The release rate can be controlled by simply adjusting the degree of oxidation to achieve drug release at different locations and periods. A higher degree of oxidation corresponds to a faster release rate, which is attributed to a higher degree of re-swelling and higher hydrophilicity of OCBs. The zero-order release kinetics of the model drugs from the OCBs suggested a constant drug release rate, which is conducive to maintaining blood drug concentration, reducing side effects and administration frequency. At the same time, the effects of different model drugs and different drug-loading solvents on the release behavior and the physical state of the drugs loaded in the beads were studied. In summary, the pH-responsive oxidized cellulose beads with good biocompatibility, low cost, and adjustable release rate have shown great potential in the field of controlled drug release.The development of controlled drug delivery systems based on bio-renewable materials is an emerging strategy. In this work, a controlled drug delivery system based on mesoporous oxidized cellulose beads (OCBs) was successfully developed by a facile and green method. The introduction of the carboxyl groups mediated by the TEMPO/NaClO/NaClO Controlled drug delivery systems that can precisely release drugs to specific sites in the human gastrointestinal tract (GIT) at a specific rate have unparalleled advantages compared with traditional oral drug delivery systems, for instance, preventing the side effects of the premature or burst drug release, maintaining a constant plasma concentration for a long time, or reducing the required dosage because of efficient delivery ,2,3. Amo10 tons, is one of the most abundant bio-renewable sources in the world [Recently, as a ubiquitous macromolecular polymer in nature, polysaccharides are attracting growing attention due to their abundance, biocompatibility, and biodegradability ,9. Celluhe world , proteinhe world , wound dhe world , and othhe world . Howeverhe world . This wiIncorporating carboxyl groups on the cellulose beads not only increases the hydrophilicity of the beads but also improves their reswelling capacity due to the pH-dependent electrostatic repulsive forces between carboxylate groups. This is particularly at pH values above the pKa value of the carboxylic acid, which are encountered in the small intestine. There are several approaches to functionalize carboxyl groups on cellulose, but in recent years, the TEMPO-mediated cellulose oxidation has gained considerable interest as it selectively converts the C6 primary hydroxyl groups of cellulose into carboxyl groups in aqueous conditions ,17.In this work, we report a pH-responsive and rate-controllable drug delivery system based on mesoporous oxidized cellulose beads. We studied the effect of the primary oxidant content on the introduction of anions and simultaneously achieved a controlled drug release rate by changing the degree of oxidation. Two model drugs, indomethacin (IND) and fenofibrate (FNB), were used as model drugs to investigate the release kinetics from different oxidized cellulose beads in simulated gastric fluid (pH = 1.2) and phosphate buffer (pH = 7.0). The release curves were fitted to both zero- and first-order kinetic models.The results of the carboxylate content analysis in four oxidized cellulose beads measured by conductometric titration are summarized in 2 adsorption results of the four samples did show differences.Taking OCBs-4 as a representative, the size of beads before and after drug loading is shown in 2/g, which is considerably higher compared to the areas obtained for polysaccharides by Borisova and coworkers. The key difference is that in their work, gels are formed in a TBA-water mixture, while here, prefabricated beads are washed with a TBA solution before drying [2/g. Although with the increasing degree of oxidation, the decrease in surface area was not dramatic, we still believe that the influence of the oxidation on the beads’ structure exists. N2 adsorption results confirm the presence of a mesoporous structure inside the beads. The average pore diameter of the four OCBs is ca. 20–25 nm, although small pores of a few nanometers and macropores over 100 nm are also observed in the pore size distribution  before the samples are frozen, substantially higher areas can be observed . Unmodife drying . Compareribution .For controlled drug delivery carriers, the swelling degree is a crucial parameter. The swelling degree of dried porous oxidized cellulose beads is mainly controlled by two important factors, the hydrophilicity of OCBs and the electrostatic repulsion between the carboxylate groups. Swelling studies are carried out at pH 7.0, which is higher than the pKa of –COOH (≈4.0), so the carboxylic acid groups in OCBs are highly deprotonated. As the oxidation degree rises, the stronger electrostatic repulsion between cellulose augments water diffusion into the beads and raises the swelling degree of the beads . After 2θ = 14.7°, 20.1°, and 21.6° as the empty OCBs, which belong to the crystal form of regenerated cellulose II [The results of mDSC and XRPD showed the physical state of the IND and FNB in the cellulose beads. The broad endothermic peak, which appears in the DSC results of IND and FNB, is due to the loss of adsorbed water. In the DSC results of the IND-loaded beads a, no melulose II ,23. AlthDifferent results were obtained in the case of FNB. The DSC thermograms of FNB show a melting peak around 75 °C, which is characteristic for polymorph II of FNB . As the The release profile of OCBs-1 to 4 loaded with IND solution in acetone is shown in −. At the same time, the carboxylic acid of IND would be ionized into carboxylate at pH 7. Thus, the release of IND from OCBs at pH 7.0 is the combined effect of beads re-swelling and the electrostatic repulsion between –COO− on beads and IND.The appearance of the beads before and after the drug release test is shown in At the same time, we also considered the impact of drug-loading solvents on beads’ morphology and drug release behavior. Dichloromethane (DCM), which has a faster evaporation rate than acetone at room temperature, was applied to investigate the influence of the solvent on drug loading and drug release. Apparently, the beads loaded with IND in DCM exhibited c a lowerIn addition, we also evaluated the capacity of OCBs for drugs with different chemical properties on drug loading and controlled release. Different from the high solubility of indomethacin at pH 7.0, fenofibrate is a neutral drug that exhibits extremely low solubility at any pH value. Similarly, the acetone solution of FNB was used for drug loading, and the release experiment was carried out for 5 h at pH 1.2 and 7.0. The release profiles  show tha0 and K1) values with their corresponding determination coefficient R2 values are presented in 2, OCBs-1 to 3 are better fitted by a first-order release model, while OCBs-4 is closer to a zero-order release, indicating that OCBs-4 achieves a nearly constant release rate of IND. It is worth noting that the release profiles of the beads loaded by IND/DCM displays a difference. The R2 value of the first-order model (R2 = 0.9788) is much higher than in the case of the zero-order model (R2 = 0.8686). Meanwhile, from the visual observation, the curve of OCBs-4_IND_DCM is more in line with the “fast then slow” first-order release, and the larger K1 indicates a faster release rate than OCBs-4_IND_Acetone. Therefore, the release kinetics of OCBs loaded with IND/acetone is more conducive to maintaining a constant blood concentration and is more conducive to achieving targeted delivery and controlled release of the lower intestinal tract. For the beads loaded with FNB, the R2 values of zero-order equations for all samples are higher than that of the first-order equation. Although the difference is minor, these results support that the release of FNB from four OCBs is closer to a zero-order release.The release data of four OCBs at pH 7.0 were fitted to the zero-order and first-order kinetic models, and the different release rate constants , and tert-butanol (99%) were obtained from ThermoFisher . Sodium lauryl sulfate (SLS) was obtained from Merck KGaA . Urea (99.5%), sodium acetate acid (99%+), NaClO (10–15%), NaClO2 (80%), acetone (99%), methyl sulfoxide , acetonitrile , and dichloromethane  were purchased from ACROS Belgium .Dissolving pulp from birch wood, obtained from Stora Enso , was used as a cellulose source and pretreated as described earlier . Sodium Cellulose beads were prepared according to Trygg et al. . Briefly2/NaClO was added in different ratios. As the primary oxidant, the proportion of NaClO2 is critical to the degree of oxidation. Therefore, different amounts of NaClO2 were added while keeping the molar ratio of the other two reagents constant  were immersed in 100 mL phosphate buffer  and kept overnight. Subsequently, TEMPO/NaClOconstant  to synthThe carboxylate content of oxidized cellulose beads was determined by conductometric titration based on the procedure reported by Paula et al. . In shorCOOH is the total acidic group content, in μmol/g;CC is the concentration of the NaOH solution, in μmol/L;V is the volume of the NaOH consumed in the whole process, and V1 is the consumption of protons neutralization liberated by NaCl, in liters;m is the weight of dry sample, in grams.To maintain the pore structure and surface area of porous cellulose beads, an optimized freeze-drying process was used to prepare dry beads. Specifically, a tert-butanol (TBA)/water cosolvent system was used due to the smaller surface tension of TBA and the “gentle” solidification of 90% TBA/10% water eutectic crystals formed during freeze drying . Using aThe surface and internal morphology of the freeze-dried beads were characterized by SEM. To prepare the samples, both intact beads and cross sections were attached to carbon tape and coated with platinum . The particles were then visualized with a Phillips XL30 SEM-FEG  including a Schottky emission electron gun. A beam of 20 kV and a conventional Everhart–Thornley-type detector was used.2 as the sorption gas at −196 °C. All samples were first degassed at 60 °C for 6 h under nitrogen flushing before analysis. The specific surface area was calculated by the Brunauer–Emmett–Teller (BET) method.The specific surface area of all freeze-dried cellulose beads was measured by a 3P meso 222 sorption analyzer  with NThe swelling degree of freeze-dried cellulose beads with different degrees of oxidation was determined by monitoring the weight gain of beads in phosphate buffer (pH = 7.0), using nonoxidized cellulose beads (CBs) as a reference. The freeze-dried beads were weighted (denoted as Wdry) and then immersed in a phosphate buffer solution at room temperature for 24 h. After removal of the excess water on the surface, the beads were weighed (denoted as Wwet) and the swelling degree (SD) was calculated according to Equation (2). Each sample was tested in triplicate.The incipient wetness procedure was used to load ca. 20% of model drugs into oxidized cellulose beads : 1 mL ofThe drug load was determined by the difference in weight of the cellulose beads before and after loading and confirmed by assay. Immersing the beads into DMSO overnight after the dissolution test and measuring the content of drugs by HPLC, the total amount of drug was the sum of the released drug during dissolution and the drugs remaining in the beads.The physical state of model drugs in beads was investigated with mDSC using a Q2000  with a heating rate of 2 °C/min and purged with dry nitrogen at a flow rate of 50 mL/min. Approximately 10 mg beads (≈2 mg drugs) were accurately weighed into large aluminum DSC pans , and the beads were gently pressed with lids to make better thermal contact with the DSC pans. The heating range in the case of IND was from 25 to 180 °C and from −20 to 135 °C in the case of FNB. Universal Analysis 2000 software  was used for data processing.XRPD experiments were carried out using an automated X’Pert PRO diffractometer  with a Cu tube (Kα λ = 1.5418 Å) and a generator set at 45 kV and 40 mA. Measurements in transmission mode were performed at room temperature (RT), using Kapton Polyimide thin films . A continuous scan mode from 4 to 40° 2θ with a 0.0167° step size and 400 s counting time was applied. X’Pert Data Viewer  was used to analyze the obtained diffractograms.