Airborne transmission represents a primary route of influenza virus dissemination. In animal infection models, conventional intranasal instillation fails to recapitulate the physiological features of natural respiratory infection. Existing aerosol-based challenge methods—including whole-body inhalation and oronasal exposure—face limitations in precise quantification of the delivered infectious dose. Moreover, oronasal exposure systems require specialized, high-cost instrumentation. This study established an economical and dosing-controlled aerosol challenge method utilizing a lung fluid quantitative nebulizer, which can deliver aerosolized virus directly to the lower respiratory tract via endotracheal intubation. The count median aerodynamic diameter (CMAD) of aerosols generated by the nebulizer was 2.28 μm. We compared pulmonary deposition efficiency and pathogenicity between lung-delivery aerosol inhalation and conventional intranasal instillation of PR8 influenza virus in mice. The pulmonary deposition rate for the aerosol inhalation was 63.1%, with an LD50 of 31.1 EID50; in contrast, intranasal instillation achieved a pulmonary deposition rate of 20.0% and an LD50 of 115.0 EID50. Histopathological analysis revealed significantly more severe lesions in lungs of mice challenged via lung-delivery aerosol inhalation relative to those receiving intranasal instillation. Furthermore, using a guinea pig model and a recombinant PR8 virus expressing Gaussia luciferase (PR8-Gluc), we demonstrated that pulmonary aerosol inhalation resulted in both more homogeneous spatial distribution and higher viral loads across lung tissues. Our results demonstrate that animals are more sensitive to lung-delivery inhaled virus than virus delivered by intranasal instillation. This study provides an optimized, economical, and technically straightforward aerosol challenge method with controlled administered dosing for viral infection models.

The respiratory viral shedding pattern of Mpox patients and the airborne transmissibility remain unclear. It is often challenging to determine the initial exposure time and infective dose at the time of exposure in Mpox patients, making it impractical to track the viral shedding pattern in human Mpox cases. Therefore, we monitored respiratory viral shedding in macaques experimentally infected with Clade IIb Mpox virus (MPXV), as well as the contamination of the ambient air. Our study shows MPXV-infected macaques had obvious symptoms, high respiratory viral shedding and continuous virus-laden particle release. The viral load in both exhaled particles and air within the isolator peaked at 11 d post infection (1.58 × 1010 copies/macaque/hour and 1.9 × 107 copies/L respectively). Most virus-laden particles (from macaques and air within the isolator) were coarse particles (>7 μm); during peak viral emission, 94%-97% of exhaled particle viral copies and 87%-97% of air within the isolator ones concentrated here. Infectious viruses were present in the exhaled coarse particles of macaques and, in some cases, in particles from the air within the isolator, but not in fine particles. This study provides evidence supporting the airborne transmission of MPXV and suggests that respiratory coarse particles may play a more significant role than fine particles in driving this mode of transmission.

The monkeypox virus (MPXV) has spread globally, posing a severe challenge to global public health. This study systematically evaluated the aerosol shedding dynamics of the epidemic Clade IIb MPXV strain in infected young rabbits, along with its direct contact and airborne transmission potential among them. We found that young rabbits could be experimentally infected with MPXV, exhibiting distinct pathogenic features and viral shedding patterns. Young rabbits infected with MPXV shed the virus through nasal secretions and exhaled aerosols, peaking at 7 dpi. In total, 89–95.8% of virus-laden respiratory particles had a diameter ≥4.7 μm. Notably, MPXV can be efficiently shed and transferred among young rabbits through direct contact and airborne routes. The nasal secretions and exhaled virus particles from donor rabbits can be contacted or inhaled by recipient rabbits. Large amounts of viral DNA were detected in the nasal wash of rabbits exposed to contact or airborne exposure. Furthermore, virus particles invade the lungs, causing pathological changes and disseminating them to multiple organs. However, no infectious virus was successfully recovered from these recipient rabbits, as their exposed or inhaled MPXV dose might have been below the MPXV’s minimum infectious dose for young rabbits. These findings indicate that although the airborne transmissibility of the current MPXV strain is relatively limited, inhalation of viral particles following airborne exposure can still result in bodily damage. Continuous monitoring of MPXV transmissibility and mutation evolution is imperative to prevent efficient respiratory aerosol transmission, which guides global monkeypox prevention and control strategies.

