The fully automated system enables virologists to take a high-accuracy approach to virus diagnosis, prevention of mutations, and life cycle and improvement of diagnostic reagents and drugs, adding a point of view to the advancement of medical virology.The SARS-CoV-2 virus is responsible for the rapid global spread of the COVID-19 disease. As a result, it is critical to understand and collect primary data on the virus, infection epidemiology, and treatment. Despite the speed with which the virus was detected, studies of its cell biology and architecture at the ultrastructural level are still in their infancy. Therefore, we investigated and analyzed the viral morphometry of SARS-CoV-2 to extract important key points of the virus’s characteristics. Then, we proposed a prediction model to identify the real virus levels based on the optimization of a full recurrent neural network (RNN) using transmission electron microscopy (TEM) images. Consequently, identification of virus levels depends on the size of the morphometry of the area . The results of our model were an error score of training network performance 3.216 × 10 In 2002–2003, an outbreak of SARS-CoV-1 in China occurred, with 8000 confirmed cases and 800 deaths. In 2012, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was responsible for recurrent outbreaks on the Arabian Peninsula. SARS-CoV and MERS-CoV were determined to have infected civets and camels, respectively ,5. DevelViral particles are typically too minute to observe with a conventional light microscope. Therefore, electron microscopy is commonly utilized in the field of virology. In many scenarios, such as when trying to diagnose a virus in a clinical setting or to understand how it enters and assembles its particles, morphological analysis of the virus is required. Furthermore, if a virus is propagated in cell culture, particularly if the viral genome has been altered, quality control of virus particle integrity is necessary. In addition, conventional analysis methods are laborious and time consuming. In addition, it can reduce the misjudgments of biologists to some extent, and their efficiency can be increased. The aim of this paper is to improve the accuracy of virus detection and help virologists and medical teams provide effective attention to the diagnostic process. In this paper, we proposed a fully automated mixed-attention algorithms based on an optimized recurrent neural network (RNN) that helps advance medical virology by enabling virologists with a high-accuracy approach to the identification and detection of the SARS-CoV-2 virus. Thus, it is helpful to assist professionals in their laborious work by combining approaches of the attention mechanism for recognition and categorization.The viruses are divided into two categories: First are the types of viruses that are helpful. Some benefits include that they can break down things and provide nutrients to plants, they can decompose organic material, improve the quality of freshwater, and they can help fix nitrogen and provide nourishment to humans . Second,As a result of its remarkable performance in a wide range of image analysis and processing domains , such asArtificial intelligence (AI) describes approaches that allow a machine to replicate or surpass human intelligence, primarily in cognitive capacities. Machine learning (ML), computer vision, and natural language processing are the main subdomains of AI ,48. ML tj, as shown in Equation (1).There has been considerable interest in artificial neural networks (ANNs) as a method for simulating complex real-world problems computationally. Analyzing non-linear data with ANNs is a statistical method. Non-linear functions are predetermined at the beginning of the process. Output data are mapped to input data ,52. The In computing, recurrent neural networks (RNNs) represent a diverse collection of models that attempt to mimic the functions of the human brain. Multiple “conceptual neurons,” or “processing elements,” in an RNN are linked together through synaptic connections. An RNN’s ability to generate self-sustained temporal activation dynamics along its recurrent connection pathways, even without input, sets it apart from the more general feedforward neural networks. Since feedforward networks are functions, this mathematically demonstrates that an RNN is a dynamic system. In response to an input signal, an RNN maintains, in its internal state, a nonlinear transformation of the input history. It allows the RNN to interpret temporal context information and gives the network a dynamic memory . Figure Here,  network .The generalized regression neural network (GRNN) is a well-known statistical technique for predicting functions and is dependent on nonlinear regression . The arcn is the total number of training patterns, m is the number element of the input vector, Here, Researchers have been trying to figure out the structure of viruses since the late 19th century after realizing that viruses were the underlying cause of disease. Electron microscopy (EM) has the potential to provide direct images of viruses for diagnosis and study, which has a high resolution power that allows investigations at the nanoscale . ElectroPatients with COVID-19 isolated in Italy/INMI were divided into nine groups in the dataset acquired from the Zenodo website. The SARS-CoV-2 dataset consists of 519 TEM images in Vero cell cultures via plastic discs that are so thin they are barely visible (60–70 nm). Additionally, a 16-bit image viewer was required to view the images. All image files have been calibrated to fit so that they can be opened in ImageJ or Fiji using the Bio-formats importer .Many investigations have found that the virus has a spherical shape with a diameter between 60 and 140 nm, giving the virus the appearance of a solar corona under an electron microscope. Virus particles, free and encapsulated in membrane-bound vesicles, were found in ultrathin sections of the human airway epithelium ,62. The TEM is a valuable technology that directly identifies virus species; it reveals species-specific morphological details and quantifies all viral particles, infected or not. However, despite its usefulness, identifying between virus subtypes has no practical application due to their overlap. On the other hand, TEM may be used in other situations. Furthermore, we can potentially use it in the study of antiviral medications. To further investigate viral pathogenesis and determine the cellular target of invasion, TEM may help observe the mechanism by which antiviral drugs inhibit viral invasion of cells . Differe−9, and the actual turbidity level is on the Y axis, with the regression estimate on the X axis. Moreover, in this paper, the results demonstrate the accuracy of the virus level prediction model.The relationship between two variables is determined using linear regression. There are two factors at play here: a dependent variable that acts as a brake on the transformation and an independent variable that acts as a catalyst. This method aims to focus on the link between the two variables rather than calculating their dependence. The activation function in each layer of an ANN allows it to understand the intricate connection between the characteristics and the target. −5, and the output and target response were between −0.5 and 0.5, which concluded that this was the best model to use.The plot target and output time-series data of the network training response are used to show the results of virus predictions in the training stages. The time-series response is where the output is the prediction, and the goal refers to accurate data in the three stages of training and the error related to the data, as shown in −11 at epoch 639 indicates a network with a high ability to anticipate virus levels.−8, as shown in −9 at epoch 639. In addition, the following figure shows the training state, including the gradient function, the training gain (Mu), and the validation check.In the backpropagation process after a batch of data training, the neural networks estimate the error contribution of each neuron. Technically, the neural network computes the loss function’s gradient to explain each chosen neuron’s error contribution. The gradient value of 9.6852 × 10Studies have shown that understanding and interpretation of the characteristics of SARS-CoV-2 is important, as follows: The structure of the spike portion enables the development of a new class of drugs for the treatment of COVID-19 ,67. AnalThe geometric properties of coronaviruses are highly similar; consequently, it is imperative that fine-grained images be differentiated between critical portions so that they can be identified by their geometric properties. Additionally, the results of this study may contribute to a better classification of viruses based on the information obtained from this study. Furthermore, to improve the deep neural network’s virus detection capabilities, we suggest a fully automated attention network to help with fine-grained classification. Traditional approaches to analysis are tedious and time consuming. In addition, the characterization of the SARS-CoV-2 particles is essential, and maybe a reference guide is needed to test various cell types and infection schemes. The particle size is most likely dependent on the cell’s type of viral generation and the specific infection conditions, such as the number of viruses infecting a cell. In summary, we provide a new viewpoint to assist medical teams and virologists by providing a high-accuracy technique to recognize viruses and prevent virus mutations.