® O-45/15 MS PTFE filter , and quickly replaced with the same volume of fresh medium. The filtered samples were used to determine the concentration of IND by HPLC. For beads loaded with 20% of FNB, the drug release tests were carried out separately in SGF with 0.25% SLS (pH 1.2) and PBS with 0.25% SLS (pH 7.0) for 5 h. Samples were taken at different time points and filtered with the same filter.The release behavior of the model drugs from OCBs-1 to OCBs-4 beads was investigated at 37 °C under nonsink conditions using a SR8PLUS dissolution station  with paddle apparatus II, and a rotation speed of 50 rpm. Each sample was tested in triplicate. More specifically, the release from beads loaded with 20% of IND was first performed in 150 mL simulated gastric fluid (SGF) containing 0.5% sodium lauryl sulfate  for 2 h, and subsequently in 150 mL phosphate buffer  for another 4 h. Then, 3 mL samples were taken at different time points, filtered through a CHROMAFILv/v), resulting in a retention time of 5.5 min. For FNB, the mobile phase consisted of 0.85% phosphoric acid (pH 1.50)-ACN , resulting in a retention time of 5 min.HPLC-analysis of IND was performed using a Merk-Hitachi LaCrom system equipped with a Nucleodur C18 column  obtained from Macherey-Nagel . For all samples, the flow rate of the mobile phase was 1 mL/min and the injection volume of samples was 20 µL. The mobile phase for analysis of IND consisted of 20 mM sodium acetate buffer (pH 3.50)-ACN  and first-order Equation (4) kinetic equations ,(3)Mt/Mp values < 0.05 were considered statistically significant. Statistical analysis was carried out using Graph Pad Prism 9 software .All release experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD) and indicated as error bars in the release profiles. The final release data of IND and FNB at pH 1.2 and 7.0 were statistically analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. 2-mediated oxidation process. Four OCBs with different oxidation degrees were successfully obtained by adjusting the amount of NaClO, resulting in an increased carboxylate content in the beads. The morphological characterization of the four OCBs confirmed the existence of a dense porous structure, with a specific surface area above 200 m2/g. The specific surface area decreased with the degree of oxidation. The swelling capacity of OCBs increased with the degree of oxidation due to the higher hydrophilicity of the beads’ surface. The larger quantities of carboxylic acid groups enhanced electrostatic repulsion between the different surfaces.Cellulose beads were prepared and anionic groups were introduced by the TEMPO/NaClO/NaClOThe drug release experiments of the two model drugs proved that OCBs had a pH-responsive release ability, i.e., almost no release at pH 1.2, but a controlled release rate at pH 7. As the degree of oxidation increased, the release rate became faster. The statistical analysis showed that there were significant differences in the release properties of beads with different oxidation degrees. Due to the difference in chemical properties, the release of IND was higher than that of FNB. It was found from the kinetics studies that the release behavior of OCBs loaded with IND/acetone solution and FNB/acetone solution was closer to a zero-order process, indicating a constant release rate. However, OCBs loaded with IND/DCM solution exhibited a first-order profile, which suggests a rapid release rate at the early stage followed by a continuously slowing down of the rate. In summary, the drug release from pH-responsive OCBs was affected by the degree of oxidation, the chemical properties of the model drugs, and the drug-loading solvent.


Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS-CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus–host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The  Upon a viral outbreak, it is important to rapidly establish whether the outbreak is caused by a new or a previously known virus Box , as this46. Viral phenotypic features include those that, like a disease, are predominantly shaped by virus–host interactions including transmission rate or immune correlates of protection, and others that are largely virus-specific, such as the architecture of virus particles. These features are of critical importance to control, and respond to medically and economically important viruses — especially during outbreaks of severe disease — and dominate the general perception of viruses.Understanding the cause of a specific disease that spreads among individuals of the same host species (infectivity) was the major driving force for the discovery of the first virus in plants, and subsequently many others in all forms of life, including humans. Historically, the range of diseases and hosts that specific viruses are associated with have been the two key characteristics used to define viruses, given that they are invisible to the naked eye48. These studies have identified huge numbers of viruses that circulate in nature and have never been characterized at the phenotypic level. Thus, the genome sequence is the only characteristic that is known for the vast majority of viruses, and needs to be used in defining specific viruses. In this framework, a virus is defined by a genome sequence that is capable of autonomous replication inside cells and dissemination between cells or organisms under appropriate conditions. It may or may not be harmful to its natural host. Experimental studies may be performed for a fraction of known viruses, while computational comparative genomics is used to classify (and deduce characteristics of) all viruses. Accordingly, virus naming is not necessarily connected to disease but rather informed by other characteristics.However, the host of a given virus may be uncertain, and virus pathogenicity remains unknown for a major (and fast-growing) proportion of viruses, including many coronaviruses discovered in metagenomics studies using next-generation sequencing technology of environmental samplesIn view of the above advancements and when confronted with the question of whether the virus name for the newly identified human virus should be linked to the (incompletely defined) disease that this virus causes, or rather be established independently from the virus phenotype, the CSG decided to follow a phylogeny-based line of reasoning to name this virus whose ontogeny can be traced in the figure in Box 1 or norovirus2 infections, well-established and internationally approved methods, standards and procedures are in place to identify and name the causative agents of these infections and report this information promptly to public health authorities and the general public. In outbreaks involving newly emerged viruses, the situation may be different, and appropriate procedures to deal with these viruses need to be established or refined with high priority.For many human virus infections such as influenza virus3, provisionally named 2019-nCoV, to known coronaviruses, and detail the basis for (re)naming this virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which will be used hereafter. Given the public interest in naming newly emerging viruses and the diseases caused by these viruses in humans, we will give a brief introduction to virus discovery and classification — specifically the virus species concept — and the roles of different bodies, such as the World Health Organization (WHO) and the International Committee on Taxonomy of Viruses (ICTV), in this process. We hope this will help readers to better understand the scientific approach we have taken to arrive at this name, and we will also discuss implications of this analysis and naming decision.Here, we present an assessment of the genetic relatedness of the newly identified human coronavirus4. Viruses are clustered in taxa in a hierarchical scheme of ranks in which the species represents the lowest and most populous rank containing the least diverged groups (taxa) of viruses  or even stigmatizing, which should be avoided. Establishing a universal nomenclature for viruses was one of the major tasks of the ICTV when it was founded more than 50 years agoBox 2 Identifying viral speciesThe terms strain and isolate are commonly used to refer to virus variants, although there are different opinions as to which term should be used in a specific context. If a candidate virus clusters within a known group of isolates, it is a variant of this group and may be considered as belonging to this known virus group. In contrast, if the candidate virus is outside of known groups and its distances to viruses in these groups are comparable to those observed between viruses of different groups (intergroup distances), the candidate virus is distinct and can be considered novel.This evaluation is usually conducted in silico using phylogenetic analysis, which may be complicated by uneven rates of evolution that vary across different virus lineages and genomic sites due to mutation, including the exchange of genome regions between closely related viruses (homologous recombination). However, given that the current sampling of viruses is small and highly biased toward viruses of significant medical and economic interest, group composition varies tremendously among different viruses, making decisions on virus novelty group-specific and dependent on the choice of the criteria selected for this assessment.5. To decide on whether a virus represents a new species—that is, the least diverged (and most populated) group of viruses—taxonomists use the results of different analyses. Taxonomical classification is hierarchical, using nested groups (taxa) that populate different levels (ranks) of classification. Taxa of different ranks differ in their intra-taxon pairwise divergence, which increases from the smallest at the species rank to the largest at the realm rank30. They may also be distinguished by taxon-specific markers that characterize natural groupings. Only the species and genus ranks need to be specified to classify a new virus; filling other ranks is optional. If a virus prototypes a new species, it will be regarded as taxonomically novel. If (within this framework) a virus crosses a host barrier and acquires novel properties, its classification will not change  even if the virus establishes a permanent circulation in the new host, which likely happened with coronaviruses of the four species that circulate in humans and display seasonal peaks (reviewed in ref. 50). Importantly, the criteria used to define a viral species in one virus family such as Coronaviridae may not be applicable to another family such as Retroviridae, and