INTRODUCTION:Sepsis is a systemic immune dysregulation syndrome triggered by secondary infection in the host, with diagnosis based on the updated Phoenix criteria and characterized by multi-organ failure as its core pathological manifestation. It is a significant global health challenge due to its increasing incidence and high mortality rates. Recent advancements in biomarker research provide promising tools for improving early diagnosis and timely intervention, essential for better patient outcomes. AREAS COVERED:This review examines the latest developments in pediatric sepsis biomarkers, categorized by inflammation, metabolism, organ damage, and non-coding RNAs (miRNAs, LncRNAs). We discuss the advancements in each category, highlighting the integration of diverse biomarkers and advanced technologies to enhance diagnostics, personalize therapy, and improve patient stratification. EXPERT OPINION:Given the limited specificity and sensitivity of current markers like CRP and PCT, multicenter studies are crucial for validating new biomarkers and for developing comprehensive panel markers that combine multiple diagnostic indicators. It is also important to consider the variability in host responses to different pathogens when identifying biomarkers based on host-pathogen interactions. To advance personalized medicine, future research may prioritize the identification of specific diagnostic biomarkers for pediatric sepsis, tailored to different pathogens.

Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals.

Coinfection with multiple viruses is a common phenomenon in clinical settings and is a crucial driver of viral evolution. Although numerous studies have demonstrated viral recombination arising from coinfections of different strains of a specific species, the role of coinfections of different species or genera during viral evolution is rarely investigated. Here, we analyzed coinfections of and recombination events between four different swine enteric coronaviruses that infect the jejunum and ileum in pigs, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), and a deltacoronavirus, porcine deltacoronavirus (PDCoV). Various coinfection patterns were observed in 4,468 fecal and intestinal tissue samples collected from pigs in a 4-year survey. PEDV/PDCoV was the most frequent coinfection. However, recombination analyses have only detected events involving PEDV/TGEV and SADS-CoV/TGEV, indicating that inter-species recombination among coronaviruses is most likely to occur within the same genus. We also analyzed recombination events within the newly identified genus Deltacoronavirus and found that sparrows have played a unique host role in the recombination history of the deltacoronaviruses. The emerging virus PDCoV, which can infect humans, has a different recombination history. In summary, our study demonstrates that swine enteric coronaviruses are a valuable model for investigating the relationship between viral coinfection and recombination, which provide new insights into both inter- and intraspecies recombination events among swine enteric coronaviruses, and extend our understanding of the relationship between coronavirus coinfection and recombination.

Abstract Background The epidemiological advantage of Omicron variant is evidenced by its rapid spread and the ability to outcompete prior variants. Among Omicron sublineages, early outbreaks were dominated by BA.1, while BA.2 has gained dominance since February 2022. The relative pathogenicity and transmissibility of BA.1 and BA.2 have not been fully defined. Methods We compared viral loads and clinical signs in Syrian hamsters after infection with BA.1, BA.2, or D614G variant. A competitive transmission model and next-generation sequencing were used to compare the relative transmission potential of BA.1 and BA.2. Results BA.1 and BA.2 caused no apparent clinical signs, while D614G caused more than 10% weight loss. Higher viral loads were detected in nasal wash samples and nasal turbinate and lung tissues from BA.1-inoculated hamsters compared with BA.2-inoculated hamsters. No aerosol transmission was observed for BA.1 or BA.2 under the experimental condition in which D614G transmitted efficiently. BA.1 and BA.2 were able to transmit among hamsters via direct contact; however, BA.1 transmitted more efficiently than BA.2 under the competitive transmission model. No recombination was detected from direct contacts exposed simultaneously to BA.1 and BA.2. Conclusions Omicron BA.1 and BA.2 demonstrated attenuated pathogenicity and reduced transmission potential in hamsters compared with early SARS-CoV-2 strains.

