Infectious Virus Research Articles

Transmission of a human isolate of clade 2.3.4.4b A(H5N1) virus in ferrets.

Since 2020, there has been unprecedented global spread of highly pathogenic avian influenza A(H5N1) in wild bird populations with spillover into a variety of mammalian species and sporadically humans1. In March 2024, clade 2.3.4.4b A(H5N1) virus was first detected in dairy cattle in the U.S., with subsequent detection in numerous states2, leading to over a dozen confirmed human cases3,4. In this study, we employed the ferret model, a well-characterized species that permits concurrent investigation of viral pathogenicity and transmissibility5 in the evaluation of A/Texas/37/2024 (TX/37) A(H5N1) virus isolated from a dairy farm worker in Texas6. Here, we show that the virus has a remarkable ability for robust systemic infection in ferrets, leading to high levels of virus shedding and spread to naïve contacts. Ferrets inoculated with TX/37 rapidly exhibited a severe and fatal infection, characterized by viremia and extrapulmonary spread. The virus efficiently transmitted in a direct contact setting and was capable of indirect transmission via fomites. Airborne transmission was corroborated by the detection of infectious virus shed into the air by infected animals, albeit at lower levels compared to the highly transmissible human seasonal and swine-origin H1 subtype strains. Our results show that despite maintaining anavian-like receptor binding specificity, TX/37 displays heightened virulence, transmissibility, and airborne shedding relative to other clade 2.3.4.4b virus isolated prior to the 2024 cattle outbreaks7, underscoring the need for continued public health vigilance.

Metavirome Insights into the Diversity and Potential Pathogenic Infection of Chlamys farreri in the Coastal Seas of the Republic of Korea

Chlamys farreri is primarily cultivated in Japan, China, and South Korea. Although mass mortality of scallops has been occurring recently, likely caused by high temperatures or infectious diseases, the underlying cause remains unclear. Little is known regarding the viral diseases affecting them. Therefore, we explored DNA virus diversity in the mid-gut gland of C. farreri and compared it with that of seawater. C. farreri was cultivated at depths below 5 m from the sea surface in the coastal waters of South Korea and sampled from May to August 2018. Different DNA viral communities were observed in both C. farreri and seawater. In C. farreri, prevalent groups included Mimiviridae (7%), Poxviridae (6%), and Phycodnaviridae (5%). Conversely, the dominant groups in seawater were Autographiviridae (20%), Kyanoviridae (12%), and Zobellviridae (10%). We identified C. farreri-specific viral communities and potentially infectious viruses, such as Ostreid herpesvirus 1 and Abalone herpesvirus Victoria/AUS/2009. Furthermore, C. farreri acts as a reservoir for various viruses, which impact microbial community dynamics and disease transmission in marine ecosystems. Understanding these viral communities is crucial to protecting and restoring coastal ecosystems by highlighting their role in the transmission of potential avian- and bivalve-specific viruses.

Molecular characterization and phylogenetic analysis of infectious laryngotracheitis virus isolates from commercial chicken flocks in Turkey.

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence.

The combination of infectious bronchitis virus BR1 and Mass vaccines provides broad protection

ABSTRACT Two vaccination-challenge trials were performed using a commercial infectious bronchitis virus (IBV) BR1 vaccine, given alone or combined with a commercial IBV Mass vaccine against challenges with IBV M41, 793B, D388 (QX), Q1, Brasil-1 or Variant 2 challenge viruses, which includes the IB viruses that are dominant in South America. The efficacy of the vaccines against the challenge viruses was investigated by determination of the ciliary activity of the tracheal epithelium after challenge. The level of protection induced by the IBV BR1 vaccine alone against the six IBV challenge strains, of which five were of heterologous genotypes, varied from 50% to 100% with an average of 80%. The level of protection induced by the combination of the IBV BR1 and IBV Mass vaccines against the six IBV challenge strains, of which four were of heterologous genotypes, varied from 80% to 100% with an average of 92%. Vaccination with IBV BR1 alone provided a high level of protection against most tested challenge viruses, though the combination of IBV BR1 and IBV Mass was more consistent, showing less variation and compliance with the criterium mentioned in the European Pharmacopoeia 10th edition (at least 80% protection) for all tested challenge viruses. These trials show that vaccination with a combination of IBV BR1 and IBV Mass vaccines provides high levels of protection against the circulating IBV strains in South America.