vice versa, since Study Groups are independent in their approach to virus classification.These challenges are addressed in the framework of virus taxonomy, which partitions genomic variation above strain or isolate level and develops a unique taxon nomenclature under the supervision of the ICTV8—have been confronted several times with the need to define whether a newly emerged virus causing a severe or even life-threatening disease in humans belongs to an existing or a new (yet-to-be-established) species. This happened with SARS12 and with Middle East respiratory syndrome (MERS)14 a few years later. Each time, the virus was placed in the taxonomy using information derived from a sequence-based family classification16.Researchers studying coronaviruses—a family of enveloped positive-strand RNA viruses infecting vertebratesCoronaviridae, suborder Cornidovirineae, order Nidovirales and realm Riboviria19 , a working group of the ICTV20. The CSG is responsible for assessing the place of new viruses through their relation to known viruses in established taxa, including placements relating to the species Severe acute respiratorysyndrome-related coronavirus. In the classification of nidoviruses, species are considered biological entities demarcated by a genetics-based method21, while generally virus species are perceived as man-made constructs22. To appreciate the difference between a nidoviral species and the viruses grouped therein, it may be instructive to look at their relationship in the context of the full taxonomy structure of several coronaviruses. Although these viruses were isolated at different times and locations from different human and animal hosts , they all belong to the species Severe acute respiratorysyndrome-related coronavirus, and their relationship parallels that between human individuals and the species Homo sapiens 24 24 Box  to identSevere acute respiratory syndrome-related coronavirus  and genome region. Accordingly, there is no agreement yet on the exact taxonomic position of SARS-CoV-2 within the subgenus Sarbecovirus. When we included SARS-CoV-2 in the dataset used for the most recent update (May 2019) of the coronavirus taxonomy currently being considered by ICTV19, which includes 2,505 coronaviruses, the species composition was not affected and the virus was assigned to the species Severe acute respiratory syndrome-related coronavirus, as detailed in Box Consistent with previous reports, SARS-CoV-2 clusters with SARS-CoVs in trees of the species rus Fig.  and the rus Fig. 25–27. DiBetacoronavirus16. These two informal subgroups were recently recognized as subgenera Sarbecovirus and Merbecovirus29 when the subgenus rank was established in the virus taxonomy30. Being the first identified representatives of a new species, unique names were introduced for the two viruses and their taxa in line with the common practice and state of virus taxonomy at the respective times of isolation. The situation with SARS-CoV-2 is fundamentally different because this virus is assigned to an existing species that contains hundreds of known viruses predominantly isolated from humans and diverse bats. All these viruses have names derived from SARS-CoV, although only the human isolates collected during the 2002–2003 outbreak have been confirmed to cause SARS in infected individuals. Thus, the reference to SARS in all these virus names  acknowledges the phylogenetic  grouping of the respective virus with the prototypic virus in that species (SARS-CoV). The CSG chose the name SARS-CoV-2 based on the established practice for naming viruses in this species and the relatively distant relationship of this virus to the prototype SARS-CoV in a species tree and the distance space 31, the WHO recently introduced a rather unspecific name ) to denote this disease. Also, the diagnostic methods used to confirm SARS-CoV-2 infections are not identical to those of SARS-CoV. This is reflected by the specific recommendations for public health practitioners, healthcare workers and laboratory diagnostic staff for SARS-CoV-2 . By uncoupling the naming conventions used for coronaviruses and the diseases that some of them cause in humans and animals, we wish to support the WHO in its efforts to establish disease names in the most appropriate way . The further advancement of naming conventions is also important because the ongoing discovery of new human and animal viruses by next-generation sequencing technologies can be expected to produce an increasing number of viruses that do not (easily) fit the virus–disease model that was widely used in the pre-genomic era , an isolate or strain number, and the time of isolation (year or more detailed) in the format virus/host/location/isolate/date; for example, SARS-CoV-2/human/Wuhan/X1/2019. This complete designation along with additional and important characteristics, such as pathogenic potential in humans or other hosts, should be included in the submission of each isolate genome sequence to public databases such as GenBank. In publications, this name could be further extended with a sequence database ID—for example, SARS-CoV-2/human/Wuhan/X1/2019_XYZ12345 —when first mentioned in the text. We believe that this format will provide critical metadata on the major characteristics of each particular virus isolate (genome sequence) required for subsequent epidemiological and other studies, as well as for control measures.To facilitate good practice and scientific exchange, the CSG recommends that researchers describing new viruses  in this species adopt a standardized format for public databases and publications that closely resembles the formats used for isolates of avian coronavirusesBox 3 Classifying coronaviruses51. The CSG currently analyses 3CLpro, NiRAN, RdRp, ZBD and HEL1 (ref. 52) . According to our current knowledge, these five essential domains are the only ones conserved in all viruses of the order Nidovirales52. They are thus used for the classification by all ICTV nidovirus study groups (coordinated by the NSG).Initially, the classification of coronaviruses was largely based on serological (cross-) reactivities to the viral spike protein, but is now based on comparative sequence analyses of replicative proteins. The choice of proteins and the methods used to analyse them have gradually evolved since the start of this century52) Fig. , two dom24. Importantly, the involvement of all coronavirus genome sequences available at the time of analysis allows family-wide designations of demarcation criteria for all ranks, including species, regardless of the taxa sampling size, be it a single or hundreds of virus(es). DEmARC delineates monophyletic clusters (taxa) of viruses using weighted linkage clustering in the PPD space and according to the classification of ranks defined through clustering cost (CC) minima presented as PPD thresholds ). In the DEmARC framework, the persistence of thresholds in the face of increasing virus sampling is interpreted to reflect biological forces and environmental factors21. Homologous recombination, which is common in coronaviruses55, is believed to be restricted in genome regions encoding the most essential proteins, such as those used for classification, and to members of the same virus species. This restriction promotes intra-species diversity and contributes to inter-species separation. To facilitate the use of rank thresholds outside of the DEmARC framework, they are converted into PD and expressed as a percentage, which researchers commonly use to arrive at a tentative assignment of a given virus within the coronavirus taxonomy following conventional phylogenetic analysis of selected viruses.Since 2011, the classification of coronaviruses and other nidoviruses has been assisted by the DivErsity pArtitioning by hieRarchical Clustering (DEmARC) software, which defines taxa and ranksBox 4 Classifying SARS-CoV-2Coronaviridae is defined by viruses whose PPD(s) may cross the inter-species demarcation PPD threshold (threshold ‘violators’). Due to their minute share of ~10–4 of the total number of all intra- and inter-species PPDs, these violators may not even be visually recognized in a conventional diagonal plot clustering viruses on a species basis (panel a of the figure in Box 4). Furthermore, they do not involve any virus of the species Severe acute respiratory syndrome-related coronavirus, as is evident from the analysis of maximal intraspecies PPDs of 2,505 viruses of all 49 coronavirus species (of which 39 are established and 10 are pending or tentative) (panel b of the figure in Box 4) and PDs of 256 viruses of this species (panel c of the figure in Box 4). Thus, the genomic variation of the known viruses of the species Severe acute respiratory syndrome-related coronavirus is smaller compared to that of other comparably well-sampled species—for example, those prototyped by MERS-CoV, human coronavirus OC43 (HCoV-OC43) and infectious bronchitis virus (IBV) (panel b of the figure in Box 4)—and this species is well separated from other known coronavirus species in the sequence space. Both of these characteristics facilitate the unambiguous assignment of SARS-CoV-2 to this species.The species demarcation threshold  in the family 56 (panel c of the figure in Box 4), representing two basal lineages within the species Severe acute respiratory syndrome-related coronavirus that constitute very few known viruses . This small median PD of human SARS-CoVs also dominates the species-wide PD distribution .Intra-species PDs of SARS-CoV-2 belong to the top 25% of this species and also include the largest PD between SARS-CoV-2 and an African bat virus isolate (SARSr-CoV_BtKY72)ses Fig. . These rc of the figure in Box Historically, public health and fundamental research have been focused on the detection, containment, treatment and analysis of viruses that are pathogenic to humans following their discovery (a reactive approach). Exploring and defining their biological characteristics in the context of the entire natural diversity as a species has never been a priority. The emergence of SARS-CoV-2 as a human pathogen in December 2019 may thus be perceived as completely independent from the SARS-CoV outbreak in 2002–2003. Although SARS-CoV-2 is indeed not a descendent of SARS-CoV Fig. , and theSevere acute respiratory syndrome-related coronavirus may be as (poorly) representative for this particular species as the few individuals that we selected to represent H. sapiens in Fig. Severe acute respiratory syndrome-related coronavirus limits our current understanding of fundamental aspects of the biology of this species and, as a consequence, our abilities to control zoonotic spillovers to humans. Future studies aimed at understanding the ecology of these viruses and advancing the accuracy and resolution of evolutionary analyses41 would benefit greatly from adjusting our research and sampling strategies. This needs to include an expansion of our current research focus on human pathogens and their adaptation to specific hosts to other viruses in this species. To illustrate the great potential of species-wide studies, it may again be instructive to draw a parallel to H. sapiens, and specifically to the impressive advancements in personalized medicine in recent years. Results of extensive genetic analyses of large numbers of individuals representing diverse populations from all continents have been translated into clinical applications and greatly contribute to optimizing patient-specific diagnostics and therapy. They were instrumental in identifying