N-linked and O-linked glycosylation to perform critical biological functions. The diverse functions of glycosylation on viral proteins during viral infections, including Dengue, Zika, influenza, and human immunodeficiency viruses as well as coronaviruses have been reported. N-linked glycosylation is the most common form of protein modification, and it modulates folding, transportation and receptor binding. Compared to N-linked glycosylation, the functions of O-linked viral protein glycosylation have not been comprehensively evaluated. In this review, we summarize findings on viral protein glycosylation, with particular attention to studies on N-linked glycosylation in viral life cycles. This review informs the development of virus-specific vaccines or inhibitors.Glycans are among the most important cell molecular components. However, given their structural diversity, their functions have not been fully explored. Glycosylation is a vital post-translational modification for various proteins. Many bacteria and viruses rely on  The high number of infectious diseases caused by viruses has posed a major threat to global public health . MoreoveN-, O-, and C-linked glycosylation, glypiation (GPI anchor attachment), and phosphoglycosylation have been reported [N-linked glycosylation, where glycans are attached to the amide nitrogen of asparagine (Asn), is the most common and extensively studied form of protein glycosylation [Viruses are obligate intracellular infectious agents that exploit host machinery to modify their viral proteins for survival. One of the key modifications is protein glycosylation. Glycosylation is the attachment of glycans to proteins and is a critical post-translational modification (PTM). To date, various glycosidic linkages such as reported . Among tsylation .N-glycans of the viral glycoproteins have multiple functions, which include promotion of expression, transport, fusion, binding to cell surface receptors, and prevention of antibody neutralization. For instance, glycosylation of envelope proteins of the human immunodeficiency virus (HIV), Japanese encephalitis virus (JEV) and West Nile virus (WNV) is essential for membrane fusion and virus infectivity [Recent studies have shown that many viral proteins, especially structural proteins are glycosylated during viral infection cycles. The ectivity . Similarectivity  , a member of the flavivirus genus of the Flaviviridae family, causes the most common arthropod-borne viral diseases in humans, and poses a huge threat to the public social, health, and economic status . DENV hae to DENV is an emerged mosquitoe-borne flavivirus that causes severe human diseases, which include neurodevelopmental malformations  and Guillain-Barre syndrome . The prMin vitro , and alsin vitro   of the WNV E protein is located between amino acid positions 154–156 [West Nile virus (WNV) is also an arthropod-transmitted virus that belongs to the Flaviviridae family. Although the prM proteins of all the WNV strains contain  protein . The N-l 154–156 . Martina 154–156 . In othe 154–156 ,38. Once 154–156 . The prMN-linked glycosylation site at positions N15 and N154, respectively [N-glycosylation resulted in a sharp decline in viral virulence in mice after virus inoculation [N-linked glycosylation site of prM, the growth of JEV is inhibited in a cell-type-specific manner [in vivo , prM and E, contain an ectively . In animc manner . In addic manner . Compare[in vivo . TherefoN-linked glycosylation sites on E1 and E2, respectively [Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2. There are 4 and 11 ectively ,43. Theyectively . Mutatioectively . Moreoveectively .For most flaviviruses, glycosylation of the envelope protein affects the adaptability, infectivity, replication, and virulence of the virus ,11,46–51N-linked glycans on the surface of gp120 are essential for proper protein folding. HIV gp120 contains nine disulfide bonds and is highly glycosylated, carrying an average of 24 N-linked glycans. Among them, the Asn260 glycosylation site defines correct expression of gp120 and gp41 [N-linked glycans of the gp120 are preferentially located near the disulfide bridge. Removal of disulfide bonds has been shown to significantly affect the ability of the DC-SIGN to bind to HIV. On the other hand, in a variety of virus strains lacking N-glycans, introduction of new N-glycans upstream of several disulfide bonds could result in complete loss of viral infectivity [The human immunodeficiency virus (HIV) envelope glycoprotein (Env) is composed of surface subunit gp120 and transmembrane subunit gp41 [and gp41 . Moreoveectivity . The abiectivity . In addiectivity . Given tN-glycosylation sites ranging from 5 to 11, most of which are located in the globular head of the HA molecule [N-linked glycan in HA was associated with the related to the resistance to collection and increased virulence in mice [N-linked glycosylation is also important in the NA functions [Influenza A virus (IAV) encodes two main viral surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA). The HA protein determines the antigenicity of the IAV. The HA of most human H1N1 has multiple molecule ,56. Glycmolecule . Other smolecule . Glycosymolecule ,64. Furt in mice . N-linkeunctions . Lack ofunctions .N-glycosylation sites, while ACE2 contains 7 N-glycosylation sites [N-linked glycans of the SARS-CoV-2 Spike protein are less important in regulating the direct binding of the Spike and ACE2, but inhibit the virus entry [SARS-CoV-2 is a novel virus that has caused the global pandemic “Coronavirus Disease2019” (COVID-19), a severe acute respiratory disease . The viron sites . The O-lus entry . Studiesus entry . HoweverN-linked glycosylated [N-glycosylation site at N4 [Some coronaviruses have E1 and E2 envelope proteins. Previous data showed that E1 protein is an O-linked glycoprotein while E2 is osylated . The M pte at N4 ,77. Althte at N4 .N-glycosylation sites. A previous study showed that loss of any of N-glycosylation sites does not affect the expression of GP but enhance pseudovirion transduction [N-glycans does not affect the binding of pseudoviral particles to the cell surface, but increases protease sensitivity of the cathepsin B [Ebola virus (EBOV) belongs to the filovirus family, and its surface glycoprotein (GP) is composed of trimers of GP1/GP2 heterodimers. The GP1 subunit is important for receptor binding, while the transmembrane-related GP2 subunit is necessary for membrane fusion. EBOV GP1 contains 15 sduction . Removalhepsin B . In antihepsin B ,79. The N-linked glycosylation sites, at Asn 33 and Asn 63 [N-linked glycosylation sites in the extracellular domain of SFTSV Gc: Asn 853, Asn 914, and Asn 93682. DC-SIGN and Non-muscular myosin heavy chain IIA (NMMHC-IIA) have been reported to be the initial binding receptor for SFTSV [N-linked glycans in Gn and Gc are critical for virus binding and entry remains to be defined.Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-transmitted bunyavirus with a segmented, negative-strand RNA genome, which includes large (L), medium (M), and small (S) segments. The M segment encodes a membrane protein precursor that matures into two glycoproteins, Gn and Gc. Recent studies have shown that SFTSV-specific neutralizing antibodies, mainly induced by Gn and Gc, play a vital role in the protective immune response . Gn has d Asn 63 . SFTSV Gd Asn 63 . Analysior SFTSV ,84. WhetN-linked glycosylation sites at N134, N235, N347, N399, and N609 while Gc contains a potential N-linked glycosylation site at N928 [N-glycosylation sites of Gn reduce the immunoreactivity of Gc protein. Conversely, lack of N-glycans on Gc has been shown to reduce immunoreactivity of the Gn protein [Hantaan virus (HTNV) also encodes two glycoproteins, Gn and Gc. Gn contains five potential  at N928 . All gly at N928 . N134 is at N928 . In addi at N928 . Besides protein .N-glycosylation sites, located at N438 of the Gn protein, and N794, N829, N1035, and N1077 of Gc protein, respectively [Rift Valley Fever is a disease caused by Rift Vally Fever virus (RVFV) and is transmitted by mosquitoes. The RNA genome of the RVFV consists of large (L), medium (M), and small (S) segments. The M segment encodes the Gn and Gc glycoproteins. RVFV has five putative ectively . Glycosyectively . A previectively . Besidesectively ,89.N-linked glycosylation sites, four of which are highly glycosylated [N-glycans on F protein has little effect on protein processing and expression, but F3 and F5 N-glycan mutants have a higher fusion level compared to the wild-type NiV [Nipah virus (NiV) is an emerging paramyxovirus with deadly consequences. NiV infects the respiratory and nervous systems and leads to fatal encephalitis, the main cause of human death ,91. The osylated . Removaltype NiV . Removaltype NiV . There atype NiV . The funtype NiV . Like Nitype NiV . The HeVtype NiV . To dateN-glycosylation sites, which are N27 and N70 located in the F2 subunit, N116 and N126 in the p27 peptide, as well as N500 in the F1 subunit. Every single N-glycosylation site in the RSV F protein is not dispensible for virus replication, but they have synergistic contribution to the efficiency of viral infection [N-glycans in the RSV F protein are removed. Data have demonstrated that the N-glycan at the N500 position is essential for syncytium formation in RSV-infected Hep-2 cells [Human Respiratory Syncytial Virus (HRSV) belongs to the Pneumovirus genus, Paramyxoviridae. The virus envelope has two main glycoproteins, G and F, which are responsible for virus attachment to cells and membrane fusion . The glynfection . The vir-2 cells . In addi-2 cells . The G p-2 cells .N-linked glycosylation sites on the LCMV glycoprotein: 6 on GP1 and 3 on GP2 [N-glycosylation sites on GP2 are conserved in all the members of this virus. In contrast, the number and location of N-glycosylation sites in GP1 are highly diverse. In addition, folding of the glycoprotein complex (GPC) precursor requires N-linked glycosylation in GP1 [Lymphocytic choroid meningitis virus (LCMV) belongs to the arenavirus family. There are three main structural proteins: one nucleocapsid protein (NP) and two glycoproteins (GP1 and GP2) . AS prev3 on GP2 . The thrn in GP1 . N-linken in GP1 . Removaln in GP1 . N-glycon in GP1 . All thrn in GP1 . These dN-glycosylation motifs in GPC, including 7 N-glycans on GP1 and 4 N-glycans on GP2 [rd (N99), 5th (N119), 6th (N167), 8th (N365) and 9th (N373) sites can mask key epitopes in the GPC and escape humoral immune response [Lassa virus (LASV), a member of the arenavirus family, causes Lassa fever . LASV GPs on GP2 . The glys on GP2 ,104,105.s on GP2 . N-glycos on GP2 . In pseus on GP2 . Besidess on GP2 . The rolGP2 [rd N9, 5th (NN-glycosylation site at Asn-146 (N146) of their S domain [N-glycan modification on the ER has a major impact on the production of infectious virus particles [N-glycosylation may also be a predictive marker for increased risk for cancer in patients with chronic HBV infection, which deserves more evaluation [Hepatitis B virus (HBV) is a human DNA virus that encodes three envelope glycoproteins, small (S), medium (M), and large (L). These three proteins share a potential S domain ,109. Eararticles ,110. Thealuation . AlthougN-linked oligosaccharides as well as 11 sites for O-linked glycosylation [N-linked oligosaccharides and 3 potential sites for O-linked glycosylation [N-linked glycosylation and numerous clustered O-linked glycans in a peptide stretch delimited by amino acids 30 and 124 [Herpes virus is a large enveloped double-stranded DNA virus, which include many important human and animal pathogens. Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpes virus, which encode 12 envelope glycoproteins and play important roles in the viral replication . Glycoprsylation . gL contsylation ,121,123. and 124 . Moreove and 124 . Two conN-glycosylation of viral envelope proteins [N-glycosylation of HSV- 1 envelope glycoproteins was associated with the production of noninfectious viral particles [in vitro [Retinoic acid was shown to suppress viral yield in Vero cells and altered proteins . The usearticles ,127. Sevarticles . Of the in vitro . Twenty-in vitro .Varicella-zoster virus (VZV), a human alphaherpesviruses, encodes various structural glycoproteins that are important for its assembly and trafficking ,130. At N-linked glycans, which play important roles in infectivity, protein folding, tropism, proteolytic processing, as well as immune escape.Other viruses, such as Rabies virus , Human PNonstructural viral proteins can also be glycosylated. The flavivirus genome encodes seven NS proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . DENV NSGlycosylation at N130 plays an important role in viral proliferation, NS1 protein secretion, and cytopathic effects. Mutations at the N130 glycosylation site of DENV1 and DENV2 NS1 have been reported to prevent DENV from producing live viruses in mammalian and mosquito cells ,145. In N-linked glycosylation sites at N130 and N207 [th amino acid position [N-linked glycosylation sites in NS1, located at Asn-106, Asn-130, Asn-207, and Asn-326 [N-linked glycosylation sites at residues 85, 207, and 223 [Like DENV, JEV, and ZIKV NS1 have two and N207 . Yellow and N207 . In addiposition . Entebbe Asn-326  while th and 223 . In brai and 223 . Multipl and 223 . These fN-linked glycosylation in ER. For instance, mouse hepatitis virus MHV nsp3 has a glycosylation site at N1525. The glycosylation site of nsp4 in gamma-coronavirus bronchitis virus (IBV) is at residue N48. For the nsp4 of MHV, two glycosylation sites have been predicted at residues N176 and N237 [The nsp3 and nsp4 nonstructural proteins of coronavirus undergo and N237 . These tand N237 .In addition, some other viral nonstructural proteins, such as Rotavirus NSP4  and HCV N-glycosylation modifications, which confers various advantages on viral survival and virulence, have become a research hotspot. Targeting glycans may be a promising approach for inhibition of viral infections. Not only is glycosylation important for pathogens, it is also vital for hosts. Enveloped viruses hijack host glycosylation mechanisms and take advantage of host-derived glycans to modify their own glycoproteins, which benefits many aspects of viral pathogenesis, including entry into host cells, membrane fusion, and infectivity. Moreover, N-linked glycosylation of envelope proteins play important roles in neutralizing antibody sensitivity and immune escape. These glycoproteins are the only antigens expressed on viral surfaces; therefore, they are essential for vaccine development. For some viruses, glycans in nonstructural proteins are vital to their life cycles. Even though the involved mechanisms are yet to be established, their roles should not be underestimated. Elucidating the mechanisms of viral protein glycosylation during viral infection and replication will inform the development of specific antiviral therapies and vaccines.Glycomics is an important study field in cell biology. Viral-related


Airborne transmission of viral pathogens is dependent on the generation, exhalation, and deposition of virus-laden aerosols from infected to susceptible hosts. Rigorous evaluations of virus transmissibility in laboratory settings have provided critical insight into viral and host features that contribute to this property, but historically have not included concurrent evaluations of viral load in the air. Similarly, investigation of respiratory pathogens in an aerosol state has been understudied relative to other areas focused on transmission dynamics between mammalian hosts. What makes collection and quantification of virus-laden aerosols in the laboratory so challenging? Here, we discuss practical obstacles and limitations on performing this work in laboratory environments, the additional challenges posed by conducting these experiments concurrent with in vivo experimentation, and how continued investment in this work will provide greater understanding of the role aerosols play in viral transmission.Humans release respiratory droplets following various activities, from breathing and speaking, to coughing and sneezing, and even singing and playing wind instruments. These droplets vary in size (from submicron diameters to droplets visible to the human eye) and are emitted at variable levels and momentum depending on the respiratory action and individual person . In geneNumerous small mammalian models are employed in laboratory settings to evaluate virus transmissibility by the airborne route ; these mAerosols can be generated from liquid suspensions of virus in controlled laboratory settings but can vary widely based on equipment and established procedures between laboratories. The choice of nebulizer and sampler influences the properties of aerosols generated and recovered, respectively, as well as preservation of virus viability ,9. The mIn laboratory settings, aerosolization of liquids containing high concentrations of infectious virus permits the recovery of high levels of infectious virus in a sampler. In the context of in vivo experimentation, infectious virus is emitted from infected animals at much lower titers, necessitating collection methods that permit sampling for prolonged periods of time, employing low volume suspensions for collection to concentrate virus, maintaining cooler temperatures in the sampler, and employing other design features to preserve virus viability. Therefore, measurement of airborne virus shedding from infected animals during transmission assessments most often involves reporting viral nucleic acid and not infectious virus –7.There are numerous practical, logistical, and methodological reasons that contribute to reporting of viral genome copies and not infectious virus . Levels Collection of infectious viruses in aerosols is most desirable, but as discussed above, there are a multitude of practical issues that preclude their routine quantification, especially during in vivo settings. For sampling devices that require animal restraint and/or sedation for optimal collection , sampling durations are limited by anesthesia schedules. Staging sensitive collection devices within reach of an awake and alert animal can be impractical or impossible if the device is not designed for such wear-and-tear ; not all caging or animal restraint systems can support close staging of samplers that require a dedicated resting surface while concurrently prohibiting direct interaction between the device and curious animals. Transmission experiments are frequently conducted with 3 to 4 pairs of donor:contact animals housed in different cages ,6,14; shAlthough multiple samplers have been shown to efficiently preserve infectivity following collection of laboratory-generated aerosols containing high levels of virus , collectDespite the challenges highlighted above, there is a need to better define the role of aerosols in laboratory-based viral transmission assessments to discover pearls of wisdom that can translate into substantive benefits to public health. To this end, recent comparative studies have assessed the relative impact strain-specific, diluent-specific, environmental condition–specific, and device-specific changes confer to virus aerosolization ,19. LaboWider adoption of aerosol collection during in vivo assessments of virus transmissibility would be of benefit. Beyond current efforts of quantifying airborne virus released from infected animals and linking these data to virus transmissibility –6, futur


