Construction of chimeric viruses based on pepper mild mottle virus using a modiffed Cre/loxP system

Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

Indoor Inoculation and Identification Technology of Sweet (Hot) Pepper Virus

[Objective] The paper was to establish an affordable indoor virus inoculation and identification technology. [Method] Taking Tobacco Mosaic Virus （TMV） and Cucumber Mosaic Vires （CMV） of sweet （hot） pepper as the sources of virus, an affordable indoor virus inoculation and identification technology was developed in the paper. [ Result] The suitable inoculation concentration of CMV was five to ten times, and the best seedling age for inoculation was five to six leav- es. Suitable inoculation concentration of TMV was 20 to 30 times, and the best seedling age for inoculation was three to six leaves. Single inoculation technology was mainly used for indoor virus inoculation and identification of sweet （hot） pepper, and complex inoculation technology could also be adopted with first, st inoculation of CMV and late inoculation of TMV. For mixed inoculation technology, CMV： TMV should be 1： 1. Complex inoculation and mix inoculation should base on the tech- nology of single inoculation. Disease resistant materials, AID1-W22-dg176, ABgl-W22-48123, AB91-DL-6428, HY031-2-8-1-6, BYT-4-1-3-6-8, JFG-2-1-2-6, JF8S-1-1-5-4-8 and I＇502-1-1-3-5, were identified by this method. [ Conclusion] This research provided scientific basis for standardization of indoor inoculation of sweet （hot） pepper virus.

Ambispective epidemiological observational study of varicella-zoster virus infection: An 18 year-single-center Bulgarian experience

BACKGROUND Varicella(chickenpox)and herpes zoster(shingles)are outcomes of varicella-zoster virus(VZV)infection,and understanding their incidence trends is vital for public health planning.AIM To conduct an ambispective epidemiological study by analyzing the main epidemiological characteristics of VZV infection during an 18 year-period(2000-2018).METHODS We used descriptive and epidemiological methods to characterize chickenpox in Bulgaria,the city of Plovdiv and the region for a period of 18 years(2000-2018).RESULTS The average incidence of varicella-zoster infection for the period 2000–2018 in the Plovdiv region was estimated at 449.58‰.The highest relative share of the infection was assessed in the month of January at 13.6%,and the lowest in the months of August and September at 2.9%(both months).The age group most affected by the infection was 1-4 years,followed by 5-9 years.This corresponds to the so-called"pro-epidemic population"-a phenomenon typical for airborne infections,confirming their mass impact on the perpetuation of VZV infection.CONCLUSION Our findings reveal significant insights into VZV epidemiology,including age-specific incidence rates,clinical manifestations,and vaccination impact.This comprehensive analysis contributes to the broader understanding of VZV infec-tion dynamics and may inform evidence-based preventive measures.

Hepatotropic viruses:Is Roma population at risk?

Roma people make up a significant ethnic minority in many European countries,with the vast majority living in Central and Eastern Europe.Roma are a vulnerable population group in social,economic,and political terms.Frequent migrations,life in segregated communities,substandard housing,poverty,and limited access to quality health care,including low immunization coverage,affect their health status and predispose them to various diseases,including viral hepatitis.Hepatitis A,B,and E are highly prevalent among Roma and mainly associated with low socioeconomic status.In contrast,hepatitis C does not seem to be more frequent in the Roma population.Enhanced efforts should be directed towards the implementation of screening programs,preventive measures,and treatment of viral hepatitis in Roma communities throughout Europe.

High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay

Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors(HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference(HT-RNAi) screens has led to the identification of several key factors involved in the viral lifecycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested(such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase Ⅲα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HTRNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.

Enigmatic origin of hepatitis B virus:An ancient travelling companion or a recent encounter?

Hepatitis B virus(HBV)is the leading cause of liver disease and infects an estimated 240 million people worldwide.It is characterised by a high degree of genetic heterogeneity because of the use of a reverse transcriptase during viral replication.The ten genotypes(A-J)that have been described so far further segregate into a number of subgenotypes which have distinct ethno-geographic distribution.Genotypes A and D are ubiquitous and the most prevalent genotypes in Europe(mainly represented by subgenotypes D1-3 and A2);genotypes B and C are restricted to eastern Asia and Oceania;genotype E to central and western Africa;and genotypes H and F(classified into 4 subgenotypes)to Latin America and Alaska.This review summarises the data obtained by studying the global phylodynamics and phylogeography of HBV genotypes,particularly those concerning the origin and dispersion histories of genotypes A,D,E and F and their subgenotypes.The lack of any consensus concerning the HBV substitution rate and the conflicting data obtained using different calibration approaches make the time of origin and divergence of the various genotypes and subgenotypes largely uncertain.It is hypothesised that HBV evolutionary rates are time dependent,and that the changes depend on the main transmission routes of the genotypes and the dynamics of the infected populations.