reliable predictive markers for specific diseases as well as genomic sites that are under selection. It thus seems reasonable to expect that genome-based analyses with a comparable species coverage will be similarly insightful for coronaviruses. Also, additional diagnostic tools that target the entire species should be developed to complement existing tools optimized to detect individual pathogenic variants (a proactive approach). Technical solutions to this problem are already available; for example, in the context of multiplex PCR-based assays42. The costs for developing and applying (combined or separate) species- and virus-specific diagnostic tests in specific clinical and/or epidemiological settings may help to better appreciate the biological diversity and zoonotic potential of specific virus species and their members. Also, the further reduction of time required to identify the causative agents of novel virus infections will contribute to limiting the enormous social and economic consequences of large outbreaks. To advance such studies, innovative fundraising approaches may be required.The currently known viruses of the species 43. With this change in place, the CSG is resolved to address the existing significant overlap between virus and species names that complicates the appreciation and use of the species concept in its application to coronaviruses.Although this Consensus Statement focuses on a single virus species, the issues raised apply to other species in the family and possibly beyond. A first step towards appreciation of this species and others would be for researchers, journals, databases and other relevant bodies to adopt proper referencing to the full taxonomy of coronaviruses under study, including explicit mentioning of the relevant virus species and the specific virus(es) within the species using the ICTV naming rules explained above. This naming convention is, regretfully, rarely observed in common practice, with mixing of virus and species names being frequently found in the literature . The adoption of accurate virus-naming practices should be facilitated by the major revision of the virus species nomenclature that is currently being discussed by the ICTV and is being planned for implementation in the near future


Routine maternal immunisation against influenza and pertussis are recommended by the WHO to protect mother and child, and new vaccines are under development. Introducing maternal vaccines into national programmes requires an understanding of vaccine delivery costs – particularly in low resource settings.We searched Medline, Embase, Econlit, and Global Health for studies reporting costs of delivering vaccination during pregnancy but excluded studies that did not separate the vaccine purchase price. Extracted costs were inflated and converted to 2018 US dollars.Sixteen studies were included, of which two used primary data to estimate vaccine delivery costs. Costs per dose ranged from $0.55 to $0.64 in low-income countries, from $1.25 to $6.55 for middle-income countries, and from $5.76 to $39.87 in high-income countries.More research is needed on the costs of delivering maternal immunisation during pregnancy, and of integrating vaccine delivery into existing programmes of antenatal care especially in low and middle-income countries. During the 2009 influenza H1N1 pandemic it was recognised that pregnant women experienced a higher risk of severe complications and death, which led the WHO’s Strategic Advisory Group of Experts (SAGE) on immunisation to identify pregnant women as the highest priority group for influenza vaccination There is growing interest in the potential for maternal immunisation using other vaccines such as pneumococcal conjugate (PCV), Haemophilus influenzae type B (HiB) and meningococcal vaccines, as well as the potential for new vaccines against Cytomegalovirus, Zika virus and malaria The objective of this study was to review the literature on the financial and economic non-vaccine costs of delivering maternal immunisation and incorporating it into routine, nation-wide vaccination programmes. The findings from this review can be used to inform economic evaluations of maternal immunisation, including current vaccines such as influenza and PCV, and prospective vaccines against RSV and GBS.2We performed a systematic search of the global literature to identify studies that reported the costs of delivering maternal immunisations during pregnancy. Searches were performed across Medline, Embase, Econlit, and Global Health bibliographic databases. The search strategy incorporated a mixture of index terms and key words which combined the concepts of: (i) vaccination; (ii) pregnancy; (iii) costs, and (iv) diseases with existing or pipeline vaccines recommended for maternal immunisation. The list of included vaccines was based on a recent review by Omer We included any English language articles that reported estimates of the non-vaccine cost of vaccinating pregnant women and excluded studies that reported only a vaccine purchase price, or for which the vaccine delivery and purchase costs could not be separated. We excluded articles where cost estimates were not specific to our population of interest, including estimates for vaccinating women of childbearing age that did not distinguish women who were pregnant. Studies were included regardless of the data source for cost estimation including estimates based on primary and secondary data, government tariffs, expert opinion, and assumption.Search results were combined using reference management software (Mendeley) and any duplicate records were removed. Two reviewers (OS and SP) screened articles using titles and abstracts, followed by full text screening of potentially eligible studies to determine the final list of included articles. We recorded descriptive study characteristics including the country, currency and year of cost estimates, type of vaccine, and details of source data and methodology for cost estimation. We extracted reported non-vaccine delivery costs related to immunisation of pregnant women – this included total programme costs, cost per dose, and any component costs which were separately reported. In addition we also recorded the reported price of purchasing a vaccine dose.Extracted costs were inflated to 2018 currency values using national GDP deflators 3Our initial search resulted in 1523 records across the different databases, and 1053 unique articles once duplicates were removed. Following the first stage of screening we identified 98 potentially eligible articles, and after full-text screening there were 16 articles that met the inclusion criteria. The flow diagram summarising the results of our search and screening is shown in Key characteristics of the articles included are summarised in Globally the estimated cost per dose of delivering maternal immunisation in 2018 USD ranged from $0.55 in Malawi Only two studies undertook detailed costing of maternal vaccine programmes through the collection of primary data on resource use and costs. Berman and co-workers estimated the financial cost to the government of $0.64 per dose for an on-going tetanus maternal immunisation programme provided as part of routine antenatal care (ANC) in Aceh province, Indonesia The majority of the articles described economic evaluations where detailed estimation of vaccination delivery costs was not a primary research objective and differed in their approaches to cost estimation. In addition to the studies by Berman and Pecenka, only three other studies by Kim 4Methodological differences between studies may have lead to important differences in estimated costs. For example, where estimates were based on tariffs or fees these service payments might reflect the marginal cost from the perspective of the healthcare payer. However, they may not necessarily represent the true economic cost of vaccine delivery, which will depend on the actual resources used in the providing the service. As an illustration, Jit et al. estimated the cost of delivering maternal influenza vaccine in the UK to be $11.87 per dose based on a National Health Service (NHS) tariff payment A further consideration is that estimates based only on tariffs and fees are likely to neglect any costs of introducing a new vaccine programme that are borne centrally by the government or health service. Two of the studies that used an ingredients-based approach reported costs of programme introduction including expenditure on planning, training, social mobilisation and capital investment in cold chain capacity The relative contribution of fixed costs – which do not change in the short-run – and variable costs that increase with the number of vaccinations can be an important determinant of programme efficiency and the overall cost per dose. For example, in their base case analysis, Pecenka et al. found the financial cost per dose was $0.55, but due to the effect of fixed programme costs this increased to $0.83 per dose in a sensitivity analysis which assumed lower vaccine coverage. Similarly, Berman et al. reported an average vaccine delivery cost of $0.64 per dose, but this ranged across sub-districts from $0.44 to $2.80 per dose. The authors found that this variation was primarily driven by the amortisation of fixed programme costs across substantially different numbers of women vaccinated in each sub-district. These fixed costs did not depend on how many women were vaccinated and included both capital investment in equipment as well as recurrent expenditure on wages and allowances for certain personnel.For the five studies in our sample conducted in LMICs our findings for maternal immunization are broadly similar to the results of a recent review on the costs of immunisation delivery in LMICs more generally, which reported costs per dose ranging from $0.16 to $2.54 (in 2016 USD) For decision makers the relative importance of non-vaccine delivery costs may be influenced by the vaccine purchase price. Amongst our included studies we found non-vaccine costs as a proportion of the total cost ranged from 15% to 70% in LICs, 6% to 22% in MICs, and 14% to 93% in HICs. The broad ranges largely reflect the variation in vaccine purchase price , which iOf the studies we found only one used a dedicated costing tool Another limitation in the literature was the lack of studies examining costs from the perspective of pregnant women and their families. The costs of transport and time spent attending appointments may act as a barrier to the uptake of maternal immunisation, but these additional costs may be avoided where maternal vaccines can be delivered as part of existing ANC visits. In this case the health benefits of introducing new maternal vaccines could even act as an incentive helping to boost coverage of wider antenatal care packages.Our study is subject to several limitations. First, we have not formally appraised the quality of the included studies for factors which may bias the reported cost estimates. However, we do report study methodology - there is a generally accepted hierarchy in which estimates using ingredients-based costing are usually considered better than those based on tariffs 5We found few studies in the academic literature that reported estimates of the costs of delivering maternal immunisation to pregnant women, and only two studies that collected primary data on resource use. This highlights the need for further research to identify the economic and financial costs of delivering vaccinations during pregnancy. More detailed costing studies are needed that use primary data and micro-costing to establish whether the non-vaccine costs of maternal immunisation delivery differ from other immunisations programmes, and the extent to which such differences may vary by setting. Studies should consider the start-up costs of introducing new maternal immunisation programmes, and the costs of integrating delivery within existing antenatal care services. A good understanding of these costs is critical to informing the cost-effectiveness and financial impact of introducing new maternal vaccines, particularly in low-resource settings.10.13039/100000865The Bill & Melinda Gates Foundation, Seattle, WA (OPP1180644).MJ, JEL, OS and SRP conceptualised the study. SRP, OS, MJ, and CP developed the methodology. OS and SRP conducted the review, analysed the results and drafted the original manuscript. All authors critically reviewed the manuscript and approved the final version.Hutubessy R and Lambach P work for the World Health Organization. The authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions, policy or views of the WHO.