Optimization of Mixed Micelles Based on Oppositely Charged Block Copolymers by Machine Learning for Application in Gene Delivery.

The COVID-19 mRNA vaccines represent a milestone in developing non-viral gene carriers, and their success highlights the crucial need for continued research in this field to address further challenges. Polymer-based delivery systems are particularly promising due to their versatile chemical structure and convenient adaptability, but struggle with the toxicity-efficiency dilemma. Introducing anionic, hydrophilic, or "stealth" functionalities represents a promising approach to overcome this dilemma in gene delivery. Here, two sets of diblock terpolymers are created comprising hydrophobic poly(n-butyl acrylate) (PnBA), a copolymer segment made of hydrophilic 4-acryloylmorpholine (NAM), and either the cationic 3-guanidinopropyl acrylamide (GPAm) or the 2-carboxyethyl acrylamide (CEAm), which is negatively charged at neutral conditions. These oppositely charged sets of diblocks are co-assembled in different ratios to form mixed micelles. Since this experimental design enables countless mixing possibilities, a machine learning approach is applied to identify an optimal GPAm/CEAm ratio for achieving high transfection efficiency and cell viability with little resource expenses. After two runs, an optimal ratio to overcome the toxicity-efficiency dilemma is identified. The results highlight the remarkable potential of integrating machine learning into polymer chemistry to effectively tackle the enormous number of conceivable combinations for identifying novel and powerful gene transporters.

Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

Improving Gene Delivery: Synergy between Alkyl Chain Length and Lipoic Acid for PDMAEMA Hydrophobic Copolymers.

In the field of gene delivery, hydrophobic cationic copolymers hold great promise. They exhibit improved performance by effectively protecting genetic material from serum interactions while facilitating interactions with cellular membranes. However, managing cytotoxicity remains a significant challenge, prompting an investigation into suitable hydrophobic components. A particularly encouraging approach involves integrating nutrient components, like lipoic acid, which is known for its antioxidant properties and diverse cellular benefits such as cellular metabolism and growth. In this study, a copolymer library comprising 2-(dimethylamino)ethyl methacrylate (DMAEMA) and lipoic acid methacrylate (LAMA), combined with either n-butyl methacrylate (nBMA), ethyl methacrylate (EMA), or methyl methacrylate (MMA), is synthesized. This enables to probe the impact of lipoic acid incorporation while simultaneously exploring the influence of pendant acyclic alkyl chain length. The inclusion of lipoic acid results in a notable boost in transfection efficiency  while maintaining low cytotoxicity. Interestingly, higher levels of transfection efficiency are achieved in the presence of nBMA, EMA, or MMA. However, a positive correlation between pendant acyclic alkyl chain length and cytotoxicity is observed. Consequently, P(DMAEMA-co-LAMA-co-MMA), emerges as a promising candidate. This is attributed to the optimal combination of low cytotoxic MMA and transfection-boosting LAMA, highlighting the crucial synergy between LAMA and MMA.

Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.

Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.

Exploiting whole genome sequence data to fine map and characterize candidate genes within a quantitative trait loci region affecting androstenone on porcine chromosome 5.

Male piglets are routinely castrated to eliminate boar taint. However, this treatment is undesirable, and alternative approaches, including genetic strategies to reduce boar taint, are demanded. Androstenone is one of the causative agents of boar taint, and a QTL region affecting this pheromone has previously been reported on SSC5: 22.6-24.8 Mb in Duroc. The QTL region is one of the few reported for androstenone that does not simultaneously affect levels of other sex steroids. The main objective of this study was to fine map this QTL. Whole genome sequence data from 23 Norwegian Duroc boars were analyzed to detect new polymorphisms within the QTL region. A subset of 161 SNPs was genotyped in 834 Duroc sires and analyzed for association with androstenone in adipose tissue and testosterone, estrone sulphate and 17ß-estradiol in blood plasma. Our results revealed 100 SNPs significantly associated with androstenone levels in fat (P < 0.001) with 94 of the SNPs being in strong linkage disequilibrium in the region 23.03-24.27 Mb. This haplotype block contains at least four positional candidate genes (HSD17B6, SDR9C7, RDH16 and STAT6) involved in androstenone biosynthesis. No significant associations were found between any of the SNPs and levels of testosterone and estrogens, confirming previous findings. The amount of phenotypic variance explained by single SNPs within the haplotype block was as high as 5.4%. As the SNPs in this region significantly affect levels of androstenone without affecting levels of other sex steroids, they are especially interesting as genetic markers for selection against boar taint.