Background: Transmission of SARS-CoV-2 from humans to other mammals, including pet animals, has been reported. However, with the exception of farmed mink, there is no previous documentation that these infected animals can infect humans, nor of further onward spread among humans. Following a confirmed SARS-CoV-2 infection of a pet store worker, animals in the store and the warehouse supplying it were tested for evidence of SARS-CoV-2 infection.Methods: Viral swabs and blood samples from pet animals were collected in a pet shop and the warehouse supplying it and tested by SARS-CoV-2 RT-PCR and serological assays, respectively. SARS-CoV-2 RT-PCR positive samples were studied by full genome sequencing analysis.Findings: Over 50% of individually tested Syrian hamsters in the pet shop (8/16) and warehouse (7/12) were positive for SARS-CoV-2 infection in RT-PCR or serological tests. None of dwarf hamsters (n=77), rabbits (n=246), Guinea pigs (n=66), chinchilla (n=116) and mice (n=2) were confirmed positive in RT-PCR tests. SARS-CoV-2 viral genomes deduced from human and hamster cases in this incident all belong to Delta variant of concern (AY.127) that had not been circulating locally prior. These sequences are highly similar, but distinct. The viral genomes obtained from hamsters are phylogenetically related with some sequence heterogeneity and phylogenetic dating suggest infection in these hamsters occurred around 21 November 2021. Two separate transmission events to humans are documented, one leading to onward household spread.Interpretation: Pet hamsters can be naturally infected in “real-life” settings. The virus can circulate within hamsters and lead to human infections. Both genetic and epidemiological results strongly suggest that there were two independent hamster-to-human transmission and that such events can lead to onward human transmission. Importation of infected hamsters was the most likely source of virus infection.Funding Information: This work is supported by US National Institutes of Health (U01AI151810 and 75N93021C00016), RGC theme-based research schemes (T11-712/19-N and T11-705/21-N), InnoHK grants for C2I and D24H, and Health and Medical Research Fund (COVID190205). Declaration of Interests: None of the authors had competing financial or non-financial interests.Ethical Approval: Animal samples were collected for clinical diagnosis during a public health investigation led by the Hong Kong Government (AFCD). Specimens from humans were collected and tested by RT–qPCR as part of routine clinical care and the viruses genetically sequenced as part of the routine public health response (Institutional Review Board approval UW20-168).