Epidemiology of hepatitis B virus in Bangladeshi general population

BACKGROUND:Hepatitis B virus(HBV)is encountered sporadically the year round in Bangladesh.It results in a wide range of liver diseases,with asymptomatic acute hepatitis at one end to hepatocellular carcinoma(HCC)at the other end of the spectrum. METHODS:All 1018 individuals of different age groups and sex with varied religious,educational and social backgrounds were tested for HBsAg by ELISA.The positive samples were further tested by ELISA for HBeAg.Before testing,blood samples were preserved at-20℃.The study was conducted in a semi-urban location on the outskirts of Dhaka. RESULTS:Of the 1018 individuals,5.5%tested positive for HBsAg.None were tested positive for anti-HCV.Among the HBsAg-positive population,58.93%were HBeAg- positive and the rest 41.07%HBeAg-negative.There was a male predominance and those who were tested positive were mostly between 16 and 50 years of age.Major risk factors for exposure to HBV appeared to be injudicious use of injectable medications,treatment by unqualified, traditional practitioners,mass-vaccination against cholera and smallpox,barbers and body piercing. CONCLUSION:HBV remains a major cause of morbidity and mortality in Bangladesh and we have a long way to go before we may bid farewell to this deadly menace.

Lurking epidemic of hepatitis C virus infection in Iran: A call to action

Despite having a relatively low prevalence in the Iranian general population, the burden of hepatitis C virus(HCV) infection is on the rise, and hepatitis C is predicted to be the most important leading cause of viral hepatitis-related mortality in the near future in Iran. The recent population-based epidemiological studies have revealed the predominant role of injecting drug use in increasing prevalence of HCV infection. Undoubtedly, new management paradigm is required to drive down the rising wave of hepatitis C in Iran. Priority should be given to young injecting drug users as the cornerstone of the lurking epidemic of HCV infection in Iran.

Prevalence of West Nile virus in Mashhad,Iran:A population-based study

Objective:To evaluate the prevalence of West Nile virus seropositivity in the general population of Mashhad.Northeast of Iran.Methods:One hundred and eighty two individuals living in the city of Mashhad were studied using cluster sampling method.Both IgM and IgG antibodies against WNV were detected by ELISA method.Results:In this study,the overall IgG seroprevalence of positive West Nile virus was 11%;however.IgM antibody was not found in the participants.Conclusions:Our study suggested that the prevalence rate of West virus is considerable in Mashhad city.It seems necessary for clinicians and health care workers to be aware of WNV infection in the Northeast Iran.

Comment on review article:Chronic hepatitis C virus infection cascade of care in pediatric patients

An enhanced cascade of care should include a younger population,helping to achieve the goal of the World Health Organization with a focus on elimination in the pediatric population.Furthermore,enhanced screening and awareness efforts and continued education of health care providers will improve the outcomes of chronic hepatitis C virus(HCV)infection in the pediatric population.The present work discusses and comments on the topic"cascade of care in HCV chronic pediatric patients".

Epidemiology of hepatitis E virus in Iran

Iran is known as an endemic country for hepatitis E virus(HEV) infection, while there are variations in the epidemiology of HEV infection throughout the country. The available epidemiological studies in different regions of Iran show HEV seroprevalence of 1.1%-14.2% among general population, 4.5%-14.3% among blood donors, 6.1%-22.8% among injecting drug users, 6.3%-28.3% among hemodialysis patients, 1.6%-11.3% among patients infected with other hepatitis viruses, 27.5% among patients with chronic liver disease, 30.8% among kidney transplant recipient patients, and 10%-16.4% among human immunodeficiency virus-infected patients. These variations reflect differences in the status of public health and hygiene, risk factors, and routes of transmission in different regions and groups. Therefore, it is necessary to review the epidemiology of HEV infection to determine the most prevalent risk factors and routes of transmission, and to evaluate the effectiveness of preventive strategies employed in the public health services of the country. Moreover, the other epidemiological aspects of HEV, including the genotypic pattern, extra hepatic manifestations, and incidence of chronic infection need to be investigated among Iranian population to expand the current knowledge on the epidemiology of HEV and to clarify the real burden of HEV infection. Therefore, this review was performed to provide a general overview regarding the epidemiology of HEV in Iran.