Identification of quantitative genetic components of fitness variation in farmed, hybrid and native salmon in the wild.

Feral animals represent an important problem in many ecosystems due to interbreeding with wild conspecifics. Hybrid offspring from wild and domestic parents are often less adapted to local environment and ultimately, can reduce the fitness of the native population. This problem is an important concern in Norway, where each year, hundreds of thousands of farm Atlantic salmon escape from fish farms. Feral fish outnumber wild populations, leading to a possible loss of local adaptive genetic variation and erosion of genetic structure in wild populations. Studying the genetic factors underlying relative performance between wild and domesticated conspecific can help to better understand how domestication modifies the genetic background of populations, and how it may alter their ability to adapt to the natural environment. Here, based upon a large-scale release of wild, farm and wild x farm salmon crosses into a natural river system, a genome-wide quantitative trait locus (QTL) scan was performed on the offspring of 50 full-sib families, for traits related to fitness (length, weight, condition factor and survival). Six QTLs were detected as significant contributors to the phenotypic variation of the first three traits, explaining collectively between 9.8 and 14.8% of the phenotypic variation. The seventh QTL had a significant contribution to the variation in survival, and is regarded as a key factor to understand the fitness variability observed among salmon in the river. Interestingly, strong allelic correlation within one of the QTL regions in farmed salmon might reflect a recent selective sweep due to artificial selection.

Mapping and validation of a major QTL affecting resistance to pancreas disease (salmonid alphavirus) in Atlantic salmon (Salmo salar).

Pancreas disease (PD), caused by a salmonid alphavirus (SAV), has a large negative economic and animal welfare impact on Atlantic salmon aquaculture. Evidence for genetic variation in host resistance to this disease has been reported, suggesting that selective breeding may potentially form an important component of disease control. The aim of this study was to explore the genetic architecture of resistance to PD, using survival data collected from two unrelated populations of Atlantic salmon; one challenged with SAV as fry in freshwater (POP 1) and one challenged with SAV as post-smolts in sea water (POP 2). Analyses of the binary survival data revealed a moderate-to-high heritability for host resistance to PD in both populations (fry POP 1 h(2)~0.5; post-smolt POP 2 h(2)~0.4). Subsets of both populations were genotyped for single nucleotide polymorphism markers, and six putative resistance quantitative trait loci (QTL) were identified. One of these QTL was mapped to the same location on chromosome 3 in both populations, reaching chromosome-wide significance in both the sire- and dam-based analyses in POP 1, and genome-wide significance in a combined analysis in POP 2. This independently verified QTL explains a significant proportion of host genetic variation in resistance to PD in both populations, suggesting a common underlying mechanism for genetic resistance across lifecycle stages. Markers associated with this QTL are being incorporated into selective breeding programs to improve PD resistance.

Genetic differentiation of southeast Baltic populations of sea trout inferred from single nucleotide polymorphisms.

