Mechanisms of Natural Killer Cell Evasion Through Viral Adaptation.

The continuous interactions between host and pathogens during their coevolution have shaped both the immune system and the countermeasures used by pathogens. Natural killer (NK) cells are innate lymphocytes that are considered central players in the antiviral response. Not only do they express a variety of inhibitory and activating receptors to discriminate and eliminate target cells but they can also produce immunoregulatory cytokines to alert the immune system. Reciprocally, several unrelated viruses including cytomegalovirus, human immunodeficiency virus, influenza virus, and dengue virus have evolved a multitude of mechanisms to evade NK cell function, such as the targeting of pathways for NK cell receptors and their ligands, apoptosis, and cytokine-mediated signaling. The studies discussed in this article provide further insights into the antiviral function of NK cells and the pathways involved, their constituent proteins, and ways in which they could be manipulated for host benefit.

Silica-based nanosystems against antibiotic-resistant bacteria and pathogenic viruses.

Today, with the intensity of antibiotic abuse and self-medication, the need for the use of novel systems with high efficiency and biosafety for targeted drug delivery against antibiotic-resistant bacteria and their infections should be highly considered by researchers. Silica-based nanosystems with unique physicochemical properties such as large surface area, tuneable pore diameter, drug loading capacity, controlled particle size/morphology, and good biocompatibility are attractive candidates against antibiotic-resistant bacteria and pathogenic viruses. They can be loaded with antiviral and antimicrobial drugs or molecules through their exclusive internal porous structures or different surface linkers. In this context, smart nanosystems can be produced via suitable surface functionalization/modification with a variety of functional groups to act against different clinical pathogenic microbes or viruses, offering great opportunities for controlling and treating various infections. However, important criteria such as the ability to degrade, biocompatibility, biodegradability, cytotoxicity, stability, clearance from targeted organs should be systematically analysed to develop nanosystems or nanocarriers with high efficiency and multifunctionality. Herein, recent advancements pertaining to the application of silica-based nanosystems against antibiotic-resistant bacteria and pathogenic viruses are deliberated, focussing on important challenges and future perspectives.

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses.

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.

Nanophotocatalysts against viruses and antibiotic-resistant bacteria: recent advances.

Photocatalysis has attracted great attention because of its direct utilisation of sunlight to obtain various chemical reactions, causing water splitting, organic pollutant degradation, and water disinfection. Nanophotocatalysts can be employed for various applications, including hydrogen storage, green diesel production, fuel cell applications, industrial manufacturing methods, pharmaceutical industries, and catalytic degradation of contaminants/hazardous materials. Photocatalytic inactivation and removal of pathogenic viruses, antibiotic-resistant bacteria and antibiotic resistance genes can be considered as simple and effective technique with low-cost, eco-friendliness, and low energy consumption features. The high specific surface areas, abundant functional groups, large amounts of active sites are some advantages of the nanostructures for photocatalytic activity with high efficiency. However, some important limitations/drawbacks of nanophotocatalysts for industrial and commercial applications such as the low selectivity, aggregation/sedimentation, difficult separation, low-usage of visible light, fast charge recombination, and low migration potential of photogenerated electrons/holes are need to be comprehensively and analytically investigated and addressed by researchers. This critical review highlighted the recent advancements in photocatalytic disinfection of pathogenic viruses and antibiotic-resistant bacteria, focussing on the development of highly efficient nanophotocatalysts and their underlying mechanisms of inactivation/removal of these pathogens.

Photo-catalyzed TiO2 inactivates pathogenic viruses by attacking viral genome.

Previous observations have been reported that viruses were inactivated using strong irradiation. Here, new evidence was disclosed by studying the effects of nanosized TiO2 on viral pathogens under a low irradiation condition (0.4 mW/cm2 at UVA band) that mimics the field setting. We showed that photo-activated TiO2 efficiently inhibits hepatitis C virus infection, and weak indoor light with intensity of 0.6 mW/cm2 at broad-spectrum wavelength and around 0.15 mW/cm2 of UVA band also lead to partial inhibition. Mechanistic studies demonstrated that hydroxyl radicals produced by photo-activated TiO2 do not destroy virion structure and contents, but attack viral RNA genome, thus inactivating the virus. Furthermore, we showed that photo-activated TiO2 inactivates a broad range of human viral pathogens, including SARS-CoV-2, a novel coronavirus responsible for the ongoing COVID-19 pandemic. In conclusion, we showed that photo-catalyzed nanosized TiO2 inactivates pathogenic viruses, paving a way to its field application in control of viral infectious diseases.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses.

Medical research is changing into direction of precision therapy, thus, sophisticated preclinical models are urgently needed. In human pathogenic virus research, the major technical hurdle is not only to translate discoveries from animals to treatments of humans, but also to overcome the problem of interspecies differences with regard to productive infections and comparable disease development. Transgenic mice provide a basis for research of disease pathogenesis after infection with human-specific viruses. Today, humanized mice can be found at the very heart of this forefront of medical research allowing for recapitulation of disease pathogenesis and drug mechanisms in humans. This review discusses progress in the development and use of transgenic mice for the study of virus-induced human diseases towards identification of new drug innovations to treat and control human pathogenic infectious diseases.

