Identification of novel mammalian viruses in tree shrews(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer,infectious diseases,metabolic disorders,and mental health conditions.Herein,we used metatranscriptomic sequencing to analyze plasma,as well as oral and anal swab samples,from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses.In total,eight mammalian viruses with complete genomes were identified,belonging to six viral families,including Flaviviridae,Hepeviridae,Parvovirinae,Picornaviridae,Sedoreoviridae,and Spinareoviridae.Notably,the presence of rotavirus was recorded in tree shrews for the first time.Three viruses-hepacivirus 1,parvovirus,and picornavirus-exhibited low genetic similarity(<70%)with previously reported viruses at the whole-genome scale,indicating novelty.Conversely,three other viruses-hepacivirus 2,hepatovirus A and hepevirus-exhibited high similarity(>94%)to known viral strains.Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants.These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews,highlighting the necessity for further research into their potential for crossspecies transmission.

Anti-herpes simplex virus activities of monogalactosyl diglyceride and digalactosyl diglyceride from Clinacanthus nutans, a traditional Thai herbal medicine

Objective: To evaluate the monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride(DGDG) from Clinacanthus nutans(C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1(HSV-1) and type 2(HSV-2) by plaque reduction assay.Methods: MGDG and DGDG were extracted with chloroform from C. nutans leaves.MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and posttreatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay.Results: MGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100%inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC50 values of 36.00 and 40.00 mg/m L, and HSV-2 at 41.00 and 43.20 mg/mL,respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have antiherpes simplex activity at the same level as standard synthetic compounds. In contrast, pretreatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30.Conclusions: MGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.

Characterization of three African swine fever viruses from diferent clinical settings revealed a potential attenuation mechanism

African swine fever(ASF)is an acute and fatal hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus(ASFV)that currently threatens the pig industry worldwide.Since the 2018 ASF outbreak in China,ASFV has evolved and caused diverse clinical manifestations,such as chronic and asymptomatic infections.Therefore,it is important to understand the molecular mechanisms underlying ASFV attenuation in the feld.Here,we isolated three ASFVs from one diseased and two asymptomatic pigs by using primary porcine alveolar macrophages(PAMs)from both domestic pigs and Bama minipigs.The three ASFVs exhibited similar phenotypes in cell culture,includ‑ing cytopathic efects(CPEs),hemadsorptions(HADs),viral protein expressions and growth curves.Genome sequenc‑ing revealed that all three ASFVs were genotype II strains.Genomic comparisons suggested that the disruption of the viral genes MGF360 and MGF110,rather than EP402R and EP153R,is likely involved in the potential attenuation of ASFV via the upregulation of innate immune responses.

Genetic and biological properties of H10Nx influenza viruses in China

H10 subtype avian influenza viruses(AIV)have been circulating in China for 40 years.H10 AIVs in China have expanded their host range from wild birds to domestic poultry and mammals,even human.Most of the H10 subtype AIVs reported in China were isolate from the southeast part.We isolated an H10N3 AIV,A/Chicken/Liaoning/SY1080/2021(SY1080),from live poultry market(LPM)in Liaoning Province of the Northeast China.SY1080replicated efficiently in mice lungs and nasal turbinates without prior adaptation.We systematically compared SY1080 with other H10 subtype isolates in China.Phylogenetic analysis showed that SY1080 and most of the H10strains belonged to the Eurasian lineage.H10 AIVs in China have formed 63 genotypes.SY1080 as well as the H10N3 strains from human infections belonged to G60 genotype.H10Nx AIV acquired multiple mammalian adaptive and virulence related mutations during circulation and the recent reassortants derived internal genes from chicken H9N2 AIVs.The H10Nx subtypes AIVs posed potential threat to public health.These results suggested we should strengthen the surveillance and evaluation of H10 subtype strains.

