Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log Aphthovirus of the family Picornaviridae [Aphthovirus also includes equine rhinitis A virus (ERAV), bovine rhinitis A virus (BRAV) and foot and mouth disease virus (FMDV), the last being one of the most significant livestock viruses worldwide to which BRBV is closely related. Bovine rhinitis A virus consists of two serotypes, BRAV1 and BRAV2, while only a single serotype of BRBV has been described [The species Bovine rhinitis B virus (BRBV) is a non-enveloped, single-stranded, positive-sense RNA virus belonging to the genus aviridae . The genescribed . The incescribed , the impFormerly bovine rhinovirus, BRAV was first isolated in Germany in 1962 and has been reported from the USA, England, Japan, Italy, Sudan and Mexico . OriginaMannheimia haemolytica, Histophilus somni and Pasteurella multicoda, and viruses included are bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BHV1), bovine viral diarrhoea virus (BVDV) and parainfluenza virus 3 (PI3) [Bovine respiratory disease (BRD) is the most economically significant disease of the cattle industry, accounting for more than $1 billion in annual losses in the USA alone . In the  3 (PI3) . Despite 3 (PI3) .Despite being established aetiological agents of BRD, BRAV and BRBV have been neglected for decades; however, more widespread use of NGS in recent years suggests that bovine rhinitis viruses play a clinical role in BRD . A viralin situ hybridization revealed BRBV infection in the epithelial cells of nasal mucosa, trachea and brain. We also studied the prevalence of common bovine respiratory pathogens in nasal swab and lung samples sent for BRD diagnosis and found that BRBV was the most common virus identified in nasal swabs from symptomatic cattleIn this study, we isolated BRBV from a nasal swab collected from a calf with clinical respiratory disease. Following intranasal inoculation of naïve calves, the virus was isolated up to 8 days post-inoculation (dpi) from nasal swabs, neutralizing antibody was detected after 7 dpi, and Mycoplasma bovis, H. somni, Mannheimia haemolytica, Bibersteinia trehalosi and P. multicoda. Viral pathogens included in the panel were BVDV, BHV1, BRSV, BCV and IDV. Samples were additionally screened for BRBV [The prevalence of common bovine respiratory pathogens was studied in nasal swab and lung samples submitted for BRD PCR panel testing at the Kansas State Veterinary Diagnostic Laboratory as part of routine herd management and veterinary care. Samples were collected from farms in Kansas between 1 October 2020 and 27 January 2021. Quantitative reverse transcriptase PCR (qRT-PCR) was used to detect viral and bacterial pathogens. Bacterial pathogens included in the PCR panel were n=22). Insufficient sample quantities prevented virus isolation from samples used for the BRBV prevalence study. Primary bovine tracheal epithelial cells developed in an earlier study were used to isolate BRBV [2. Confluent cells were inoculated at a volumetric 1 : 1000 dilution of 0.22 µm filtered nasal swabs resuspended in PBS. Infected cells were cultured at 33 °C in 5 % CO2 for 5 days. Virus isolation was confirmed by cytopathic effects (CPEs), qRT-PCR and genome sequencing.Nasal swabs submitted to the South Dakota State Animal Disease Research and Diagnostic Laboratory for BRD testing were screened for BRBV by quantitative RT-PCR. Virus isolation was attempted on positive samples with Ct values <25 (ate BRBV . Cells wde novo in CLC Genomics and identified by blastx using the integrated Blast2Go plugin and the GenBank non-redundant protein database.Metagenomic sequencing was performed as previously described . Paired n=7) and isolated virus BRBV strain 6900. Complete coding DNA sequences and partial non-coding regions of the genomes were assembled.Metagenomic sequencing was performed on BRD submissions used for the BRBV prevalence study with qRT-PCR Ct values <25 for BRBV  were mock-inoculated with PBS.Five calves were used for the study. Three calves  were challenged intranasally with 10 ml of 6.0 logCalves were observed daily and clinical scores were recorded as previously described . Beginni °C for 30 min then serially diluted in DMEM/F12 media. Equal volumes of sera were combined with 100 TCID50 of virus at 33 °C for 1 h. The serum and virus suspension was used to inoculate primary bovine tracheal epithelial cells, which were observed for 5 days for CPEs. The neutralizing antibody