Culex, Aedes and Anopheles mosquito genera and report their global distribution. In addition, we highlight the challenges of extracting and integrating published virome data and we propose that a standardized reporting format will facilitate data interpretation and re-use by other scientists. We expect our comprehensive database, summarizing mosquito virome data collected over 20 years, to be a useful resource for future studies.Mosquitoes are vectors for emerging and re-emerging infectious viral diseases of humans, livestock and other animals. In addition to these arthropod-borne (arbo)viruses, mosquitoes are host to an array of insect-specific viruses, collectively referred to as the mosquito virome. Mapping the mosquito virome and understanding if and how its composition modulates arbovirus transmission is critical to understand arboviral disease emergence and outbreak dynamics. In recent years, next-generation sequencing as well as PCR and culture-based methods have been extensively used to identify mosquito-associated viruses, providing insights into virus ecology and evolution. Until now, the large amount of mosquito virome data, specifically those acquired by metagenomic sequencing, has not been comprehensively integrated. We have constructed a searchable database of insect-specific viruses associated with vector mosquitoes from 175 studies, published between October 2000 and February 2022. We identify the most frequently detected and widespread viruses of the  When viruses were not assigned to the species level, but only the closest viral match was reported, these were included in the database. No thresholds were taken into account for the minimal number of reads and genome coverage required for the accurate detection of viruses, with the exception of the study by Hameed et al.   and therefore absent from the ICTV Master Species List 2021 . For ent2.3Initial analysis of the contribution of individual publications to the database indicated that one study dominated the dataset, supplying 4169 of the 8378 (50%) total unique virus entries  ’, ‘Location (Specific)’ and ‘Species’ for the analyses of virus families, or the columns ‘Study’, ‘Virus (clean)’, ‘Location (Specific)’ and ‘Species’ for the analyses at the virus species level. In-house R-scripts were used for data analyses.3n = 4591) and use it as a metric for the abundance of viruses and virus families.We performed a review of the literature on virus identification in wild-caught mosquitoes. Based on a total of 175 publications, we generated a database consisting of 11,261 rows, each entry representing a virus detected in a specific sample . The num3.1n = 70) used next-generation sequencing-based approaches to characterize the virome, whereas PCR-based approaches (n = 67) were frequently used to specifically detect viruses from genera known to contain arboviruses and/or ISVs, such as flaviviruses, alphaviruses, phleboviruses, orthobunyaviruses, densoviruses and rhabdoviruses  or next-generation sequencing (n = 38) to detect in vitro replicating viruses.The number of mosquito virome studies has gradually risen over the years, with two studies published between 2000 and 2008 and a total of 27 studies published in 2021 A. A sligoviruses A. In fewUnique virus entries were not equally distributed across virome studies, with the majority of entries (88.1%) derived from metagenomic sequencing studies and only a small percentage from PCR studies (7.2%) and culturing studies (4.7%). Furthermore, over 50% of the total number unique virus entries were derived from only 10 out of 175 studies A.Mosquitoes were sampled across the globe B,C with 3.2Culex, Aedes, and Anopheles genera were most frequently found to harbor viruses, likely because these vector mosquitoes are sampled more often for their importance in pathogen transmission  and Cell fusing agent virus (20%) for the Aedes genus, and Anopheles flavivirus (23%) for the Anopheles genus. These viruses were among the most abundant and widespread in our dataset and have been detected in multiple species within and across mosquito genera .We analyzed the contribution of virus families to the virome of and ISVs ,72. In oo genera . A signio genera . Strikinsquitoes . While fMesoniviridae is a recently established taxon of mosquito-infecting positive-sense RNA viruses [Mesoniviridae virus entries in both Culex (74%) and Aedes (70%) mosquitoes were derived from alphamesonivirus 1, the founding species of the family that includes several closely related variants, such as Nam Dinh virus, Houston virus and Cavally virus [The family of  viruses . The larly virus ,74. AlphNodaviridae and Iflaviridae are both well-established families containing insect-associated viruses [Culex and Aedes mosquitoes. The majority of these virus entries was derived from only two studies, that sampled in California and China [Nodaviridae and Iflaviridae sequences, although not classified at the virus species level, were detected in 12 mosquito species [Nodaviridae  were detected in several species of Culex mosquitoes, representing 29% of the unique virus entries for Iflaviridae and 51% for Nodaviridae [Culex mosquitoes in Brazil, China, Belgium and Serbia [Table 2). viruses ,76, whicnd China ,78. In C species . In Calid Serbia  , Genomodetected ; Fig. 4Aoviruses ,61,67. Fe genera ,78,81.Genomoviridae and Circoviridae, novel species have mostly been detected through metagenomic sequencing [Circoviridae, and specifically the Cyclovirus genus, arthropods  have been suggested to be the primary host [Genomoviridae viral family, was found to infect the mycophagous mosquito species Lycoriella ingenua under experimental conditions [. These studies suggest that vector mosquitoes could be a part of the host range of genomoviruses and circoviruses.For both quencing ,92. As aquencing ,92. Howeary host ,93. Morenditions . These s3.8Negevirus [Culex (53 unique virus entries) and Aedes (32 unique virus entries) mosquitoes. Negeviruses have been reported in multiple Culex and Aedes species and across at least four continents. Among these, the most abundant virus was the eponymous Negev virus, which was detected in Aedes aegypti [Culex species across three continents [The majority of virus entries in our database are unclassified at the family level . This large group included 114 unique virus entries from the genus egevirus , a taxon aegypti  and seventinents ,65,95,963.9Aedes and Culex, the broad global distribution of these viruses was well supported, being detected in multiple countries in two to five continents in at least three independent studies, lending support to the validity of these observations. Surprisingly, all or nearly all top 20 viruses for Culex and Aedes respectively, were detected in multiple mosquito species, suggesting that vertical transmission is not the sole transmission route for these viruses. In line with the more limited sampling of Anopheles mosquitoes (Anopheles viruses were detected less frequently (between 2 and 9 unique virus entries) and at fewer places across the globe  the viruses identified along with accession numbers, nucleotide and amino acid identity scores, genome coverage, numbers of reads/contigs mapping to the viral genome, ii) information on the sample, such as pool identifier, number of mosquitoes per sample, mosquito species, sex, and life stage, iii) the date and location of sampling, along with geographic coordinates and type of habitat, and iv) sequencing information, including the sequencing platform, method for library preparation, sequencing depth per library, and accession number of the repository in which the raw sequence data have been deposited.During our study, we noticed that virome data are often impractically reported for interpretation and re-use by other scientists, due to unreported critical variables or an impractical format to present results  without accompanying presentation in a reusable data format. We propose that reporting can be improved by the standard inclusion of a supplementary table containing per virus positive sample, Retroviridae [Herpesviridae [Hepadnaviridae  [Retroviridae, it remains possible that these sequences derive from unidentified mosquito retroviruses or from retroelements in the mosquito genome.Next-generation sequencing is a relatively unbiased approach that successfully detects both RNA and DNA viruses in mosquitoes. However, the detection of sequences of well-known mammalian viruses of the oviridae ,65,66,82sviridae ,45,65 (eB virus)  indicateDetection of viral sequences does not provide direct support of active replication in the mosquito host and, indeed, bacteriophage sequences were frequently detected in mosquito virome studies ,65,67,69Aedes aegypti and Aedes albopictus genomes contain a large number of non-retroviral EVEs [Small RNAs may also help to distinguish replicating viruses from another source of viral sequences, EVEs. The ral EVEs , transcrral EVEs . Small Rral EVEs ,108.The composition of the mosquito virome is likely shaped by the environment, virus transmission modes, and restrictive factors in specific mosquito species or genera. Moreover, changing biotic and abiotic factors associated with global warming and increasing globalization may further affect the mosquito virome. Our database is an up-to-date, comprehensive overview of primary literature on mosquito-associated viruses from the last 22 years. As such, our study forms a solid foundation to study inter- and intra-species pathogen transmission from a One Health perspective. A future challenge will be to understand how virome dynamics affect mosquito-borne disease outbreaks.et al. recently reported that Phasi Charoen-like virus and Humaita Tubiacanga virus were highly abundant and widespread in Ae. aegypti worldwide .In agreement with our analyses, Olmo Final selection of 175 articles used for the analyses and their references.Supplementary file 1Sample-structured database of viruses identified in mosquito virome studies.Supplementary Table S1Virus positive mosquito species, with the corresponding number of continents, countries and studies in which they are sampled.Supplementary Table S2Unique virus entries in mosquito virome studies. (A) Number of unique virus entries reported for the top 50 studies that contributed most entries in our database. Unique virus entries for Hameed et al. (2020) before (dark red) and after  data curation are indicated. (B) Number of unique virus entries corresponding to the individual virus families for all mosquito species in our database. Bacteriophage families are indicated with salmon fill color; viruses not classified at the family level in light blue. (C) Number of unique virus entries for the top 10 most frequently detected families, according to detection method.Supplementary Fig. S1The following are the supplementary data related to this article.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.


Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the  Culicoides biting midge and therefore the global and seasonal distribution of AHSV is dependent on climate conditions of this insect vector [African horse sickness (AHS) is a severe and fatal disease of equids that is caused by African horse sickness virus (AHSV). AHS has been classified as a notifiable OIE-listed disease, resulting in the implementation of significant constraints on international trade and increased disease surveillance . AHSV hat vector ,6.Orbivirus genus and part of the Reoviridae family, African horse sickness virus (AHSV) is a complex non-enveloped virus with a characteristic double-stranded (ds) RNA genome consisting of ten segments contained within an icosahedral core particle that is encapsidated by an outer protein layer [Belonging to the in layer ,8,9,10. in layer ,12. The in layer ,14,15.Orbivirus genus and their functional significance, if any, remains to be discovered and is the focus of this study. AHSV VP7 is highly conserved among all nine serotypes [Culicoides-derived insect cell line (KC cells) infected with AHSV [During replication, certain hallmarks of infection start to appear such as the formation of structures and protein inclusions within the cytoplasm. NS1 self-assembles into tubule structures of unknown function within the cytoplasm of AHSV-infected cells ,17. Theserotypes . To dateerotypes ,22,23. Tith AHSV . The finith AHSV . Anotherith AHSV ,26,27. MOnce assembled, the core particles are released from VIBs and join the host exocytosis pathway with the aid of NS3/NS3A. It is during this virus egress stage that the outer capsid proteins VP2 and VP5 are incorporated onto the core particle to form a mature virion which then exits the cell via further NS3/NS3A facilitation ,29,30,31Currently, the only method of protection against the virus is a polyvalent live-attenuated vaccine that confers broad protection against all nine AHSV serotypes. With the recent developments in reverse genetics technology, it has become possible to produce recombinant viruses containing defined mutations and/or markers in their viral genomes resulting in the rapid production of safe attenuated and “Differentiation of Infected from Vaccinated Animals” (DIVA)-compliant vaccines, respectively ,36,37. TAHS can cause up to 95% mortality in susceptible horses . The outSeveral viruses have been shown to cause neurodegeneration in humans due to protein aggregation in the brain ,48,49. FGiven that AHSV pathogenicity and disease is more severe than that of BTV , it is pIn this study, we set out to use this soluble version of AHSV VP7 to study the impact of VP7 crystalline particle formation on virus replication by using a plasmid-based reverse genetics system to generate recombinant AHSV that does not form VP7 crystalline particles. Using the backbones of virulent (AHSV5) and attenuated (AHSV4) serotypes, we replaced wild-type segment 7 with a gene encoding our soluble mutant VP7, and used these recombinant viruses to study the effect of crystalline particle formation on virus yield and release in mammalian and insect cells. The interaction of soluble VP7 with other AHSV proteins during the replication cycle was also examined. The results obtained here allowed us to further understand the role of AHSV VP7 on replication, and possibly cellular pathogenesis. This study sheds light on the potential role of viral protein inclusions in disease.®, Merck, Darmstadt, Germany), 5% foetal bovine serum  and 1% penicillin and streptomycin . Geneticin  was added every third passage at 1 mg/mL. Cells were grown at 37 °C in the presence of 5% CO2. KC cells (derived from Culicoides variipennis) were obtained from the Onderstepoort Veterinary Institute  and cultured at 28 °C in TC-100 insect medium  with nonessential amino acids supplemented with 10% FBS , 1% penicillin and streptomycin , and antifungals .BSR-T7/5 cells are a clone of baby hamster kidney-21 cells that constitutively express T7 polymerase, and were used with permission from Ulla Buchholz, Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Tubingen, Germany . The celA plasmid-based reverse genetics system described previously  was usedACACTTGGCACGCATTACGCGCTGTCATTTTTCAGCAGATGAATATGCAGCCTATTAATCCGCCGATTTTTCCACCGACTGAAAGGAATGAAATTGTTGCGTATCTATTAGTAGCTTCTTTAGCTGATGTGTATGCGGCTTTGAGACCAGATTTCGCGATGAATGGTGTTAATCCGATGCCAGGGCCGATTAACAGAGCTCT-3′). This dsDNA fragment was inserted into the pJAD-S7 backbone using the In-Fusion® HD Cloning kit  cloning strategy. The dsDNA fragment was designed to include 15 bp homologous ends to the VP7 sequence and inserted into the VP7 plasmid backbone linearized by inverse PCR using primers that contained the same 15 bp homologous ends in their 5′ regions by In-Fusion® reaction according to manufacturer’s instructions. The sequence of the resulting plasmid, pJAD-S7-276, was confirmed by Sanger sequencing.To construct reverse genetics plasmids that encode the mutant soluble VP7 (sVP7), the In-Fusion cloning strategy  was usedACACCTGGCACGCCCTACGCGCAGCCATCTTTCAACAGATGAATATGCAGCCAATTAATCCGCCAATTTTTCCACCAACAGAAAGAAATGAGATTGTTGCATATTTGCTAGTAGCTTCTTTGGCAGACGTGTATGCGGCTTTAAGACCAGACTTCGCGATGAATGGTGTTAATCCGATGCCAGGGCCGATCAACAGAGCTCT-3′) and inserted into the S7 pSMART-T7 transcription vector backbone using In-Fusion® HD Cloning kit as described above. The sequence of the resulting plasmid, pSMART-T7-sVP7, was confirmed by Sanger sequencing.To construct the AHSV5-sVP7 transcription plasmid, the pSMART-T7 transcription vector containing a cDNA copy of AHSV5 S7 (GenBank KM886350) was used as a template. A dsDNA fragment containing the mutations that encode for sVP7 (underlined in sequence) in the AHSV5 backbone was synthesised by GenScript  (5′-ATGCGTATGTTTCGC50/mL (50% tissue infective dose per millilitre) [Recombinant viruses were rescued by plasmid-based reverse genetics system as described previously ,68. Monolilitre)  and convw/v) paraformaldehyde/PBS for 30 min at room temperature (RT). After fixation, cover slips were incubated for 15 min at RT in 0.5% Triton X-100/PBS to permeabilize the cells. BSR cells were blocked in 5% blocking solution (5% milk powder in PBS) directly after fixation followed by incubation with primary antibodies for 1 h. The cells were washed three times with wash buffer (0.5% (v/v) Tween20 in PBS) and incubated for 1 h with appropriate secondary antibody. Cells were washed and stained with 10 μg/mL DAPI  in 1% blocking solution for 10 min at RT. Coverslips were mounted onto glass slides using Vectashield Mounting Medium . Slides were then viewed using a Zeiss LSM 880 Laser Scanning Confocal Microscope (LSCM) coupled to an Airyscan detector. Images were processed and analysed for colocalisation by using Zeiss ZEN 3.3 Blue edition software package .Immunofluorescence microscopy was performed as previously described . Cell moFor the identification of AHSV VP7 and NS2 proteins, anti-VP7 guinea pig  and anti50/mL (50% tissue culture infective dose per millilitre) and converted to pfu/mL by multiplying by 0.7 [t-test was applied to statistical differences amongst the wild-type and mutant groups for each time point separately. In all the experiments, p ≤ 0.05 was considered statistically significant with the following denotations used: * (p ≤ 0.05) and ** (p ≤ 0.01).Virus growth kinetics was carried out in BSR and KC cells. Cells were grown to ~80% confluency in 24-well plates and each well was infected with appropriate virus at a MOI of 0.1, in triplicate, and incubated at 37 °C (BSR) or 27 °C (KC cells). After 1 h, BSR cells were washed and media replaced with 0.7 mL complete media to remove unbound virus. Cells were incubated for a further 12, 24, 36, 48 or 72 h. At each time point, both released and cell-associated virus were harvested separately. The supernatant of each well was carefully collected (representing released virus fraction) and the remaining cells harvested in 0.7 mL clean media by scraping cells and homogenizing through a 22G needle several times (representing cell-associated virus fraction). Debris was removed by centrifugation at 2500 rpm for 10 min and virus fractions stored at 4 °C. Virus titre was determined by endpoint dilution in BSR cells for each triplicate fraction, i.e., cell-associated virus, released virus, or total virus. Briefly, cells were infected with 10-fold dilutions of each sample (6 repeats), plates were sealed, incubated for 72 h and wells were scored according to presence or absence of visible CPE observed with an inverted light microscope. Titres were expressed as TCIDg by 0.7 . All expTo understand the role of AHSV VP7 crystal formation during AHSV replication, we aimed to generate recombinant ‘synthetic’ AHSV that contains a modified segment 7 that encodes a soluble VP7 protein (sVP7), by reverse genetics. To also determine whether AHSV VP7 crystalline particles could play a role in virulence or cellular pathogenesis, this study set out to generate both recombinant AHSV4 (attenuated) and AHSV5 (virulent) viruses that do not form VP7 crystalline particles, i.e., rAHSV4-sVP7 and rAHSV5-sVP7, to compare their properties with their wild-type counterparts. Therefore, four recombinant viruses  were generated by reverse genetics technology.A plasmid-based reverse genetics system was used to recover wild-type and mutant viruses. Here, recombinant AHSV4 (rAHSV4), representing wild-type AHSV serotype 4 virus, was derived using segments 1–10 representing the live attenuated AHSV4 strain known as “LP” . This avirulent AHSV4 LP strain is used as a vaccine in South Africa and its origin has been described previously by Baltus Erasmus . RecombiThe AHSV4 LP strain is an attenuated virus; it is avirulent in horses and has reduced CPE in mammalian cells. Therefore, we included analysis of a highly virulent OIE reference strain of AHSV5 (HS 30/62), also known as “Fourie” or FR , to alloSuccessful virus rescue from transfection was confirmed by the presence of CPE, positive labelling of AHSV proteins and titration determination of passaged viruses. Furthermore, growth curves of rescued rAHSV and rAHSV5 and their respective wild-type reference strains AHSV4 (LP)  and AHSVThe detection of virus proteins following rescue and after multiple passage events confirmed the ability of the mutant rAHSV4-sVP7 and rAHSV5-sVP7 viruses to replicate, indicating that