Sea trout (Salmo trutta m. trutta) is a migratory form of brown trout common in the Baltic Sea. Nine populations from the southeast Baltic (Poland; Lithuania; Denmark, Bornholm; Estonia and Russia) were genotyped using iPLEX Gold technology (Sequenom) with 62 informative SNPs. A diagnostic panel of 23 SNPs was applied to estimate genetic differentiation and assess the population structure of Baltic sea trout. The highest level of pairwise FST differences was observed between the Russian (East Gulf of Finland) and Polish (Baltic main basin) populations. The lowest differences were between the two Polish and the Polish and Lithuanian populations. A genetic similarity was noted between the Estonian Riguldi River and Danish Bornholm populations, and this finding was supported by a Bayesian and factorial correspondence analysis. Diversity within populations was highest for populations from Estonia and lowest for the Lithuanian population. Genetic structure analysis indicated that individuals from the nine populations were clustered into four groups.

Tailoring Gene Transfer Efficacy through the Arrangement of Cationic and Anionic Blocks in Triblock Copolymer Micelles.

The arrangement of charged segments in triblock copolymer micelles affects the gene delivery potential of polymeric micelles and can increase the level of gene expression when an anionic segment is incorporated in the outer shell. Triblock copolymers were synthesized by RAFT polymerzation with narrow molar mass distributions and assembled into micelles with a hydrophobic core from poly(n-butyl acrylate). The ionic shell contained either (i) an anionic segment followed by a cationic segment (HAC micelles) or (ii) a cationic block followed by an anionic block (HCA micelles). The pH-responsive anionic block contained 2-carboxyethyl acrylamide (CEAm), while the cationic block comprised 3-guanidinopropyl acrylamide (GPAm). Increasing the molar content of CEAm in HAC and HCA micelles from 6 to 13 mol % improved cytocompatibility and the endosomal escape property, while the HCA micelle with the highest mol % of anionic charges in the outer shell exhibited the highest gene expression. It became evident that improved membrane interaction of the best performing HCA micelle contributed to achieving high gene expression.

Harnessing Guanidinium and Imidazole Functional Groups: A Dual-Charged Polymer Strategy for Enhanced Gene Delivery.

Histidine and arginine are two amino acids that exhibit beneficial properties for gene delivery. In particular, the imidazole group of histidine facilitates endosomal release, while the guanidinium group of arginine promotes cellular entry. Consequently, a dual-charged copolymer library based on these amino acids was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The content of the N-acryloyl-l-histidine (His) monomer was systematically increased, while maintaining consistent levels of methyl N-acryloyl-l-argininate hydrochloride (ArgOMe) or N-(4-guanidinobutyl)acrylamide hydrochloride (GBAm). The resulting polymers formed stable, nanosized polyplexes when complexed with nucleic acids. Remarkably, candidates with increased His content exhibited reduced cytotoxicity profiles and enhanced transfection efficiency, particularly retaining this performance level at lower pDNA concentrations. Furthermore, endosomal release studies revealed that increased His content improved endosomal release, while ArgOMe improved cellular entry. These findings underscore the potential of customized dual-charged copolymers and the synergistic effects of His and ArgOMe/GBAm in enhancing gene delivery.

A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes.

Agouti and extension are two genes that control the production of yellow-red (phaeomelanin) and brown-black (eumelanin) pigments in the mammalian coat. Extension encodes the melanocyte-stimulating hormone receptor (MC1R) while agouti encodes a peptide antagonist of the receptor. In the mouse, extension is epistatic to agouti, hence dominant mutants of the MC1R encoding constitutively active receptors are not inhibited by the agouti antagonist, and animals with dominant alleles of both loci remain darkly pigmented. In the fox the proposed extension locus is not epistatic to the agouti locus. We have cloned and characterized the MC1R and the agouti gene in coat colour variants of the fox (Vulpes vulpes). A constitutively activating C125R mutation in the MC1R was found specifically in darkly pigmented animals carrying the Alaska Silver allele (EA). A deletion in the first coding exon of the agouti gene was found associated with the proposed recessive allele of agouti in the darkly pigmented Standard Silver fox (aa). Thus, as in the mouse, dark pigmentation can be caused by a constitutively active MC1R, or homozygous recessive status at the agouti locus. Our results, demonstrating the presence of dominant extension alleles in foxes with significant red coat colouration, suggest the ability of the fox agouti protein to counteract the signalling activity of a constitutively active fox MC1R.

Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

Self-perceived health and return to work following work-related hand injury.

BACKGROUND: Return to work is an important outcome following traumatic work-related hand injuries. It is unclear how psychosocial factors affect the time to return to work following traumatic work-related hand injury. AIMS: To investigate the relationships between hand injury severity, self-perceived health, demographics and time off work (TOW) following traumatic work-related hand injuries and the influence of psychosocial factors on the readiness of return to work. METHODS: Data from 120 traumatic work-related hand injured patients were gathered. The Modified Hand Injury Severity Score (MHISS) and Short Form Health Survey (SF-36) were used to assess the severity of hand trauma and self-perceived health, respectively. The relationships between MHISS, SF-36, demographics and TOW were analysed by multiple regression analysis. RESULTS: Mean duration of TOW was 127 days for patients with a mild MHISS, 108 days for a moderate score, 160 days for a severe score and 236 days for those with a major score. A positive correlation between MHISS and duration of TOW was identified. Self-perceived physical functioning was found to have a negative correlation with TOW, whereas self-perceived mental health was positively correlated with TOW. CONCLUSIONS: This study highlights the importance of self-perceived health in considering return to work following traumatic work-related hand injury.

Quantitative trait loci for clinical mastitis on chromosomes 2, 6, 14 and 20 in Norwegian Red cattle.

Mastitis is the most frequent and costly disease in dairy production and solutions leading to a reduction in the incidence of mastitis are highly demanded. Here a genome-wide association study was performed to identify polymorphisms affecting susceptibility to mastitis. Genotypes for 17 349 SNPs distributed across the 29 bovine autosomal chromosomes from a total of 2589 sires with 1 389 776 daughters with records on clinical mastitis were included in the analysis. Records of occurrence of clinical mastitis were divided into seven time periods in the first three lactations in order to identify quantitative trait loci affecting mastitis susceptibility in particular phases of lactation. The most convincing results from the association mapping were followed up and validated by a combined linkage disequilibrium and linkage analysis. The study revealed quantitative trait loci affecting occurrence of clinical mastitis in the periparturient period on chromosomes 2, 6 and 20 and a quantitative trait locus affecting occurrence of clinical mastitis in late lactation on chromosome 14. None of the quantitative trait loci for clinical mastitis detected in the study seemed to affect lactation average of somatic cell score. The SNPs highly associated with clinical mastitis lie near both the gene encoding interleukin 8 on chromosome 6 and the genes encoding the two interleukin 8 receptors on chromosome 2.

Genome-wide association mapping in Norwegian Red cattle identifies quantitative trait loci for fertility and milk production on BTA12.

Reproductive performance is a critical trait in dairy cattle. Poor reproductive performance leads to prolonged calving intervals, higher culling rates and extra expenses related to multiple inseminations, veterinary treatments and replacements. Genetic gain for improved reproduction through traditional selection is often slow because of low heritability and negative correlations with production traits. Detection of DNA markers associated with improved reproductive performance through genome-wide association studies could lead to genetic gain that is more balanced between fertility and production. Norwegian Red cattle are well suited for such studies, as very large numbers of detailed reproduction records are available. We conducted a genome-wide association study for non-return rate, fertility treatments and retained placenta using almost 1 million records on these traits and 17 343 genome-wide single-nucleotide polymorphisms. Genotyping costs were minimized by genotyping the sires of the cows recorded and by using daughter averages as phenotypes. The genotyped sires were assigned to either a discovery or a validation population. Associations were only considered to be validated if they were significant in both groups. Strong associations were found and validated on chromosomes 1, 5, 8, 9, 11 and 12. Several of these were highly supported by findings in other studies. The most important result was an association for non-return rate in heifers in a region of BTA12 where several associations for milk production traits have previously been found. Subsequent fine-mapping verified the presence of a quantitative trait loci (QTL) having opposing effects on non-return rate and milk production at 18 Mb. The other reproduction QTL did not have pleiotropic effects on milk production, and these are therefore of considerable interest for use in marker-assisted selection.