The H7N9 influenza viruses, which are capable of causing severe respiratory syndrome in humans, were first discovered to infect humans in 2013 and continue to pose a persistent public health threat. Quail has been proposed as a potential intermediate host that may facilitate the emergence of novel reassorted influenza A viruses with the capacity to infect humans across species barriers; however, information on the biological characterization of quail H7N9 remains limited. In this study, we isolated and identified an avian H7N9 influenza virus from quails, designated as A/quail/Hebei/CH06-07/2018 (H7N9) and abbreviated as CH06-07, in Hebei, China. Phylogenetic analyses revealed that both the HA gene and the NA gene of CH06-07 were clustered in the Eurasian lineage. Furthermore, CH06-07 exhibited binding affinity for both α2,3-linked and α2,6-linked sialic acid receptors and demonstrated high pathogenicity in both quails and mice. Notably, transmission studies revealed that CH06-07 not only exhibited efficient inter-quail transmission and inter-guinea pig transmission but also demonstrated effective cross-species transmission. Importantly, infected quails and guinea pigs generated significant quantities of viral aerosols (≥18,998 ± 1672 copies per liter of air at 3 days post-infection), and infectious viruses were successfully recovered from environmental aerosols. These findings highlight the necessity for continuous surveillance of the prevalence of quail-origin H7N9 influenza A viruses in poultry populations due to their potential threat to human health.

The global spread of the monkeypox virus (MPXV) poses a significant challenge to public health, yet reliable and consistent animal models for evaluating MPXV remain limited, which to some extent restricts the advancement of treatment strategies and transmission-blocking technologies. As natural hosts, African dormice ( Graphiurus spp. ) represent promising candidates. However, the biological characteristics of MPXV in dormice remain largely unexplored. This study systematically evaluated the pathogenicity and transmissibility of MPXV in dormice. Experimental results demonstrated that dormice are highly susceptible to MPXV infection. Following intranasal inoculation, MPXV induced significant weight loss, lethal infections, and multi-organ pathological damage, with robust viral replication in respiratory and liver tissues. Notably, MPXV can efficiently transmit among dormice through direct contact, with one-third of the contact-exposed dormice shedding infectious viruses and two-thirds exhibiting seropositivity. In addition, one-third of the airborne exposure dormice show seropositivity, yet no infectious viruses were detected in their respiratory tissues, indicating that the airborne transmissibility of MPXV among dormice is relatively restricted. Furthermore, it was observed that infected dormice continuously released large quantities of virus-laden aerosols, with emissions peaking on 12 dpi (3.78±1.01×106 copies/dormouse/hour of respiration). Particle size analysis revealed that the viral copies in ≥7 μm coarse particles accounted for >90.9% of exhaled viral aerosols during peak shedding (8–14 dpi). These findings demonstrate that the African dormice serve as an effective animal model for assessing MPXV infection, pathogenicity, and transmission dynamics. Simultaneously, enhanced monitoring of wild dormouse populations is critical due to their potential role in MPXV transmission chains.

Lentinan (LNT) was found to reduce the aerosol transmission rate between golden hamsters from 100% (9/9) to 44.4% (4/9). The viral loads in the respiratory system, including the nasal turbinate, trachea, and lung, were significantly reduced in the infected golden hamsters that received LNT treatment. Furthermore, the amount of exhaled virus aerosols in hamsters treated with LNT was significantly lower than that in untreated hamsters throughout the entire disease progression. In detail, the amounts of virus-laden particles with aerodynamic diameters less than 5 µm exhibited a significant decreasing trend following LNT treatment. Moreover, the detection rate of infectious SARS-CoV-2 in each stage of the Anderson-6 sampler exhibited a decreasing trend following LNT treatment post-infection. In summary, our findings indicate that LNT therapy represents a promising therapeutic candidate for the treatment of COVID-19 patients. Meanwhile, during the course of treatment, LNT has the potential to reduce viral infectivity in affected individuals.

Somatic mutations are the cause of cancer and have been implicated in other, noncancerous diseases and aging. While clonally expanded mutations can be studied by deep sequencing of bulk DNA, very few somatic mutations expand clonally, and most are unique to each cell. We describe a detailed protocol for single-cell whole-genome sequencing to discover and analyze somatic mutations in tissues and organs. The protocol comprises single-cell multiple displacement amplification (SCMDA), which ensures efficiency and high fidelity in amplification, and the SCcaller software tool to call single-nucleotide variations and small insertions and deletions from the sequencing data by filtering out amplification artifacts. With SCMDA and SCcaller at its core, this protocol describes a complete procedure for the comprehensive analysis of somatic mutations in a single cell, covering (1) single-cell or nucleus isolation, (2) single-cell or nucleus whole-genome amplification, (3) library preparation and sequencing, and (4) computational analyses, including alignment, variant calling, and mutation burden estimation. Methods are also provided for mutation annotation, hotspot discovery and signature analysis. The protocol takes 12–15 h from single-cell isolation to library preparation and 3–7 d of data processing. Compared with other single-cell amplification methods or single-molecular sequencing, it provides high genomic coverage, high accuracy in single-nucleotide variation and small insertions and deletion calling from the same single-cell genome, and fewer processing steps. SCMDA and SCcaller require basic experience in molecular biology and bioinformatics. The protocol can be utilized for studying mutagenesis and genome mosaicism in normal and diseased human and animal tissues under various conditions.

Substantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification. We tested this in cell culture, which avoids the limitation that an increased mutation burden in vivo typically leads to cancer. We performed single-cell whole-genome sequencing of primary fibroblasts from DNA mismatch repair (MMR) deficient Msh2-/- mice and littermate control animals after long-term passaging. Apart from analyzing somatic mutation burden we analyzed clonality, mutational signatures, and hotspots in the genome, characterizing the complete landscape of somatic mutagenesis in normal and MMR-deficient mouse primary fibroblasts during passaging. While growth rate of Msh2-/- fibroblasts was not significantly different from the controls, the number of de novo single-nucleotide variants (SNVs) increased linearly up until at least 30,000 SNVs per cell, with the frequency of small insertions and deletions (INDELs) plateauing in the Msh2-/- fibroblasts to about 10,000 INDELS per cell. We provide evidence for negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots. Overall, our results provide evidence that increased somatic mutation burden drives significant cell evolutionary changes in a dynamic cell culture system without significant effects on growth. Since similar selection processes against mutations preventing organ and tissue dysfunction during aging are difficult to envision, these results suggest that increased somatic mutation burden can play a causal role in aging and diseases other than cancer.

IntroductionInfluenza A viruses (IAVs) are important pathogens of respiratory infections, causing not only seasonal influenza but also influenza pandemics and posing a global threat to public health. IAVs infection spreads rapidly, widely, and across species, causing huge losses, especially zoonotic IAVs infections that are more harmful. Fast and sensitive detection of IAVs is critical for controlling the spread of this disease.MethodsHere, a real-time reverse transcription recombinase-aided amplification (real-time RT-RAA) assay targeting conserved positions in the matrix protein gene (M gene) of IAVs, is successfully established to detect IAVs. The assay can be completed within 20 min at 42°C.ResultsThe sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability, which was comparable to the sensitivity of the RT-qPCR assay. The specificity assay showed that the real-time RT-RAA assay was specific to IAVs, and there was no cross-reactivity with other important viruses. In addition, 100%concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 120 clinical specimens.DiscussionThe results suggested that the real-time RT-RAA assay we developed was a specific, sensitive and reliable diagnostic tool for the rapid detection of IAVs.

Poultry farms are a complex environment for close contact between humans and animals. Accumulating evidence has indicated that pathogens and drug resistance genes in chicken houses may pose a serious threat to public health and economic concerns. However, insufficient knowledge of the indoor aerosol microbiome and resistome profiles of layer hen houses hampers the understanding of their health effects. Environmental surveillance of antibiotic resistance may contribute to a better understanding and management of the human exposure risk of bioaerosols under the environmental conditions of chicken houses. In addition, the chicken house has a long operation cycle, and the bacterial diversity and antibiotic resistance genes of aerosols in different periods may be different. In this study, air samples were collected from 18 chicken houses on three farms, including the early laying period (EL), peak laying period (PL), and late laying period (LL). 16S rRNA gene sequencing and metagenomics were used to study the composition of the bacteria and resistome in aerosols of layer hen houses and the results showed that they varied with laying period. The highest alpha diversity of bacteria was observed in PL bioaerosols. The dominant bacterial phyla included Firmicutes, Bacteroidetes and Proteobacteria. Three potential pathogenic bacterial genera (Bacteroides, Corynebacterium and Fusobacterium) were found. The most abundant ARG type was aminoglycosides in all laying periods. In total, 22 possible ARG host genera were detected. ARG subtypes and abundance were both higher in LL. Network analysis also showed higher co-occurrence patterns between the bacteria and resistome in bioaerosols. The laying period plays an important role in the bacterial community and resistome in layer house aerosols.