Abstract Background Using a computational approach, NL-CVX1 was developed by Neoleukin Therapeutics, Inc. to create a de novo protein that both blocks SARS-CoV-2 infection and is highly resilient to viral escape. In this study we evaluated the efficacy of NL-CVX1 against variants of the original SARS-CoV-2 strain, including important viral variants of concern (VOC) such as B.1.1.7, B.1.351, and P.1. Methods The relative binding affinity of NL-CVX1 to the SARS-CoV-2 viral spike protein of VOC was measured using biolayer interferometry (Octet). A competitive ELISA measured the ability of NL-CVX1 to compete with hACE2 for binding to the receptor binding domain (RBD) of the SARS-CoV-2 S protein from the original strain and VOC. The activity of NL-CVX1 in preventing viral infection was assessed by evaluating the cytopathic effects (CPE) of SARS-CoV-2 in a transmembrane protease, serine 2-expressing Vero E6 cell line (Vero E6/TMPRSS2) and determining the viral load using quantitative real-time reverse transcriptase polymerase chain reaction in infected cells. A K18hACE2 mouse model of SARS CoV-2 infection was used to study the dose-response of NL-CVX1 anti-viral activity in vivo. Results NL-CVX1 binds the RBD of different VOC of SARS-CoV-2 at low nanomolar concentrations (Fig 1; Kd < 1-~5 nM). When competing with hACE2, NL-CVX1 achieved 100% inhibition against hACE2 binding to the RBD of different VOC with IC50s values ranging from 0.7-53 nM (Fig 2). NL-CVX1 neutralized the B.1.1.7 variant as efficiently as the original strain in Vero E6/TMPRSS2 cells, with EC50 values of 16 nM and 101.2 nM, respectively (Fig 3). In mice, we found that a single intranasal dose of 100 µg NL-CVX1 prevented clinically significant SARS-CoV-2 infection and protected mice from succumbing to infection. Results from additional in vitro and in vivo experiments to be conducted this summer will be presented. Figure 1. NL-CVX1 binds the RBD from multiple strains of SARS-CoV-2 at low nanomolar concentrations. Figure 2. NL-CVX1 achieves 100% inhibition against all strains tested, including SARS-CoV-2 VOC. Figure 3. NL-CVX1 neutralizes the B.1.1.7 variant as efficiently as the original SARS-CoV-2 strain. Conclusion In vitro and in vivo data (Fig 4) demonstrate that NL-CVX1 is a promising drug candidate for the prevention and treatment of COVID-19. As a hACE2 mimetic, it is resilient to antibody escape mutations found in SARS-CoV-2 VOC. NL-CVX1 further demonstrates the power and utility of de novo protein design for developing proteins as human therapeutics. Figure 4. NL-CVX1 is effective in preventing clinically significant SARS-CoV-2 viral infection in a K18hACE2 mouse model. Disclosures Matthew Walker, PhD, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Laurie Tatalick, DVM, PhD, DACVP, Neoleukin Therapeutics, Inc. (Consultant, Other Financial or Material Support, Ownership options and stock.) Marianne Riley, BS, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Kevin Yu, BS, MS, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Luis M. Blancas-Mejia, PhD, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Daniel-Adriano Silva, PhD, Neoleukin Therapeutics, Inc. (Advisor or Review Panel member, Other Financial or Material Support, Ownership of Neoleukin options and stock) David Shoultz, PhD, MBA, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Goncalo Bernardes, PhD, Neoleukin Therapeutics, Inc. (Consultant, Advisor or Review Panel member, Shareholder) Hui-Ling Yen, PhD, Neoleukin Therapeutics, Inc. (Grant/Research Support)Saiba AG (Other Financial or Material Support, Received donation from Saiba AG)

BACKGROUND:A(H1N1)pdm09 influenza viruses replicate efficiently in respiratory epithelia and are transmitted via respiratory droplets and aerosols expelled by infected hosts. The relative onward transmission potential of influenza viruses replicating in the upper and lower respiratory epithelial cells has not been fully defined.METHODS:Wild-type and barcoded A(H1N1)pdm09 viruses that differed by 2 synonymous mutations per gene segment were inoculated into ferrets via intranasal and intratracheal routes. Naive recipients were exposed to the exhaled breath of inoculated donors for 8 hours on day 2 postinoculation. Onward transmission potential of wild-type and barcoded genotypes were monitored by next generation sequencing.RESULTS:Transmissible airborne particles were respired from the upper but not the lower respiratory epithelial cells of donor ferrets. There was limited mixing of viral populations replicating in the upper and lower respiratory tissues.CONCLUSIONS:The ferret upper respiratory epithelium was mapped as the anatomic site that generated influenza virus-laden particles mediating onward transmission by air. Our results suggest that vaccines and antivirals should aim to reduce viral loads in the upper respiratory tract for prevention of influenza transmission.

The segmented genome of influenza A virus has conferred significant evolutionary advantages to this virus through genetic reassortment, a mechanism that facilitates the rapid expansion of viral genetic diversity upon influenza co-infections. Therefore, co-infection of genetically diverse avian influenza viruses in poultry may pose a significant public health risk in generating novel reassortants with increased zoonotic potential. This study investigated the reassortment patterns of a Pearl River Delta-lineage avian influenza A(H7N9) virus and four genetically divergent avian influenza A(H9N2) viruses upon co-infection in embryonated chicken eggs and chickens. To characterize "within-host" and "between-host" genetic diversity, we further monitored the viral genotypes that were subsequently transmitted to contact chickens in serial transmission experiments. We observed that co-infection with A(H7N9) and A(H9N2) viruses may lead to the emergence of novel reassortant viruses in ovo and in chickens, albeit with different reassortment patterns. Novel reassortants detected in donor chickens co-infected with different combinations of the same A(H7N9) virus and different A(H9N2) viruses showed distinct onward transmission potential to contact chickens. Sequential transmission of novel reassortant viruses was only observed in one out of four co-infection combinations. Our results demonstrated different patterns by which influenza viruses may acquire genetic diversity through co-infection in ovo, in vivo, and under sequential transmission conditions.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main target for neutralizing antibodies. These antibodies can be elicited through immunization or passively transferred as therapeutics in the form of convalescent-phase sera or monoclonal antibodies (MAbs).