Genome characterization of the Caprine arthritis-encephalitis virus in China:A retrospective genomic analysis of the earliest Chinese isolates

Caprine arthritis-encephalitis virus(CAEV) is an under-studied virus infecting caprines and ovines worldwide. Over the last four decades, CAEV has spread in China, obtaining genomic data on CAEV strains circulating in China is of importance for developing diagnostic methods and eradicating associated diseases. However, there is limited information on the genome, including characterizations, and the probable origin. This work aimed to characterize Chinese CAEV genomes and population structures. Five CAEV strains isolated from infected dairy goats between 1989and 1994 in Gansu, Guizhou, Shaanxi, Shandong and Sichuan provinces were cloned and sequenced. The Chinese CAEV had a 58–93% genome similarities to strains outside of China, and they belonged to subgenotype B1. The highest similarity levels(98.3–99.3%) were with two other Chinese strains, and they shared a 91.8–92.3% similarity with the strain Clements(GenBank accession no. NC_001463.1) from outside of China. The Chinese CAEV strains isolated from different provinces over five years were still highly homologous and contained unique ancestral population components,indicating that these Chinese strains had a common origin that differed from other known strains. Our results provide genomic data on circulating Chinese CAEV strains and will be useful for future epidemiological investigations and CAEV eradication programs.

Hepatitis C virus among blood donors and general population in Middle East and North Africa:Meta-analyses and meta-regressions

BACKGROUND Despite the Middle East and North Africa(MENA)Region reported to have the highest prevalence of hepatitis C virus(HCV)globally,HCV infection levels in the majority of MENA countries remain inadequately characterized.Blood donor data have been previously used as a proxy to assess levels and trends of HCV in the general population,however,it is unclear how comparable these populations are in MENA and whether blood donors provide an appropriate proxy.AIM To delineate HCV epidemiology among blood donors and the general population in the MENA.METHODS The data source was the systematically gathered MENA HCV Epidemiology Synthesis Project Database.Random-effects meta-analyses and meta-regressions were conducted.For comparison,analyses were conducted for Europe,utilizing the Hepatitis C Prevalence Database of the European Centre for Disease Prevention and Control.RESULTS One thousand two hundred and thirteen HCV antibody prevalence measures and 84 viremic rate measures were analyzed for MENA.Three hundred and seventyseven antibody prevalence measures were analyzed for Europe.In MENA,pooled mean prevalence was 1.58%[95%confidence interval(CI):1.48%–1.69%]among blood donors and 4.49%(95%CI:4.10%–4.90%)in the general population.In Europe,pooled prevalence was 0.11%(95%CI:0.10%–0.13%)among blood donors and 1.59%(95%CI:1.25%–1.97%)in the general population.Prevalence in the general population was 1.72-fold(95%CI:1.50–1.97)higher than that in blood donors in MENA,but it was 15.10-fold(95%CI:11.48–19.86)higher in Europe.Prevalence was declining at a rate of 4%per year in both MENA and Europe[adjusted risk ratio:0.96(95%CI:0.95–0.97)in MENA and 0.96(95%CI:0.92–0.99)in Europe].Pooled mean viremic rate in MENA was 76.29%(95%CI:67.64%–84.02%)among blood donors and 65.73%(95%CI:61.03%–70.29%)in the general population.CONCLUSION Blood donor data provide a useful proxy for HCV infection in the wider population in MENA,but not Europe,and could improve HCV burden estimations and assess progress toward HCV elimination by 2030.

Human leukocyte antigen-DP loci are associated with the persistent infection of hepatitis B virus in Chinese population