titre was calculated as the reciprocal of the highest antibody dilution capable of neutralizing 100 TCID50 of virus.Sera were heat-inactivated at 56Viral RNA was isolated using a commercially available kit . qRT-PCR was performed using a one-step qRT-PCR kit  following the manufacturer’s protocol. The Taqman assay used in the study was previously reported . For ampA DNA fragment encompassing the region targeted by the qRT-PCR assay flanked by a 5′-T7 promoter sequence was synthesized and used to generate a synthetic RNA transcript using a MAXIscript kit following the manufacturer’s instructions. The concentration of the resulting RNA was determined using a Qubit RNA kit following digestion with DNase I. Serial 10-fold dilutions of synthetic RNA were used to generate a standard curve to correlate RNA copies to the qRT-PCR Ct value .Tissues were fixed overnight in 10 % neutral buffered formalin. Tissues were routinely processed at the South Dakota State University Animal Disease Research and Diagnostic Laboratory. Haematoxylin and eosin (H&E)-stained slides were used for histopathological examination.In situ hybridization was performed on formalin-fixed paraffin-embedded (FFPE) tissue using a commercially available kit . Slides were deparaffinized, re-hydrated and blocked with hydrogen peroxide. Following antigen retrieval, sections were hybridized with the probe designed against the 3D polymerase gene region of the BRBV genome. The presence of BRBV in the tissue section was visualized using the fast red substrate. In situ hybridization was performed in duplicate on four sections of nasal turbinates, three sections of trachea, six sections of lung and two sections of cerebrum.g for 5 min, filtered through a 0.22 µm filter and stored at −80 °C. For titration, primary bovine tracheal epithelial cells were seeded in 96-well plates and upon 70 % confluency 10-fold serial dilutions of the tissue homogenates were used to inoculate cells. Cells were cultured at 33 °C and 5 % CO2 for 5 days. Cells were observed for visible CPEs. Virus titre was determined using the Reed and Muench method of determining 50 % endpoints [Tissue homogenates were prepared from the nasal mucosa, upper and lower trachea, lungs, mediastinal lymph node, tonsil and brain collected on day 5. Tissue samples were homogenized in Hanks’ balanced salt solution, pH 7.4, centrifuged at 12 000 ndpoints .A collection of 49 nasal swabs and 153 lung samples submitted for BRD diagnostic testing were analysed for bacteria and viruses associated with BRD by qRT-PCR. Bovine rhinitis virus was detected in 20 of 49 (40.8 %) nasal swabs but only in 18 of 153 (11.8 %) lung samples . BRBV waMetagenomic sequencing was performed on seven of the BRBV-positive samples that had the lowest Ct values for BRBV. Complete BRBV genomes were assembled for strains D2 and B10. These sequences were submitted to GenBank under accessions OL410605 and OL410606.Bos taurus, mapped to BRBV and we were able to obtain a near complete BRBV genome sequence of 7466 nt. Strain 6900 was submitted to GenBank as accession MZ574106. A blastn search of the NCBI nucleotide database identified a maximum of 89.5 % identity to a BRBV genome from Mexico (GenBank accession KU159357). The isolated BRBV strain 6900 was 79.7 % identical to the reference BRBV strain EC-11.Nasal swabs collected from cattle with clinical BRD and submitted for diagnostic testing were used for virus isolation. BRBV strain 6900 was isolated from a nasal swab collected from a calf in California with acute respiratory disease on primary bovine tracheal epithelial cells. Five days post-inoculation, a CPE was evident and virus growth was confirmed by qRT-PCR (Ct <20), and NGS was used to determine the viral genome sequence and to confirm the absence of any exogenous organisms. All of the sequencing reads, excluding those derived from the host cell n=19) and the three genomes determined here. Strains 6900 and B10 clustered with four genomes recovered from calves with BRD from a large calf ranch in California, designated BSRI2  on day 2 post-challenge which resolved by day 3. No other clinical signs were observed.Three 7-day-old calves were inoculated intranasally with 10Nasal swabs were analysed by qRT-PCR. All calves were negative for BRBV on the day of challenge. BRBV RNA was detected in nasal swabs from day 1 to 11 for BRBV-challenged calves . Mock chTissues collected at 5 dpi from two BRBV and one mock-challenged