Susceptibility of Influenza A, B, C, and D Viruses to Baloxavir1.

Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater.

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Occurrences of similar viral diversity in campus wastewater and reclaimed water of a university dormitory.

Water reuse from wastewater sources still remain some critical safety concerns associated with treacherous contaminants like pathogenic viruses. In this study, viral diversities in campus wastewater (CWW) and its reclaimed water (RCW) recycled for toilet flushing and garden irrigation of a university dormitory were assessed using metagenomic sequencing for acquisition of more background data. Results suggested majority (>80%) of gene sequences within assembled contigs predicted by open reading frame (ORF) finder were no-hit yet believed to be novel/unrevealed viral genomic information whereas hits matched bacteriophages (i.e., mainly Myoviridae, Podoviridae, and Siphoviridae families) were predominant in both CWW and RCW samples. Moreover, few pathogenic viruses (<1%) related to infections of human skin (e.g., Molluscum contagiosum virus, MCV), digestion system (e.g., hepatitis C virus, HCV), and gastrointestinal tract (e.g., human norovirus, HuNoV) were also noticed raising safety concerns about application of reclaimed waters. Low-affinity interactions of particular viral exterior proteins (e.g., envelope glycoproteins or spike proteins) for disinfectant ligand (e.g., chlorite) elucidated treatment limitations of current sewage processing systems even with membrane bioreactor and disinfectant contactor. Revolutionary disinfection approaches together with routine monitoring and new regulations are prerequisite to secure pathogen-correlated water quality for safer reuse of reclaimed waters.

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater.

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 µM and 100 µM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 µM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed -0.52-1.15, 0.9-1.51, and 0.31-1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.

Application of a high-throughput quantitative PCR system for simultaneous monitoring of SARS-CoV-2 variants and other pathogenic viruses in wastewater.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.

Capsid integrity detection of pathogenic viruses in waters: Recent progress and potential future applications.

Waterborne diseases caused by pathogenic human viruses are a major public health concern. To control the potential risk of viral infection through contaminated waters, a rapid, reliable tool to assess the infectivity of pathogenic viruses is required. Recently, an advanced approach (i.e., capsid integrity (RT-)qPCR) was developed to discriminate intact viruses (potentially infectious) from inactivated viruses. In this approach, samples were pretreated with capsid integrity reagents (e.g., monoazide dyes or metal compounds) before (RT -)qPCR. These reagents can only penetrate inactivated viruses with compromised capsids to bind to viral genomes and prevent their amplification, but they cannot enter viruses with intact capsids. Therefore, only viral genomes of intact viruses were amplified or detected by (RT-)qPCR after capsid integrity treatment. In this study, we reviewed recent progress in the development and application of capsid integrity (RT-)qPCR to assess the potential infectivity of viruses (including non-enveloped and enveloped viruses with different genome structures [RNA and DNA]) in water. The efficiency of capsid integrity (RT-)qPCR has been shown to depend on various factors, such as conditions of integrity reagent treatment, types of viruses, environmental matrices, and the capsid structure of viruses after disinfection treatments (e.g., UV, heat, and chlorine). For the application of capsid integrity (RT-)qPCR in real-world samples, the use of suitable virus concentration methods and process controls is important to control the efficiency of capsid integrity (RT-)qPCR. In addition, potential future applications of capsid integrity (RT-)qPCR for determining the mechanism of disinfection treatment on viral structure (e.g., capsid or genome) and a combination of capsid integrity treatment and next-generation sequencing (NGS) (capsid integrity NGS) for monitoring the community of intact pathogenic viruses in water are also discussed. This review provides essential information on the application of capsid integrity (RT-)qPCR as an efficient tool for monitoring the presence of pathogenic viruses with intact capsids in water.

Pathogenic Virus Detection by Optical Nanobiosensors.

The novel coronavirus pandemic is sweeping the world and causing global crises. The lack of effective methods of early diagnosis and accurate detection may result in severe infection as well as mortality. Therefore, it is urgently required that rapid, selective, and accurate techniques for detecting pathogenic viruses are developed. Nanotechnology-based biosensors are finding many applications in biological detection, which may address these issues and realize direct detection of molecular targets in real time. Among various nanoplatforms, optical nanobiosensors have aroused much interest due to their inherent advantages of high sensitivity and direct readout. In this review, a summary of recent progress on the optical biosensors based on nanotechnology for pathogenic virus detection is provided, with focus on quantum dots (QDs), upconversion nanoparticles (UCNPs), noble metal nanoparticles, and organic fluorescent molecules-based nanoprobes and chemiluminescence assays. These representative studies demonstrate appealing performance as biosensors and hold great promise for clinical diagnosis.

Recent advancements in the measurement of pathogenic airborne viruses.