VP7 crystalline particle formation is not vital for virus fitness.As observed previously, when expressed alone, sVP7 has a homogenous intracellular distribution . TherefoThe small VP7 foci observed to be present for wild-type and mutant viruses in To compare the replication properties of wild-type and mutant rAHSV, next we examined the association of VP7 with VIBs. VIBs are virus factories which act as a scaffold for viral replication and assembly. VP7 foci have previously been shown to associate alongside VIBs . To compResults thus far indicated the presence of unique sVP7 tubule-like structures in rAHSV4-sVP7 LSCM data. To determine if the tubule-like distribution was due to association with characteristic AHSV NS1 tubules, dual labelling of VP7 and the tubule-forming NS1 protein was performed and analysed. Results showed that the tubule-like structures formed by sVP7 fully colocalised with the NS1 signal , indicatTaken together, results thus far show that soluble VP7 associates with VP2 and NS2 proteins similarly to wild-type VP7. The notable difference observed can be seen for the unique colocalisation and potential interaction of soluble VP7 with NS1 tubules.To determine the impact of VP7 solubility on AHSV replication, in vitro time trials were performed in mammalian cells to determine the replication kinetics of each virus, i.e., attenuated (rAHSV4 versus rAHSV4-sVP7) and virulent (rAHSV5 versus rAHSV5-sVP7). To obtain a clear picture of the virus replication kinetics, released versus cell-associated virus was determined, in triplicate, at 12 hourly intervals over 3 days for each virus. Virus titre means were plotted and represented on a logarithmic scale to illustrate growth curves. Total virus was reported from the sum of titres from released and cell-associated fractions and illustrated to scale to show proportional differences between each virus at each time point.Released virus titre was determined from collecting media separately from cell monolayers prior to cell lysis. Results showed that rAHSV4-sVP7 released significantly less virus throughout the 3-day period, culminating with a decreased yield of released progeny virus at 72 hpi A. Cell-aLooking at the overall wild-type rAHSV4 replication kinetics C, total Percentage of released progeny virus, calculated as percentage of released fraction over total virus yield, indicated that rAHSV4 virus release increased rapidly from 36 to 72 hpi D, while Similarly to rAHSV4-sVP7, the released fraction of the virulent strain rAHSV5-sVP7 also had reduced virus titres over the 72 h period compared to rAHSV5 E. IntereComparing attenuated versus virulent serotypes overall revealed that both rAHSV4-sVP7 and rAHSV5-sVP7 had a general reduction in virus release compared to their wild-type virus counterparts D,H. InteSince AHSV is a vector-borne disease, it was decided to repeat this part of the study in the insect vector cell line (KC cells) to determine if VP7 solubility affects replication differently in these cells. Time trials were conducted as above, except the released and cell-associated virus fractions could not be collected separately as the cell integrity of the fragile KC cells could not be guaranteed at time of collection. Instead, at time of virus harvest, total virus was collected and titred as described in In KC cells, both rAHSV4 and rAHSV5 wild-type virus titres increased continuously from 12 to 72 hpi  with no Insoluble aggregates or inclusions that are formed during virus infection have been evidenced to have various functions within the cell . For exaThis study aimed to understand the nature of the implied effects of crystalline particle formation on cellular biology and AHSV replication. A reverse genetics approach was used to generate a recombinant AHSV that contains a mutated segment 7 that encodes the soluble VP7 in its genome and expresses soluble VP7 during replication. Successful rescue of rAHSV-sVP7 for both attenuated and virulent strains confirmed that VP7 crystalline particle formation is not required for successful virus replication. The impact of this genetic knock-in of a soluble VP7 was analysed by observing virus protein-protein interactions and replication kinetics compared to wild-type rAHSV.The soluble VP7 protein interacted similarly to wild-type VP7 with outer capsid protein VP2 and VIB mediator NS2 in infected cells, indicating that sVP7 forms cores as usual during replication. This served to confirm that wild-type VP7 is required in its soluble form to assemble into cores during replication as previously suggested ,70. IntePrevious studies have shown that increasing the solubility of AHSV VP7 increases the yield of CLPs —likely dReleased and cell-associated virus fractions were collected to obtain a more detailed view of intracellular progeny virus production versus released virus. Initial infection rates were also compared by examining virus yield at 12 hpi. It was revealed that both wild-type and mutant viruses (serotypes 4 and 5) had similar initial replication rates and, following initial infection, differences in virus production and release were observed.AHSV is typically released from the mammalian cell via disruption of the plasma membrane in a lytic manner mediated by NS3 ,32. WhenWhen comparing rAHSV4 with rAHSV4-sVP7 replication kinetics in BSR cells, it was found that rAHSV4-sVP7 replicates slower than rAHSV4 with significantly reduced virus yield overall. The decreased total virus yield over 72 h was due to a decrease in intracellular virus production. Interestingly, there was also a significant reduction in rAHSV4-sVP7 release throughout the 72 h period. It is likely that the delay in virus replication observed for rAHSV4-sVP7 is due to reduced released progeny virus particles available for reinfection. VP7 crystalline particles therefore play a role in AHSV4 release and intracellular virus production in mammalian cells.Replication kinetics of rAHSV5-sVP7 revealed that similarly to rAHSV4-sVP7, there was decreased virus release throughout the 72 h period. Therefore, VP7 crystalline particles also play a role in AHSV5 release in mammalian cells. Most noteworthy for serotype 5 was that although at early hours post-infection rAHSV5-sVP7 had lower yields than rAHSV5, instead of declining after 36 hpi the mutant virus continued to produce intracellular virus causing exponential growth from 48 hpi resulting in a significant increase in total virus yield at 72 hpi. The exponential growth of rAHSV5-sVP7 in BSR cells was similar to what is observed of AHSV growth in KC cells due to lack of cell death (CPE) as a result of non-lytic release mechanisms. It is therefore likely that, like KC cells, the infected BSR cells were not damaged by rAHSV5-sVP7 virus replication due to reduced CPE resulting in the increased total virus yield observed for rAHSV5-sVP7. For this virulent serotype, it is therefore possible that VP7 crystalline particles contribute toward cellular pathogenesis by disrupting the cellular membrane to aid virus release therefore causing CPE in mammalian cells. These results therefore implicate a potential role of AHSV VP7 crystalline particles in rAHSV5 cellular pathogenesis.Replication kinetics of recombinant viruses in KC cells revealed similar results for both serotype 4 and 5. Since virulence factors would typically only play a role in host mammalian cells, it is expected that no notable differences be revealed in KC cells. The decreased virus yield of mutant viruses in KC cells at 72 hpi further indicates a potential role of VP7 crystalline particles in efficient intracellular virus production as observed for mutant viruses in mammalian cells. Since no CPE is observed for insect cells, VP7 crystalline particles are not required for non-lytic release therefore resulting in similar replication kinetics of wild-type and mutant AHSV4 and AHSV5 in KC cells observed here. Furthermore, virus replication kinetics in mammalian cells revealed that virus release was significantly reduced for both mutant viruses rAHSV4-sVP7 and rAHSV5-sVP7, further indicating that AHSV VP7 crystalline particles play a role in mammalian, i.e., lytic, virus release mechanisms for these strains.Virus protein aggregates or structures that disrupt cellular membranes to promote pathogenesis and human disease have been previously reported. For instance, the accessory protein PB1-F2 of influenza A virus oligomerises into amyloid fibres, and has been shown to rupture cell membranes and cause cell death at late time points in human lung epithelial and monocyte cells ,80. As oProtein aggregates that mediate virus pathogenesis have also been shown for the nuclear export protein of a highly mutating H1N1 influenza A virus. This protein intrinsically forms aggregates which have been correlated with its role in virion budding ,81. HepaSince no exponential growth of rAHSV4-sVP7 was observed, it would be suggested that VP7 crystalline particles do not contribute toward rAHSV4 cellular pathogenesis as they may for rAHSV5. The cellular pathogenicity mechanisms of these viruses differ considerably since rAHSV4 is an attenuated virus with lowered CPE in mammalian cells while rAHSV5 is a virulent virus and unattenuated in mammalian cells. Therefore the role of VP7 crystalline particles in each virus may differ. A possible explanation could arise from our LSCM data that revealed the presence of sVP7-NS1 tubules in rAHSV4-sVP7 infected cells. NS1 has previously been linked to virus morphogenesis and cellular pathogenesis ,85. PerhThis study has revealed that AHSV VP7 crystalline particles play a role in virus release during AHSV replication. Furthermore, AHSV VP7 crystalline particles are required for efficient intracellular virus production in mammalian and insect cells. It was further revealed that VP7 crystalline particles may play a role in the lytic release of AHSV5 which may contribute to cellular pathogenesis in mammalian cells. To confirm the role of AHSV VP7 crystalline particles in pathogenesis, further cell viability and in vivo studies should be performed. To further elucidate the role of VP7 crystalline particles in virus release, localisation analysis should be carried out with exit mediator protein NS3, and the association of NS1 with VP7 should be further explored in the context of virus production. This study advances the understanding of AHSV replication.