Enhanced Recovery and Detection of Highly Infectious Animal Disease Viruses by Virus Capture Using Nanotrap® Microbiome A Particles

This study reports the use of Nanotrap® Microbiome A Particles (NMAPs) to capture and concentrate viruses from diluted suspensions to improve their recovery and sensitivity to detection by real-time PCR/RT-PCR (qPCR/RT-qPCR). Five highly infectious animal disease viruses including goatpox virus (GTPV), sheeppox virus (SPPV), lumpy skin disease virus (LSDV), peste des petits ruminants virus (PPRV), and African swine fever virus (ASFV) were used in this study. After capture, the viruses remained viable and recoverable by virus isolation (VI) using susceptible cell lines. To assess efficacy of recovery, the viruses were serially diluted in phosphate-buffered saline (PBS) or Eagle’s Minimum Essential Medium (EMEM) and then subjected to virus capture using NMAPs. The NMAPs and the captured viruses were clarified on a magnetic stand, reconstituted in PBS or EMEM, and analyzed separately by VI and virusspecific qPCR/RT-qPCR. The PCR results showed up to a 100-fold increase in the sensitivity of detection of the viruses following virus capture compared to the untreated viruses from the same dilutions. Experimental and clinical samples were subjected to virus capture using NMAPs and analyzed by PCR to determine diagnostic sensitivity (DSe) that was comparable (100%) to that determined using untreated (-NMAPs) samples. NMAPs were also used to capture spiked viruses from EDTA whole blood (EWB). Virus capture from EWB was partially blocked, most likely by hemoglobin (HMB), which also binds NMAPs and outcompetes the viruses. The effect of HMB could be removed by either dilution (in PBS) or using HemogloBind™ (Biotech Support Group; Monmouth Junction, NJ), which specifically binds and precipitates HMB. Enhanced recovery and detection of viruses using NMAPs can be applicable to other highly pathogenic animal viruses of agricultural importance.

The host tropism of current zoonotic H7N9 viruses depends mainly on an acid-labile hemagglutinin with a single amino acid mutation in the stalk region.

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability.

Early Steps of Individual Multireceptor Viral Interactions Dissected by High-Density, Multicolor Quantum Dot Mapping in Living Cells.

Viral capture and entry to target cells are the first crucial steps that ultimately lead to viral infection. Understanding these events is essential toward the design and development of suitable antiviral drugs and/or vaccines. Viral capture involves dynamic interactions of the virus with specific receptors in the plasma membrane of the target cells. In the last years, single virus tracking has emerged as a powerful approach to assess real time dynamics of viral processes in living cells and their engagement with specific cellular components. However, direct visualization of the early steps of multireceptor viral interactions at the single level has been largely impeded by the technical challenges associated with imaging individual multimolecular systems at relevant spatial (nanometer) and temporal (millisecond) scales. Here, we present a four-color, high-density quantum dot spatiotemporal mapping methodology to capture real-time interactions between individual virus-like-particles (VLPs) and three different viral (co-) receptors on the membrane of primary living immune cells derived from healthy donors. Together with quantitative tools, our approach revealed the existence of a coordinated spatiotemporal diffusion of the three different (co)receptors prior to viral engagement. By varying the temporal-windows of cumulated single-molecule localizations, we discovered that such a concerted diffusion impacts on the residence time of HIV-1 and SARS-CoV-2 VLPs on the host membrane and potential viral infectivity. Overall, our methodology offers the possibility for systematic analysis of the initial steps of viral-host interactions and could be easily implemented for the investigation of other multimolecular systems at the single-molecule level.