Air-transmissible pathogenic viruses, such as influenza viruses and coronaviruses, are some of the most fatal strains and spread rapidly by air, necessitating quick and stable measurements from sample air volumes to prevent further spread of diseases and to take appropriate steps rapidly. Measurements of airborne viruses generally require their collection into liquids or onto solid surfaces, with subsequent hydrosolization and then analysis using the growth method, nucleic-acid-based techniques, or immunoassays. Measurements can also be performed in real time without sampling, where species-specific determination is generally disabled. In this review, we introduce some recent advancements in the measurement of pathogenic airborne viruses. Air sampling and measurement technologies for viral aerosols are reviewed, with special focus on the effects of air sampling on damage to the sampled viruses and their measurements. Measurement of pathogenic airborne viruses is an interdisciplinary research area that requires understanding of both aerosol technology and biotechnology to effectively address the issues. Hence, this review is expected to provide some useful guidelines regarding appropriate air sampling and virus detection methods for particular applications.

Towards the Forest Virome: High-Throughput Sequencing Drastically Expands Our Understanding on Virosphere in Temperate Forest Ecosystems.

Thanks to the development of HTS technologies, a vast amount of genetic information on the virosphere of temperate forests has been gained in the last seven years. To estimate the qualitative/quantitative impact of HTS on forest virology, we have summarized viruses affecting major tree/shrub species and their fungal associates, including fungal plant pathogens, mutualists and saprotrophs. The contribution of HTS methods is extremely significant for forest virology. Reviewed data on viral presence in holobionts allowed us a first attempt to address the role of virome in holobionts. Forest health is dependent on the variability of microorganisms interacting with the host tree/holobiont; symbiotic microbiota and pathogens engage in a permanent interplay, which influences the host. Through virus-virus interplays synergistic or antagonistic relations may evolve, which may drastically affect the health of the holobiont. Novel insights of these interplays may allow practical applications for forest plant protection based on endophytes and mycovirus biocontrol agents. The current analysis is conceived in light of the prospect that novel viruses may initiate an emergent infectious disease and that measures for the avoidance of future outbreaks in forests should be considered.

Antiviral Strategies Using Natural Source-Derived Sulfated Polysaccharides in the Light of the COVID-19 Pandemic and Major Human Pathogenic Viruses.

Only a mere fraction of the huge variety of human pathogenic viruses can be targeted by the currently available spectrum of antiviral drugs. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the urgent need for molecules that can be deployed quickly to treat novel, developing or re-emerging viral infections. Sulfated polysaccharides are found on the surfaces of both the susceptible host cells and the majority of human viruses, and thus can play an important role during viral infection. Such polysaccharides widely occurring in natural sources, specifically those converted into sulfated varieties, have already proved to possess a high level and sometimes also broad-spectrum antiviral activity. This antiviral potency can be determined through multifold molecular pathways, which in many cases have low profiles of cytotoxicity. Consequently, several new polysaccharide-derived drugs are currently being investigated in clinical settings. We reviewed the present status of research on sulfated polysaccharide-based antiviral agents, their structural characteristics, structure-activity relationships, and the potential of clinical application. Furthermore, the molecular mechanisms of sulfated polysaccharides involved in viral infection or in antiviral activity, respectively, are discussed, together with a focus on the emerging methodology contributing to polysaccharide-based drug development.

Pathogenic viruses: Molecular detection and characterization.

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Virological Quality of Irrigation Water Sources and Pepper Mild Mottle Virus and Tobacco Mosaic Virus as Index of Pathogenic Virus Contamination Level.

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

A comprehensive comparison of the fifth-wave highly pathogenic and low-pathogenic H7N9 avian influenza viruses reveals potential threat posed by both types of viruses in mammals.

Before 2013, zoonotic influenza infections were dominated by H5N1 viruses in China. However, the emergence of the H7N9 viruses in early 2013 changed this dominance greatly, and more than 1,600 laboratory-confirmed human cases of H7N9 infections have been reported since then. To understand the underlying mechanism of the emergence of the fifth epidemic wave that shows an unexpected sharp increase, we systematically investigated the biological characteristics of the highly pathogenic (HP) and low-pathogenic (LP) H7N9 AIVs during this period. We first systematically analysed the haemagglutination assay gene of all the isolates available from the website and found that the HP and LP viruses differed a little in the well-established receptor binding sites and in other potentially important sites. Phylogenetic analysis showed that both the HP and LP viruses belong to the branch of the Yangtze River Delta, whereas they diverged to different small branches. To further compare the biological variations in the HP and LP viruses, we selected six HP and six LP strains for in-depth analysis, including receptor binding characteristics, thermal stability, viral replication and virulence in mice. The three major findings of this study were as follows: (a) Other potential site/sites may affect the receptor binding property of the H7N9 viruses; (b) the HP viruses displayed a higher thermostability than did the LP viruses, quite consistent with the epidemiological data during the summer period; and (c) one-third of the HP viruses were moderately pathogenic in mice, whereas all the LP viruses were nonpathogenic in this animal model. However, the LP viruses replicated more efficiently in the mouse lung and can spread to the extrarespiratory organs (spleen, kidney and brain). Taken together, our results suggest that both the HP and LP H7N9 viruses can pose a potential threat to public health, highlighting the importance of the continual surveillance of the H7N9 AIVs.