Role of viruses in periodontitis:An extensive review of herpesviruses,human immunodeficiency virus,coronavirus-19,papillomavirus and hepatitis viruses

Periodontitis is the inflammation of the supporting structures around the dentition.Several microbial agents,mostly bacteria,have been identified as causative factors for periodontal disease.On the other hand,oral cavity is a rich reservoir for viruses since it contains a wide variety of cell types that can be targeted by viruses.Traditionally,the focus of research about the oral flora has been on bacteria because the most widespread oral diseases,like periodontitis and dental caries,are outcomes of bacterial infection.However,recently and especially after the emergence of coronavirus disease 2019,there is a growing tendency toward including viruses also into the scope of oral microbiome investigations.The global high prevalence of periodontitis and viral infections may point out to a concomitant or synergistic effect between the two.Although the exact nature of the mechanism still is not clearly understood,this could be speculated through the manipulation of the immune system by viruses;hence facilitating the furthermore colonization of the oral tissues by bacteria.This review provides an extensive and detailed update on the role of the most common viruses including herpes family(herpes simplex,varicella-zoster,Epstein-Barr,cytomegalovirus),Human papillomaviruses,Human immunodeficiency virus and severe acute respiratory syndrome coronavirus 2 in the initiation,progression and prognosis of periodontitis.

The identities and anti-herpes simplex virus activity of Clinacanthus nutans and Clinacanthus siamensis

Objective:To distinguish the difference among the Clinacanthus nutans(Burm.f.)Lindau(C.nutans)and Clinacanthus siamensis Bremek(C.siamensis)by assessing pharmacognosy characteristics,molecular aspect and also to evaluate their anti-herpes simplex virus(HSV)type 1 and type 2 activities.Methods:Macroscopic and microscopic evaluation were performed according to WHO Geneva guideline.Stomatal number,stomatal index and palisade ratio of leaves were evaluated.Genomic DNA was extracted by modified CTAB method and ITS region was amplified using PGR and then sequenced.Dry leaves were subsequently extracted with n-hexane,dichloromethane and methanol and antiviral activity was performed using plaque reduction assay and the cytotoxicity of the extracts on Vero cells was determined by MTT assay.Results:Cross section of midrib and stem showed similar major components.Leaf measurement index of stomatal number,stomatal index and palisade ratio of C.nutans were 168.32±29.49,13.83±0.86 and 6.84±0.66,respectively,while C.siamensis were 161.60±18.04,11.93±0.81and 3.37±0.31,respectively.The PCR amplification of ITS region generated the PGR product approximately 700 bp in size.There were 34 polymorphisms within the ITS region which consisted of 11 Indels and 23 nucleotide substitutions.The IC_(50)values of C.nutans extracted with n-hexane,dichloromethane and methanol against HSV-1 were(32.05±3.63)μg/mL,(44.50±2.66)μg/mL,(64.93±7.00)μg/mL,respectively where as those of C.siamensis were(60.00±11.61)μg/mL,(55.69+4.41)μg/mL,(37.39±5.85)μg/mL,respectively.Anti HSV-2 activity of n-hexane,dichloromethane and methanol C.nutans leaves extracts were(72.62±12.60)μg/mL,(65.19±21.45)μg/mL,(65.13±2.22)μg/mL,respectively where as those of C.siamensis were(46.52±4.08)μg/mL,(49.63±2.59)μg/mL,(72.64±6.52)μg/mL,respectively.Conclusions:The combination of macroscopic,microscopic and biomolecular method are able to authenticate these closely related plants and both of them have a potency to be an anti-HSV agent.

Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A:Impacts on rescue of sequence-modifying viruses

Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.

Development and application of a SYBR Green I fuorescent PCR assay for the diferentiation of genotypes I and II African swine fever viruses

African swine fever(ASF)is a highly fatal hemorrhagic disease afecting domestic pigs caused by African swine fever virus(ASFV).Genetic analysis of ASFV isolates to date has identifed 24 geographically related genotypes with various subgroups,but only genotype I and II ASFVs have been reported outside Africa.ASFV genotype II and genotype I viruses were reported in China in 2018 and 2021,respectively.In this study,unique and highly conserved noncoding regions were found between MGF_505-9R and MGF_505-10R in the 188 genomes of ASFV genotypes I and II.A pair of primers was designed on the basis of this region.By optimizing the reaction system and conditions,a SYBR Green I fuorescence PCR assay that can distinguish between ASFV genotypes I and II was established,and the sensitivity,reproducibility and specifcity were evaluated.The detection limit was 1 TCID_(50)/0.1 mL for both genotypes,with no cross-reactivity observed with other common pig pathogens.The intra-and interbatch variation coefcients were both less than 1.2%.Clinical sample detection analysis revealed 47 positive cases out of 100,including 3 for genotype I and 44 for genotype II,aligning with results from the WOAH-recommended and national standard methods.The method developed in this study allows for the diferentiation of ASFV genotypes I and II without the need for genome sequencing,ofering a convenient and rapid approach for ASFV detection and genotype identifcation.