Comparative animal modeling has long served as a cornerstone for understanding the biological effects of infection by many DNA and RNA viruses. Over the years, conventional and unconventional animal models have been developed for this purpose. The goal of the Special Issue, “Comparative Animal Models of Human Viral Infections” is to gather the latest information on recent advances in the development of animal models in order to understand human viral infections and the diseases caused by them. The successful development of comparative animals of human viral infections has important implications in helping to determine the mechanisms of disease pathogenesis and pathologies and immune correlates of protection against virus infection, as well as in informing relevant strategies for vaccination, immunotherapeutic, and antiviral developments to benefit humans.This Special Issue consists of five published articles (two reviews and three primary research articles), which were contributed by some of the leading researchers in the field. A comprehensive review article  written Hemorrhagic fevers can be caused by some significant human viruses, such as Ebola virus (EBOV), Marburg virus (MARV), and Lassa virus (LASV). These and other emerging viruses, such as the Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), can cause severe vascular leakage and other symptoms. In an original research article , WestoveLASV is another virus  that can cause lethal VHF disease. Because there are currently no FDA-approved vaccines or therapeutics against this deadly virus, the development of animal models that can recapitulate clinical and pathological features of arenavirus-induced hemorrhagic fever (AHF) diseases in humans is necessary. Laboratory mice are refractory to AHF infections, and NHPs, while being a good animal model for AHFs, are limited by their high cost and ethical and other constraints . As an a, which serve as the natural reservoir for these viral pathogens [Another study published in this Special Issue  also attathogens . Likewisathogens . The autathogens  found thathogens . These fAs described in a comprehensive review article in this Special Issue by B. M. Warner , a resea


Cryptococcus tetragattii fungus is rare and has been found in specific geographic regions. We report a case of meningitis caused by C. tetragattii (molecular type VGIV) in an immunocompetent patient in Taiwan. The patient had traveled to Egypt and was positive for granulocyte-macrophage colony-stimulating factor autoantibody.Meningitis caused by  Cryptococcusgattii species complex is found in specific geographic or climatic regions (C.gattii–caused meningitis is much lower than that caused by C.neoformans sensu stricto. We report a case of meningitis caused by C. tetragattii in an immunocompetent patient in Taiwan.Meningitis caused by members of https://www. astrazeneca.com) 4 months before admission. She had no contact history with pets, birds, or other animals. She frequently traveled abroad before the COVID-19 pandemic and had visited Italy, Egypt, and mainland China during 2018–2019. A 59-year-old female taxi driver with no systemic diseases sought care at China Medical University Hospital in Taichung, Taiwan, because she had experienced intermittent headache with unsteady gait for 2 months. She had received 1 dose of the ChAdOx1 nCoV-19 vaccine . We detected C. neoformans and C. gattii by using a multiplex PCR panel for meningitis . Fungal culture of the CSF specimen yielded Cryptococcus species. We identified the organism as C. gattii; its score value was 1.92 by Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system . We performed multilocus sequence typing (MLST) and phylogenetic analysis MLST for 7 genetic loci, including CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, and the URA5 region, as previously described (C. gattii ST576. We deposited sequence data of the 7 foci from MLST into GenBank (accession nos. OP828681–87). The URA5 gene restriction fragment length polymorphism of the C. gattii isolate performed as previously described (C. tetragattii (CMU-3028)  on the second hospitalization day. Studies on the CSF were positive for yeasts by India ink staining and showed a cryptococcal antigen titer of 1:40×  all showed negative results, but the result for the anti-granulocyte-macrophage colony-stimulating factor autoantibody (anti-GM-CSF AAb) was positive (titer 0.84), as measured using ELISA as previously described , HIV-1/2 antibodies, and free HIV-1 p24 antigen  plus oral flucytosine  for 21 days. A follow-up CSF culture revealed negative growth on hospital day 15. Oral voriconazole (250 mg/12 h) was prescribed as consolidation treatment for 15 days. The patient was discharged in stable condition and followed up at the outpatient department. She received oral fluconazole (400 mg/d) for the subsequent 6 months and remained well.C. gattii species complex has different geographic distribution, and C. tetragattii is mostly isolated in Africa (C. tetragattii in Zimbabwe (C. gattii infection, as well as those with C. neoformans infection (In this case, the patient is an immunocompetent host who had previously received 1 dose of the ChAdOx1-S vaccine and had a travel history to Egypt 2 years before symptom onset of meningitis. The members of the C. tetragattii fungal infection in patients who have traveled to meningitis-endemic regions.Whether COVID-19 vaccines have a trigger effect in unmasking underlying diseases, such as that in our patient, warrants further study. To our knowledge, no studies demonstrate the relationship between anti-GM-CSF AAb and COVID-19 vaccines. However, research showed that autoantibody such as anti–smooth muscle antibody was triggered by COVID-19 vaccine as autoimmune hepatitis (


Acanthamoeba host infected with bacterial symbionts. Subsequently, coinfection experiments were carried out, using the fresh environmental isolates as well as additional amoeba laboratory strains. Employing fluorescence in situ hybridization and qPCR, we show that the bacterial symbiont, identified as Parachlamydia acanthamoebae, represses the replication of the sympatric Viennavirus in both recent environmental isolates as well as Acanthamoeba laboratory strains. In the presence of the symbiont, virions are still taken up, but viral factory maturation is inhibited, leading to survival of the amoeba host. The symbiont also suppressed the replication of the more complex Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean (Mimiviridae). Our work provides an example of an intracellular bacterial symbiont protecting a protist host against virus infections. The impact of virus–symbiont interactions on microbial population dynamics and eventually ecosystem processes requires further attention.Protists are important regulators of microbial communities and key components in food webs with impact on nutrient cycling and ecosystem functioning. In turn, their activity is shaped by diverse intracellular parasites, including bacterial symbionts and viruses. Yet, bacteria–virus interactions within protists are poorly understood. Here, we studied the role of bacterial symbionts of free-living amoebae in the establishment of infections with nucleocytoplasmic large DNA viruses (Nucleocytoviricota). To investigate these interactions in a system that would also be relevant in nature, we first isolated and characterized a giant virus  and a sympatric potential  Protists are ubiquitous microbial eukaryotes found in virtually any environment ranging from natural aquatic or terrestrial ecosystems to engineered environments . As primViruses shape protist populations, with major impact on entire ecosystems as in the case of algal blooms and demise . A wides3In contrast to the lytic giant viruses, bacterial symbionts of protists can either be mutualists, parasites, or commensals. While protists are frequently associated with diverse bacterial endosymbionts, functions and potential benefits of these intracellular bacteria are largely unknown –11. ImpoAcanthamoeba castellanii as a surrogate host, a lytic virus was isolated from activated sludge of a wastewater treatment plant previously shown to contain diverse giant viruses (Marseillevirus viennavirus (Viennavirus) (SI Appendix). An Acanthamoeba hatchettii isolate was obtained from the same sample. The amoeba contained bacterial symbionts, identified by genome sequencing as a novel Parachlamydia acanthamoeba strain  and leading to demise of the host population within 55 h , which led to the establishment of the symbionts in these amoeba cultures. When subsequently infected with Viennavirus, no viral factories were observed in both Acanthamoeba species that now contained the symbiont. Viennavirus replication was significantly repressed, mirroring our findings for the naturally cooccurring host amoeba. In contrast, symbiont-free controls showed viral factory formation and viral replication   D indicatabolites . When inabolites D. Notablabolites D. OveralWe infected amoebae simultaneously with the giant virus and the symbiont. This experimental setup led to the formation of viral factories observed at 12 and 24 hpi, notably along with the presence of intracellular symbionts E and F. The symbiont was next added to naïve amoebae 12 h prior to virus infection, a time point which coincides with major changes in the host cell and the establishment of the inclusion, the phagosome-derived vacuole containing the bacteria . Viral fIn summary, these findings, together with the notion that Marseilleviruses exploit different entry paths , indicatA. castellanii carrying the Parachlamydia symbiont, with Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean. While Viennavirus belongs to the Marseilleviridae, Mimivirus and Tupanvirus are Mimiviridae. Mimivirus was the first described giant virus and serves as a “model system”  were done as described , 10. DetSupplementary File