A genome-wide association study recently showed that genetic variants in human leukocyte antigen (HLA)-DP loci were strongly associated with a risk of persistent infection of hepatitis B virus (HBV) in Japanese and Thai individuals and variants in interleukin 28B (IL-28B) have been associated with responses to anti-hepatitis C virus (HCV) treatment. The aim of this study was to investigate whether the HLA-DP loci and IL-28B were associated with different outcomes of chronic HBV infection (CHB) in Chinese subjects. The rs9277535 near HLA-DPB1,rs3077 near HLA-DPA1, and rs12979860 near IL-28B were genotyped by direct sequencing in 185 CHB patients and 193 self-limited hepatitis B virus (SLHBV)-infected subjects who recovered from HBV infection. The rs9277535 near HLA-DPB1 was strongly associated with CHB (P=0.000 018 1, OR=1.905). This association was observed independent of HBV e antigen (HBeAg) status and HBV viral loads in HBeAg-positive CHB patients (P=0.000 4, OR=1.956), in HBeAg-negative CHB patients (P=0.000 9, OR=1.857), and in HBeAg-negative CHB individuals without detectable levels of HBV DNA in serum (P=0.001 1, OR=2.05). The rs3077 near HLA-DPA1 was associated with CHB (P=0.020 6, OR=0.686 5) and HBeAg-positive CHB infection status (P=0.014 3, OR=0.604 7). Meanwhile, a genetic variation of insertion-deletion (INDEL) polymorphism (rs361527, -/ATAAATGTTGA) near HLA-DPA1 was found to be associated with CHB (P=0.030 7, OR=0.702 8) and HBeAg-positive CHB infection status (P=0.023 3, OR=0.619). However,the rs12979860 genotype near IL-28B had no correlation with CHB. This study demonstrated that in the Han Chinese populations, HLA-DP loci, but not IL-28B, were associated with persistence of infection in different outcomes of HBV-infected patients; however, the mechanism needs to be further investigated.

Evolutionary dynamics of the severe acute respiratory syndrome coronavirus 2 genomes

The coronavirus disease 2019(COVID-19)pandemic has caused immense losses in human lives and the global economy and posed significant challenges for global public health.As severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of COVID-19,has evolved,thousands of single nucleotide variants(SNVs)have been identified across the viral genome.The roles of individual SNVs in the zoonotic origin,evolution,and transmission of SARS-CoV-2 have become the focus of many studies.This review summarizes recent comparative genomic analyses of SARS-CoV-2 and related coronaviruses(SC2r-CoVs)found in non-human animals,including delineation of SARS-CoV-2 lineages based on characteristic SNVs.We also discuss the current understanding of receptor-binding domain(RBD)evolution and characteristic mutations in variants of concern(VOCs)of SARS-CoV-2,as well as possible co-evolution between RBD and its receptor,angiotensin-converting enzyme 2(ACE2).We propose that the interplay between SARS-CoV-2 and host RNA editing mechanisms might have partially resulted in the bias in nucleotide changes during SARS-CoV-2 evolution.Finally,we outline some current challenges,including difficulty in deciphering the complicated relationship between viral pathogenicity and infectivity of different variants,and monitoring transmission of SARS-CoV-2 between humans and animals as the pandemic progresses.

Evolutionary study of hepatitis C virus envelope genes during primary infection

Background Hepatitis C virus （HCV） envelope genes encoding glycoproteins E1 and E2 exhibits a high degree of variability that gives rise to differing phenotypic traits; including alterations in receptor-binding affinity and immune recognition and escape. This study aims to elucidate the relationship of the evolutionary patterns for HCV envelope glycoproteins to viral persistence. Methods HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5＇ half of E2; including hypervariable region 1 （HVR1）. To detect representative variants for sequencing thirty-three cloned cDNAs representing each specimen were assessed by a method that combined analysis of a single-stranded conformational polymorphism （SSCP） method and heteroduplex analysis （HDA）. For each patient, the rates of both synonymous and nonsynonymous substitutions for the El, HVR1 and E2 regions outside HVR1 were evaluated. The amino acid sequences and predicted antigenic profiles were analyzed. Results The genetic diversity within HVR1 was consistently higher than that in the E1 and E2 regions outside HVR1 in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region predominated and gradually increased, compared to that in the E1 and E2 regions outside HVR1. By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions, including HVR1, were consistently lower in individuals with clearance of viremia. HVR1 had a higher antigenic variable and lower positive charge in subjects with persistent viremia. All cysteine residues and N-linked glycosylation sites, some of which were known to play a major role in protein folding and others play a role in HCV entry, were 100% conserved among the sequenced cloned cDNAs from the two outcome groups. Conclusion HCV persistence may be associated with positive selection pressures on HVR1, rather than functional constraints in the envelope reaion.