In Europe, half of people living with HIV (PLWH) present late to care, with associated higher morbidity and mortality. This study aims to assess short- and long-term costs of HIV-care based on time of presentation and identify other factors contributing to higher costs in the first and fifth year after antiretroviral therapy (ART) initiation.We included ATHENA cohort data which prospectively includes 98% of PLWH in the Netherlands. PLWH who initiated ART in 2013 were included and followed over five years. PLWH were divided in three categories based on CD4 cell-count at time of ART initiation: timely presentation (CD4>350cells/μL), late presentation (CD4 200-350cells/μL or >350cells/μL with AIDS-defining illness) and very late presentation (CD4<200cells/μL). The total HIV-care cost was calculated distinguishing ART medication and non-ART medication costs .From 1,296 PLWH, 273 (21%) presented late and 179 (14%) very late. Nearly half of those who entered HIV-care in a very late stage were of non-Dutch origin, with 21% originating from sub-Saharan Africa. The mean cost per patient in the first year was €12,902 , of which about two-thirds due to ART ). ART costs in the first and fifth year were comparable regardless of time of presentation. During the first year on treatment, non-ART medication costs were substantially higher among those with late presentation ) and very late presentation ), compared with timely presentation ). Higher non-ART costs were attributable to hospitalization and co-medication. The total non-ART costs incurred across five years on treatment were 56% and 246% higher for late and very late presentation respectively as compared to timely presentation.Very late presentation is associated with substantial costs, with non-ART costs nearly seven times higher than for those presenting timely. Hospitalization and co-medication costs are likely to continue to drive higher costs for individuals with late presentation into the future. Programs that identify individuals earlier will therefore likely provide significant short- and long-term health cost savings. Late presentation, or accessing care with a CD4 cell-count <350 cells/μL or with an AIDS-defining illness, has several consequences on an individual clinical, public health, and health systems level . On the In 2018, 5.6 per 100,000 people were newly infected with HIV in the European region . DespiteThe comprehensive cost of late presentation has not been assessed with a longer duration of follow-up since the World Health Organization recommended to initiate ART at CD4 cell-count <500μL . We therIn the Netherlands, HIV-care is provided by designated treatment centres As an integral part of HIV-care, the HIV Monitoring Foundation is responsible for prospectively collecting demographic data and relevant HIV and treatment data, as well as data on viral hepatitis coinfection, from PLWH in the Netherlands and receiving care in one of these 27 treatment centres .This data collection is known as the ATHENA cohort which was initiated in 1998 and captures data from >98% of all patients with a diagnosed HIV-infection who are in care in the Netherlands . Th. Th20]. Costs are reported in 2018 Euro and presented as cost per patient per year (using means and standard deviations).At initiation, the cohort was approved by the institutional review board of all participating centers. People entering HIV care receive written material about participation in the ATHENA cohort and are being informed by their treating physician of the purpose of collection of data, after which they can consent verbally or elect to opt-out. Data are pseudonymized before being provided to investigators and may be used for scientific purposes. A designated quality management coordinator safeguards compliance with the European General Data Protection Regulation .In total 1529 PLWH initiated ART between 1 January 2013 and 31 December 2013. After exclusion of PLWH who enrolled the SHM database > 6 months after start of ART and adolescents (<18 years of age), a full year of follow-up was available for 1316 of PLWH . Of PLWHThe majority of PLWH included in our analysis were male, comparable to the overall PLWH population in the Netherlands that year . The majHIV is mainly transmitted among MSM . IndividDuring the first year after ART initiation PLWH with very late presentation used the most healthcare resources . The strAfter the first year of ART initiation the total HIV-care cost per individual is €12,902 , of which €8,250  was for ART medication and €4,652  for all non-ART medication costs . There iSimilar as to timely presentation non-ART costs were mostly attributed to the use of co-medications. However, co-medication costs were twice the amount ) for presentation late and ten times the amount ) for presenting very late. Additionally, hospitalization costs are more common among individuals with late presentation ) and very late presentation ), while negligible for those who presented timely ) .Over the years, the total annual HIV-care cost per individuals remained similar for PLWH with a timely presentation  . AlthougOur results showed that apart from higher morbidity, mortality, and prolonged possibility of transmission, late presentation- and more specifically very late presentation- resulted in considerably higher short-term HIV-care costs. The higher cost per patient that initiated timeously compared to those with a late presentation persisted through to the end of our follow-up period at five years post ART initiation. Although the cost of ART medication comprises the greatest proportion of the total annual HIV-care costs, non-ART costs drive the increasing cost of late presentation. The short-term non-ART costs were nearly twice as high for late presentation and nearly seven-times higher for very late presentation as compared with those who presented timely in HIV-care. Over a five-year period, the total HIV-care cost for late presentation were 56% higher for late presentation and 246% higher for very late presentation as compared to timely presentation.Cost components driving the higher short-term non-ART cost among PLWH presenting late in care not surprisingly mainly included hospitalization and co-medication costs. Given the likelihood of such individuals suffering from conditions requiring frequent and prolonged hospitalization and the use of multiple co-medications. Unfortunately, after five years this increase in costs is still present mainly in the form of increased co-medication costs. This suggests that even though the clinical condition of PLWH presenting later in care improved, over time their health state remains worse than those presenting timely. Similar findings were found by Krentz et al. who found higher continuing medical costs for PLWH presenting later in care related to non-ART drugs .We identified several patient characteristics associated with late presentation such as heterosexual transmission, elderly, and origin in line with other studies , 15, 21.Another strategy which may help in earlier identification of HIV disease, would be through HIV indicator condition related testing , 25. HIVMigrants, from low- and middle-income countries, are a subgroup which are disproportionately affected by HIV in high-income countries and frequently present late for care High levels of HIV-related stigma, unawareness, and limited access to care result in insufficient engagement with HIV prevention and treatment services. Therefore migrants are placed at risk of poor HIV related clinical outcomes . In ordeFree and easy to access self-tests could also lower the barrier for stigma among several risk groups . In addiApart from the individual clinical benefits of timely presentation into HIV-care, our findings stress the importance of earlier identification of HIV-infection from a cost perspective in both the short and long run. Moreover, investing in earlier identification will also results in a decrease of transmission. Importantly, given that an individual presenting late incurs €8,463 and very late incurs €26,404 of additional costs over the first five years on treatment compared to those presenting timeously, these financial resources could be redirected to novel testing and HIV screening programmes. If a new HIV-infection could be identified for that cost, then those testing or screening programmes would quickly become cost-saving, particularly since the cost savings that we calculate here do not include the cost of onward transmission.A major strength of our study is that the population studied can be considered to be highly representative of the PLWH and in care in the Netherlands, as more than 98 percent of those in care consent to their data being collected as part of the ATHENA cohort . As 91% Our study was the first study analyzing the long-term follow-up after ART treatment is recommended for patients with CD4 cell-counts <500 μL . The guiOne of the limitations of this study is that we did not have access to all costs of HIV-care as appointments or procedures performed by a general practitioner are not included. This could result in a slight under-estimation of costs for timely presentation, since they possibly use more care provided by general practitioners. Care provided by a general practitioner is, however, less expensive than hospital care. Moreover, our resource utilization showed that PLWH with timely presentation only have slightly less outpatient visits. Therefore, it would be unlikely that visits to the general practitioner office would make a substantial difference. Secondly, we did not include cost to the patient. If considering costs from the patient perspective, those presenting very late, due to the greater number of inpatient days and outpatient visits incurred, are likely to have incurred more personal costs than those presenting on time. Including costs from the patient perspective would only have further strengthened our conclusions. Lastly, we excluded PLWH who died during the study period as we calculated a full year and consecutive full four years of HIV health care utilization. The number of PLWH who died that presented very late in care was much higher compared to individuals with timely and late presentation even after five years. Although these PLWH utilized less health care costs, due to short hospital admission stay and often not receiving ARTs, they have the worst health care outcome, death. This further strengthens the need for programs focused on early identification of new HIV infections.In conclusion, late presentation can result in substantial additional non-ART-medication costs over one- and five-year time periods. Several patient characteristics as country of origin, transmission route, and age are predictors of presenting late in care highlighting populations where aggressive testing strategies should be implemented, and awareness should be increased, to maximize both individual benefit and reduce costs. Investment in these strategies could improve clinical outcomes, prevent onward HIV transmission, and avoid these long-term excess patient costs.S1 TableCosts are presented per unit in €.(DOCX)Click here for additional data file.S2 TableThe cost of co-medications per day in €. Costs are calculated based on the average of the lowest and highest price of the available medication for a 70kg adult and 2018 list price.(DOCX)Click here for additional data file.S3 TableThe cost of ART per day in €. Costs are calculated based on the average of the lowest and highest price of the available medication and 2018 list price.(DOCX)Click here for additional data file.