A. sinensis were reported. According to the phylogenetic analysis, they were classified as nege-like virus, iflavirus, chu-like virus, and ollusvirus. The complete genome sequence of four RNA viruses were obtained with RACE. Moreover, the analysis of virus-derived small interfering RNAs showed that most of the RNA viruses were targeted by the host antiviral RNA interference pathway.The present work focused on the RNA virome analysis of an agricultural pest. In this study, the four novel RNA viruses in Atractomorpha sinensis (Orthoptera: Pyrgomorphidae), an important agricultural pest known as the pink-winged grasshopper. These insect viruses were classified as the nege-like virus, iflavirus, ollusvirus, and chu-like virus using HTS and phylogenetic analyses. Meanwhile, the full sequences of four novel RNA viruses were obtained with RACE and named Atractomorpha sinensis nege-like virus 1 (ASNV1), Atractomorpha sinensis iflavirus 1 (ASIV1), Atractomorpha sinensis ollusvirus 1 (ASOV1), and Atractomorpha sinensis chu-like virus 1 (ASCV1), respectively. Moreover, the analysis of virus-derived small interfering RNAs showed that most of the RNA viruses were targeted by the host antiviral RNA interference pathway. Moreover, our results provide a comprehensive analysis on the RNA virome of A. sinensis.A large number of RNA viruses have been discovered in most insect orders using high-throughput sequencing (HTS) and advanced bioinformatics methods. In this study, an RNA virome of the grasshopper was systematically identified in  With the aid of large-scale metatranscriptomic sequencing, an increasing number of novel RNA viruses have been discovered in insects, which can broaden our horizons of RNA viruses’ diversity ,2. TheseHematophagous and sap-sucking insects play an important role in the transmission of arthropod-borne viruses (arboviruses). Previous studies have shown that several IRVs isolated from hematophagous insects interact with arboviruses and affect vector competence for arboviruses. For example, IRVs in the Flaviviridae modulate arboviruses’ replication and transmission in mosquitoes and sandflies ,7,8. NegAtractomorpha sinensis, commonly known as the pink-winged grasshopper (Orthoptera: Pyrgomorphidae) [A. sinensis has not attracted wide attention to its outbreak and potential damage over the past several decades.rphidae) ,18, is arphidae) . In termrphidae) ,20. NeveA. sinensis. As revealed through analysis on sRNA data, RNAi-mediated antiviral immunity was active during the infection of these RNA viruses.In this study, four novel RNA viruses were identified in A single adult pink-winged grasshopper was collected in the field of Huzhou, Zhejiang, China, in September 2019. This sample was delivered to our laboratory and stored at −80 °C until further analysis. Then, insect morphology was identified under a dissecting microscope and verified with Sanger sequencing using a pair of universal primers to amplify mitochondrial cytochrome c oxidase subunit 1 (COI) genes.The single sample was homogenized in liquid nitrogen, and total RNA with rRNA was extracted using TRIzol reagent  following the manufacturer’s instructions. High throughput sequencing (HTS) and small RNA sequencing were conducted after evaluating the quality of total RNA with rRNA through Nanodrop . The Illumina TruSeq Total RNA with rRNA Sample Preparation Kit was adopted to construct the cDNA library. A small RNA library was established using the Truseq Small RNA Library Preparation Kit  and sequenced based on the Illumina HiSeq 2500 platform, which resulted in 19 Mbp of raw data. Furthermore, raw RNA reads were subject to quality control, adapter trimming, and contig assembly using Trinity program (Version 2.8.5)  with def−20 for each comparison. Based on blast hit results, unassembled overlaps and same viral reads were merged by using SeqMan program of DNAstar software package to form partial/complete virus genomes. All candidate viral reads were verified through RT-PCR following Sanger sequencing. The first strand of complementary DNA (cDNA) from 1000 ng of total RNA was synthesized with HiScript ®II Q RT SuperMix for PCR (+gDNA wiper)  in line with the manufacturer’s protocol. RT-PCR was performed in 10 µL-reaction agents containing 0.5 µL template cDNA, 5 µL of 2 × Phanta Max Buffer, 0.2 µL dNTP mix, 0.2 µL of Phanta Max Super-Fidelity DNA Polymerase , 0.2 µL of 1 µM forward and reverse primers, and 3.7 µL of ddH2O. The thermal cycling conditions were 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 70 °C for 30 s. Abundance of each virus genome was estimated by mapping quality-checked raw reads to virus genomes with Bowtie 2 and Samtools programs [To identify viral reads, the assembled RNA-seq contigs were queried against the non-redundant nucleotide (nt) and non-redundant protein (nr) sequence databases in NCBI using Blastn and Blastx algorithm, respectively. Furthermore, the candidate viral reads were compared against the Viral RefSeq database downloaded from GenBank in order to avoid false positive reads. E-value cut-off was set at 2 × 10programs ,23. Thenprograms .® RACE 5′/3′ Kit . Both 5′ and 3′ PCR products were amplified with sequence-specific primers with nest-PCR and cloned into a pMD19-T clone vector individually  after Sanger sequencing, to obtain the complete virus genome sequence. The primers used for identification of virus-like sequences are listed in To obtain the full-length genome of the candidate virus, the rapid amplification of cDNA ends (RACE) assay was performed as described . BrieflyA. sinensis, the clean small RNA (sRNA) reads (18- to 30-nt) were mapped back to full-length virus genome sequences using Bowtie 2 with zero mismatches. In addition to that, virus-derived siRNAs were further vegetated using the Linux bash scripts and the custom perl scripts.To explore the small interfering RNA (siRNA) triggered by viruses in https://www.ncbi.nlm.nih.gov/cdd/, accessed on 5 March 2022) and InterProScan Search .The potential open reading frames (ORFs) in the virus genomes were predicted using the ORF Finder program in NCBI. The function annotation of each ORF was conducted through the NCBI Conserved Domain Database  were generated from the obtained cDNA library, which had deep sequencing on the Illumina HiSeq 4000 platform (150-bp paired-end reads) of Novogene. In total, 86,784,590 reads were generated and assembled de novo into 464,844 reads in the library. Through a Blastn search against the nucleotide database in NCBI, it was found that the insect COI sequence showed a high homology (99% identities) to that of A. sinensis (accession number: MW085548.1). In total, 17,356 virus-like reads were found in the library. After a BLASTn search against the NCBI nucleotide (nt) and the entire viral reference database, four novel RNA viruses were identified in the assembled contigs of A. sinensis, which were classified as nege-like virus, iflavirus, ollusvirus, and chu-like virus, respectively. The sequences of four viruses were uploaded to NCBI, and the accession numbers include OL672484, OL672485, OL672486, and OL672487. It was shown that three reads were almost identical to those of the Aphid lethal paralysis virus (ALPV), suggesting that these reads were derived from a strain of ALPV  was 12,278 nt (excluding polyA tail), which contained a 69 nt 5′ untranslated region (UTR), a 457 nt 3′ UTR, and four predicted ORFs that further included ORF1 , ORF2 , ORF3 , and ORF4 . ORF1 was featured by four conserved domains, namely, Vmethyltransf super family (vMet) domain (253–1089 nt), FtsJ-like methyltransferase (FtsJ) domain (2206–2775 nt), Viral_helicase1 (Hel) domain (5689–6483 nt), and RdRP_2 super family (RdRP) domain (6994–8448 nt) A, whereave sense C.The analysis of the phylogenetic relationship showed that ASNV1 was the cluster with Ganwon-do negev-like virus 1, together with Ingleside virus, Nephila clavipes virus 4, and Hubei virga-like virus 13, forming unclassified group1, showing a close relationship with the Kitaviridae B. AnalysThe sRNA library was constructed to analyze virus-derived small RNA (vsiRNA) of ASNV1; as a result, ASNV1 triggered vsiRNA with peaks of 22 nt related to RNAi-mediated antiviral immunity C. The vsA. sinensis replicated in an efficient way (The full-length sequence of Atractomorpha sinensis iflavirus 1 (ASIV1) contains 9, 412 nt with the 791 nt 5′ UTR, the 118 nt 3′UTR, and a predicted ORF (792–9293 nt), where the ORF contained two rhv_like (rhv) domains (1827–2060 nt and 2631-2954 nt), the CRPV_capsid super family (CRPV) domain (3648–4304 nt), the Viral_helicase1 (Hel) domain (5208–5531 nt), and the RdRP domain (7761–9254 nt) A. Meanwhient way A.Moreover, phylogenetic analysis results demonstrated that ASIV1 displayed a close relationship with another virus in Iflaviridae, especially Hubei coleoptera virus 1 and Tribolium castaneum iflavirus B. FurtheFor vsiRNA, ASIV1 triggered vsiRNA with peaks of 22 nt C, which A. sinensis (The full-length sequences of Atractomorpha sinensis ollusvirus 1 (ASOV1) and Atractomorpha sinensis chu-like virus 1 (ASCV1) possess 14,715 nt and 7673 nt, accordingly. The sequence structures including an 85 nt 5′UTR, a 252 nt 3′UTR and three predicted ORFs were included in ASOV1, where the three predicted ORFs were ORF1 (86-3406 nt) encoding 1107 amino acids, ORF2 (4440-6805 nt) encoding 789 aa, and ORF3  encoding 2475 aa. Meanwhile, the InterProScan was employed to predict the conserved domain of ASOV1. The ORF1 and ORF2 had no conserved domain, while ORF3 possessed three conserved domains, namely, RdRP domain , mRNA_cap domain , and FtsJ-like methyltransferase (FtsJ) domain  A. For ASsinensis A,B.Furthermore, the evolutionary relationship of ASOV1 and ASCV1 was explored with other viruses with phylogenetic analysis. ASOV1 had a close relationship with Culverton virus and Scaldis River bee virus, both of which were classified into the ollusvirus in the Aliusviridae with the aa sequence identity of 28.5–52.2%. ASCV1 clustered with Blattodean chu-related virus, which belonged to an unclassified group (Group 2) of Chuviridae, with 61.3% aa and 64.2% nt sequence identities .ASOV1 triggered the peaks of 22 nt D, and unA. sinensis. Negeviruses have a broad host range as a group of insect-specific viruses, including the orders of Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Odonata, Orthoptera, and Thysanoptera [A large number of RNA viruses were discovered using HTS and advanced bioinformatics methods, thus, gaining a better understanding of insect viromes and viral evolution. Recently, numerous RNA viruses have been identified in multiple grasshopper species . In thisanoptera . In the anoptera ,35. Iflaanoptera . In addianoptera ,38. The anoptera . In our A. albopictus C6/36 cell line [A. sinensis responded strongly during the infection of three RNA viruses.As potential biocontrol agents, ISVs have attracted more attention due to their effects on vectoring ability. In addition, they impede some arboviruses’ infections in vivo and in vitro in mosquitos, such as Nhumirim virus and Zika virus, participate in reducing West Nile virus transmission when co-infecting the mosquito vector , and areell line . Otherwiell line ,40,41. IA. sinensis and classified as nege-like virus, iflavirus, ollusvirus, and chu-like virus according to a phylogenetic analysis. Except for ASCV1, other RNA viruses were targeted by the antiviral RNA interference pathway in A. sinensis.In conclusion, ASNV1, ASIV1, ASOV1, and ASCV1 were identified in


