the field.

The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the Rad17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate Rad17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between Rad17 and the 9‒1‒1 complex. In human Rad17, the Y665 and S667 residues are conserved in iVERGE. The Rad17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of Rad17, T670 in human Rad17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. Rad17-T670 is phosphorylated by casein kinase 1δ/e. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis, a malignant B-cell lymphoma in cattle. The DNA sequence polymorphisms of bovine leukocyte antigen (BoLA)-DRB3 have exhibited a correlation with BLV-induced lymphoma in Holstein cows. However, the association may vary between different cattle breeds. Furthermore, little is known about the relationship between BLV-induced lymphoma and DRB3 at the amino acid and structural diversity levels. Here, we comprehensively analyzed the correlation between BLV-induced lymphoma and DRB3 at DNA, amino acid, and binding pocket property levels, using 106 BLV-infected asymptomatic and 227 BLV-induced lymphoma Japanese black cattle samples. DRB3*011:01 was identified as a resistance allele, whereas DRB3*005:02 and DRB3*016:01 were susceptibility alleles. Amino acid association studies showed that positions 9, 11, 13, 26, 30, 47, 57, 70, 71, 74, 78, and 86 were associated with lymphoma susceptibility. Structure and electrostatic charge modeling further indicated that binding pocket 9 of resistance DRB3 was positively charged. In contrast, alleles susceptible to lymphoma were neutrally charged. Altogether, this is the first association study of BoLA-DRB3 polymorphisms with BLV-induced lymphoma in Japanese black cattle. In addition, our results further contribute to understanding the mechanisms regarding how BoLA-DRB3 polymorphisms mediate susceptibility to BLV-induced lymphoma.

Elastin-like polypeptides (ELPs) are thermally responsive biopolymers that consist of a repeated amino acid motif derived from human tropoelastin. These peptides exhibit temperature-dependent phase behavior that can be harnessed to produce stimuli-responsive biomaterials, such as nanoparticles or injectable drug delivery depots. As ELPs are genetically encoded, the properties of ELP-based biomaterials can be controlled with a precision that is unattainable with synthetic polymers. Unique ELP architectures, such as spherical or rod-like micelles or injectable coacervates, can be designed by manipulating the ELP amino acid sequence and length. ELPs can be loaded with drugs to create controlled, intelligent drug delivery systems. ELPs are biodegradable, nonimmunogenic, and tolerant of therapeutic additives. These qualities make ELPs exquisitely well-suited to address current challenges in drug delivery and have spurred the development of ELP-based therapeutics to treat diseases-such as cancer and diabetes-and to promote wound healing. This review focuses on the use of ELPs in drug delivery systems.

BACKGROUND The insect cuticle protects against environmental stresses such as insecticides, physical injury, dehydration and pathogenic microorganisms. Structural cuticular proteins (CPs) are the primary components, and are of interest for physiology-based pest management methods. Anopheles sinensis CPs are poorly characterised at present, therefore we performed whole genome sequence analysis and re-analysed available transcriptome data to identify potential insecticide resistance-associated CPs. RESULTS Genome analysis revealed 238 putative CPs belonging to 11 different families; 136 proteins possessing a Rebers and Riddiford motif (CPR), 12 CPs analogous to peritrophins with a single chitin-binding domain (CPAP1), eight CPs analogous to peritrophins with three chitin-binding domains (CPAP3), four proteins with a 44 amino acid motif (CPF), five CPF-like (CPFL) proteins, nine Tweedle proteins, three proteins of low-complexity with alanine residues (CPLCA), 26 proteins of low-complexity with conserved glycine residues (CPLCG), seven proteins of low-complexity with invariant W residues (CPLCW), 27 proteins of low-complexity with proline residues (CPLCP), and one protein with two or three copies of a C-X(5)-C motif (CPCFC). Two CPs belonging to CPR and CPAP3 families were over-expressed in a deltamethrin-resistant strain. CONCLUSION These results establish an information framework for An. sinensis CPs that provides a basis for further molecular research.

Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials., Jessica Garb et al. report the transcriptome of the major ampullate silk gland of Darwin’s bark spider, known for its extraordinarily tough silk. They identify a novel predicted protein, MaSp4, which is highly divergent compared to related proteins in other species and has properties predicted to confer silk extensibility.