Previous studies have suggested associations between addictive behavior and gallstone disease (GSD) risk, yet conflicting results exist. It also remains unclear whether this association is causal or due to confounding or reverse associations. The present study aims to systematically analyze the epidemiological evidence for these associations, as well as estimate the potential causal relationships using Mendelian randomization (MR).N = 126,906–4,584,729 participants) in this meta-analysis based on longitudinal studies. The two-sample MR was conducted using summary data from genome-wide associations with European ancestry .We analyzed four common addictive behaviors, including cigarette smoking, alcohol intake, coffee, and tea consumption , P = 0.004], problematic alcohol use (PAU) , decaffeinated coffee intake , as well as caffeine-metabolism , and tea consumption , respectively.An observational association of GSD risk was identified for smoking [RR: 1.17 (95% CI: 1.06–1.29)], drinking alcohol [0.84 (0.78–0.91)], consuming coffee [0.86 (0.79–0.93)], and tea [1.08 (1.04–1.12)]. Also, there was a linear relationship between smoking (pack-years), alcohol drinking (days per week), coffee consumption (cups per day), and GSD risk. Our MRs supported a causality of GSD incidence with lifetime smoking [1.008 (1.003–1.013), Our study suggests cigarette smoking, alcohol abuse, and decaffeinated coffee are causal risk factors for GSD, whereas tea consumption can decrease the risk of gallstones due to the effect of caffeine metabolism or polyphenol intake. Addictive behavior causes a major public health concern, and it has a massive, long-term impact on human suffering and societal costs . GallstoN = 4,213,482) has provided evidence that smokers have an estimated 11% increased risk of GSD per 10 cigarettes per day compared to non-smokers  has identified a significant non-linear trend of GSD risk reduction associated with the increment of drinking alcohol (up to about 30 g per day). In addition, one meta-analysis based on six studies  has observed a dose-dependent association of coffee consumption with GSD   fills the gap of making causal inferences by using single nucleotide polymorphism (SNP) as an instrumental variable (IV) since SNPs are usually established before the development of disease and therefore independent of confounders . Indeed,The current study aims to comprehensively evaluate the relationship between these common addictive behaviors and the GSD risk. We first summarized the evidence in one updated meta-analysis only including a longitudinal study. Data from the meta-analysis was further tested for potential dose–response relationships and by trial sequential analysis (TSA) to check if the present evidence is conclusive. We then explored a putative causal association of tobacco smoking, alcohol use, caffeine intake, and tea consumption with the risk of GSD using a two-sample MR design.Our meta-analysis has been registered at PROSPERO (CRD42020179076) and following PRISMA checklists. We searched PubMed and Embase databases for studies published before January 2021, and references to the retrieved articles were manually searched for additional information . The floI1 exceeded 50%; otherwise, a fixed-effect meta-analysis was conducted  and with R2 < 0.1 identified by LDlink2 were used as IVs for lifetime smoking  (SNPs showing genome-wide significance (account) . The selaccount) , 25, 26.account) . DetailsNg = 462,933, 7682/455251) obtained from UKB, MRC-IEU (id: ukb-b-18700). Third, the symptomatic GSD with a history of cholecystectomy from the UKB .GSD cases and controls were obtained from the UK Biobank, a cohort of about 500,000 adults recruited during 2006–2010 in the United Kingdom . Three sp-value of the MR-Egger intercept was used to indicate potential horizontal pleiotropy, and Cochrane’s Q-value was used to evaluate the heterogeneity among those SNPs for each addictive behavior . Here, the causal association would be considered statistically significant when a Bonferroni corrected P-value was less than 0.013 . A p-value < 0.05 was regarded as the marginal significance.For our MR study, the multiplicative random-effect inverse variance weighted (IVW) method was used to estimate the causal associations between addictive behaviors and GSD risk. In sensitivity analysis, the MR-Egger regression was used to identify and correct for the horizontal pleiotropy, the weighted median method provides the estimates when SNPs accounting for more than half of the weight are valid, and the maximum likelihood method maximizes the likelihood of the model based on the causal association –31. The behavior . In thisN = 4,584,729), alcohol intake , coffee consumption , and tea consumption . A positive significant association of incident GSD was observed with smoking [RR: 1.17 (95% CI: 1.06–1.29)] and tea consumption [1.08 (1.04–1.12)]; while a negative significant association of intaking alcohol [0.84 (0.78–0.91)] or coffee [0.86 (0.79–0.93)] with GSD was found , P = 0.08] (P ≤ 0.01)] per 5 units. We further found an increment of days per week of alcohol drinking decreased the GSD risk with a linear inverse association . As for caffeine consumption, a potential linear association was detected between coffee cups per day and GSD risk . Despite a non-linear relationship between GSD risk with consuming tea-cups per day (P = 0.01), we found no significant association. In addition, the risk of GSD increased by 4% and 8% with every 5 and 10 pack-years increments in cigarettes-smoking; while the risk was reduced by 20% and 23% per five units increment in alcohol-drinking days per week and coffee-cups per day ,  = 0.08] . However per day .Z-curve crossed trial sequential monitoring and/or conventional boundary and penalized tests adjusted Z-curves also presented similar results, denoting that this evidence was robust and conclusive  and patients underwent cholecystectomy , but not for self-reported gallstones  when compared with Padjusted < 0.013. The estimates remained directional and consistent observed in MR-Egger regression, despite the causalities were not significant. Then, by using the intercept of MR-Egger, we observed no evidence of horizontal pleiotropy , and the weighted median and maximum likelihood methods yielded similar results, which illustrated the high stability of this causality.In the IVW method, using lifetime-smoking associated 120 independent SNPs as IVs, we found that it had a causal effect on diagnosed cholelithiasis . As for ever-smoking, it was associated with an increased risk of diagnosed cholelithiasis , self-reported gallstones , and cholecystectomy  in IVW. There was no detected horizontal pleiotropy by using MR-Egger , and similar effects were observed using the weighted median or maximum likelihood method. For the subgroup analyses, current-smoking also significantly increased the risk of diagnosed cholelithiasis , whereas it disappeared in smoking-cessation . This study, for the first time, provided an explanation of the pathogeny of GSD with addictive behavior, the results of our MR showed that nicotine dependence was a major risk factor for GSD , and ETS was also causally associated with the risk of cholecystectomy  , no causal association of GSD risk was found, and this result might be due to the presence of heterogeneity . In this MR, we further found that PAU was potentially associated with the risk of diagnosed cholelithiasis  and self-reported gallstones , but not cholecystectomy  in IVW. Moreover, the effects attenuated slightly in MR-Egger regression with the intercept  confirming that pleiotropy was not detected in the three outcomes. Similarly, the positive estimate was identified for the risk of GSD with PAU in the maximum likelihood method.Regarding common alcohol use , P = 0.016] for cholecystectomy. Although all of the PQ ≤ 0.001 and heterogeneity existed, no horizontal pleiotropy was detected by MR-Egger with the intercept of 0.136, 0.164, and 0.113 in our outcomes. Furthermore, we analyzed the associations between the metabolism of caffeine and the incidence of GSD , P = 0.013], and the GSD risk; whereas decaffeinated coffee  or instant coffee  was related with the GSD risk increased.No significant causal association of GSD risk with coffee consumption was found in this IVW. Even though removing an SNP (rs2472297), the null effect on GSD with consuming coffee was not altered . While genetically predicted caffeine-intake significantly decreased the GSD risk in the weighted median , self-reported gallstones  and cholecystectomy . Although an association was not significant, the estimate remained directional and consistent as observed in MR-Egger; and similar estimates were also obtained in the weighted median and maximum likelihood methods. For other sensitivity analyses, no heterogeneity existed with the funnel plot presented symmetrically in Consuming tea  was significantly negatively associated with GSD risk in IVW . For diaAs summarized in this study, the meta-analysis based on a longitudinal study indicates that addictive behaviors are significantly associated with the incidence of GSD. Compared with never smokers, current smokers have a positive dose-dependent response to GSD risk, and the evidence is further verified by the MR analysis. There are negative dose–response relationships between common alcohol use, coffee intake, and the GSD risk. However, the results of MR do not confirm the causal relationship between them. The novel finding of this study is that alcohol abuse may be causally associated with an increment in GSD risk, whereas tea consumption has a protective effect on the GSD risk in Europe.Smoking has been shown to alter lipid metabolism, and the abnormal synthesis of bile may cause cholesterol supersaturation for the formation of gallstones . ConsistTo date, evidence linking alcohol drinking with GSD is controversial . In thisInterestingly, high coffee consumption was associated with a decrement in GSD risk. An MR also suggested a causal relationship between them in the Danish cohort . But, ouTea consumption as another addictive behavior with caffeine intake (including black and green tea) has been associated with a GSD risk decreased in both genders within the population of Asia, and caffeine can stimulate cholecystokinin secretion and release bile acids into the intestine , 41. OurHere, some plausible mechanisms are explored for the causal associations between addictive substance use and GSD risk. For the association between active smoking or ETS and the risk of GSD, the nicotine-dependence may be a key factor in this relationship. Of note, electronic cigarette has not been reported in GSD-related research. In addition, caffeine and tea polyphenols are the most commonly consumed psychostimulants, and they both causally decrease the risk of GSD in our MR. However, coffee consumption (including decaffeinated coffee or other beverage) is not associated with GSD, which may weaken the effect of caffeine.There is high heterogeneity in our meta-analysis, and existing research regarding this topic is relatively fewer, which may have yielded publication bias. For example, the positive association between tea consumption and GSD in the American population might be due to the smaller number of studies included, which is an important limitation of our study. The second limitation is vertical pleiotropy, which could be shown to mediate the effect within a relationship between exposure and outcome. Another limitation is that we could not explore a non-linear relationship using this MR approach. As for the heterogeneity in a different population, our IVs were all identified in GWAS of a European-origin sample, although these instruments can only explain the percent of 0.24–1.72  in total estimated heritability, which limits the generalizability of our finding to diverse populations.In conclusion, tobacco smoking, PAU, and decaffeinated coffee directly confer high risks of GSD; nonetheless, habitual caffeine intake and tea consumption may have a protective role against GSD due to an effect of caffeine metabolism or polyphenol intake. Accordingly, we infer that changing addictive behavior may be necessary for reducing the risk of GSD.The original contributions presented in this study are included in the article/BZ managed the project and study design. XW and YB read and abstracted the studies included in the meta-analysis. XJ and YB analyzed the data in the Mendelian randomization study. MZ and YB prepared the tables and figures. DG, MT, and YB did the statistical analyses. YB drafted the manuscript with HC, XS, YW, XW, and XJ. All authors reviewed and approved the article.


Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50–100 μM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.At present, there is no effective experimental method for detecting whether the  Pseudorabies (PR) is caused by the suid herpesvirus 1 (SHV-1), a common infectious disease in animals  and cultured in RPMI-1640  medium containing 10% heat-inactivated fetal bovine serum  and 1% penicillin/streptomycin  at 37°C and 5% CO2. The suid herpesvirus 1 (SHV-1) was provided by South China Agricultural University and inoculated into PK-15 cells, followed by culture at 37°C and 5% CO2. After 3–4 days of culture, cell cytopathic effects (CPE) were observed. The viral liquid was filtered and then diluted by 10-fold.Propidium monoazide was purchased by Biotium . The porcine kidney cell line PK-15 cell was obtained from the American Type Culture Collection  was used as a negative control.The virus was boiled in a water bath for 30 min for heat inactivation. For UV inactivation, the virus suspension (1 ml) was evenly spread on the bottom of a 10-mm petri dish and exposed to ultraviolet radiation for 4 h. The UV source was a Xin Yate ZWS-type UV ultraviolet lamp , purchased from Suzhou Xin Yate Light Source Factory. The test irradiation intensity was 90 uW/cmPropidium monoazide (1 mg) dye was dissolved in 500 μL of dimethyl sulfoxide (DMSO) to obtain a 4-mM storage solution, which was stored at −20°C in the dark. PMA working solution was added to the diluted virus solution to make the final concentrations of 100, 50, 20, and 10 μM and allowed to react in the dark for 13–15 min. The solution was placed under a 100-W blue light for 18–20 min and shaken once every 3–5 min.SHV-1-infective, SHV-1-heat-inactivated, SHV-1-UV-inactivated, and SHV-1 nucleic acid solutions and PMA. In group two, PBS was added to the SHV-1-infective, SHV-1-heat-inactivated, SHV-1-UV-inactivated, and SHV-1 nucleic acid solutions. Distilled H2O (dH2O) was used as a negative control in qPCR amplification. The experimental design is given in To determine the effectiveness of PMA, we prepared the following experimental groups based on the PMA concentration of 50 μM recommended in ref. . In grou2O was included as a negative control in qPCR amplification.To determine the PMA working concentration, the infective virus and heat-inactivated virus were treated with 100, 50, 20, and 10 μM PMA as the experimental groups. dHSHV-1 reported in the NCBI, and different primers were screened. The primer sequences and amplicon lengths are listed in Two pairs of primers were designed according to the envelope glycoprotein E (gE) gene of 2O used as a negative control. The reaction system contained 2 × AceQ qPCR SYBR Green Master Mix (10.0 μL), 10 μM upstream primer (0.4 μL), 10 μM downstream primer (0.4 μL), 50 × ROX Reference Dye 1 (0.4 μL), and template cDNA (2.0 μL), and deionized water was added to a total reaction volume of 20 μL. The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 40 cycles at 95°C for 10 s and 60°C for 30 s, followed by 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. A CFX96TM real-time PCR instrument  was used for this procedure.To verify the specificity of the primer amplification products, the two pairs of specific primers designed were identified by qPCR for the SHV-1 virus, with dHAfter the virus solution was treated with different concentrations of PMA, the Ct value of the virus samples was quantified by qPCR using two pairs of gene-specific primers.SHV-1-gE gene fragment amplified by primer 1 was inserted into pUC19 to form a recombinant plasmid. Recombinant pUC19-primer 1 was extracted using a Qiagen Plasmid Plus Kit  and stored at −20°C. The plasmid concentration was measured, and a 10-fold dilution of this sample was used for qPCR.The SHV-1, swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, and porcine epidemic diarrhea virus were extracted and verified by qPCR. dH2O was included in the qPCR step as a negative control.To test the specificity of the primer, the nucleic acids of SHV-1, the virus was diluted by 10-, 100-, and 1,000-fold and amplified with the appropriate primer and PMA concentrations using qPCR. dH2O was included in the qPCR step as a negative control.To clarify the effective concentration range for successfully detecting SHV-1 infectivity, we used this method to evaluate 50 infective SHV-1 virus samples and 50 inactivated SHV-1 copies. PBS was set as the negative control group. dH2O was included in the qPCR step as a negative control.To verify whether PMA-qPCR can detect A two-way analysis of variance was used to assess differences between groups, and Pearson's correlation coefficient  was used for correlation analysis when required. The results were judged as follows: For a sample Ct value ≤ 35, the sample was judged as positive; 35 < sample Ct value ≤ 40, if the amplification curve was logarithmic, the sample was judged as suspiciously positive and otherwise the sample was judged as negative; when the sample was suspected as positive, it was retested. If the Ct value of the retested sample was ≤ 35, the sample was judged as positive; otherwise, the sample was judged as negative. When the sample had no Ct value or if the Ct > 40, the sample was judged as negative.SHV-1 (blank control group), and group four contained cells infected with SHV-1 (cytopathic group). The heat-inactivated and UV-inactivated groups and blank control exhibited no cytopathic effects  are presented in SHV-1, different viruses were detected with primer 1 using qPCR. The SHV-1-infective virus group showed positive results .In the PMA treatment group, of the 50 inactivated virus samples, 48 samples showed negative Ct values >35, indicating negative results. The Ct values of the 50 infective samples ranged from 26 to 30, corresponding to copy numbers of 6Pseudorabies has caused significant economic losses to the global pig industry. SHV-1 is prevalent in China, which severely restricts the development of the breeding industry. The timely, rapid, and accurate diagnosis of SHV-1 infection is necessary for effective prevention and control of swine PR.PR mainly include pathogenic identification and serological testing. Pathogen identification shows high sensitivity and specificity and is rapid, simple, and suitable for clinical detection of SHV-1; however, this method cannot distinguish between live and dead viruses, that is, whether the virus is infectious. Serological testing has a high coincidence rate and specificity, making it suitable for laboratory sample inspection, quarantine and origin analysis, epidemiological investigation, and screening and establishment of healthy pigs without disease. However, this method is time- and labor-intensive. To improve detection methods, we combined PMA and qPCR, which simplified the detection process and enabled the determination of virus infectivity.In the OIE Manual of OIE Handbook of Diagnostic Tests and Vaccines for Terrestrial Animals, methods for identifying SHV-1. Thus, we established a method for identifying SHV-1 infectivity.Propidium monoazide is a photosensitive dye with a high affinity for nucleic acids. When exposed to strong visible light, PMA binds covalently to DNA or RNA , 29. PMASHV-1 infectivity, we first tested the effect of PMA on SHV-1 detection using qPCR. The results showed that the addition of PMA did not affect the SHV-1 detection results. PMA can only penetrate viruses with damaged capsids . According to the average CT, the loss of about 104 copies due to PMA treatment can be quantified. The higher the virus concentration, the loss of more. When the virus stock solution is diluted more than 1,000 times, that is, when the virus concentration is low, the PMA concentration is relatively supersaturated, resulting in false negative results (In addition, we examined the effect of virus dilution on PMA-qPCR; the results showed that when the virus was diluted by 10–1,000-fold, it was possible to determine whether  results . It may  results . Follow-SHV-1 gE gene as a detection target and established a PMA-qPCR method for rapidly and quantitatively determining the infectious ability of PR. This method shows high specificity, sensitivity, and repeatability and can efficiently, quickly, and accurately identify SHV-1 infection. Our PMA-qPCR method can be applied in clinical virus isolation and to establish the effectiveness of the disinfection of farms. It is critical to note that PMA treatment of samples shows promise as a method of capsid protein-damaged viral DNA amplification. However, for UV-inactivated cells, PMA treatment is ineffective (In summary, we used the ffective , which pffective  speculatSHV-1. After confirming that the addition of PMA did not affect the detection of live viruses, we established that primer gE, a PMA concentration of 50–100 μM, and a virus solution diluted by 10–1,000-fold were optimal experimental conditions. We determined whether heat-inactivated SHV-1 was infectious. Our method showed high specificity, sensitivity, and repeatability and can efficiently, quickly, and accurately identify SHV-1 infectivity. Our PMA-qPCR method can be applied for clinical virus isolation and to establish the efficacy of disinfection of farms.A combination of PMA and qPCR was used to detect the infectivity of The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.LY designed and conducted experiments, analyzed data, and wrote the manuscript. YL, QL, WS, YH, GL, DY, MZ, GX, and YC conducted the experiment. CH and XT participated in the experiment and conducted data analysis. All authors contributed to the article and approved the submitted version.This study received funding from Wuhan Keqian Biology Co., Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.Authors LY, YL, QL, WS, YH, GL, DY, MZ, GX, CH, and XT were employed by Wuhan Keqian Biology Co., Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tobamoviruses are among the most well-studied plant viruses and yet there is still a lot to uncover about them. On one side of the spectrum, there are damage-causing members of this