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Astroviridae, Caliciviridae and Coronaviridae. Some of the viruses may have potential cross-species transmissibility, while others indicated a wide genetic diversity of duck viruses within a genus. The study also showed evidence of potential transmission of viruses along the East Asian—Australasian Flyway; potentially facilitated by migrating shorebirds. The detection and characterisation of several avian viruses not previously described, and causing asymptomatic but potentially also symptomatic infections suggest the need for more virus surveillance studies for pathogenic and potential zoonotic viruses in wildlife reservoirs.Birds, notably wild ducks, are reservoirs of pathogenic and zoonotic viruses such as influenza viruses and coronaviruses. In the current study, we used metagenomics to detect and characterise avian DNA and RNA viruses from wild Pacific black ducks, Chestnut teals and Grey teals collected at different time points from a single location. We characterised a likely new species of duck aviadenovirus and a novel duck gyrovirus. We also report what, to the best of our knowledge, is the first finding of an avian orthoreovirus from Pacific black ducks and a rotavirus F from Chestnut teals. Other viruses characterised from the samples from these wild ducks belong to the virus families  These viruses could become potential threats to other wild animals, poultry and possibly humans under the right set of circumstances2. Virome (virus community) analysis of potential reservoir bird hosts is expected to unveil the role of viruses causing acute or long-term asymptomatic infections and the diversity and range of viruses that infect wild birds. Virome analysis is also expected to aid in understanding the transmission and possible role of viruses on host health in the wild. Metagenomics of viruses, utilises the power of NGS for the discovery, analysis and characterisation of viruses, thereby enabling virome analysis and providing insight into viruses for which these birds act as a natural reservoir. The use of the metagenomic technique in bird samples can lead to the determination of the virome from an individual bird or a population, and the identification of various novel avian viruses6. Nevertheless, only a few studies have explored the viruses circulating among wild ducks in Australia6.Birds, notably wild ducks, can be reservoirs for zoonotic viruses including, but not limited to, influenza viruses3. We previously focused on avian parvoviruses and picornaviruses from these duck samples as they were found to be abundant compared to other avian viruses6. We also reported a reassorted low pathogenicity H9N2 avian influenza virus with intercontinental gene segments in Chestnut teal samples collected on August 20181. It was the first report of an H9N2 avian influenza virus in resident wild birds in Australia1. In the current study, we focus on the sequence characterisation of additional avian DNA and RNA viruses from multiple virus families that were present in the Australian wild ducks. We aimed to determine the abundance of the different viruses in Australian wild ducks and the factors that might influence the ecology of these viruses in relation to host species.In the current study, we collected fresh duck faecal samples from a single location at various time points. Metagenomics of viruses was carried out to detect and characterise avian viruses present in the sample of Australian wild ducks using a protocol optimised in our laboratory and described earlierParvoviridae and Picornaviridae from our duck samples as they had the most abundant virus reads generated6. Avian viruses belonging to virus families other than Parvoviridae and Picornaviridae are described here and were detected and characterised in samples from Pacific black ducks , Chestnut teals (CT08.18 and CT11.18) and Grey teals (GT11.18)  and Wood duck (WD08.18) samples collected in May and August 2018, respectively, no other avian viruses were detected other than those previously described viruses belonging to Parvoviridae and Picornaviridae6.We have previously described viruses belonging to the virus families 6, were detected and characterised from the duck samples, one belonging to the virus family Adenoviridae and another to Anelloviridae.Two DNA viruses, in addition to the parvoviruses already described3. However, more nucleotide sequences were generated by resequencing the sample at a greater depth. A total of 37,832 nucleotides (out of ~ 43 kb) of adenovirus sequence was generated from seven consensus sequences of the virus with a coverage ranging from 2 to 4456 , and only 81.5% (1024/1255 amino acids) identical to JF510462.1 Goose adenovirus 4 from Hungary8 , genomic organisation, RFLP analysis, host range, pathogenicity, cross-neutralization and ability to recombine9. From the PBDAdV/PBD12.16 consensus sequences generated here and based on the DNA polymerase phylogenetic analysis and host species; this virus can be considered potentially as a new species. BLASTP analysis of these protein sequences showed that some of them are more identical but still distant to their counterparts expressed by the KJ469653.1 Muscovy duck adenovirus 2 or the KR135164.1 Muscovy duck adenovirus 2, while others to that of JF510462.1 Goose adenovirus 4  to be located in the central region of the Pacific black duck adenovirus genome. Thirty genes or ORFs (out of 34–36 genes) were found in total from the seven consensus sequences of the virus. The motifs and other sequence or amino acid features found such as bipartite nuclear localisation signals have been given in the Supplementary material Anelloviridae, and the species demarcation criteria of some of the genera in the family Anelloviridae are based on ORF1/VP1 (capsid) protein with a nucleotide divergence of > 35%10. Hence, based on the phylogenetic analysis of the VP1 protein sequence the Grey teal gyrovirus can be considered as a new virus species. The GTGV/GT11.18 was relatively most identical, but still distant, to MH378452.1 gyrovirus from Ashy storm petrel faecal sample from the US in 2012 with 55.2% (159/288 amino acids) identity only. The GTGV/GT11.18 was also distantly related to the Southern screamer gyrovirus (MH016740.1) from the US in 200711 with 52.4% identity only in the VP1 protein sequence.A novel gyrovirus was found in the single Grey teal sample GT11.18). Real-time PCR amplification and Sanger sequencing were carried out and were successful for the Grey teal gyrovirus for the region from 264th to 761st nucleotide. A near-complete genome of 1549 nucleotides long consensus sequences encoding complete VP1, VP2 and hypothetical VP3 protein of GTGV/GT11.18 was generated from the NGS reads of the sample with a coverage ranging from 2 to 105  superfamily CX5R (46CSCGNFR52) and the motif 34WX7HX3CXCX5H54 was found in the VP2 protein, suggesting that this protein may exhibit PTPase function13. It is also to be noted that the VP3 protein did not have any apoptin conserved protein domains.The VP1 protein of the GTGV/GT11.18 contained the motif 6, detected and characterised from the duck samples belonged to the virus families Reoviridae, Astroviridae, Caliciviridae and Coronaviridae.The RNA viruses, in addition to the picornaviruses already describedReoviridae, was detected and characterised. All the 10 segments were detected and a total of 21,170 nucleotides (out of ~ 23 kb) of the consensus sequence of the Pacific black duck orthoreovirus genome were generated with a coverage from 2 to 336  than to any other orthoreovirus sigma C proteins  of the consensus sequence of the Pacific black duck rotavirus G genome were generated with a coverage of 2–512  identity.From the Chestnut teal 08.18 sample, a few good quality NGS reads of Chestnut teal rotavirus F were detected and characterised. The NGS reads were mapped to segment 7 of the virus encoding partial NSP3 protein. Further analysis led to the generation of a 647 nucleotides long consensus sequences with a coverage of 2–11 with a minimum mapping quality of 80 or higher  of the consensus sequence of the Pacific black duck avastrovirus genome was generated. In comparison, a total of 4185 nucleotides of the consensus sequence for the Chestnut teal avastrovirus genome was generated  identity only  identity  of the consensus sequence of the virus genome were generated. Five consensus sequences of 305 nucleotides, 1596 nucleotides, 331 nucleotides, 1129 nucleotides, and 1112 nucleotides encoding partial ORF1 were generated with a coverage from 2–90  and the RdRp region (PBDCV/1129nt/PBD12.16) showed these to be 100% identical to the MK204392.1 Grey teal calicivirus ORF1 protein. In comparison, the VP1 region (PBDCV/1112nt/PBD12.16) was 99.4% (368/370 amino acids) identical to the MK204392.1 Grey teal calicivirus ORF1 protein  might indicate active infection and shedding of the virus from these birds. Analysis of the virus proteins showed that some of them were more identical but still distant to their counterparts expressed by the KJ469653.1 Muscovy duck adenovirus 2, while others to that of JF510462.1 Goose adenovirus 4. Nevertheless, the Pacific black duck aviadenovirus was almost equidistant from both the viruses in the maximum likelihood tree generated. This analysis is consistent with our previous analysis of the PBDAdV/PBD12.16 virus, as reported from the very short sequences generated3 before resequencing of the sample. From the central region of the partial Pacific black duck aviadenovirus genome, 18 genes (IVa2–fibre) were found in total which are common to all adenoviruses and said to be inherited from a common ancestor of all known adenoviruses8. However, only a partial fibre gene sequence was generated from the NGS data, and hence at this point, it is uncertain if the PBDAdV/PBD12.16 have one fibre gene like the Fowl adenovirus 825 or two fibre genes like the Goose adenovirus 48. Among the ORFs, ORF2 contained Parvovirus NS1 superfamily domain at 2nd–191st amino acids, as seen in some of the other waterfowl aviadenoviruses8. As reported earlier6, in the PBD12.16 sample, we detected an adeno-associated virus which may utilise the Pacific black duck aviadenovirus as a helper virus for its replication. However, currently, how the ORF2 protein assists the adeno-associated virus replication is uncertain.The Pacific black duck aviadenovirus characterised here belongs