In early 2017, the University Medical Center Groningen, the Netherlands, had an outbreak of 2 strains of vancomycin-resistant enterococci (VRE) that spread to various wards. In the summer of 2018, the hospital was again hit by a VRE outbreak, which was detected and controlled early. However, during both outbreaks, fewer patients were admitted to the hospital and various costs were incurred. We quantified the costs of the 2017 and 2018 VRE outbreaks.Using data from various sources in the hospital and interviews, we identified and quantified the costs of the 2 outbreaks, resulting from tests, closed beds (opportunity costs), cleaning, additional personnel, and patient isolation.The University Medical Center Groningen, an academic hospital in the Netherlands.The total costs associated with the 2017 outbreak were estimated to be €335,278 ; the total costs associated with the 2018 outbreak were estimated at €149,025 .The main drivers of the costs were the opportunity costs due to the reduction in admitted patients, testing costs, and cleaning costs. Although the second outbreak was considerably shorter, the costs per day were similar to those of the first outbreak. Major investments are associated with the VRE control measures, and an outbreak of VRE can lead to considerable costs for a hospital. Aggressively screening and isolating patients who may be involved in an outbreak of VRE may reduce the overall costs and improve the continuity of care within the hospital. The number of resistant isolates varies by country. In continental Europe, low resistance generally is found in northern and western countries, while high resistance in found in the east and south. In the Netherlands, the prevalence of VRE is low compared to most European countries, ranging from 0% to 2% of clinical isolates. VRE is mainly transmitted through contaminated surfaces and the hands of healthcare workers; hence, VRE transmission can be reduced by strictly isolating patients carrying the resistant bacterium and by adhering to hygiene guidelines, such as frequent handwashing. An important tool to adequately isolate VRE-carrying patients concerns the screening of high-risk patients, such as patients suffering from gastrointestinal diseases or patients who have been admitted to hospitals in regions with a high prevalence of VRE. Most patients carrying VRE will not become ill due to the resistant bacterium; however, when the VRE causes an infection, it may more difficult to treat compared to susceptible enterococci and result in worse health outcomes, especially for patients with comorbid conditions. A recent study estimated the global number of deaths caused by various enterococci to be almost 100,000 in 2019.Enterococci are bacteria normally present in the human gastrointestinal system. Especially in healthcare settings, enterococci resistant to certain antibiotics are transmitted, most importantly vancomycin-resistant enterococci (VRE).Early in 2017, the University Medical Center Groningen (UMCG) in the Netherlands had an outbreak of VRE, defined as transmission of a specific subtype of VRE to several patients in a defined period and location. Due to an incidental finding during routine screening from a patient who was hospitalized for ∼10 days, patients who shared the same hospital room were screened. Prior to the outbreak, patients previously admitted to hospitals abroad or patients admitted to the gastrointestinal ward were screened. In the months leading up to the outbreak, on average, 9 patients per day were screened, with a positivity rate of 0.6%. During the outbreak, 68 tests were performed per day and 2.8% were positive for VRE. Typing with next-generation sequencing (NGS) showed that 2 strains were causing the outbreak. One of these strains was isolated first from a patient who had previously been admitted to a German hospital and tested positive in November 2016. Despite isolation measures, the VRE strain could spread during December 2016 and the first weeks of January 2017. Due to the movements of patient to various wards and intensive care units, VRE spread to several locations.At UMCG, 2 wards with many positive patients were closed completely and the patients had to be moved to a temporary ward that was only used for VRE-positive or high-risk patients . In UMCG, most rooms are shared rooms. To prevent further transmission and to ensure adequate capacity in the intensive care for acute care, new admissions to the hospital were stopped. In total, 38 patients tested positive for VRE during this outbreak, with 2 separate strains. During the summer of 2018, 27 patients tested positive in another outbreak, all of whom could be traced back to a single VRE-carrying patient. Again, despite isolation measures, the VRE spread. To contain the spread of VRE, new patients were temporarily rejected, current patients were moved to an outbreak ward and the original ward, was completely disinfected using hydrogen peroxide vapor decontamination. Because the outbreak was detected in an early stage and aggressive measures were taken to clean the affected ward, the outbreak was controlled quickly without spreading to other wards within the hospital. During the first outbreak, no invasive VRE infections occurred in the patients who tested positive for VRE. During the second outbreak, the index patient had a positive blood culture, indicating an invasive VRE infection. and in a study in a French university hospital the total costs of a VRE outbreak amongst 13 patients was estimated at €171,439 , with the opportunity costs being a major driver.Few studies previously assessed the costs associated with VRE outbreaks in hospitals. A VRE outbreak in the UMCG in 2013 was estimated to cost ∼€3800  per dayDuring these outbreaks, various costs were incurred by the hospital, such as the costs of cleaning, personnel costs, laboratory costs and lost costs due to closed beds. In this research, we quantified the costs directly attributable to the 2017 and 2018 VRE outbreaks at UMCG. In a prior study, in which costs associated with several outbreaks in the UMCG were quantified, 5 main categories of costs were identified: diagnostics, closed beds, cleaning, additional personnel, and patient isolation. These categories were also assessed in this study. To calculate the costs associated with each category, we estimated volumes of the various categories and multiplied them with the unit costs. To estimate the volumes of the various items, we used clinical data and data collected during interviews with representatives of various departments that were affected by the outbreaks.The UMCG is an academic hospital in the Netherlands with >28,000 admissions annually, a workforce of >11,000 full-time equivalents, and an annual revenue of €1.6 billion (US $1.7 billion).The first outbreak started January 10, 2017, and ended February 21, 2017. The second outbreak started August 21, 2018, and ended September 8, 2018. For both outbreaks, transmission occurred before the starting date. We considered only the periods when hospital staff were aware of the outbreak. For the analyses, the VRE status of patients is important, and we used 3 categories: (1) VRE positive, for patients confirmed to carry VRE, having positive results in both a polymerase chain reaction (PCR) test and a culture; (2) VRE suspect, for patients at high risk to carry VRE, including patients sharing rooms with VRE-positive patients, and in case VRE was spread across several rooms, everyone admitted to an affected ward for >24 hours; and (3) VRE negative, for patients not suspected of carrying VRE.The methods for sampling and culturing VRE in the UMCG are described in detail elsewhere.Interviews. Overall, 8 interviews with representatives of relevant departments were conducted to get an overview of the relevant costs. We interviewed staff from the infection prevention unit, the microbiological and viral laboratories, the most severely affected department , facility services, procurement, and business intelligence. These interviews were used to get an expert opinion on various costs, but interviewees were also requested to provide data sources where available.Data sources. Various sources of clinical input data were used to assess the unit volumes used for the cost analysis. Additional hours worked by nurses were registered by the planning department. Patient movement data were used to estimate the number of times a room had to be cleaned. Cleaning staff was estimated to need 1–2 hours to complete cleaning per room in which a VRE-positive patient was admitted. The VRE decontamination procedure for these rooms included spraying the room with a chlorine solution, disinfecting the beds and medical devices, removing unwrapped disposables, and replacing the curtains. Some patients were admitted to rooms where >1 VRE-positive patient was admitted; therefore, we assumed that the cleaning time per VRE-positive patient was 1 hour. Patient isolation data were used to estimate the number of days VRE-positive or VRE-suspect patients were put in isolation to prevent further spread through the hospital. Ward occupancy data were used to estimate the opportunity costs due to closed beds. Laboratory data were used to estimate the volume of VRE tests performed, from both patient and environmental samples. were used where possible; otherwise, data from previously published literature were used. Table  Internal cost calculations from the microbiology department were used for the unit prices of the various tests. These are the costs that are offset to the clinical departments; they include staff costs but do not reflect commercial prices as all tests were performed by the internal laboratory of the UMCG. Cleaning costs per hour were used as published in previous research. For the 2018 outbreak, the affected ward was cleaned using hydrogen peroxide; the invoice totals were used in the analysis.To estimate the unit costs, Dutch reference prices For each ward, the best-fitting ARIMA model was automatically determined using the Hyndman-Khandakar algorithm. To estimate the opportunity costs, we used the difference between the measured occupancy and the bootstrapped 95% prediction interval of the forecast occupancy. For example, if the lower bound of the ARIMA model predicted an occupancy of 10, but only 6 patients were admitted to a ward, the difference (10 – 6 = 4) was used to calculate the opportunity costs due to closed beds. The result was then multiplied by the ward stay costs to estimate the opportunity costs  models.During both