genus: such as the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV) and cucumber green mottle mosaic virus (CGMMV), on the other side, there are members which cause latent infection in host plants. New technologies, such as high-throughput sequencing (HTS), have enabled us to discover viruses from asymptomatic plants, viruses in mixed infections where the disease etiology cannot be attributed to a single entity and more and more researchers a looking at non-crop plants to identify alternative virus reservoirs, leading to new virus discoveries. However, the diversity of these interactions in the virosphere and the involvement of multiple viruses in a single host is still relatively unclear. For such host–virus interactions in wild plants, symptoms are not always linked with the virus titer. In this review, we refer to latent infection as asymptomatic infection where plants do not suffer despite systemic infection. Molecular mechanisms related to latent behavior of tobamoviruses are unknown. We will review different studies which support different theories behind latency. Tobamovirus comprises positive-sense, single-stranded RNA (+ssRNA) viruses belonging to the family Virgaviridae and contains 37 recognized and several tentative species [The genus  species . These v species .Solanaceae. PMMoV usually causes milder symptoms on leaves but is more severe on fruits causing a reduction in size and number, uneven ripening, corky or necrotic rings and internal necrosis. Tomato brown rugose fruit virus (ToBRFV) was first described by Salem et al. [Tobamovirus disease symptoms can vary depending on the host plant, virus species and environmental conditions. Leaves of infected plants appear to be deformed, mottled or display a mosaic pattern . Sometimm et al.  and, sinm et al. . The symm et al. . The leam et al. ,5,7.Hoya spp. showing chlorotic ring spots and irregular leaf chlorosis. [Apart from the agriculturally important crop plants, several new tobamoviruses have been detected infecting horticultural crops. Two tobamoviruses have been identified from hibiscus (Hibiscus latent Singapore virus (HLSV) and hibiscus latent fort pierce virus (HLFPV) ,8) Simillorosis. ,10. Tobamoviruses are one of the most studied viruses due to their availability and ease of use in biotechnological studies . A singlThe extreme stability of tobamoviruses and their presence in the environment has been attributed to their structure . TMV parPurified TMV can be infectious even after 50 years of storage at 4 °C . TMV hasThe +ssRNA genome is approximately 6300 nt to 6800 nt in length. N. benthamiana and may be associated with cellular movement [The next ORF encodes the 28–34 kDa movement protein, which has RNA-binding activity and is required for cell-to-cell movement ,18. A pomovement . The 3′ movement ,21. 7G-capped G residue, which is followed by a ~70 nt G-deficient sequence, called an omega sequence (Ω), that contains multiple CAA repeats. The sg RNAs also contain these motifs [The 5′-UTR region of TMV starts with an me motifs . Severale motifs . The 3′-e motifs . These Ue motifs ,23,24,25On the basis of genome organization, tobamoviruses have been divided into three subgroups ,27,28. TThe spread of tobamoviruses is mainly driven by mechanical transmission. Seed transmission is also possible. Tobamoviruses do not infect the embryo but can be transmitted to the seedling from the surrounding tissue during germination. Tobamoviruses can be found in the seed coat and sometimes in the endosperm . Seed trOther sources of primary inoculum include the juice of infected fruits, as well as plants grown in infected soil or grafted with infected material . BumblebAs a part of the disease triangle, host, virus, and environment equally play a role in disease establishment. Therefore, the viral symptoms are dependent on the right host and a suitable environment. Over the last few decades, there have been various meanings and explanations for asymptomatic infection and virus latency. This terminology is loosely used in the context of a host–virus interaction that does not give rise to visible disease symptoms as shown in (1)Infection with a very mild virus strain,(2)A tolerant host plant,(3)Leaves that escape infection because of their age and position on the plant,(4)‘Recovery’ from the disease symptoms in newly formed leaves,(5)Dark green areas in a mosaic pattern,(6)Plants that are infected with cryptic viruses.The existence of several definitions of latent infection gives rise to even more explanations of mechanisms that may cause this phenomenon. According to Roger Hull , there cViral populations encompass different genetic variants, and their distribution is shaped by selection and genetic drift. In addition to virus replication and maintenance, properties of the host plant play an important role for the selection of viruses. This includes the adaptation to plant resistance, plant defense mechanisms and also the relationship between virus replication and plant fitness. An interesting aspect of viral genome organization are the multifunctional proteins and overlapping open reading frames. This results in a trade-off regarding selection for different functionalities. Although RNA viruses can show high mutation rates, their proteins are not more variable than those of DNA-viruses ,36. An iGenetic variation can lead to differences of symptom severity in plants infected with different strains. For example, it has long been known that attenuated strains of TMV exist, which cause little or no symptoms in tobacco plants and may even interfere with the development of symptoms in a mixed infection with a more severe strain ,40. In sMost studies on tobamoviruses focus on virus multiplication as the criteria for trade-off between host–virus interactions. However, when it comes to host range determination, there are two important components of viral fitness: particle stability and infectivity . As showAnother reason that might explain latency can be the balance between host defense and viral counter defense. Tobamoviruses have co-evolved with their hosts  and haveThe beneficial interaction between plant viruses and hosts have been discussed in more detail in previous studies ,52. ExpeN. benthamiana [Many wild plants naturally harbor more than one virus. Synergistic or antagonistic interactions between two or more viruses in a host can alter symptom development. A synergistic interaction between two viruses can improve vial replicability and fitness inside the host. A detailed discussion about antagonism and synergism between plant viruses is available in previous reviews ,56. Antathamiana . HTS of thamiana . If an aN. benthamiana plants against the U1 strain of TMV [On the other side, viruses that cause mild or no symptoms have been widely studied for their potential use in mild strain cross-protection. It is a well-known phenomenon that plants are protected from severe virus strains due to the pre-infection with a mild strain of the same virus ,59. The n of TMV . hoya plants using a combined EM- HTS approach [Orthotospovirus genus. HoTV-2, however, did not cause symptoms when N. benthamiana plants were mechanically inoculated with the virus (case study Ilyas 2021). Despite the above-mentioned benefits of asymptomatically present plant viruses, their potential to spread to a different host and cause diseases in a new environment cannot be ignored. Viral disease symptoms are often host- and environment-dependent. Therefore, when inoculated on a susceptible host in a new environment, an asymptomatic virus can potentially induce severe symptoms . The thrapproach . The plaWhile most common members of the tobamovirus genus cause severe disease symptoms in host plants, there are other members, which do not cause obvious symptoms in wild host plants. Examples of latent tobamoviruses include Hibiscus latent Fort-Pierce virus , HibiscuN.tabacum, which were systemically infected [Tobacco latent virus belongs to the subgroup I (solanaceous-infecting) tobamoviruses. The virus was found in Nigeria, in field-grown tobacco from a mixed infection with several other viruses . It remainfected , while iinfected . Brugmansia is an ornamental plant belonging to the Solanaceae family. During an examination of the Brugmanisa plant collection in the Royal Botanical Garden Kew, for Columbian datura virus (CDV), Brugmansia latent virus was detected in plants with no obvious viral symptoms [symptoms . The virMalvaceae family have not been known as common hosts for tobamoviruses until the detection of two latent viruses from hibiscus plants [Hibiscus is used as an ornamental plant mostly in tropical and subtropical climates for hedges and flowers. Plants from s plants . Hibiscus plants ,65. HSFPN. benthamiana [Hibiscus suggest that this is a common feature among the malvaceous-infecting tobamoviruses. Niu et al. [These two viruses are unique among the other members of this genus because of their internal poly-(A)-tract (IPAT) in the 3′-UTR region ,23. Thisthamiana . The preu et al.  speculatN. benthamiana. [Viruses that possess an IPAT seem to cause milder or no symptoms in their hosts . This evhamiana. . It is pThe phylogeny in  of the aAlthough latency has been observed for many tobamoviruses, the mechanisms underlying this phenomenon are still elusive. While there are multiple definitions of latent infection in the literature, there are even more explanations that use this umbrella term as mentioned in i.There is a need to combine HTS with biological, serological, electron microscopy and molecular detection and characterization methods to reliably detect the asymptomatic viruses.ii.Further studies are important to understand why some tobamoviruses cause latent infection and if that latency is host dependent.iii.For crop protection, it is important to analyze if latent viruses from ornamentals can infect crop plants in different climatic and geographical conditions.iv.Latent tobamoviruses can be used as a model for studying host–virus interaction and virus–virus interaction in a cross-protection scenario.


