TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps.

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach.

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Discovering Fragile Clades and Causal Sequences in Phylogenomics by Evolutionary Sparse Learning.

Phylogenomic analyses of long sequences, consisting of many genes and genomic segments, reconstruct organismal relationships with high statistical confidence. But, inferred relationships can be sensitive to excluding just a few sequences. Currently, there is no direct way to identify fragile relationships and the associated individual gene sequences in species. Here, we introduce novel metrics for gene-species sequence concordance and clade probability derived from evolutionary sparse learning models. We validated these metrics using fungi, plant, and animal phylogenomic datasets, highlighting the ability of the new metrics to pinpoint fragile clades and the sequences responsible. The new approach does not necessitate the investigation of alternative phylogenetic hypotheses, substitution models, or repeated data subset analyses. Our methodology offers a streamlined approach to evaluating major inferred clades and identifying sequences that may distort reconstructed phylogenies using large datasets.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Extensive remodeling of sugar metabolism through gene loss and horizontal gene transfer in a eukaryotic lineage.

BACKGROUND: In yeasts belonging to the subphylum Saccharomycotina, genes encoding components of the main metabolic pathways, like alcoholic fermentation, are usually conserved. However, in fructophilic species belonging to the floral Wickerhamiella and Starmerella genera (W/S clade), alcoholic fermentation was uniquely shaped by events of gene loss and horizontal gene transfer (HGT). RESULTS: Because HGT and gene losses were first identified when only eight W/S-clade genomes were available, we collected publicly available genome data and sequenced the genomes of 36 additional species. A total of 63 genomes, representing most of the species described in the clade, were included in the analyses. Firstly, we inferred the phylogenomic tree of the clade and inspected the genomes for the presence of HGT-derived genes involved in fructophily and alcoholic fermentation. We predicted nine independent HGT events and several instances of secondary loss pertaining to both pathways. To investigate the possible links between gene loss and acquisition events and evolution of sugar metabolism, we conducted phenotypic characterization of 42 W/S-clade species including estimates of sugar consumption rates and fermentation byproduct formation. In some instances, the reconciliation of genotypes and phenotypes yielded unexpected results, such as the discovery of fructophily in the absence of the cornerstone gene (FFZ1) and robust alcoholic fermentation in the absence of the respective canonical pathway. CONCLUSIONS: These observations suggest that reinstatement of alcoholic fermentation in the W/S clade triggered a surge of innovation that goes beyond the utilization of xenologous enzymes, with fructose metabolism playing a key role.

Evidence of Reduced Virulence and Increased Colonization among Pneumococcal Isolates of Serotype 3 Clade II Lineage in Mice.

Recent phylogenetic profiling of pneumococcal serotype 3 (Pn3) isolates revealed a dynamic interplay among major lineages with the emergence and global spread of a variant termed Clade II. The cause of Pn3 clade II dissemination along with epidemiological and clinical ramifications are currently unknown. Here, we sought to explore biological characteristics of dominant Pn3 clades in a mouse model of pneumococcal invasive disease and carriage. Carriage and virulence potential were strain dependent with marked differences among clades. We found that clinical isolates from Pn3 clade II are less virulent and less invasive in mice compared to clade I isolates. We also observed that clade II isolates are carried for longer and at higher bacterial densities in mice compared to clade I isolates. Taken together, our data suggest that the epidemiological success of Pn3 clade II could be related to alterations in the pathogen's ability to cause invasive disease and to establish a robust carriage episode.

A Laboratory-Developed Assay for Clade II Human Mpox Virus on the Panther Fusion Open Access System.

This paper describes the methods for developing and optimizing a laboratory-developed assay (LDA) for detecting clade II human mpox virus using the automated Panther Fusion platform and Open Access software. Various concentrations of reagents in a primer-probe mix were tested to optimize the LDA. The LDA was validated using 10 previously characterized positive and 10 negative human mpox samples, resulting in 95% accuracy and 100% precision. The LDA resulted in 100% specificity among previously tested HSV1-, HSV2-, and VZV-positive human samples. Several spiked media extensions were also validated and achieved 98% accuracy and 100% precision across all collection media types. The assay's limit of detection was calculated to be 1.475 copies/reaction, and the polymerase chain reaction efficiency resulted in 89.87% (slope, -3.5911; R2 = 0.9947). The methods described here can be applied to the rapid optimization and development of LDAs for many possible pathogens of public health importance.