A genome wide association study for QTL affecting direct and maternal effects of stillbirth and dystocia in cattle.

Dystocia and stillbirth are significant causes of female and neonatal death in many species and there is evidence for a genetic component to both traits. Identifying causal mutations affecting these traits through genome wide association studies could reveal the genetic pathways involved and will be a step towards targeted interventions. Norwegian Red cattle are an ideal model breed for such studies as very large numbers of records are available. We conducted a genome wide association study for direct and maternal effects of dystocia and stillbirth using almost 1 million records of these traits. Genotyping costs were minimized by genotyping the sires of the recorded cows, and using daughter averages as phenotypes. A dense marker map containing 17,343 single nucleotide polymorphisms covering all autosomal chromosomes was utilized. The genotyped sires were assigned to one of two groups in an attempt to ensure independence between the groups. Associations were only considered validated if they occurred in both groups. Strong associations were found and validated on chromosomes 4, 5, 6, 9, 12, 20, 22 and 28. The QTL region on chromosome 6 was refined using LDLA analysis. The results showed that this chromosome most probably contains two QTL for direct effect on dystocia and one for direct effect on stillbirth. Several candidate genes may be identified close to these QTL. Of these, a cluster of genes expected to affect bone and cartilage formation (i.e. SPP1, IBSP and MEPE) are of particular interest and we suggest that these genes are screened in candidate gene studies for dystocia and stillbirth in cattle as well as other species.

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon.

A better understanding of the genotype-phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.

Caprine CSN1S1 haplotype effect on gene expression and milk composition measured by Fourier transform infrared spectroscopy.

The Norwegian dairy goat population has a high frequency of a CSN1S1 (alphaS1-casein) haplotype with negative effects on protein and fat content. It is characterized by a single point deletion in exon 12 of CSN1S1, leading to a truncated protein and hence a low content of alphaS1-casein in the milk. This haplotype together with another haplotype with a deletion in exon 9 are called "weak" haplotypes. "Strong" haplotypes, on the other hand, have positive effects on important milk production traits. We show that expression of CSN1S1 in the mammary gland of lactating goats is significantly lower in animals with 2 weak haplotypes. Moreover, the effects of defective alleles were not detected in animals having 1 strong and 1 weak haplotype. Expression levels of other genes in the casein cluster were not affected by the CSN1S1 haplotypes investigated. Milk samples from goats with 2 weak haplotypes could be distinguished from the other milk samples using Fourier transform infrared (FTIR) spectroscopy and partial least squares discriminant analysis (PLS-DA). The PLS-DA components were related to spectra of pure caseins and whey proteins, hence FTIR has a potential for identifying milk samples with low alphaS1-casein content and different protein composition. The results indicate that FTIR-based measurements can be incorporated into breeding plans, or for selection of milk samples with high casein content, which in turn may improve cheese-making properties of the milk.

Liquid crystal alignment on carbonaceous surfaces with orientational order.

We used near-edge x-ray absorption fine structure (NEXAFS) spectroscopy to link the orientational bond order at three carbonaceous surfaces-rubbed polyimide, ion beam-irradiated polyimide, and ion beam-irradiated diamondlike carbon films-with the direction of liquid crystal (LC) alignment on these surfaces. We show that, in general, LC alignment can be created on any carbonaceous substrate by inducing orientational order at its surface. Our results form the scientific basis for LC alignment layers consisting of amorphous carbon films in which orientational order near the surface is induced by a directional low-energy ion beam.

Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004.

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5-99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.