Canine distemper virus as a threat to wild tigers in Russia and across their range

Canine distemper virus(CDV)has recently been identified in populations of wild tigers in Russia and India.Tiger populations are generally too small to maintain CDV for long periods,but are at risk of infections arising from more abundant susceptible hosts that constitute a reservoir of infection.Because CDV is an additive mortality factor,it could represent a significant threat to small,isolated tiger populations.In Russia,CDV was associated with the deaths of tigers in 2004 and 2010,and was coincident with a localized decline of tigers in Sikhote-Alin Biosphere Zapovednik(from 25 tigers in 2008 to 9 in 2012).Habitat continuity with surrounding areas likely played an important role in promoting an ongoing recovery.We recommend steps be taken to assess the presence and the impact of CDV in all tiger range states,but should not detract focus away from the primary threats to tigers,which include habitat loss and fragmentation,poaching and retaliatory killing.Research priorities include:(i)recognition and diagnosis of clinical cases of CDV in tigers when they occur;and(ii)collection of baseline data on the health of wild tigers.CDV infection of individual tigers need not imply a conservation threat,and modeling should complement disease surveillance and targeted research to assess the potential impact to tiger populations across the range of ecosystems,population densities and climate extremes occupied by tigers.Describing the role of domestic and wild carnivores as contributors to a local CDV reservoir is an important precursor to considering control measures.

A unified global genotyping framework of dengue virus serotype-1 for a stratified coordinated surveillance strategy of dengue epidemics

Background: Dengue is the fastest spreading arboviral disease, posing great challenges on global public health. A reproduceable and comparable global genotyping framework for contextualizing spatiotemporal epidemiological data of dengue virus (DENV) is essential for research studies and collaborative surveillance.Methods: Targeting DENV-1 spreading prominently in recent decades, by reconciling all qualified complete E gene sequences of 5003 DENV-1 strains with epidemiological information from 78 epidemic countries/areas ranging from 1944 to 2018, we established and characterized a unified global high-resolution genotyping framework using phylogenetics, population genetics, phylogeography, and phylodynamics.Results: The defined framework was discriminated with three hierarchical layers of genotype, subgenotype and clade with respective mean pairwise distances 2-6%, 0.8-2%, and ≤ 0.8%. The global epidemic patterns of DENV-1 showed strong geographic constraints representing stratified spatial-genetic epidemic pairs of Continent-Genotype, Region-Subgenotype and Nation-Clade, thereby identifying 12 epidemic regions which prospectively facilitates the region-based coordination. The increasing cross-transmission trends were also demonstrated. The traditional endemic countries such as Thailand, Vietnam and Indonesia displayed as persisting dominant source centers, while the emerging epidemic countries such as China, Australia, and the USA, where dengue outbreaks were frequently triggered by importation, showed a growing trend of DENV-1 diffusion. The probably hidden epidemics were found especially in Africa and India. Then, our framework can be utilized in an accurate stratified coordinated surveillance based on the defined viral population compositions. Thereby it is prospectively valuable for further hampering the ongoing transition process of epidemic to endemic, addressing the issue of inadequate monitoring, and warning us to be concerned about the cross-national, cross-regional, and cross-continental diffusions of dengue, which can potentially trigger large epidemics.Conclusions: The framework and its utilization in quantitatively assessing DENV-1 epidemics has laid a foundation and re-unveiled the urgency for establishing a stratified coordinated surveillance platform for blocking global spreading of dengue. This framework is also expected to bridge classical DENV-1 genotyping with genomic epidemiology and risk modeling. We will promote it to the public and update it periodically.

Cooperative system analysis of the Ebola virus epidemic model

This paper aims to study the global stability of an Ebola virus epidemic model.Although this epidemic ended in September 2015,it devastated several West African countries and mobilized the international community.With the recent cases of Ebola in the Democratic Republic of the Congo(DRC),the threat of the reappearance of this fatal disease remains.Therefore,we are obligated to be prepared for a possible re-emerging of the disease.In this work,we investigate the global stability analysis via the theory of cooperative systems,and we determine the conditions that lead to global stability diseases free and endemic equilibrium.

不同药剂处理对辣椒病毒病防效试验研究

为筛选出辣椒病毒病的有效防治药剂,开展了辣椒病毒病防治药剂筛选试验。结果表明,施用维大力健有机水溶肥(灌根:5 kg/667 m^(2))+维大力康微量元素水溶肥800倍液、5%氨基寡糖素水剂1000倍液、8%宁南霉素水剂1200倍液、30%毒氟磷可湿性粉剂1200倍液能有效防治辣椒病毒病,防控率均在60%以上;维大力健有机水溶肥+维大力康微量元素水溶肥、5%氨基寡糖素水剂1000倍液、8%宁南霉素水剂1200倍液、30%毒氟磷可湿性粉剂1200倍液可为云南辣椒病毒病防治首选药剂,生产上该4种方式可交替使用。