calves were analysed by qRT-PCR. Nasal turbinates and trachea samples from both BRBV inoculated calves were positive . For calMetagenomic sequencing of pooled nasal swabs collected on the day of challenge unexpectedly identified BCV. Despite transfer of calves to BSL2 isolation immediately after birth, the calves were naturally infected with BCV, presumably due to exposure to BCV-contaminated faecal material during birth. To confirm this finding, qRT-PCR for BCV was performed on individual nasal swabs. All calves were positive with Ct values of 18.1–28.2 on day 0. Likewise, nasal swabs from all calves were positive for BCV on day 1 post-challenge. By day 5, only calves 206 and 203 remained positive for BCV. Complete genomes with >99 % identify to BRBV strain 6900 were assembled from nasal swabs from all BRBV-challenged calves on days 1 and 5 post-inoculation. Apart from BCV, no other pathogens were identified.Both mock-challenged calf 206 and BRBV-challenged calf 205 were negative for neutralizing antibodies at days 0 and 5 post-challenge. Calf 206 remained seronegative to day 21. In contrast, calf 205 had measurable serum neutralizing titres to BRBV strain 6900 of 20, 60, 160 and 80 on days 7, 10, 14 and 21, respectively.Examination of routine H&E-stained slides showed no obvious microscopic lesions in tissue from both BRBV- and mock-inoculated calves. There were small numbers of resident lymphocytes, plasma cells and histiocytes in the mucosa of the upper respiratory tract with no differences between groups.in situ hybridization (ISH) signals were observed in small clusters and individual epithelial cells, covering approximately 10–25 % of the upper respiratory tract . Although BCV is a known aetiologic agent of BRD, it is also commonly carried in asymptomatic animals . BCV wasReproduction of significant clinical disease with BRBV is challenging. Clinical disease was not apparent in our pathogenesis study, with histological lesions relatively minor and limited to the upper respiratory tract. The ability of BRBV to serve as an initiator of BRD is unknown but warrants further study. In addition to costs directly attributed to clinical disease, economic losses from subclinical BRD are estimated at around $50 and 16 kg of carcass per animal . InclusiClick here for additional data file.


Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.Sunflowers ( Helianthus annuus L.) production is an economically important crop. In China, the total sunflower acreage planted, mainly for the non-oilseed types, was relatively stable with fluctuations from 1978 to 2018. The average yield, however, increased steadily from 1077 kg/ha to 2707 kg/ha, so the overall trend of sunflower production is increasing. In the USA, the yield and production show a stable trend due to the improved hybrid varieties, despite declining sunflower acreage [Plasmopara halstedii, Phomopsis helianthi, and Puccinia helianthi, respectively, and symptoms are readily identifiable in the field. Unfortunately, existing controls based on host resistance or fungicides have not provided sufficient control to date, which calls for developing alternative control methods. Annual sunflower . Fiiewed in ,8. Howevalstedii ,10, and etically  and can etically . Crypticetically . TherefoMacrophomina phaseolina tobamo-like virus 1 [Sometimes, lab isolates of fungal cultures have sectoring growth and are discarded by the pathologists when the transferred isolates grow slowly and become hard to maintain. Therefore, candidates of biopesticide or biotechnological use could be eliminated in the culturing/serial passage process, e.g.,  virus 1 ,14. More virus 1 ,15 bypas virus 1 .In this study, we directly identify novel viruses from the diseased tissues of sunflowers caused by rust and phomopsis. This direct approach of sequencing diseased tissue without first culturing bypasses the possibility of selecting for the mildly attenuating or asymptomatic mycovirus infections and allows us to characterize the plant viromes of the pathogens to include the ones that cannot be easily cultured in the lab.To identify potential mycoviruses from the field, tissue samples of diseased sunflowers were collected in the summer of 2017. Symptomatic leaf samples from four diseased plants, each for phomopsis and rust, from Brookings County and Sully County in South Dakota, USA, were sequenced, respectively, in this study. The two diseased plant samples were collected separately