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD. Keywords: α-synuclein, Disulfide bond, Amyloid, Protein aggregation, Parkinson's disease

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Vanda tricolorLindey var.Suavisis an endemic orchid from Mt.Merapi, Yogyakarta Special Region, Indonesia. This orchid has beautiful flowers with unique patterns of white and purple spots, fragrant and can live in the slopes of Mt.Merapi which is a very active volcano. UGM in collaboration with the Netherlands carried out ex situ conservation of theV. tricolorMerapi through the self-pollination of orchids by the Queen of the Netherlands, Queen Maxima on March 11, 2020 at the UGM campus, Yogyakarta. In honor, the new generation of crosses is namedVanda tricolorvarSuavis‘Queen Maxima’. This study aims to produce mass quantities of the orchid and characterize theHSP70gene in it. Methods: Standardin vitroculture for Vanda on MS, NP and VW medium, isolation of plant gDNA, PCR with V. tricolor HSP70primers and sequencing of amplicon DNA. The results showed that > 90 % ofV. tricolor‘Queen Maxima’ seeds germinated well in all media and the best on VW medium.V. tricolorhas the HSP70 protein with PTZ00009 super family amino acid motif that 87 % similar to the HSP70 protein from thePhalaenopsis equestrisorchid, which is probably the reasonV. tricolorbecome superior to high temperature environments.

We propose a novel method that predicts binding of G-protein coupled receptors (GPCRs) and ligands. The proposed method uses hub and cycle structures of ligands and amino acid motif sequences of GPCRs, rather than the 3D structure of a receptor or similarity of receptors or ligands. The experimental results show that these new features can be effective in predicting GPCR-ligand binding (average area under the curve [AUC] of 0.944), because they are thought to include hidden properties of good ligand-receptor binding. Using the proposed method, we were able to identify novel ligand-GPCR bindings, some of which are supported by several studies.

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-beta-catenin pathway and for drug discovery.

Background: We discovered that lipopolysaccharide (LPS, an initiating factor in sepsis) and hypoxia-reoxygenation (H/R, a confounding factor) reduce electrical coupling between microvascular endothelial cells from wild-type (WT) but not Cx40-/- mice. Because Cx40 knockout could result in nonspecific effects, this discovery may not establish the causal relationship between Cx40 and reduced coupling. Using the same cell culture model, we aimed to address this uncertainty by using the rescue-of-function approach. Methods/Results: Electrical coupling between endothelial cells (hind-limb muscle origin) was determined by electrophysiology. LPS, H/R and concurrent LPS + H/R reduced coupling between WT but not Cx40-/- cells. The defect in Cx40-/- cells was rescued by ectopic expression of Cx40, after infecting the cells with adenovirus encoding Cx40. Cx40-/- cells were also engineered to express mutant Cx40 that lacked the carboxyl terminal domain beginning at residue 236 (Cx40Δ237-358) or 344 (Cx40Δ345-358). No response to inflammatory stimuli was observed in cells expressing either of these 2 mutants. Conclusion: Our data establish the causal relationship between Cx40 and reduced coupling and suggest that the 345-358 amino acid motif of the Cx40 carboxyl terminal is required for reduced coupling. Cx40 may participate in compromised conducted response in the microvasculature during sepsis.

An H4N6 subtype avian influenza virus was isolated from a pockmark duck in southern China in November 2013 and named A/duck/Guangxi/149D24/2013 (H4N6). All eight gene segments of the strain were sequenced. Sequence analysis indicated that this H4N6 virus was a natural reassortant virus. This H4N6 virus has two basic amino acids in the cleavage site of hemagglutinin 1 (HA1) and HA2, and the amino acid motif of cleavage site was PEKASRGLF, which is the typical characteristic of the low-pathogenic avian influenza virus. This study will help understand the epidemiology and molecular characteristics of avian influenza virus in pockmark ducks.

Norovirus (NV) RNA-dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer-template duplex was investigated using X-ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back-tracked state of the enzyme, in which the (Formula presented.) -end of the primer occupies the position expected for the post-incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.

The evolution of the pituitary hormone, ACTH, the melanocortin receptors, a family of G protein-coupled receptors, and the melanocortin receptor accessory proteins, MRAP1 and MRAP2, are intertwined, and parallel the evolution of the chordates. While all melanocortin-related peptides (i.e., ACTH, αMSH, βMSH, γMSH, δMSH) have the canonical HFRW amino acid motif required for activation of melanocortin receptors (i.e., MC1R, MC2R, MC3R, MC4R, MC5R), during the radiation of the gnathostomes (jawed vertebrates) the ligand selectivity of melanocortin-2 receptor orthologs has diverged between cartilaginous fishes and bony vertebrates in a manner that has implications for the Hypothalamus/Pituitary/Adrenal-Interrenal axis.