WNV and SLEV coinfection in avian and mosquito hosts: impact on viremia, antibody responses, and vector competence.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown.IMPORTANCEWest Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related viruses that are transmitted by the same mosquitoes and infect the same birds in nature. Both viruses circulate in the same regions and have caused concurrent outbreaks in humans. It is possible that mosquitoes, birds, and/or humans could be infected with both WNV and SLEV simultaneously, as has been observed with Zika, chikungunya, and dengue viruses. To study the impact of coinfection, we experimentally infected vertebrate and invertebrate cells, American robins, and two Culex species with WNV and/or SLEV. Robins were efficiently coinfected, with no impact of coinfection on virus levels or immune response. Similarly, in mosquitoes, coinfection did not impact infection rates, and mosquitoes could transmit both WNV and SLEV together. These results reveal that WNV and SLEV coinfection in birds and mosquitoes can occur in nature, which may impact public health and human disease risk.

Translation of HIV-1 unspliced RNA is regulated by 5' untranslated region structure.

HIV-1 must generate infectious virions to spread to new hosts and HIV-1 unspliced RNA (HIV-1 RNA) plays two central roles in this process. HIV-1 RNA serves as an mRNA that is translated to generate proteins essential for particle production and replication, and it is packaged into particles as the viral genome. HIV-1 uses several transcription start sites to generate multiple RNAs that differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. The virus relies on host machinery to translate its RNAs in a cap-dependent manner. Here, we demonstrate that the 5' context of HIV-1 RNA affects the efficiency of translation both in vitro and in cells. Although both RNAs are competent for translation, 3G RNA is translated more efficiently than 1G RNA. The 5' untranslated region (UTR) of 1G and 3G RNAs has previously been shown to fold into distinct structural ensembles. We show that HIV-1 mutants in which the 5' UTR of 1G and 3G RNAs fold into similar structures were translated at similar efficiencies. Thus, the host machinery translates two 99.9% identical HIV-1 RNAs with different efficiencies, and the translation efficiency is regulated by the 5' UTR structure.IMPORTANCEHIV-1 unspliced RNA contains all the viral genetic information and encodes virion structural proteins and enzymes. Thus, the unspliced RNA serves distinct roles as viral genome and translation template, both critical for viral replication. HIV-1 generates two major unspliced RNAs with a 2-nt difference at the 5' end (3G RNA and 1G RNA). The 1G transcript is known to be preferentially packaged over the 3G transcript. Here, we showed that 3G RNA is favorably translated over 1G RNA based on its 5' untranslated region (UTR) RNA structure. In HIV-1 mutants in which the two major transcripts have similar 5' UTR structures, 1G and 3G RNAs are translated similarly. Therefore, HIV-1 generates two 9-kb RNAs with a 2-nt difference, each serving a distinct role dictated by differential 5' UTR structures.

Role of RNA G-Quadruplexes in the Japanese Encephalitis Virus Genome and Their Recognition as Prospective Antiviral Targets.

G-quadruplexes (GQs) have been primarily studied in the context of cancer and neurodegenerative pathologies. However, recent research has shifted focus to their existence and functional roles in viral genomes, revealing GQ-regulated key pathways in various human pathogenic viruses. While GQ structures have been reported in the genomes of emerging and re-emerging viruses, RNA viruses have been understudied compared to DNA viruses, including notable examples such as human immunodeficiency virus-1, hepatitis C virus, Ebola virus, Nipah virus, Zika virus, and SARS-CoV-2. The flavivirus family, comprising the Japanese encephalitis virus (JEV), poses a significant global threat due to recurring outbreaks yet lacks approved antivirals. In this study, we identified and characterized eight putative G-quadruplex-forming motifs within essential genes involved in genome replication, assembly, and internalization in the host cell, conserved across different JEV isolates. The formation and stability of these motifs were validated through a multitude of biophysical and cell-based assays. The interaction and binding affinity of these motifs with the known GQ-binding ligand BRACO-19 were supported by biophysical assays, confirming the capability of these motifs to form GQ structures. Notably, BRACO-19 also exerted antiviral properties through reduction of viral replication and infectious virus titers as well as inhibition of viral protein expression, as evaluated by the cell-based assays. This comprehensive molecular characterization of G-quadruplex structures within the JEV genome highlights their potential as promising antiviral targets for intervention strategies against JEV infection through GQ-specific ligands.