Abstract Single-cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single-cell somatic mutation analysis remains to be developed. We address the two largest sources of artifacts, i.e., DNA denaturation-related cytosine deamination and allelic bias-driven whole genome amplification errors. We first reconfigured multiple displacement amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single-cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP-based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA-amplified single cells with unamplified clones derived from single cells from the same population. Using this highly accurate single-cell whole-genome sequencing method we analyzed human B lymphocytes from donors varying in age from birth to over 100 year, studying both genome distribution and functional impact of base substitution mutations. Mutations per cell were found to increase with age from less than 500 in cord blood to over 3,000 in cells from individuals over 100. While overall mutations were randomly distributed across the genome with all chromosomes equally affected, 24 hotspot regions were identified, five of which were part of immunoglobulin variable regions subject to somatic hypermutation. Age-related mutation accumulation was found to be significantly slower in genomic sequences directly involved in cellular function, such as exons and gene regulatory sequences. Still, on average, B lymphocytes from aged individuals contained 3-15 damaging mutations in the transcribed part of the exome identified by RNA-seq, plus 15-50 mutations in transcription factor binding sites identified by ATAC-seq. Analysis of the spontaneous mutation spectra in these normal cells revealed signatures similar to those previously found associated with B cell leukemia and other cancers. These results indicate that cancer risk is already part of age-related mutation accumulation in normal cells. Taken together, our single-cell sequencing method provides a firm foundation for analyzing cellular genetic heterogeneity in normal human tissues. Citation Format: Xiao Dong, Lei Zhang, Moonsook Lee, Alexander Y. Maslov, Jan Vijg. Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 440.

On-line virtual games and community have becoming a new way of entertainment and networking. As people exhibit different activities in the virtual world as the real world, obsession in virtual world enjoyment might seems to reduce people’s time on real life consumption, job, which seems to jeopardize their job performance and curtail the revenue generated from the real world. This article tried to analyze the effect of virtual activities on the macro-economy. We find that virtual activities, which include in-world activities and inter-world activity, do generate economic value and make contribution to the GDP for the whole country. We also develop a method on how to measure the net replacement effect of the virtual economic activity on real GDP, and the net effect on Mega-GDP. Data from Second Life demonstrates that the virtual game and online community has positive effect on the Mega-GDP and is beneficial to the economy.

On-line games have become a new way of entertainment. In order to analyze why people choose to enter an online game which might harm their real life and job, and how they allocate time between real world and virtual world, this paper proposed a Multi-Extended Meta-Utility Function where people get physiological, psychological and monetary satisfaction from the real world (main utility function) and the virtual world (auxiliary utility function). People will balance their time allocation between the two worlds to maximize their meta-utility. We also proposed a method of survey on the lowest monetary compensation for people cutting online game-play time, to indirectly measure the relative psychological satisfaction and utility value from virtual games compared with that from the real world. Results from survey show that pure psychological utility from virtual activities is much higher than that from the real world, which drives people into the virtual games.

To construct the fused expression vector of ctxA gene and ctxB gene of Vibrio cholerae and to realize the expression of ctxAB gene of Vibrio cholerae in E. coli and to lay basis for future research on the values of immunogenicity and immunoadjuvant, in this study, the fusion gene ctxAB containing ctxA and ctxB gene was obtained from DNA of Vibrio cholera by overlap PCR, and to construct recombinant plasmid pET-ctxAB the fusion gene ctxAB was cloned into prokaryotic expressed vector pET32a( + ) containing thioredoxin gene Trx, and pET-ctxAB was transformed into E. coli strain BL21(DE3). pET-ctxAB was analyzed with restriction-endonuclease digestion, PCR and DNA sequencing analysis, and was induced with isopropy-β-D-thiogalactoside (IPTG) to express fusion protein Trx-CTAB, and Trx-CTAB was examined with SDS-PAGE and Western blot techniques. Restriction endonuclease digestion, PCR and DNA sequencing analysis showed that the ctxAB gene of 1 158 bp was amplified from Vibrio cholerae DNA, and the recombinant plasmid pET-ctxAB was constructed and its expression in prokaryotic cell was detected successfully with SDS-PAGE and Western blot techniques. Itis concluded that the ctxA gene and ctxB gene of Vibrio cholerae were fused together by overlap PCR, and the fusion ctxAB gene has been highly expressed in E. coli.

We cloned cholera toxin subunit B gene from 569B and M045 strain of Vibrio cholerae with polymerase chain reaction, constructed recombinant plasmid pCTB, and transformed pCTB into the prokaryotic cell strain JM109. The indentification was made by means of restriction enzyme analysis, polymerase chain reaction, DNA sequencing, SDS-polyacrylamine gel electrophoresis analysis and Western blot. The results indicate that we have amplified cholera toxin subunit B gene of 376 bp from Vibrio cholerae and hve constructed the recombinant plasmid pCTB, and we have affained the object amied at successful expression of 12 KD in the prokaryotic cell strain.