Emerging evidence supports the role for the intracellular domains of amyloid precursor protein (APP) in the physiology and function of APP. In this short report, I discuss the hypothesis that mutation of Tyr682 on the Y682 ENPTY687 C-terminal motif of APP may be directly or indirectly associated with alterations in APP functioning and activity, leading to neuronal defects and deficits. Mutation of Tyr682 induces an early and progressive age-dependent cognitive and locomotor decline that is associated with a loss of synaptic connections, a decrease in cholinergic tone, and defects in NGF signaling. These findings support a model in which APP-C-terminal domain exerts a pathogenic function in neuronal development and decline, and suggest that Tyr682 potentially could modulate the properties of APP metabolites in humans.

Avian influenza viruses in wild birds at the Jeziorsko reservoir in Poland in 2008-2010 During a 3-year surveillance study for avian influenza virus (AIV) infections at the Jeziorsko reservoir in central Poland, 549 oropharyngeal or cloacal swabs from 366 birds of 14 species belonging to 3 orders (Anseriformes, Charadriiformes and Gruiformes) were tested. AIV was detected in 14 birds (3.8%): Common Teals (12x), Mallard (1x) and Garganey (1x). Three potentially dangerous H5 AIV were detected in Common Teals (2x) and Garganey (1x) but all of them revealed a low pathogenic pathotype. A unique cleavage site amino acid motif PQREIR*GLF was found in one H5 isolate from a Garganey.

Spiders have developed specialized silks with outstanding biophysical properties over millions of years of evolution. As biopolymers composed by highly repetitive amino acid motifs, spider silks have been the focus of research for years. Due to recent advances in genetic engineering, recombinant spider silks have been produced, revealing the relationships between their protein structure and their mechanical properties. Each amino acid motif present in the silk adopts a particular secondary structure responsible for conferring a specific mechanical property to it. This feature has opened up the possibility to produce recombinant silks with controlled properties for various biotechnological applications. Moreover, spider silks are biocompatible and biodegradable biomaterials, which also allow their application in medicine. Accordingly, the relationship between molecular composition, secondary structure, and mechanical properties of spider silks is described in this chapter, along with a discussion of the current strategies for the production of recombinant spider silks, their importance in new biotechnological applications, and the current status of the field.

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold, and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S, and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic α-helix is especially important since it has been found in all dehydrins. Since more than 20 years, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made towards the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins, leading to their degradation and the liberation of nuclear factor κB for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor, at a resolution of 3.6 A. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix–loop–helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity and instead is required for IKKβ activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function. IKK (inhibitor of κB kinase) is an important element in the activation of nuclear factor κB (NF-κB) transcription factors — master regulators of inflammatory, immune and apoptotic responses. Here Wu and colleagues describe the long awaited X-ray structure of a member of the IKK family in combination with an inhibitor. They show that IKKβ has a tri-molecular architecture that contains the kinase domain, a ubiquitin-like domain and an extended scaffolding and dimerization domain, and discuss the functional implications.

Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the alpha3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.

In 2002-2003, velogenic Newcastle Disease Virus outbreaks, closely related to the Mexican isolates, were confirmed in the United States (U.S.) in southern California, Arizona, Nevada, and Texas. In this report, virulent NDVs isolated in Mexico between 1998 and 2006 were subjected to biologic characterization, using standard pathogenicity tests, and to phylogenetic analysis. Chicken embryo mean death time (MDT) test results ranged from 39.7 to 61.5 hours, and intracerebral pathogenicity index (ICPI) values were between 1.59 and 1.94, compared to a possible maximum value of 2.0. These isolates showed a dibasic amino acid motif at the fusion protein cleavage site sequence required for host systemic replication. Phylogenetic analysis indicated that the Mexican virulent NDVs belong to the class II, genotype V viruses and can be clearly divided in two groups as follows: isolates from 1998 to 2001 with close epidemiologic relationship with the latest U.S. NDV outbreaks, and phylogenetically distinct viruses, isolated from 2004 to 2006, which showed higher virulence. The assessment of the evolution of viruses from Mexico and other neighboring countries will aid in the U.S surveillance efforts for early detection of highly virulent NDV.