Linear epitopes of PRRSV-1 envelope proteins ectodomains are not correlated with broad neutralization

BackgroundNeutralizing antibodies against PRRSV are capable of conferring protection against viral reinfection, but they tend to be strain specific and usually have poor cross-reactivity. Nonetheless, it has been described that there are individuals capable of efficiently neutralizing viruses of different origin, so it is expected that there are conserved neutralizing epitopes relevant for broad neutralization. However, although immunodominant regions and neutralizing epitopes have been described in different envelope proteins, their role in broad neutralization is unknown. The main objective of this study was to determine whether the linear epitopes existing in the ectodomains of PRRSV envelope proteins play a role in cross-neutralization.ResultsA pepscan analysis was carried out using synthetic peptides against the ectodomains of PRRSV envelope proteins and PRRSV-hyperimmune sera of different cross-reactivity. The results obtained confirm the existence of antigenic regions in the ectodomains of the GP2, GP3, GP4 and GP5 that tend to be relatively conserved among different PRRSV isolates. Nonetheless, these antigenic regions have poor immunogenicity since they are only recognized by a limited number of sera. Furthermore, no differences were found between the reactivity of sera with broad cross-neutralization capacity and sera with poor heterologous neutralization activity, which indicate that linear epitopes existing in the ectodomains of PRRSV envelope proteins are not relevant for the development of broadly reactive neutralizing antibodies. Subsequently, some selected peptides were used in competition assays with the virus for binding to the cell receptors and in seroneutralization inhibition assays by incubation with hyperimmune sera. Firstly, some peptides that interfere with virus infectivity were identified in competition assays, but only in the case of one viral isolate, which points to the possible existence of a strain-dependent inhibition. However, the results of the seroneutralization inhibition assay indicate that, under the conditions of our study, none of the peptides used was capable of inhibiting virus neutralization by the hyperimmune sera.ConclusionsThe results obtained indicate that the linear peptides analyzed in this study do not play a major role in the induction of broadly reactive neutralizing antibodies, which could probably depend on conformational neutralizing.

Identifying the amino acid domains of ORF59 responsible for interactions with ORF57 and PAN RNA during KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.

Effects of Alkaline Solutions on the Structure and Function of Influenza A Virus

Influenza A virus (IAV) infection contributes to high annual morbidity and mortality, thus necessitating measures aimed at protecting against the disease. Alcohol-based disinfectants are commonly used to inactivate IAV, but they have several undesirable properties. In search of other means which would inactivate IAV, we focused on the effect of alkaline solutions on IAV. We found the viral infectivity remarkably decreased with treatment of an alkaline solution at pH 12.0 for 1 min, where destruction of the viral spikes was observed using an electron microscope. A more detailed examination revealed that the infectivity of IAV was remarkedly reduced by brief treatment with the alkaline solution at pH 11.75 or above, most likely due to the degradation of viral hemagglutinin protein. These results show that at a high pH, the haemagglutinin protein is degraded, resulting in very rapid inactivation of IAV.