Concurrent Clade I and Clade II Monkeypox Virus Circulation, Cameroon, 1979-2022.

During 1979-2022, Cameroon recorded 32 laboratory-confirmed mpox cases among 137 suspected mpox cases identified by the national surveillance network. The highest positivity rate occurred in 2022, indicating potential mpox re-emergence in Cameroon. Both clade I (n = 12) and clade II (n = 18) monkeypox virus (MPXV) were reported, a unique feature of mpox in Cameroon. The overall case-fatality ratio of 2.2% was associated with clade II. We found mpox occurred only in the forested southern part of the country, and MPXV phylogeographic structure revealed a clear geographic separation among concurrent circulating clades. Clade I originated from eastern regions close to neighboring mpox-endemic countries in Central Africa; clade II was prevalent in western regions close to West Africa. Our findings suggest that MPXV re-emerged after a 30-year lapse and might arise from different viral reservoirs unique to ecosystems in eastern and western rainforests of Cameroon.

Highly Pathogenic Avian Influenza Virus A(H5N1) Clade 2.3.4.4b Infection in Free-Ranging Polar Bear, Alaska, USA.

We report a natural infection with a Eurasian highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a free-ranging juvenile polar bear (Ursus maritimus) found dead in North Slope Borough, Alaska, USA. Continued community and hunter-based participation in wildlife health surveillance is key to detecting emerging pathogens in the Arctic.

Evolution and Spread of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Wild Birds, South Korea, 2022-2023.

During October 2022-March 2023, highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus caused outbreaks in South Korea, including 174 cases in wild birds. To understand the origin and role of wild birds in the evolution and spread of HPAI viruses, we sequenced 113 HPAI isolates from wild birds and performed phylogenetic analysis. We identified 16 different genotypes, indicating extensive genetic reassortment with viruses in wild birds. Phylodynamic analysis showed that the viruses were most likely introduced to the southern Gyeonggi-do/northern Chungcheongnam-do area through whooper swans (Cygnus cygnus) and spread southward. Cross-species transmission occurred between various wild bird species, including waterfowl and raptors, resulting in the persistence of HPAI in wild bird populations and further geographic spread as these birds migrated throughout South Korea. Enhanced genomic surveillance was an integral part of the HPAI outbreak response, aiding in timely understanding of the origin, evolution, and spread of the virus.

Concurrent Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza H5N6 and H5N1 Viruses, South Korea, 2023.

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

Novel Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2023.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Domestic Ducks, Indonesia, 2022.

Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses were isolated from domestic ducks in South Kalimantan, Indonesia, during April 2022. The viruses were genetically similar to those detected in East Asia during 2021-2022. Molecular surveillance of wild birds is needed to detect potential pandemic threats from avian influenza virus.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

Avian Influenza A(H5N1) Neuraminidase Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09.

We detected high titers of cross-reactive neuraminidase inhibition antibodies to influenza A(H5N1) virus clade 2.3.4.4b in 96.8% (61/63) of serum samples from healthy adults in Hong Kong in 2020. In contrast, antibodies at low titers were detected in 42% (21/50) of serum samples collected in 2009. Influenza A(H1N1)pdm09 and A(H5N1) titers were correlated.

Differential Retention of Pfam Domains Contributes to Long-term Evolutionary Trends.

Protein domains that emerged more recently in evolution have a higher structural disorder and greater clustering of hydrophobic residues along the primary sequence. It is hard to explain how selection acting via descent with modification could act so slowly as not to saturate over the extraordinarily long timescales over which these trends persist. Here, we hypothesize that the trends were created by a higher level of selection that differentially affects the retention probabilities of protein domains with different properties. This hypothesis predicts that loss rates should depend on disorder and clustering trait values. To test this, we inferred loss rates via maximum likelihood for animal Pfam domains, after first performing a set of stringent quality control methods to reduce annotation errors. Intermediate trait values, matching those of ancient domains, are associated with the lowest loss rates, making our results difficult to explain with reference to previously described homology detection biases. Simulations confirm that effect sizes are of the right magnitude to produce the observed long-term trends. Our results support the hypothesis that differential domain loss slowly weeds out those protein domains that have nonoptimal levels of disorder and clustering. The same preferences also shape the differential diversification of Pfam domains, thereby further impacting proteome composition.

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

Broadening the application of Yarrowia lipolytica synthetic biology tools to explore the potential of Yarrowia clade diversity.

Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the Golden Gate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least five members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the uncharacterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.