The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979–1983, 1984–1987 and 1988–1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor–binding specificity of the EA swine influenza viruses—that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979–1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

We report the use of environmental samples to assess avian influenza virus activity in chickens at live poultry markets in China. Results of environmental and chicken samples correlate moderately well. However, collection of multiple environmental samples from holding, processing, and selling areas is recommended to detect viruses expected to have low prevalence.

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

As a unique mammalian host for influenza A viruses, dogs support the transmission of canine influenza viruses (CIVs) of H3N8 and H3N2 subtypes and are susceptible to infection by avian and human influenza viruses. A cross-sectional serological study was performed to assess the exposure history of dogs in Hong Kong to CIV and human influenza viruses. Among 555 companion dogs sampled in 2015-2017, 1.3 per cent and 9.5 per cent showed hemagglutination inhibition (HI) antibody titre to CIV of H3N8 or H3N2 subtypes and to A(H1N1)pdm09 human influenza viruses, respectively. Among 182 shelter dogs sampled in 2017-2018, none showed HI titre to CIV and 1.1 per cent reacted to H3N2 human influenza virus. There was a poor correlation between ELISA and HI test results. The higher seropositive rates to human influenza viruses suggests that the contact dynamics of dogs under urban settings may affect the exposure risk to human influenza viruses and CIVs.

The highly pathogenic avian influenza (HPAI) A/H5N1 virus hijacks host cellular machinery to complete its life cycle; identification of the host factors involved in viral replication may facilitate antiviral drug development. Here, we first characterize a metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), and showed that it plays a crucial role in H5N1 viral replication. We found that H5N1 infection upregulated NDRG1 mRNA and protein expression. Overexpression of NDRG1 released approximately 4-fold more virions compared to the control group, whereas knockdown of NDRG1 resulted in a drop in viral RNA and protein production. Further investigation revealed that NDRG1 facilitated HPAI A/H5N1 viral replication by suppressing the canonical NF-kappa B signaling pathway. Furthermore, our results also showed that the NDRG1 mRNA level was mainly stimulated by Ml and PB1 viral proteins. Overall, our results suggest that NDRG1 plays a positive role in HPAI replication by suppressing the canonical NF-kappa B signaling pathway.

The migration of wild birds plays an important role in the transmission and spread of H5 highly pathogenic avian influenza (HPAI) virus, posing a severe risk to animal and human health. Substantial evidence suggests that altered gut microbial community is implicated in the infection of respiratory influenza virus. However the influence of H5N1 infection in gut microbiota of migratory birds remains unknown. In January 2015, a novel recombinant H5N1 virus emerged and killed about 100 migratory birds, mainly including whooper swans in Sanmenxia Reservoir Area of China. Here, we describe the first fecal microbiome diversity study of H5N1-infected migratory birds. By investigating the influence of H5N1 infection on fecal bacterial communities in infected and uninfected individuals, we found that H5N1 infection shaped the gut microbiota composition by a difference in the dominance of some genera, such as Aeromonas and Lactobacillus. We also found a decreased α diversity and increased β diversity in infectious individuals. Our results highlight that increases in changes in pathogen-containing gut communities occur when individuals become infected with H5N1. Our study may provide the first evidence that there are statistical association among H5N1 presence and fecal microbiota compositional shifts, and properties of the fecal microbiota may serve as the risk of gut-linked disease in migrates with H5N1 and further aggravate the disease transmission.