from different fields on different dates and were not coinfected by the two pathogens. The phomopsis plants were sampled early to mid-September 2017 for leaves showing symptoms but not yet completely wilted. Specifically, the green leaf tissues on the edge of the lesions caused by phomopsis were sampled because the lesion area had very little intact RNA left (data not shown). Four leaves per plant were stacked, punched into discs, divided into 150 mg per tube, flash-frozen in liquid nitrogen, and stored at −80 ℃ until further processing. Leaf discs containing rust pustules and between the pustules were both included in the RNA extraction.RNA extraction was conducted as previously described by Marzano et al. . Leaf diTotal RNA (approximately 1 μg) free of genomic DNA was depleted of rRNAs with the Ribo-Zero Plant Kit and used as a template to construct paired-end libraries with a ScriptSeq RNA sample preparation kit , and were cleaned and sequenced on an Illumina HiSeq 4000 for pair-end sequencing through the W.M. Keck Center, University of Illinois. SF1 and SF2 libraries were constructed using RNA extracts from phomopsis- and rust-infected leaves, respectively.The microbiome associated with sunflower leaves was profiled using DIAMOND  by alignPartitiviridae and 44 for Mitoviridae because the new viral contigs would not cluster with other viruses otherwise. For other viruses, the thresholds were set at 50.The predicted amino acid sequences of potential novel viruses were aligned to the contigs from NCBI using ClustalX 2.0 . PhylogeCandidatus kryptonium, Bacillus azotoformans, and Staphylococcus haemolyticus. However, the SF1 library had nearly 10X more Actinobacteria in normalized read counts than the SF2 library. In addition, most of the HaEV1 reads were aligned to the SF1 library, in contrast to the SF2 library, which had only mitoviruses and dsRNA viruses  detected using the MEGAN analysis based on short read alignments  of phomopsis stem canker of sunflower were detected from infected leaves, coinfected by other fungi under families Sclerotiniaceae, Hypocreales, and Pleosporineae. Some bacterial taxa are common to both libraries in approximately the same amount, including ignments .Alphaendornavirus genus.The predicted amino acid sequence of a known virus, HaEV1,  contained a conserved polyprotein with 99.8% identity to the previously published Helianthus annuus alphaendornavirus sequence (NC_040799.1) and is related to another alphaendornavirus (MN015676.1) with 40% identity. Besides these two viruses, the HaEV1 amino acid sequence was also grouped with other endornaviruses, including Helicobasidium mompa alphaendornavirus 1, Soybean leaf-associated endornavirus 1, Phytophthora endornavirus 1, and Vicia faba endornavirus. Therefore, based on the structure of the phylogenetic tree and the genus of known viruses from the International Committee on Taxonomy of Viruses (ICTV), HaEV1 belongs to Sclerotinia homoeocarpa mitovirus  and 43.9% identity to the RdRp of Ophiostoma mitovirus 3a (2617 nt) [Mitovirus genus  and Cronartium ribicola mitovirus 1 .HlaMV1 , discovered in this study, contained an RdRp with 43.7% identity to the RdRp-like protein of us genus . HlaMV2 Partitiviridae family. The contig named HlaPV1  contained an incomplete open reading frame (ORF) of RdRp, which shares identity with Raphanus sativus cryptic virus 1 (66.8%) (YP_656506.1), Radish partitivirus JC-2004 (66.1%) (AAU88207.1), and many other alphapartitiviruses  shows similarities to the RdRp of Medicago sativa deltapartitivirus 1 (MF443259.1) and Pyrus pyrifolia associated RNA virus (BAA34783.1) with 60.5% and 59.7% identity, respectively. The Helianthus annuus leaf-associated partitivirus 3  contig shares 76% identity with unclassified Partitiviridae: Verticillium dahlia partitivirus 1 and Botryotinia fuckeliana partitivirus 1 (NC_010349.1) through BLASTX. The phylogenetic tree shows that HlaPV3 was most closely related to members of the Gammapartitivirus genus (Three contigs were predicted to express the RdRp proteins with similarities to the viruses belonging to the iviruses . The RdRus genus .Helianthus annus leaf-associated partitivirus 4 (MZ532542), Helianthus annus leaf-associated cryptic virus 1 (MZ532543), Helianthus annus leaf-associated partitivirus 5 (MZ532544), and Helianthus annus leaf-associated cryptic virus 2 (MZ532556)  .Totiviridae family were found and named as Helianthus annuus leaf-associated totivirus 