to the genus Anelloviridae family12. Gyroviruses have been found in cloacal, oral, and blood samples from birds29 and faecal samples of carnivorous mammals such as ferrets, cats and also humans34. However, the natural host of gyroviruses is thought to be birds. The finding of the GTGV/GT11.18 contributes to the wide genetic diversity of the gyroviruses. The Southern screamer gyrovirus (MH016740.1)11, the only other gyrovirus from a bird belonging to the order Anseriformes yet characterised, was distantly related to the Grey teal gyrovirus. Despite being very different to currently known gyroviruses, genetic analysis revealed that the N-terminal region of the VP1 protein is rich in basic amino acids and is a characteristic shared by all members of the Anelloviridae family12. The motif 173WWRWA177 in the VP1 protein of the GTGV/GT11.18 also appears to be conserved for all gyroviruses30. The VP2 of the GTGV/GT11.18 has the characteristic motif of protein tyrosine phosphatases which is the CX5R motif and the WX7HX3CXCX5H motif, that is conserved in other gyroviruses and anelloviruses13. A minimal signature motif with the configuration CX5R is highly conserved in all PTPases. PTPases catalyse the removal of phosphate from phosphotyrosine35 and may be important for the replication of the virus13. The VP3 protein of many gyroviruses, such as chicken anaemia virus, has some important motifs related to the induction of apoptosis in infected cells38. However, similar to some other gyroviruses from zoo and wild birds, no such related motif was found in the GTGV/GT11.18.A novel gyrovirus was found in the Grey teal 11.18 sample and was distantly related to other gyroviruses. Gyroviruses have been recently reclassified in the 41.Reoviruses were detected and characterised in both Pacific black ducks and Chestnut teals collected in August 2018, which suggest that these reovirus infections may be more common in winter compared to other seasons. Other reovirus seasonal distribution studies in humans and chickens also show reovirus infections to be high during colder months of the year42 was only 54.5% identical to its nearest homologous avian virus protein counterpart. This may be because of a specific host adaptation and to the best of our knowledge, this is the first report of an avian orthoreovirus from Pacific black ducks and more widely from a wild duck in Australia. It is also to be noted that sigma C is the most variable protein of avian orthoreoviruses and antibodies to this protein can neutralise the virus infection43. Nevertheless, further studies are required to determine whether the Pacific black duck orthoreovirus can infect other closely related host species that can lead to reassortment. However, the analysis of the other encoded proteins of the virus suggests that this virus may infect closely related host species leading to reassortment.The sigma C protein of the Pacific black duck orthoreovirus that forms part of the outer capsid and helps in host cell attachment of the virus particle44, and the phylogenetic analysis of the VP1 protein that functions as the RdRp46 shows that rotavirus G forms two different clades. Clade B includes the rotavirus G from the Pacific black duck, Grey teal5 and Ruddy turnstone19 . In contrast, clade A includes the rotavirus G from chicken18, turkey17, avocet19  and pigeon (unpublished). The most diverse segments of PBDRVG/PBD08.18 were S3 (VP4 protein), S9 (VP7 protein) and S10 (NSP4 protein). VP4 and VP7 form part of the outer capsid and the genetic diversity may be due to host-related immune selection pressure. However, the NSP4, which act as a viral enterotoxin, is only 53.7% identical to its closest relative and could indicate the presence of a diverse group of rotavirus G in this wildlife population that may exhibit different patterns of virulence in these hosts.All the 11 segments of a Pacific black duck rotavirus G were detected and characterised from the PBD08.18 sample. The phylogenetic analysis of the VP6 protein, one of the most conserved proteins that form the intermediate layer of the capsid18, although partial sequences of the same have been detected and characterised from a pig (unpublished) and partridge47. The current study is the first to detect and characterise rotavirus F from a duck species, albeit only a partial segment being identified. This is due to the low amount of virus (0.0001% of NGS reads) present in the sample. The genetic diversity and evolution of rotavirus F are less understood due to the limited characterisation of the virus, and further studies involving more infected duck samples may help in its elucidation.The complete genome of rotavirus F has only been molecularly characterised from chickensThe phylogenetic analysis of the avastrovirus from Pacific black ducks and Chestnut teals suggest that these viruses are quite different to one another with ~ 60% difference albeit being detected from the same location at different time points. This indicates the wide genetic diversity of the avastroviruses.23 to the Chestnut teal avastrovirus suggests that the migration of birds (likely migratory shorebirds) along the East Asian—Australasian Flyway could be important in the evolution of these viruses, as similarly observed for an avian paramyxovirus 6 characterised earlier from a Pacific black duck sample collected from the same site 18 months prior to this sample3.The Pacific black duck orthoreovirus (except for the segment S1) and the Pacific black duck rotavirus G from the PBD08.18 sample, and the Chestnut teal avastrovirus from the CT11.18 sample showed conservation at the amino acid sequences while being more distantly related at the nucleotide level to their closest relatives, respectively. Taken together, this may indicate that there is significant evolutionary distance/time between a common ancestor of the viruses, but that conservation at the amino acid sequence of the protein is subjected to a strong selective pressure. The conservation of protein structure across different avian hosts may also indicate that these viruses may be capable of cross-species infection or that cross-species infection is part of their evolutionary history. The close relatedness of the Northern pintail avastrovirus detected from China5. The Pacific black duck collection site is about 375 km away from the MK204392.1 Grey teal calicivirus collection site and was taken three months prior to the sample collected from the Grey teal. Both Pacific black duck and Grey teal are dabbling ducks found in Australia that may share habitat at any particular time point. Dabbling ducks can travel about 25 km to hundreds of kilometres per day49. Caliciviruses are thought to be transmitted through direct contact with an infected host or indirectly via oral-faecal route50. Thus, the calicivirus detected in the current study may indicate cross-species transmission and infection occurs between these related duck species.The Pacific black duck calicivirus is very closely related to the MK204392.1 Grey teal calicivirus from south west Victoria, Australia52. For viruses in the genus Gyrovirus, Chicken anaemia virus is so far known to be the only pathogenic member of that genus12 causing an economically important clinical and subclinical disease in young chickens, with a worldwide distribution53. Nonetheless, a gyrovirus was also sequenced from Northern fulmar  and Southern screamers (Chauna torquata) with neurological clinical signs28, as well as from healthy birds11.It should be noted that at the collection time, no clinical signs of disease were observed in the ducks and no close physical or extended examination such as post mortem or histopathological examination, was carried out. However, the high abundance of virus reads for some of the viruses from the bird samples may indicate an active infection and shedding of the virus. Aviadenoviruses can cause gizzard erosion or severe, often fatal cases of hepatitis-hydropericardium syndrome or inclusion body hepatitis depending on the virus and the host species42. However, avian orthoreoviruses were sequenced from diseased Muscovy ducks that had severe haemorrhagic and necrotic lesions in the liver and spleen54 and from Pekin ducks that had spleen necrosis55. Rotavirus G and rotavirus F were found in chickens with the runting-stunting syndrome; however, its role in the disease remains unclear56.Although avian orthoreoviruses have been associated with various disease conditions, a direct link between the virus and disease has been demonstrated only for viral arthritis syndrome or tenosynovitis in chickens59. However, the pathogenicity of the viruses described in the current study is currently unknown, and further studies involving histopathological examinations are required for its elucidation. Nevertheless, all of the viruses described in the current study may spread through the faecal-oral route60 and may cause asymptomatic infections as observed of the wild ducks in the current study.Among birds, astroviruses have been associated with various diseases such as enteritis, hepatitis, nephritis and runting-stunning syndrome6. The generation of the partial sequences of the other avian viruses from the duck samples described here may be due to various factors, including but not limited to, virus particle quantity, stage of infection and library preparation. Also, as stated earlier, the most abundant avian virus from each of the duck samples, except for the GT11.18 sample, was either an avian parvovirus or an avian picornavirus6. Future studies involving more extensive sample collection may lead to the complete genome elucidation of the viruses described here. However, the presence of motifs at the expected location in the generated virus consensus sequences suggests that they form part of a functional virus genome, although the description of each motif functions is beyond the scope of the manuscript. Furthermore, taken together with the previously described viruses6, the detection of more than one virus from each sample indicates co-infection, which could have a more significant health impact on the host when compared to infection with an individual virus.The current study was carried out by sampling several representative bird species from a bird community at specific time points from a single location in an effort to comprehend virus diversity, abundance and seasonal prevalence. The Grey teal gyrovirus was the most abundant virus (414 reads at Q ≥ 20) from the GT11.18 sample and exceeded the two parvoviruses previously reported from this sample with 141 and 222 reads at Q ≥ 20 respectivelyDetection and characterisation of several potentially pathogenic avian viruses, some genetically novel while others similar to those detected in other avian hosts, indicates further virus surveillance studies of wild ducks are warranted. Ducks being a natural reservoir of pathogenic and zoonotic viruses should be monitored continuously to detect any potential threat to other wildlife and humans. Future pathological studies on the pathogenicity of the characterised viruses will also aid in comprehending the host–virus relationship.6. Fresh wild duck faecal samples of Pacific black ducks (PBD), Chestnut teals (CT), Grey teals (GT) and Wood ducks (WD) were collected in May 2018 (late autumn), August 2018 (late winter), November 2018 and December 2016 (late spring/early summer) from Wallington, south-eastern Victoria, Australia. Pacific black ducks were captured in May 2018 (PBD05.18), August 2018 (PBD08.18) and December 2016 (PBD12.16 (described as MAD previously3)). Chestnut teal samples were collected in May (CT05.18), August (CT08.18) and November 2018 (CT11.18). Grey teal samples were collected in November 2018 (GT11.18) only, while Wood duck samples were collected in August 2018 (WD08.18) only. All samples were stored at − 80 °C within 1–3 h of collection until processing.The current study uses the samples reported earlierBird sample collection was approved under Deakin University’s Animal Ethics Committee project number B43–2016 and Department of Environment, Land, Water and Planning permit number 1008206. The current study involving these samples were performed in accordance with relevant guidelines and regulations.3. Briefly, the faecal samples were subjected to various biophysical methods such as homogenisation (25 Hz for 2 min), centrifugation (17000×g for 3 min) and filtration using a 0.8 µm PES filter (17000×g for 1 min). The sample was then divided into two aliquots  as described earlier3. Both aliquots were then nuclease treated using benzonase and micrococcal nuclease (37 °C for 2 h), followed by nucleic acid extraction using the QIAamp Viral RNA mini kit (Qiagen), as previously described3.Virus particles enrichment and virus nucleic acid extraction was carried out as per a previously optimised protocol in our laboratory3. Libraries were then pooled and were loaded onto Ion 530 or 540 chips using the Ion Chef Instrument. Template preparation was carried out and then the chips were run on an Ion Torrent S5XL System (Thermo Fisher Scientific) as per company protocols and as described earlier3.cDNA synthesis and amplification was carried out using the SeqPlex RNA Amplification Kit (Sigma) as per the manufacturer’s instructions using the extracted virus nucleic acids from both aliquots of the samples. Next generation sequencing library preparation was performed using the Ion Fragment Library kit (Life Technologies) followed by quantification of libraries, as described earlier62. Two local BLAST datasets were created for all the available virus sequences and all RefSeq virus sequences from the NCBI GenBank genetic sequence database (Dec 2018), as described earlier6. BLASTN and TBLASTX query against the two virus reference sequence databases was performed with an e-value cut-off of 1 × 10−10 and 1 × 10–30. The query results files were converted into spreadsheet files, sorted by virus matches, a list of potential virus targets created and then viruses of interest were identified for each sample, as described earlier6.NGS data analyses were carried out as described earlier62. Contigs were generated from the AssemblerSPAdes 5.6.0 plugin on the Ion Torrent Server by using the NGS reads of each sample. The contigs were bioinformatically analysed to identify virus sequences as described above using the same e-value cut-offs. Virus contigs (> 500 nucleotides) were then used as references in TMAP plugin and trimmed to regions with a mapping quality of 80 or higher and a coverage depth of at least 2 unless specified, as described earlier6. Virus sequences were also generated by assembling overlapping sequences from contigs and consensus sequences by using MEGA7 and magicblast63. Final virus consensus sequences were then generated after subjecting the sequences again to TMAP and mapping using IGV, as described above.The NGS reads were mapped against reference virus genomes of interest using the TMAP plugin on the Ion Torrent server. The mapped reads were used to obtain full or partial consensus sequences of the viruses using Integrative Genomics Viewer software (IGV) , as described earlier6, and the region of PCR analysis had NGS reads with very low abundance. Primer-BLAST64 software was used to design two sets of primers . FP1-GTGV-347nt (5′-CCCGGAAACCTGTACGAGTC-3′) RP1-GTGV-761nt (5′-TCTCCTGTAGTGGCGTCTGA-3′) and FP2-GTGV-264nt (5′-TCCTGTGGCAACTTTCGGAG-3′) RP2-GTGV-756nt (5′-TGTAGTGGCGTCTGAATCGG-3′) were used as the forward and reverse primers. The PCR master mix was prepared with 2X AmpliTaq Gold™ 360 Master Mix with 1 mM of each primer and 2 µl of DNA in a total reaction volume of 10 µl. The PCR reaction was carried out in QuantStudio™ Flex 6 real-time thermal cycler (Applied Biosystems) using the following conditions: 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, followed by a final step of 72 °C for 3 min. This is followed by a melt curve stage of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s and a final hold at 4 °C. The two PCR products of ~ 434 bp size and ~ 512 bp size, respectively, were purified using the 2% Size Select E-Gel System . These products were sequenced using the Big Dye Terminator Cycle v3.1 on a Hitachi 3500XL Genetic Analyzer  as per the manufacture instructions.Real-time PCR amplification and Sanger sequencing were carried out for Grey teal gyrovirus from the GT11.18 sample for the region from 264th to 761st nucleotide, as a potential indication of this virus being different from other gyroviruses was observed during NGS analysis. The GTGV/GT11.18 was the most abundant virus from the sample compared to the two parvoviruses previously reported66 , NCBI ORFfinder67, ScanProsite68 and PSORT II69 were used for identifying open reading frames (ORFs), and features of the virus genomes such as promotors, motifs and nuclear-locating signals, as described earlier6. The presence of these features in their expected location was used as an additional confirmation of the generated consensus sequences.Basic Local Alignment Search Tool70 in MEGA 771 software. MEGA7/MEGACC software was used to identify the best evolutionary model and the selected model was then used for generating the maximum likelihood tree with a bootstrapping of 1000 replicates. The distance data between sequences were calculated using the same software.Relevant related sequences were selected from the NCBI GenBank database. Nucleotide and protein sequences were aligned using Clustal-WSupplementary Information.


Sri Lankan cassava mosaic virus is an emerging pathogen in Southeast Asia. Here, we report the complete genome of a Thai isolate obtained using Nanopore technology. The isolate was collected in 2019 from the northeastern province of Surin, soon after disease eradication was reported in the country. Sri Lankan cassava mosaic virus is an emerging pathogen in Southeast Asia. Here, we report the complete genome of a Thai isolate obtained using Nanopore technology. The isolate was collected in 2019 from the northeastern province of Surin, soon after disease eradication was reported in the country. Geminiviridae (genus Begomovirus). It is one of several geographically distinct virus species causing cassava mosaic disease (CMD), a major disease of cassava (Manihot esculenta Crantz), in Africa and Asia (Sri Lankan cassava mosaic virus (SLCMV) is a circular, bipartite single-stranded DNA virus belonging to the family and Asia . SLCMV wand Asia ; it alsoand Asia , 4. The The complete genome of SLCMV was obtained from an infected cassava plant collected in the province of Surin in February 2019 from a field presenting a 0.7% incidence of CMD. A phi29 rolling-circle amplification protocol  was carried out with 60 ng of total DNA, which had been extracted using cetyl trimethylammonium bromide (CTAB) . After ade novo assembly, using the raw Nanopore reads that passed quality control (fastq pass) with Canu v1.8 . A consensus was created using the reference assembly results, and the quality was checked using Qualimap v2.2.1 , i.e., anu v1.8 , and refanu v1.8  and Pilop v2.2.1 . The resSequence Demarcation Tool analysis  of the aMN544647 (DNA-A) and MN544648 (DNA-B) and in the Sequence Read Archive under accession number PRJNA587722. A phylogenetic tree based on Nextstrain analysis (https://nextstrain.org/community/pestdisplace/CMDASIA?c=virus&r=location).Genome sequences and Nanopore reads were deposited in GenBank under accession numbers analysis  is avail


We summarized the characteristics of genes and receptors, antiviral therapy, and early warning and prediction. We put forward the importance of vaccine development and recommend that relevant research be carried out in specific laboratories.Coronaviridae within the order Nidovirales. The coronavirus genome is composed of a positive-sense, single-stranded RNA of about 30Kb, which is the largest non-segmented genome among known RNA viruses. It can cause respiratory, digestive, and nervous system diseases in humans and animals  , could be effective against 2019-nCoV. The immunosuppressive drug CsA prevents the nucleocapsid protein of the virus from binding to cyclophilin A (CypA) of the host cell, which has a peptidyl prolyl cis/trans isomerase (PPIase) activity, and a combination of interferon and CsA has been shown previously to significantly inhibit the replication and tissue damage caused by coronavirus infection in bronchi and lungs of humans. Pfefferle et al. detected specific interactions between CypA and SARS-nCoV non-structural protein 1 (Nsp1) by yeast-two-hybrid and other protein-protein interaction techniques. They also tested the drug CsA against a variety of coronaviruses and identified it to be a “pan-coronavirus inhibitor” (Haagmans et al., Global public health concerns are focused against a number of known and well-characterized infections. However, the outbreak of pneumonia caused by a novel 2019-nCoV virus is a reminder that importance should be given to predicting the risk of novel virus infections in humans (Thompson, At present, complete genomes of different 2019-nCoV have been released, and the latest published information on these genomes provides peer-reviewed information that is urgently needed to improve the risk assessment and response, which occurs in real time (Chen et al., However, many other important aspects of virus biology, such as, whether the virus can travel across continents, the list of species it can infect and whether it can cause severe mortality, are much harder to forecast. Our premise is that the amount of sequencing data currently available and the latest advances in computing methods using that data will make it possible for the first time to generate virtual models of how viruses evolve in each environment and how those viruses behave. Such models have the potential to make risk assessments as they arise that can be used to inform policy and direct strategies to head off impending threats.To date, many problems remain to be solved, including the virus’ origin, extent, and duration of transmission in humans, its ability to infect animal hosts, its spectrum and pathogenesis of human infections, and an effective vaccine development.At the heart of vaccine development is the question of immunology and it is crucial to understand the immunological questions associated with viral infections. The clinical characteristics and treatment of 2019-nCoV and SARS both suggested a serious problem of immunopathology, particularly in the lung mucosa, which is complex and unique. It might be due to the fact that a systematic protective immune response is not enough to protect against viral infection. Currently, one of the most dangerous but valuable experiments is to perform tests on immune cells in the blood and lungs of infected patients, preferably during different stages of viral infection. Data on clinical immunity can lay the foundation for future vaccine development. We need to be aware of the challenges and concerns that 2019-nCoV poses to our community. Every effort should be made to understand and control the disease.