Prediction of lumpy skin disease virus using customized CBAM-DenseNet-attention model

Lumpy skin disease virus (LSDV) is an extremely infectious, viral, and chronic skin disease that is caused by the Capripox virus. This viral disease is predominantly found in cows. Mosquitoes and ticks are the primary transmitters for the spread of this virus. Recently, LSDV has been rapidly spreading all over the world, especially in several areas of Pakistan, India, and Iran. Thousands of cows have died due to this infectious virus in Pakistan and early detection of LSDV is needed to avoid further loss. The prediction and classification of LSDV are hindered by the lack of publicly available datasets. Despite a few studies using LSDV datasets, such datasets are often small, which may lead to model overfitting. In this regard, we collect the dataset from several online sources, as well as, collecting images from veterinary farms in different areas of Pakistan. Deep learning has been widely used in the medical field for disease detection and classification. Therefore, this study leverages DenseNet deep learning models for LSDV detection and classification. Experiments are performed using VGG-16, ResNet-50, MobileNet-V2, custom-designed convolutional neural network, and Inception-V3. The DenseNet architecture presents a Convolutional Block Attention Module (CBAM) and Spatial Attention (SA) for the prediction and classification of LSD. Results demonstrate that a 99.11% accuracy can be obtained on the augmented dataset while a 94.23% accuracy can be achieved with the original dataset for chicken pox, monkey pox, and LSDV. Comparison with state-of-the-art studies corroborates the superior performance of the proposed model.

The signal peptide of BmNPV GP64 activates the ERAD pathway to regulate heterogeneous secretory protein expression

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems.

Molecular characterization of reassortant infectious bursal disease virus (IBDV) strains of genogroup A3B1 detected in some areas of Britain between 2020 and 2021

Infectious bursal disease virus (IBDV) causes a major immunosuppressive disease of chickens. As part of ongoing epidemiological surveillance for IBDV, the hypervariable region (HVR) of the VP2 capsid gene encoded by segment A, and a region of the VP1 polymerase gene, encoded by segment B, were sequenced from 20 IBDV-positive bursal samples obtained in 2020 and 2021, from 16 commercial British broiler farms. Birds had received a live IBDV vaccine at 17–22 days of age, and samples were obtained at 25–55 days of age. Of the 16 farms, none contained very virulent (vv) strains, one contained a classical virulent strain, two contained vaccine strains, and five contained sequences of reassortant strains with a vv segment A and a non-vv segment B belonging to genogroup A3B1. In eight of the farms, we identified the sequences of both genogroup A3B1 reassortant strains and vaccine strains in the same samples. Therefore, the majority of the farms (13/16 (81%)) contained genogroup A3B1 reassortant viruses. Of the flocks containing reassortant strains, 5/13 (38%) had HVR mutations Q219L, G254D, D279N, and N280T, consistent with a recently described Western European clade, but the rest had other mutations or no mutations, demonstrating that multiple clades were present in the samples. Taken together, vv strains were not detected, but reassortant strains predominated in the farms, which belonged to different clades, and were frequently found together with vaccine strains.

Streptomyces fradiae Mitigates the Impact of Potato Virus Y by Inducing Systemic Resistance in Two Egyptian Potato (Solanum tuberosum L.) Cultivars

In this study, the impact of culture media filtrate of QD3 actinobacterial isolate on two potato cultivars, Spunta and Diamond, infected with potato virus Y (PVY) was investigated. Various parameters, including infection percentage, PVY virus infectivity, disease severity scoring, PVY optical density, photosynthetic and defense-related biochemical markers, enzymatic profiling, phenolic compounds, proline content, salicylic acid levels, and growth and yield parameters, were assessed to elucidate the potential of the QD3 actinobacterial isolate culture filtrate in mitigating PVY-induced damage. The physiological and biochemical characteristics of the QD3 actinobacterial isolate, including its salinity tolerance, pH preferences, and metabolic traits, were investigated. Molecular identification via 16S rRNA gene sequencing confirmed its classification as Streptomyces fradiae QD3, and it was deposited in GenBank with the gene accession number MN160630. Distinct responses between Spunta and Diamond cultivars, with Spunta displaying greater resistance to PVY infection. Notably, pre-infection foliar application of the QD3 filtrate significantly reduced disease symptoms and virus infection in both cultivars. For post-PVY infection, the QD3 filtrate effectively mitigated disease severity and the PVY optical density. Furthermore, the QD3 filtrate positively influenced photosynthetic pigments, enzymatic antioxidant activities, and key biochemical components associated with plant defense mechanisms. Gas chromatography‒mass spectrometry (GC‒MS) analysis revealed palmitic acid (hexadecanoic acid, methyl ester) and oleic acid (9-octadecanoic acid, methyl ester) as the most prominent compounds, with retention times of 23.23 min and 26.41 min, representing 53.27% and 23.25%, respectively, of the total peak area as primary unsaturated fatty acids and demonstrating antiviral effects against plant viruses. Cytotoxicity assays on normal human skin fibroblasts (HSFs) revealed the safety of QD3 metabolites, with low discernible toxicity at high concentrations, reinforcing their potential as safe and effective interventions. The phytotoxicity results indicate that all the seeds presented high germination rates of approximately 95–98%, suggesting that the treatment conditions had no phytotoxic effect on the Brassica oleracea (broccoli) seeds, Lactuca sativa (lettuce) seeds, and Eruca sativa (arugula or rocket) seeds. Overall, the results of this study suggest that the S. fradiae filtrate has promising anti-PVY properties, influencing various physiological, biochemical, and molecular aspects in potato cultivars. These findings provide valuable insights into potential strategies for managing PVY infections in potato crops, emphasizing the importance of Streptomyces-derived interventions in enhancing plant health and crop protection.