Sleepio is an automated digital program that delivers digital cognitive behavioural therapy for insomnia (dCBT-I). Sleepio has been proven effective in improving sleep difficulties; however, evidence for the possible impact of Sleepio use on healthcare costs in the UK has not, to the authors’ knowledge, previously been developed.Sleepio in terms of primary care costs in the NHS in England.To identify the effect of a population-wide rollout of Sleepio was made freely available to all residents between October 2018 and January 2020. The study relies on a quasi-experimental design, using an interrupted time series (ITS) to compare the trend in primary care costs before and after the rollout of Sleepio.The study was conducted in the Thames Valley region of England, where access to Primary care data for people with relevant characteristics from nine general practices in Buckinghamshire was used. Primary care costs include general practice contacts and prescriptions. Segmented regression analysis was used to estimate primary and secondary outcomes.Sleepio user. Secondary analyses suggest that savings may be driven primarily by reductions in prescribing.For the 10 705 patients included in the sample, the total saving over the 65-week follow-up period was £71 027. This corresponds to £6.64 per person in the sample or around £70.44 per Sleepio rollout reduced primary care costs. National adoption of Sleepio may reduce primary care costs by £20 million in the first year. The expected impact on primary care costs in any particular setting will depend on the uptake of Sleepio. Sleepio has been shown to be clinically effective in 12 randomised controlled trials and offers patients access to fully-automated dCBT-I at scale. The real-world impact of providing whole population access to dCBT-I has not previously been determined in terms of healthcare costs. Sleepio was made freely available in a large region of England, and primary care costs were evaluated before and after rollout.Therapist-delivered cognitive behavioural therapy (CBT) is a recommended first-line treatment for insomnia, but it is difficult to access, and the majority of patients who present for GP management only receive verbal advice or medications for sleep.  Insomnia is commonly managed by GPs through verbal advice (100%), sleep hygiene education (89%), and medication. Patients rarely receive access to first-line CBT for insomnia. This is due in part to a shortage of trained providers and a lack of treatment awareness. dCBT-I offers a potential solution.In England, approximately 39% of people have symptoms of insomnia. The prevalence of diagnosed insomnia has increased over time and was 5.8% of the population in 2007.Sleepio (https://www.sleepio.com) is a standardised and fully automated  digital therapeutic, which comprises the full range of cognitive and behavioural techniques used in CBT for insomnia. It is designed for adults who want to improve their sleep and is available through a website and a supporting app. There is a growing body of evidence to support the effectiveness of Sleepio. For example, Espie et al and Freeman et al demonstrated benefit in terms of the Sleep Condition Indicator and Insomnia Severity Index. Espie et al found benefits to functional health, wellbeing, sleep-related quality of life, and symptoms of anxiety and depression. and evidence suggests that treatment can be cost-saving.Sleepio has been shown to improve workplace productivity, and some evidence indicates that Sleepio is associated with reductions in self-reported prescribed sleep medication use. However, its impact on healthcare service use and prescription costs is mostly unknown. Insomnia is a public health concern, with the costs associated with insufficient sleep estimated to be around £40 billion in the UK owing to lost productivity. Further costs are thought to be generated from increased healthcare expenditure and accident risk, with higher healthcare costs for people co-presenting with insomnia and comorbidities.The societal costs of insomnia are substantial,Sleepio could be provided free of charge to the whole population, or to a subset of people who might need it, through NHS funding. Real-world evidence can provide valuable insights beyond evidence generated in a trial setting.In England, Sleepio in terms of: 1) primary care service use costs; and 2) prescription costs. The study's main objective was to identify whether providing access to Sleepio resulted in a change in the trend of total primary care costs from the perspective of the NHS in England.This study sought to address the current gap in knowledge by using real-world data to evaluate the impact of providing public access to Sleepio program consists of six 20-minute dCBT-I sessions over at least 6 weeks, with each session unlocked 1 week after completing the previous session. People in therapy can maintain a sleep diary to track progress, with advice tailored to the provided information. The sleep diary can be automatically populated with data from a wearable device. Patients can also access other online support tools, including an electronic library and user community.The Sleepio in partnership with the Oxford Academic Health Science Network (AHSN). Population rollout involved all adult residents of the Thames Valley region of England  being granted permission to access Sleepio online free of charge. Eligibility was determined by the registrants’s postcode, which must be entered when individuals first use Sleepio. Passive promotional activity, such as online and print advertising, was conducted throughout the region to encourage individuals to use Sleepio. The largest employers in the region were identified and invited to promote Sleepio to their staff and provided with promotional content.As part of a 2-year implementation experiment funded by Innovate UK, Big Health Ltd provided access to Sleepio team worked with selected general practices to offer Sleepio to patients most likely to benefit from dCBT-I. This work involved evaluating different implementation strategies at population scale and included training, implementing digital prompts for GPs, and providing patient-centred resources to each practice. During the rollout period, additional awareness material was distributed, and practices were given tailored support. This level of engagement was not employed in Oxfordshire and Berkshire.This study gave particular attention to Buckinghamshire, where additional primary care engagement was employed. The Sleepio. The ITS analysis controlled for baseline levels and trends in costs.This study adopted a before and after quasi-experimental design. An ITS approach was used to estimate the change in total primary care costs following the rollout of Sleepio (based on criteria described below) were selected to reduce noise in the sample.The cohort consisted of patients from nine general practices. Patients who were expected to be more likely to use Sleepio are not affected by Sleepio rollout. Second, people who do not satisfy the selection criteria do not use Sleepio. These assumptions imply that there would be no change in primary care use outside of the sample that is attributable to Sleepio rollout.The study design relies on two key assumptions. First, the primary care costs of people who do not use EMIS Health is a software company that provides electronic patient record systems to general practices across the UK. Nine GP practices in Buckinghamshire were recruited, from which it was aimed to extract data for ≥10 000 patients from the EMIS system.Practices were selected to represent a range of levels of deprivation. Patients needed to satisfy at least one of the following criteria within the extraction period:a diagnosis of insomnia;a diagnosis of depression or anxiety disorder;prescription of a hypnotic or anxiolytic medication; orSleepio by a GP.referral to Sleepio. It was expected that insomnia diagnosis would rarely be coded in the data. Therefore, relevant prescribing  was used as an inclusion criterion to identify people experiencing sleep problems.Individuals below the age of 18 years were excluded. The purpose of these criteria was to limit the sample to those who it was anticipated might use Sleepio rollout (from October 2017) up to 15 months after Sleepio rollout (to January 2020). This provided an adequate timeframe to capture seasonal effects within the ITS design. Notably, the timeframe incorporates three Christmas periods, which were anticipated to be a significant correlate for primary care service use. Data were extracted by an independent specialist provider .Patient-level data were aggregated as patient-weeks, except for time-invariant patient characteristics. The data extraction period was 12 months before Sleepio, either through the Sleepio website or the supporting iOS app. This sample cannot be linked to the EMIS sample. These data are used for descriptive purposes only, providing information on uptake across the region.Data were also collected from all individual users of  According to the specific medicine, dose, and pack size, prescription costs were obtained from the BNF Online.The primary outcome for the analysis is the average primary care costs per patient per week, where primary care costs include GP practice contacts and prescription costs. Unit costs were attributed to resource use using information from the Unit Costs of Health and Social Care.A segmented regression analysis of the ITS data was employed to estimate the change in the trend of the primary outcome, such that the full model was estimated asijtY is total primary care costs for individual i from practice j at time t; ttime corresponds with the number of the week in the time series at time t; interventiont is a binary indicator for time t being before (intervention = 0) or after (intervention = 1) Sleepio rollout; tpost corresponds to the number of weeks after the intervention at time t; Xijt is a vector of patient-, practice-, and time-specific confounders; ju represents a random error term at the level of the practice; and ijte represents unexplained variability.where ß0 estimates baseline costs at t = 0; ß1 estimates the pre-Sleepio trend in costs; ß2 estimates the immediate change in Y at the time of Sleepio rollout; and ß3 estimates the change in trend after Sleepio rollout compared with the pre-intervention trend. The post-intervention slope is estimated as ß1 + ß3. Seasonal effects are accounted for by including an indicator within X for the season in which week t falls, where weeks 10–22 of any calendar year are spring, 23–35 are summer, 36–48 are autumn, and 49 through to 9 are winter. ß4 estimates seasonal and other confounding effects. The rollout period for Sleepio was assumed to span 6 weeks .In this model, The segmented regression analysis used a generalised linear model, with appropriate distributions and link functions