Lymantria dispar L. adults who survived after viral infection. As a result of parental infection, the fecundity of survived females, pupae weight, and fertility were significantly different compared to the untreated insects. However, differences in these parameters between high- and low-virulence strains were not observed. The prevalence of virus strains in the offspring measured by quantitative polymerase chain reaction also did not differ. When the larvae reached the fourth instar, they were starved to activate the vertically transmitted virus. The frequency of virus activation in the experiment was not dependent on the virulence of the virus strains. These results are helpful for understanding the strategy of virus survival in nature and for the selection of the most effective strains with transgenerational effects in the years following pest treatment.Baculoviruses can persist in insect host organisms after infection and may be vertically transmitted to the next generation, in which they may be reactivated. The goal of the present study was to compare the efficiency of the vertical transmission of high- and low-virulence strains and the subsequent reactivation of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) in the offspring of  The causes of cycles in insect pest populations have long been a major focus in ecological research ,2. AmongIt is known that baculoviruses can be transmitted not only horizontally but also vertically. Horizontal transmission is thought to be a major route of baculovirus transmission . VerticaThe horizontal and vertical types of transmission strategies generally exclude one another because a virus with high virulence kills the host prior to the adult stage. This mean that the ability for vertical transmission will be selected during natural selection in high virulent strains . For thiAlphabaculovirus that has been used as the basis of biological insecticides for the control of the gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae) [L. dispar possess a more than 10-fold higher potency over three LdMNPV (LdMNPV-27/0) strains isolated from a continental Asian (Western Siberia) host population [L. dispar individuals that consumed OB could vertically transmit the virus to offspring and were detected in the following two generations by PCR [Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) is a highly pathogenic, host-specific rebidae) ,29. To drebidae) ,31,32 anrebidae) ,34,35,36s by PCR . We hypos by PCR ,24. AddiL. dispar was at the rising phase of population dynamics. The egg masses were mixed, and the result used as a single stock for treatment  in autumn 2016 and stored in a refrigerator at 2 °C. The population of m method  with somm method  for the L. dispar were placed in plastic containers  and fed with the leaves of cut branches of silver birch Betula pendula Roth. The insects were reared under laboratory conditions at a constant temperature (23 °C) and in a natural daylight regime. Then, insects were used for the treatments.In spring 2017, the eggs of the stock were sterilized using 1% sodium hypochlorite. Then, the eggs were placed in a Petri dish with 100 eggs each. The Petri dishes were kept under thermostat at a constant temperature (28 °C) for 48 hours for egg hatching. Next, neonate Larvae (1 day after fourth instar molting) were infected with two strains of LdMNPV from the collection of the Laboratory of Ecological Physiology of the Institute of Systematics and Ecology of Animals : strain 45/0 , kindly given by Dr John Podgwaite (USDA Forest Service) and strain 27/0 . 4 OBs/disc for LdMNPV-45/0 and 2 × 105 OBs/disc for LdMNPV-27/0 as described in detail by Akhanaev et al., 2017 [To initiate infection, groups of 250 fourth instar larvae were allowed to eat leaf discs (dia = 8 mm) containing a dried drop of suspension of 4 × 10l., 2017 . The dosL. dispar have clear sexual dimorphism , the analysis was performed separately between males and females. The collected egg mass was kept at room temperature for two months for postembryonic development. Then, the eggs were moved to a refrigerator at 2 °C for winter diapause. In the following year, the offspring were used for estimating success transmission of the virus. Once the adults emerged from surviving pupae, they were moved into plastic containers (1000 mL) (1♀:1♂) with filter paper for oviposition. Males and females from the same viral strain treatment were used. After mating and oviposition, egg masses were collected and marked to indicate them as belonging to parental pairs of adults. The following life-history traits were compared for those adults that survived OB treatment between treated and untreated insects: larval development time (from egg hatching to pupation), pupal weight, duration of the pupal stage, sex ratio, fecundity, and fertility. Since females of n = 320) and analyzed as described in To estimate the prevalence of transmitted LdMNPV infection in the offspring, we used the qPCR technique. For this, we randomly selected 5 eggs from each egg mass laid by females that survived after infection. Total DNA was extracted from each egg individually . The offspring were reared under laboratory conditions. When larvae reached the fourth instar, they were exposed to starvation to test for possible activation of the transmitted virus. Previously we showed that starvation is an effective stress-factor for the activation of covert to overt infection ,24. To dMortality of host treatments was analyzed using the generalized linear model with a binomial distribution and a logit link function (GLM). As no untreated insects died of virus infection, no correction factors needed to be applied. Non-viral deaths, predominantly fungal, did occur at a very low level (<1%) late in the assay. Data on the influence of the virus on the life history of surviving parents and reproduction were evaluated with the Shapiro–Wilk W test. Non-normally distributed data were analyzed with the Kruskal–Wallis test, followed by Dunn’s post hoc test. Normally distributed data were analyzed by one-way ANOVA, followed by Tukey’s post hoc test (only for data of fertility). Data of median development time of pupae were analyzed using a nonparametric Mann–Whitney U test between virus strains.A comparison of effects test of the vertically transmitted virus in the next generation between low- and high virulence strain treatments was conducted by GLM. Data were analyzed using the program PAST 3.0  and stat2= 140.1, df = 1, p < 0.0001). The percentages of NPV mortality were 49.2% for LdMNPV-45/0 and 63.5% for LdMNPV-27/0. We observed that the mortality caused by LdMNPV-45/0 was lower and the difference was statistically significant as compared with LdMNPV-27/0 . This difference was the result of different viral loads for different strains in which we tried to equalize the induced mortality of the hosts. All dead larvae were characterized by typical signs of polyhedrosis following infection by both viral strains.Host survival was severely reduced by baculovirus infection , although differences in absolute values were as low as 1 day. The duration of the pupal stage was not significantly affected for both sexes by OB treatment . Pupal weight was significantly higher among the survivors of OB treatment when compared to in the untreated insects . The surviving pupae after LdMNPV-27/0 were heavier than those surviving after LdMNPV-45/0 infection . The sex ratio of survived adults did not differ significantly between strains (p > 0.05), although the proportion of males was lower in the untreated insects when compared with the OB-treated insects (p < 0.05). The fecundity of parental females was reduced by up to 30% in OB-treated insects when compared to the untreated insects , independently of virus strains . Finally, fertility as measured by the proportion of eggs that successfully hatched was significantly lower in OB treatments than in the untreated insects  and did not differ between virus strains . The mean percentage of offspring positive for viral DNA did not exceed 30% in the OB-treated insects and did not differ significantly between virus strains . The interaction between virus strain and starvation was non-significant . In parallel, we observed a low frequency of activation of the virus in the OB treatment insects reared without starvation, which was not significantly different among virus strains , we observed more marked differences in activation rates of compared strains: 81.8% in the LdMNPV-27/0-treated insects and 55.7% in the LdMNPV-45/0-treated insects. L. dispar have previously been conducted with conflicting results. For example, Murray et al. (1991) [L. dispar [Studies of vertical baculovirus transmission in . (1991)  reported. (1991)  observed. dispar ,44. We s. dispar  showed t. dispar  demonstr. dispar  and less. dispar . In thes. dispar ,49, althL. dispar larvae by the peroral inoculation of a heterologous virus. Stress factors such as crowded rearing conditions and high relative humidity (RH) were reported to induce higher activation levels of NPVs in the offspring of Trichoplusia ni laboratory culture. The activation of Trichoplusia ni nucleopolyhedrosis disease was significantly greater at 95% to 100% RH than at RH levels of 75% or less. Crowded rearing conditions activate the covert virus more so than in uncrowded conditions [L. dispar larvae, we did not observe the activation of covert infection [Malacosoma pluviale californicum [Spodoptera exigua in the laboratory [L. dispar population using starvation [Many studies have investigated the activation of covert infections in pest populations. Hughes et al., (1993)  reportednditions . On the nfection . Food refornicum . Activatboratory . Similarboratory  previousarvation . The morarvation . Thus, wL. dispar [Many studies have shown debilitating effects on the host after consuming OBs . In the. dispar . Rothman. dispar  reportedThe development of highly sensitive molecular tools has enabled researchers to focus their attention on the vertical transmission of insect viruses and to assess the role of vertical transmission in the survival of baculoviruses in both natural and laboratory insect populations ,19,61,62Our study shows no differences in capability to vertical transmission from parent to filial host generation between low- and high virulent strains of LdMNPV when its used in the doses leaded to same lethal effect. However, low virulent strain tends to be more frequently reactivated from covert to overt form of infection by effect of such stress factor as starvation. From pest management point of view this result indicates that if there is the needs to get prolong effect after baculovirus treatment  it is reasonably to use low virulent viral strains for the application. Further investigation could be focused on the mechanisms of replication of virus within the host after the stress factors effects.