Differential Expression of Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) and Interleukin12B (IL12B) in the Bursa of Fabricius of chicken infected with Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) caused by Infectious Bursal Disease virus (IBDV), poses a significant threat to the poultry industry, causing immunosuppression and economic losses since its discovery in 1957. To understand the host response to IBDV infection, this study examined the modulation of MAP2K3 and IL12B in chickens infected with IBDV. A local IBDV isolate propagated in embryonated chicken eggs was inoculated into 18-day-old chicken. The mRNA transcription levels of MAP2K3 and IL12B in bursal tissue were quantified by real-time quantitative polymerase chain reaction on day two post-infection and compared with those of uninfected, age-matched control birds (sham inoculated). The study revealed increased expression of the MAP2K3 and IL12B genes, indicating their role in inflammatory response during IBDV infection. Thus, the present investigation sheds light on the involvement of MAP2K3 and IL12B in the inflammatory response to IBDV infection in chickens. Keywords: Infectious bursal disease virus, immunosuppression, MAP2K3, IL12B, pro-inflammatory cytokine gene expression

Dependence of aerosol-borne influenza A virus infectivity on relative humidity and aerosol composition.

We describe a novel biosafety aerosol chamber equipped with state-of-the-art instrumentation for bubble-bursting aerosol generation, size distribution measurement, and condensation-growth collection to minimize sampling artifacts when measuring virus infectivity in aerosol particles. Using this facility, we investigated the effect of relative humidity (RH) in very clean air without trace gases (except ∼400 ppm CO2) on the preservation of influenza A virus (IAV) infectivity in saline aerosol particles. We characterized infectivity in terms of 99%-inactivation time, t 99, a metric we consider most relevant to airborne virus transmission. The viruses remained infectious for a long time, namely t 99 > 5 h, if RH < 30% and the particles effloresced. Under intermediate conditions of humidity (40% < RH < 70%), the loss of infectivity was the most rapid (t 99 ≈ 15-20 min, and up to t 99 ≈ 35 min at 95% RH). This is more than an order of magnitude faster than suggested by many previous studies of aerosol-borne IAV, possibly due to the use of matrices containing organic molecules, such as proteins, with protective effects for the virus. We tested this hypothesis by adding sucrose to our aerosolization medium and, indeed, observed protection of IAV at intermediate RH (55%). Interestingly, the t 99 of our measurements are also systematically lower than those in 1-μL droplet measurements of organic-free saline solutions, which cannot be explained by particle size effects alone.

SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo.

In late 2019, SARS-CoV-2 spilled-over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions, and compare species, sex, age, virus and antibody detection assay, and types, frequency and length of clinical signs, demonstrating broad heterogeneity amongst infections. We then describe a SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for four months. Lions had significantly more viral RNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health.