fitted according to visual inspection of the data and use of a modified Park test and link tests. First-order autocorrelation was tested using the Durban–Watson test. Individual-level observations were drawn from practices, within which observations may be correlated. A multilevel regression model was implemented to account for clustering within practices.Four exploratory secondary analyses were conducted, as set out in Sleepio — at any point in the follow-up period — were included. Reducing the size of the dataset in this way made it possible to more effectively control for individual-level variation, and potentially identify effects directly attributable to Sleepio use.For analyses A and B, only individuals who were referred to Sleepio has demonstrated benefits in terms of anxiety or depression symptoms and the potential for Sleepio to be particularly effective for people with anxiety or depression. To contribute to this evidence base, the primary analysis was implemented with stratification according to the presence of a diagnosis of anxiety or depression.Previous research on Sleepio rollout, analysis D was conducted whereby referral to Sleepio is the intervention. In this case, the control group is the population of patients who were never referred to Sleepio. An analysis similar to the primary analysis was implemented, using a hierarchical generalised linear model to evaluate the effect on primary care costs, in any given week, of having been referred to Sleepio by a GP. This approach is more susceptible to selection bias, but could provide more precise identification of patients who use Sleepio and reduce noise in the sample.To further test the robustness of the findings and the extent to which any treatment effect is attributable to Sleepio at least once within each practice, of which there were 1008 patients in total.The analysis included 1 252 485 patient-week observations from 10 705 patients over 117 weeks. Of the sample, 64.41% identified as female. Age was observed in 5-year bands (except for those aged 18–25 and >90 years). The median age band was 50–55 years; 50.21% of the sample were aged 35–65 years (data not shown). Across the sample, 1655 (15.46%) people had at least one record of an insomnia diagnosis, and 5515 (51.52%) had at least one diagnosis of anxiety or depression. Inclusion criteria prescriptions (hypnotics or anxiolytics) were received by 3001 (28.03%) people, with 1919 (17.93%) receiving at least one hypnotic prescription and 1502 (14.03%) receiving at least one anxiolytic prescription (data not shown).Sleepio for different population sizes, based on the number of people recorded as being referred by their GP in the EMIS data and the actual number of patients recorded by Sleepio within the full 117 weeks of the study.In October 2018, the total number of patients registered with the nine practices in the study was 129 865, and the total population of Buckinghamshire was around 540 059.Sleepio data included all referral routes. Sleepio data relating to the nine practices rely on patients self-reporting GP referral. An individual’s county is determined by their postcode provided in Sleepio. The Thames Valley region was taken as comprising the three counties of Buckinghamshire (including Milton Keynes), Oxfordshire, and Berkshire.The EMIS data only included referrals recorded by GPs, while the The regression results from the primary analysis are shown in V (µ) = µ2 and log link. Model 5 is an ordinary least squares linear regression model. The preferred model is that which exhibited superiority in statistical tests and provided the most convincing predictions visually.All models except Model 5 are hierarchical generalised linear models using a quasi-gamma distribution with a variance function of Time shows that primary care costs increase over time, before Sleepio rollout. A negative coefficient for Intervention implies that the immediate impact of Sleepio rollout (during the 6-week rollout period) is to reduce primary care costs. A negative coefficient for Post shows that the effect of Sleepio rollout was to reduce the trend shown by the coefficient on Time. If the negative coefficient for Intervention is smaller than the positive coefficient for Time, it implies that the upward trend in primary care costs continues after Sleepio rollout, but at a slower rate.A positive coefficient for Sleepio rollout has a small but statistically significant negative impact on costs during the initial 6-week rollout period. Third, Sleepio rollout mitigates the trend of increasing primary care costs.The regression results imply three key overall findings. First, there is a slight increasing trend in primary care costs over time. Second, Intervention and results in statistical significance for Post.Comparison between the alternative model specifications reveals that seasonal adjustment is critical; accounting for seasonal effects reverses the direction of effect for Sleepio rollout, is a saving of £0.16 at week 65. This corresponds to £6.64 per person over the 65-week follow-up period, including the initial rollout period. The 95% confidence interval (CI) for this estimate is a saving of between £4.60 and £8.67. Across the observed sample of 10 705 people, Sleepio rollout reduced primary care costs by £71 027 (95% CI = £49 291 to £92 762)  .Sleepio rollout . The 3-year savings would be £139.59 (£228.07 minus £88.48). Rather than projecting a growing saving over time as trends diverge, this approach assumes a shift to the pre-rollout trend to converge on an annual saving of £90.07 per user in the long term. Based on existing evidence, these effects might be expected to be maintained for up to 3 years.Sleepio on the trend (–0.004 compared with –0.002). The coefficients for Analysis A can be used to estimate a reduction in prescription costs at 65-week follow-up of £8.62 per person (£5.40 in the first year), suggesting that the observed reductions in prescription costs of the primary analysis may significantly explain savings. Analysis B shows that Sleepio had a small but statistically significant impact in achieving a downward trend in the prescription of Z-drugs. Analysis C supports the notion that Sleepio may be more effective in reducing costs among people diagnosed with anxiety or depression (n = 5515). The average saving per person in this group, over 65 weeks, was £9.27.The coefficient for the pre-existing increasing trend in prescription costs (0.005) is greater than that for overall costs (0.002), as is the coefficient for the impact of Sleepio referral as the intervention, identified a negative but statistically non-significant effect. This is likely owing to a selection bias, whereby individuals who use Sleepio may be more likely to have higher levels of resource use, all else being equal. An exploratory analysis of the insomnia subsample did not identify any statistically significant findings. This may be owing to difficulty with coding insomnia or a lack of power associated with the sample size.The final analysis (D), treating Sleepio in the Thames Valley region reduced average primary care costs, including general practice contacts and prescriptions.The main findings of the analysis show that the rollout of Sleepio, this corresponds to a saving of £71 027 across the nine practices.Over the observed follow-up period, the average saving in the sample was £6.64 per person. Assuming that people outside of the sample were not affected by the rollout of Sleepio, collected separately from the present analysis, suggest that around 26% of people referred to Sleepio would start CBT . Based on these figures, the saving per patient associated with prescription costs may be £20.77 in the first year (£5.40/0.26), around half of the per-user saving identified across the whole sample.The secondary analyses suggest that reductions in prescription costs are a significant driver in reducing overall primary care costs. This is partly explained by reductions in the prescription of Z-drugs, which may include both off-label and on-label use. Data from n = 5515) was greater than the average saving across the whole sample. Future research should explore the potential for cost savings in different categories of expenditure and different populations.The reduction in costs observed in the subsample of people diagnosed with anxiety or depression  programme. The analysis does not provide estimates of any impact on referrals to secondary care or resources used in settings other than GP practices. Owing to the pre-specification of the primary outcome and the development of the model to suit these data, there was limited scope for the study to explore different aspects of resource use without suffering from overtesting. The design of the analysis also limits the extent to which the impact on resource use relating to GP contacts compared with that associated with prescription costs can be estimated.The study was also unable to observe any changes in resource use in areas outside of Buckinghamshire, where primary care engagement was based on passive promotional activity and not used to drive uptake. As shown in  One limitation of this study is that simulations to specify a sample size were not conducted before data collection was commenced. The sample size was determined on the basis of practicality and the authors’ expectations about uptake and the variability in healthcare costs. Nevertheless, the total number of observations is likely to provide reliable estimates.Optimal sample specifications for identifying statistically significant effects in ITS analyses are difficult to estimate reliably.An alternative study design may have enabled the study to overcome several of the limitations described here. Observation of a contemporaneous comparator, such as practices outside of the rollout area, would have enabled accounting for unobserved causes of variation in healthcare costs. However, it was not possible to establish the necessary contacts and agreements to support such a design. which may not be generalisable to primary care settings where GPs manage insomnia. This analysis supports previous research showing that dCBT-I can reduce prescribing.To the authors’ knowledge, no studies have evaluated the real-world healthcare cost impact of providing CBT for insomnia at population scale. Previous health economic evaluations of CBT for insomnia have focused on evaluations using data from controlled trials,Sleepio in the Thames Valley resulted in lower primary care costs across nine practices within 1 year. Providing NHS patients in England with access to Sleepio, while encouraging GPs to refer patients with sleep problems to register for Sleepio, is likely to result in fewer GP attendances and fewer prescriptions in the population. Therefore, these savings will partially or entirely offset direct costs associated with the adoption of Sleepio.The rollout of