1 , Helianthus annuus leaf-associated totivirus 2 , Helianthus annuus leaf-associated totivirus 3 , and Helianthus annuus leaf-associated totivirus 4 . The predicted amino acid sequence of HlaTV1 contains a complete RdRp which shares 50% identity to Puccinia striiformis totivirus 4 (KY207364). The HlaTV2 contig has 37% identity with RdRp of Puccinia striiformis totivirus 2 (KY207362). Both HlaTV3 and HlaTV4 are similar to Puccinia striiformis totivirus 5 (KY207365) with identities of 49% and 79%, respectively. It is worth noting that Puccinia striiformis is another rust fungus, which suggests that our approach was successful in identifying viruses associated with the pathogenic fungi. These four viral contigs are more closely related to the viruses belonging to the Totivirus genus than other genera in the Totiviridae family , 10 viral contigs were identified, including 9 totiviruses and a victorivirus (MZ532545~54) . Among tA potyvirus-like contig was also identified, which shares 99.5% amino acid identity to bean yellow mosaic virus (BYMV) for the 555 nt contig and has only 70% nucleotide identity to the Meadow saffron breaking virus polyprotein gene and the Butterfly flower mosaic virus. As the sequences for BYMV are very diverse, and one of the top hits (93.3% nt identity) in BLASTX for this BYMV-like sequence is a BYMV isolate from sunflowers in Iran encoding a portion of the CP, we still named it as BYMV. The naming is based on the knowledge that CPs need to form specific structures to assemble virus particles, so their amino acid sequences are often highly conserved. Moreover, based on coat protein similarity, we detected a short contig (197 nt) that came from very few reads in both libraries and is similar to the Sclerotinia sclerotiorum hypovirulence-associated DNA virus. It is divergent from the other detected CPs from dsRNA viruses, and therefore excluded from the phylogenetic analysis shown in Phomopsis spp. infection. Phomopsis stem canker in sunflowers has been reported to be associated with at least 15 species of Diaporthe that are either pathogenic or non-pathogenic [Diaporthe (Phomopsis) species [Phomopsis warrants further investigation into whether the virus replicates in P. helianthin, and the effects of HaEV1 on fungal virulence. It has been reported that a plant virus, cucumber mosaic virus, can naturally infect a fungal host, Rhizoctonia solani [Diaporthe spp., which will be reported in a separate paper. The current study has the limitation that more validation is required to cross-check with Diaporthe metatranscriptomes, for example, to make a connection to phomopsis-infecting mycoviruses. Nevertheless, this study profiled known and novel sunflower-associated viruses from the field and provided hypotheses for further laboratory determination. In addition, endornaviruses belong to a group of viruses without true virions, which will require the construction of infectious cDNA clones driven by a T7 promoter in future studies to establish cause-and-effect relationships.In this study, we identified 25 putative viral contigs using a metatranscriptomics approach, ranging from 1.6 kb to 14.6 kb for the RNA viruses, and one short DNA virus contig. Only one of the viruses, an endornavirus, had been described previously as Helianthus annuus alphaendornavirus (HaEV1). It was sampled from leaves showing typical symptoms of brown, irregular-shaped spots on infected leaves, which coalesced into large patches of dead tissue spreading in from the leaf margin, indicating  species . Althouga solani . TherefoBased on RdRps, totiviruses with less than 50% amino acid sequence identity are considered different species. Therefore, HlaTV2 and HlaTV3 are considered new species. For partitiviruses, the threshold is 40%: amino acid sequence similarity >40% between RdRps of viruses from different species in the same phylogenetic cluster and <40% between members of species in different clusters; therefore, new species are reported in this study. Based on CPs, we discovered more viral contigs than RdRps, and it will require further investigation to better characterize these newly reported viruses. Using similarities to existing CPs, we detected more contigs, probably because they need to be translated to specific protein structures to assemble virions and are more conserved. Although some viruses lack CPs, such as endornaviruses and mitoviruses, using CPs to detect new viruses is very useful for the