outbreaks, a transfer ward was established to isolate VRE-positive patients. To prevent the double counting of outbreak-related costs, we did not include the hospitalization costs of this transfer department in the analysis because they are counted using the ARIMA models.For all VRE-positive patients, we assumed 1 hour of cleaning for every movement through the hospital, from the moment at which the VRE infection was confirmed. For all VRE-suspect patients, we also assumed 1 hour of cleaning for every movement through the hospital, until they were confirmed to be VRE negative. If this exact point of time was unknown in the patient isolation records, we assumed a period of 48 hours between the start of the suspicion of VRE and the confirmation of the VRE status, either positive or negative. Patient isolation costs were applied to all patients, for each isolation day.Test costs were calculated by multiplying the number of tests performed with the costs per test. After the 2017 outbreak, an inhouse PCR test was developed, resulting in a less expensive test used in the 2018 outbreak, compared to the 2017 outbreak, where the Cepheid GeneXpert  was used. A patient’s first positive PCR result was followed by NGS. VRE tests are performed regularly in the UMCG. To correct for the baseline level of VRE tests, we used the average daily number of VRE tests from the preceding 4 months and subtracted this number from the total tests during the outbreaks. For example, if the average daily tests in the 4 months prior to the first outbreak was 9 and the average number of tests per day during the outbreak was 68, then a total of 68 − 9 = 59 daily tests were counted toward the outbreak costs. Due to the confidentiality of the unit prices for tests, only aggregated costs were reported. For time series analyses, the fable package was used. In addition to the total costs of both outbreaks, the outbreak costs per day were calculated.All analyses were performed using R version 4.1.0 software  with the dplyr package for data transformation.The Medical Ethical Committee of the University Medical Center Groningen (UMCG) exempted the present study from a full review according to the Dutch Medical Research with Human Subjects Law because it falls outside the scope of this law. This research was approved by the UMCG Central Ethics Review Board and was considered to adhere to all applicable laws, such as the Medical Treatment Agreement Act, Data Protection Act and the Code of Conduct for Responsible Use.During the first outbreak, in 2017, 38 patients tested positive for VRE. In the second outbreak in 2018, 27 patients tested positive for VRE. Table In this study, we assessed the outbreak costs of 2 VRE outbreaks in the UMCG . The total costs of the 2017 outbreak were estimated at €335,278  and the total costs of the 2018 outbreak were estimated at €149,025 . The main drivers of the costs were the opportunity costs, additional diagnostics, and cleaning costs. Although the second outbreak was considerably shorter, the costs per day were similar to those of the first outbreak. Compared to the first outbreak, cleaning costs were considerably higher during the second outbreak. However, due to the early intervention and isolation measures, fewer wards needed to be screened for VRE, resulting in a major decrease in PCR tests (by a factor of 10). Although the first outbreak was longer, fewer additional hours were worked by nurses. This difference may have been due to a constrained availability of staff because no additional nurses could be found. The overall reduction in costs for the 2018 outbreak may be due to the early, intensive response, which may have prevented a hospital-wide outbreak that could have resulted in overall costs comparable to the 2017 outbreak. However, this outbreak was smaller; 19 patients were involved and only 1 ward was affected. Like the 2017 and 2018 outbreaks, the major drivers of these costs were those of diagnostics and opportunity costs due to closed beds. The French study also estimated that the loss of income from spare isolation beds was a major driver of the overall outbreak costs. A difference in this study with ours concerns the inclusion of the costs of antibiotics, which was the second most important driver of costs. We also considered the inclusion of antibiotics; however, the main alternative for VRE patients is teicoplanin, and the price differences between vancomycin and teicoplanin in the Netherlands are negligible. Thus, we decided to not include these as extra costs.Compared to a previous study of a VRE outbreak in the UMCG, the costs per day were considerably higher in this study: ∼€7,900  compared to €3,800  in 2013, the latter amounting to €4,100  in 2020 euros.This study had several limitations. It is complex to accurately estimate the opportunity costs due to closed beds because patients are exchanged between the various wards in the hospital. Also, we were unable to determine exactly how many patients went to other hospitals during the period of no new admissions at UMCG. We tried to estimate this cost using various ARIMA models, and we considered the measured occupancy outside the 95% prediction intervals to be caused by the outbreaks. This is a conservative approach; the prediction intervals are rather wide due to the variability in the data. Hence, we may have underestimated the opportunity costs due to closed beds. In December 2017, the UMCG switched the computer system used to measure the ward occupancy and movements of patients through the hospital, resulting in poorly comparable data for the 2 periods. We trained the time-series model on the 4 months preceding both outbreaks to make sure the data cut caused by the new system did not affect the analysis, but this prevented us from fitting more advanced predictive models.Another limitation related to the cleaning costs. Although the cleaning procedures for patients in contact isolation are rather strict, no data were available regarding increased staff expenditure and cleaning materials. Instead, an approximation was used in which we counted 1 hour of cleaning time per isolated patient. The cleaning costs in the 2018 outbreak were higher compared to the 2017 outbreak. In the 2018 outbreak, hydrogen peroxide vapor decontamination was used to ensure a rapid reopening of the ward. This was very precisely accounted for in this analysis because the invoice was available. Although the costs associated with hydrogen peroxide vapor decontamination are substantial, we did not assess its effectiveness relative to standard cleaning procedures. The overall costs of the second outbreak may have been lower if cleaning staff had thoroughly cleaned the affected ward instead of the specific approach with hydrogen peroxide. The limitations concerning data collection raise an important opportunity to improve the registration and availability of data. Even for directly involved staff, high-quality data were difficult and in many cases impossible to find. Currently, the incidence of VRE is low in the Netherlands, and the stringent VRE control measures may have played a role. Our results indicate that major investments are associated with VRE control measures. However, the containment of VRE may result in lower healthcare costs overall due to a shorter length of stay and decreased mortality. Estimations from previous research show that annually ∼16,000 VRE infections occur in Europe, causing >1,000 deaths, but the burden of VRE-related morbidity and mortality in the Netherlands is very low.We did not consider clinical consequences of VRE-positive patients because we focused on the direct costs attributable to the outbreak. A meta-analysis including 12 cohort studies found increased mortality for patients with a VRE bacteremia and increased length of stay compared to vancomycin-susceptible enterococci (VSE).From this study, we can conclude that an outbreak of VRE can lead to considerable costs for a hospital. Although each outbreak differs, aggressively screening and isolating patients who may be involved in an outbreak of VRE may reduce the overall costs and improve the continuity of care within the hospital.


Among the different pathogenic microorganisms described in animals and humans, viruses show huge diversity. Their complex interactions with their host are often challenging to study, thus limiting the development of prophylactic and therapeutic treatments.The emergence of African swine fever (ASF) in China in 2018 has placed an increasing economic burden on many countries. The virus (ASFV) responsible for this disease was identified in Kenya in 1921   present the latest knowledge about the complex interplay between ASFV and innate immunity and its impact on viral pathogenesis. Ultimately, research studies deciphering the complex mechanisms of AFSV pathogenesis may facilitate the development of safe and effective live-attenuated vaccines against this terrible disease. This topic is currently hot and reviews are needed to keep everyone on track. Then, Proctor et al. (rising star Dr. Tobias Käser) investigated the vaccine efficacy and immunogenicity of a modified live virus (Prevacent® PRRS MLV vaccine) against four heterologous type 2 PRRSV (PRRSV-2) strains in 60 pigs, and showed that Prevacent elicits various degrees of efficacy and immunogenicity against these four phylogenetically distant strains.The articles featured in this Research Topic dedicated to “Bigay et al. (rising star Dr. Pauline Maisonnasse) present the mechanisms that may be associated with VAED risk and that have to be taken into consideration when assessing vaccine safety and searching for ways to define models and immunization strategies for alleviating such concerns. Predicting VAED is clearly challenging, and the authors highlight various research areas that can be improved to better deal with this issue. Then, with regard to antiviral drugs, Mao et al. (rising stars Dr. Chen and Dr. Sheng) suggest the potential use of the antipsychotic drug trifluoperazine (TFP) against viruses such as vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). They identified that TFP can inhibit virus replication through a protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis.After these two articles dealing with pig viruses, we move to other animal models and humans and the complex question of VAED. In their review, Together these original review articles represent significant contributions to the infectious agents and disease field, demonstrating that the new generation of scientists is ready to shine in our fascinating field of research.Both authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.