Current Status of Natural Products from Plants as Anti-herpes Simplex Virus 1 Agents

Nucleoside analogues have been the mainstay of clinical treatment of herpes simplex virus 1 (HSV-1) infections since their development. However, the emergence of drug resistant strains has underlined the urgency of the discovery of novel anti-HSV-1 drugs. Natural products, which provided many novel drug leads, are known to be an important source of anti-HSV-1 agents. Herein, we present an overview of natural products with anti-HSV-1 activities isolated from a variety of plants reported in recent years. Several different compounds, mainly belonging to the three groups of polysaccharides, polyphenols and terpenes, showed antiviral effects against HSV-1, indicating their potential to be promising anti-HSV-1 agents.

Implementation of the Guidelines for Oncolytic Viruses in China

Objective To study related guidelines such as“Guidelines for Clinical Trial Design of Oncolytic Viruses”“Technical Guidelines for Non-clinical Research and Evaluation of Gene Therapy Products”and“Technical Guidelines for Pharmacological Research and Evaluation of Oncolytic Viruses(OVs)Products”issued by the Center for Drug Evaluation(CDE)on OV and offer some suggestions for further improvement of the policies and regulations.Methods Literature comparison and questionnaire survey were used in this paper to investigate the difficulties encountered in the practical work of domestic companies that have conducted clinical trials,thus drawing some lessons to help the subsequent implementation of the guidelines.Results and Conclusion According to the characteristics of specific varieties and the published laws,regulations and guidelines,companies can adopt more suitable and scientific strategies to accelerate the development of anti-cancer drugs.In the future,as more clinical studies and product development for various cancers expand,regulatory requirements are expected to become more specialized and complex.Learning from the regulatory experience of developed countries and regions,we can improve the regulatory system by adapting it to national conditions and development status of China.Additionally,some ideas and useful inspirations can be provided after reviewing the content of the relevant guidelines and the obstacles in the practice of corporate R&D process can be addressed.These efforts will facilitate the speed of R&D and allow enterprises to work more smoothly and efficiently.

Interleukin 1βreceptor and synaptic dysfunction in recurrent brain infection with Herpes simplex virus type-1

Several experimental evidence suggests a link between brain Herpes simplex virus type-1 infection and the occurrence of Alzheimer’s disease.However,the molecular mechanisms underlying this association are not completely understood.Among the molecular mediators of synaptic and cognitive dysfunction occurring after Herpes simplex virus type-1 infection and reactivation in the brain neuroinflammatory cytokines seem to occupy a central role.Here,we specifically reviewed literature reports dealing with the impact of neuroinflammation on synaptic dysfunction observed after recurrent Herpes simplex virus type-1 reactivation in the brain,highlighting the role of interleukins and,in particular,interleukin 1βas a possible target against Herpes simplex virus type-1-induced neuronal dysfunctions.

Antiviral therapy for hepatitis B virus infection is beneficial for the prognosis hepatocellular carcinoma

In this editorial,we comment on the article by Mu et al,published in the recent issue of the World Journal of Gastrointestinal Oncology.We pay special attention to the immune tolerance mechanism caused by hepatitis B virus(HBV)infection,the pathogenesis of hepatocellular carcinoma(HCC),and the role of antiviral therapy in treating HCC related to HBV infection.HBV infection leads to systemic innate immune tolerance by directly inhibiting pattern recognition receptor recognition and antiviral signaling pathways,as well as by inhibiting the immune functions of macrophages,natural killer cells and dendritic cells.In addition,HBV leads to an immunosuppressive cascade by expressing inhibitory molecules to induce exhaustion of HBV-specific cluster of differentiation 8+T cells,ultimately leading to long-term viral infection.The loss of immune cell function caused by HBV infection ultimately leads to HCC.Long-term antiviral therapy can improve the prognosis of patients with HCC and prevent tumor recurrence and metastasis.

Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

Background:The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses,explicitly focusing on norovirus genogroup II(GII),astrovirus,rotavirus,Aichi virus,sapovirus,hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR(ddPCR).Enteric viruses are of significant public health concern,as they are the leading cause of diseases like gastroenteritis.Regular monitoring of environmental samples,particularly from wastewater treatment plants,is crucial for early detection and control of these viruses.This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions.Our protocol's objective is to establish a novel ddPCRbased methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal,India.Our assay is capable of accurately quantifying virus concentrations without standard curves,minimizing extensive optimization,and enhancing sensitivity and precision,especially for low-abundance targets.Methods:The study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years,ensuring comprehensive coverage and consistent data.Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater,a more advanced technique.Additionally,we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses.Conclusion:This study will aid in understanding these viruses’genetic diversity and mutation rates,which is crucial for developing tailored intervention strategies.The findings will be instrumental in shaping public health responses and improving epidemiological surveillance,especially in localities heaving sewage networks.

Insights into gastrointestinal manifestation of human immunodeficiency virus:A narrative review

Human immunodeficiency virus(HIV)modifies CD4-positive cells,resulting in immunodeficiency and a wide range of gastrointestinal(GI)manifestations.The burden of HIV-related GI illnesses has significantly evolved with the widespread use of antiretroviral therapy(ART).While ART has effectively reduced the occurrence of opportunistic infections,it has led to an increase in therapy-related GI illnesses.Common esophageal conditions in HIV patients include gastroesophageal reflux disease,idiopathic esophageal ulcers,herpes simplex virus,cytomegalovirus(CMV),and candidal esophagitis.Kaposi’s sarcoma,a hallmark of acquired immunodeficiency syndrome,may affect the entire GI system.Gastritis and peptic ulcer disease are also frequently seen in patients with HIV.Diarrhea,often linked to both opportunistic infections and ART,requires careful evaluation.Bloody diarrhea,often a sign of colitis caused by bacterial infections such as Shigella or Clostridium difficile,is prevalent.Small bowel lymphoma,although rare,is increasing in prevalence.Anorectal disorders,including proctitis,fissures,and anal squamous cell carcinoma,are particularly relevant in homosexual men,underlining the importance of timely diagnosis.This review comprehensively explores the epidemiology,pathogenesis,and treatment considerations for the various GI disorders associated with HIV,highlighting the importance of accurate diagnosis and effective treatment to improve outcomes for HIV-infected patients.

Advance of the Research on Testing of Animal Viruses by LAMP Technology in Abroad

Loop-mediated isothermal amplification（LAMP） is an amplification method developed by Notomi et al and has been applied successfully for the detection of many viruses.This paper introduced the current status of LAMP and recent developments,and the method applying in the diagnosis of animal viruses in abroad.

Molecular Characteristics of gp90 Gene of 14 Reticuloendotheliosis Viruses Isolated in China

[Objective] The paper was to study molecular characteristics of gp90 gene of 14 Reticuloendotheliosis viruses isolated in China.[Method] The surface envelop gene gp90 of 14 REV strains isolated from different commercial layer farms in China were amplified,and their nucleotide sequences were determined.[Result] Sequence analysis showes that 14 REV strains are more identical to the subtype 3 isolates than to the early Chinese REV isolates.In addition,14 REV strains have a high identity with some REV strains in US and Taiwan.[Conclusion] The study provided necessary information for further understanding the evolution of REV.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

Human immunodeficiency virus and hepatotropic viruses comorbidities as the inducers of liver injury progression

Hepatotropic viruses induced hepatitis progresses much faster and causes more liver-related health problems in people co-infected with human immunodeficiency virus(HIV). Although treatment with antiretroviral therapy has extended the life expectancy of people with HIV, liver disease induced by hepatitis B virus(HBV) and hepatitis C virus(HCV) causes significant numbers of non-acquired immune deficiency syndrome(AIDS)-related deaths in coinfected patients. In recent years, new insights into the mechanisms of accelerated fibrosis and liver disease progression in HIV/HCV and HIV/HBV co-infections have been reported. In this paper, we review recent studies examining the natural history and pathogenesis of liver disease in HIV-HCV/HBV co-infection in the era of direct acting antivirals(DAA) and antiretroviral therapy(ART). We also review the novel therapeutics for management of HIV/HCV and HIV/HBV coinfected individuals.

Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.