viruses that encode CPs.S. sclerotiorum that does not produce sclerotia is a desirable phenotype [We previously reported a survey of soybean leaf-associated virome from field-collected tissues regardless of the disease symptoms . In thishenotype , and thehenotype  or to behenotype . Again, To simultaneously compare the changes in microbiome and virome in the field or in greenhouse production, the same direct sequencing of total RNA without culturing, in a repeated sampling scheme, should be carried out in the future. Ecological modeling should be performed to include the abundances of each virus and overall microbial population, including fungal pathogens, shown in normalized read counts based on the high throughput sequencing data, in conjunction with the disease severity rating, and the environmental conditions for multiple time points. Applying such a systems approach will provide us new insights into the emergence and maintenance of viromes facing environmental variability, which would be proactive, considering their importance as ecological drivers of the pathogen population and overall microbial composition. A study to include multiple sites can be advantageous, especially if the disease in question is widespread and the pathogen is cosmopolitan. It would be interesting to monitor whether there is a correlation between the mycoviral read counts and the disease severities and yield intra-annually , since y


Porcine parainfluenza virus 1 (PPIV-1) is a recently emerged respirovirus closely related to human parainfluenza virus 1 (HPIV-1) and Sendai virus (SenV). PPIV-1 has been detected in Asia, the Americas and Europe, but knowledge on its epidemiology and genetic diversity is very limited. In the present study, the complete nucleotide sequences of the fusion (F)-protein gene obtained from samples from 12 Polish and 11 US herds were analysed and compared to previously available genetic data from the Americas, Asia and Europe. The existence of two distinct clades was observed, grouping European sequences and one Hong Kong sequence (clade 1), or one American sequence and three Asian sequences (clade 2). The mean genetic distances measured with the p-distance were 0.04  within both clades, and 0.095  between the clades. Moreover, two distinct clusters of highly similar sequences were identified, which corresponded to the geographically distant nurseries and finishing units, from three pig flows within one Polish pig-production company. The obtained data indicate that the two PPIV-1 lineages may have evolved independently in Europe and America. More studies, particularly involving Asian viruses, are necessary to understand the virus’ emergence and epidemiology and the role of carriers in the spread of PPIV-1. Paramyxoviridae, order Mononegavirales, and includes six species causing respiratory tract infections of humans and animals: bovine respirovirus 3 (virus name: bovine parainfluenza virus 3—BPIV-3), caprine respirovirus 3 (virus name: caprine parainfluenza virus 3—CPIV-3), human respirovirus 1 (virus name: human parainfluenza virus 1—HPIV-1), human respirovirus 3 (virus name: human parainfluenza virus 3—HPIV-3), murine respirovirus 1 (virus name: Sendai virus—SenV) and the most recently discovered porcine respirovirus 1 (virus name: porcine parainfluenza virus 1—PPIV-1) . The. The8]. The samples from the USA were collected for different diagnostic purposes, and involved nasal swabs, oral fluids, and a single oropharyngeal swab, from nursery pigs and one neonatal piglet. The samples originated from 11 production sites. With the exception of two farms, they belonged to different owners . The samFor sequence analysis, 17 complete or nearly complete F-gene nucleotide sequences from the USA, Hong Kong, China, Hungary, Chile and Germany previously published and available in GenBank on 15 January 2022 were downloaded. As the three sequences from a single Hungarian farm were nearly identical, only one representative sequence was kept for the analysis [The sequence analysis was performed with Geneious 10.2.6. The sequences were aligned using MUSCLE , and a mp-distance [The mean genetic distances within and between major clades of PPIV-1 were calculated with MEGA11 using the distance . The disAdditionally, the nucleotide sequences were translated and aligned using MUSCLE, and a maximum-likelihood phylogenetic tree was reconstructed with PhyML using the Blosum62 substitution model, with 1000 replicates .


Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This editing can be highly selective, affecting a specific site within a transcript, or nonselective, resulting in hyperediting. ADAR editing is important for regulating neural functions and autoimmunity, and has a key role in the innate immune response to viral infections, where editing can have a range of pro- or antiviral effects and can contribute to viral evolution. Here we examine the role of ADAR editing across a broad range of viral groups. We propose that the effect of ADAR editing on viral replication, whether pro- or antiviral, is better viewed as an axis rather than a binary, and that the specific position of a given virus on this axis is highly dependent on virus- and host-specific factors, and can change over the course of infection. However, more research needs to be devoted to understanding these dynamic factors and how they affect virus–ADAR interactions and viral evolution. Another area that warrants significant attention is the effect of virus–ADAR interactions on host–ADAR interactions, particularly in light of the crucial role of ADAR in regulating neural functions. Answering these questions will be essential to developing our understanding of the relationship between ADAR editing and viral infection. In turn, this will further our understanding of the effects of viruses such as SARS-CoV-2, as well as many others, and thereby influence our approach to treating these deadly diseases. ADAR editing plays an important role in dynamic gene regulation in animals and is a key element of the innate immune response against viral infections. Effects of editing have been documented only in a few groups of viruses, including some examples where editing contributes to viral evolution. Here we summarize available evidence of virus–ADAR interactions across multiple groups of viruses, with primary focus on RNA viruses, including two recent threats, Zika virus and SARS-CoV-2. We call attention to the need for more research on virus–ADAR interactions in light of the role that viral-activated ADARs may play in pathogenesis, including the neurological consequences of viral infections.The ongoing pandemic caused by SARS-CoV-2, a novel coronavirus that emerged in late 2019 , has alrRdRp), RNA viruses have mutation rates higher than those of other viruses . On theus [MeV]  and Pfalus [MeV] ). Althouus [MeV] ). Note tNTO3 in another herpesvirus, varicella zoster virus, where it was thought to play a role in avoiding degradation, although further studies are needed  to produce inosines (I) within double-stranded RNAs (dsRNAs) (Since the discovery of A-to-I (adenosine to inosine) RNA modification in the late 1980s , we have(dsRNAs) , leading(dsRNAs) . In vira(dsRNAs) . Moreove(dsRNAs) . For conADAR loci, of which two encode the enzymes, namely, ADAR  and ADARB1  that are responsible for editing activity. The product from the third locus, ADARB2  does not have known catalytic activity, despite encoding an intact open reading frame   isoform .ADAR1 and ADAR2 have been associated with these two types of editing  a highly selective one, where deamination occurs at a specific site within a transcript, such as the Q/R site of the glutamate receptor subunit GRIA2 , and 2)  editing , althoug editing —remian t editing . Yet, in editing —includin editing . Such ed editing . Nonethe editing  as part  editing , for exa editing  or highl editing . As described in the measles (MeV) and dengue (DENV) virus examples below, the pro-/antiviral action is likely not a dichotomy, where editing has either only pro- or antiviral consequences; but rather appears to be a situation-specific outcome that depends on the viral strain and specifics of the host immune response. For example (presumably deleterious) sequence changes introduced early in the infection by ADAR editing as part of the antiviral defense may be proviral given the selective context imposed by a different arm of the immune response, for example, in immunocompromised individuals. In the latter category, the most prominent example is ADAR1’s ability to block activation of IFN-stimulated protein kinase regulated by RNA , whether through direct binding to PKR or by editing viral dsRNA to prevent activation of dsRNA sensors. This is because activated PKR acts in an antiviral manner by triggering apoptosis, shutting down translation, and enhancing IFN-beta production .Strong (or Susceptible to ADAR editing), and the latter (A’s in the GA dinucleotides) as Weak (or Resistant) ADAR sites, respectively , replicated via reverse transcriptases, and satellite hepatitis D virus (HDV), which lacks its own RdRp and relies on an obligatory helper such as hepatitis B virus (HBV) envelope protein , to g dsRNAs . Thus, wogenesis . Similarogenesis . It has ogenesis  or that ogenesis , for exaogenesis  rather togenesis . NonetheBelow we summarize what is known about evolutionary consequences of ADAR editing across major RNA viral groups , a member of the Arenaviridae family, evidence of ADAR hyperediting has been reported within the context of the activated IFN pathway and resultant elevated levels of  isoform . Becausetivation , though tivation . Notablytivation . Furthertivation .The influenza A virus (IAV) is known to generally induce strong immune response, including activation of type I IFNs . InteresM (matrix) gene of IAV, accompanied by a higher viral load and stronger inflammation ssRNA viruses that have a broad range of hosts and includes numerous pathogens of humans and other animals, including MeV, Ebola virus (EBOV), human parainfluenza viruses (HPIV), mumps virus (MuV), vesicular stomatitis virus (VSV), as well as plant viruses . It alsoHyperediting in the MeV genome was one of the first studied examples of ADAR editing, where clustered A-to-G mutations were identified from persistent MeV infection samples isolated  even befAlthough ADAR activation has been shown to suppress apoptosis in acute MeV infection in cell culture, thus acting in a proviral manner , the impJL) vaccine has been attributed to ADAR (and/or APOBEC) action (JL vaccine (SH) gene that result in premature stop codons and/or disrupt the canonical stop codon , where ADAR-generated A-to-G and U-to-C hypermutations were associated with immune escape . Other p) action . One pos) action , where A vaccine . Similar vaccine , attribu vaccine . Recent op codon . Becauseop codon , these sGP (glycoprotein) gene, was observed when Ebola virus, a filovirus  was passaged in bat cells. Further examination revealed that these mutations accumulated within the same genome strands and were consistent with the ADAR 5′ targeting specificity, with an excess of 5′ A/U and depletion of 5′ G  mutations within a  of 5′ G . Notably of 5′ G , because of 5′ G . No such of 5′ G , althoug of 5′ G . It shou of 5′ G , but no  of 5′ G , though  of 5′ G  or be se of 5′ G , althoug of 5′ G . Nonethe of 5′ G .NP (nucleocapsid) mRNA and targeted sites had 5' A or U nucleotide neighbors, attributed to p150 activity , Sudan (SEBOV), and Bundibugyo (BEBOV) ebolaviruses in both Vero cells and primary human macrophages , where clusters of U-to-C and A-to-G changes accumulated in a 3' untranslated region of the activity . These eactivity . Althougactivity , it is iactivity . It is nrophages . The latAnother member of the Mononegavirales, Borna disease virus (BoDV), appears to rely on host ADARs activity, specifically, editing by ADAR2 (but not the IFN-regulated ADAR p150) to establish persistent infection in the cell nucleus . In thisInteractions with ADARs have also been reported in multiple members of the Rhabdoviridae family. In VSV infections, reduced levels of ADAR led to reduced viral replication, although ADAR overexpression had no detectable effect on the viral growth . This prDrosophila melanogaster sigma virus (DMelSV), another rhabdovirus that is vertically transmitted , but not in CCapSV and PAegRV viruses , indicating that these viruses may have differing dynamic relationships with their insect hosts  transcripts (Cyfip2), filamin A (Flna), and bladder cancer-associated protein (Blcap) (Ctenopharyngodon idella), an important freshwater fish, showed that the ADAR1 homolog is activated in response to viral infection, together with other immune response genes  , indicatse genes , implyinThe alphaviruses supergroup includes many zoonotic arboviruses that cycle between arthropod insect vectors and a broad range of vertebrate hosts, from fish to mammals, as well as reptiles and birds . Human pADAR overexpression in −/−STAT1 fibroblast cells in two alphaviruses, chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV)  , indicats (VEEV) . Howevers (VEEV) , it is pADAR1 (suppressing expression of p150), but not ADAR2, resulted in increased HCV expression  and ZIKV. Antiviral action of A-to-G editing by ADAR1 was first reported in IFN-stimulated Huh7 liver cells, where multiple A-to-G changes, presumed to be deleterious due to lack of replication, were detected. Subsequent siRNA knockdown of pression , indicatpression . Furtherferences . The molferences .miR-3614-5p that downregulates both ADAR p110 and p150 expression , dengue virus (DENV), yellow fever virus (YFV), and West Nile virus (WNV). To counteract degradation of hyperedited viral sequences , as an epression , suggestpression , includiSimilarly, expression of ADAR appeared to enhance replication of YFV , whereasAnother notable example of ADAR’s effects on flaviviruses is ZIKV, where several studies independently documented footprints of ADAR editing in ZIKV genomes. Specifically, an analysis of substitution patterns, including frequencies of Weak  and Strong  dinucleotide targets of ADAR editing  which, . BecauseAedes albopictus mosquitoes showed enrichment of metabolites associated with ADAR editing  and other enteroviruses, hepatitis A virus, and various rhinoviruses, among others . EvidencAlthough such events are quite rare , the facThe order Nidovirales encompasses a group of enveloped (+)ssRNA viruses that cause acute and persistent infections in mammals and birds, of which Coronaviridae represents the largest family. This family consists of coronaviruses (CoVs) that cause respiratory, gastrointestinal, and neurological diseases , includiInterestingly, unlike other RNA viruses, coronavirus genomes encode a highly conserved nonstructural NS14 protein capable of proofreading , resultiAnalyses of thousands of recently generated SARS-CoV-2 genomes, from both intra- and interpatient comparisons, showed that, overall, the pattern of mutations appears to be consistent with that expected from editing by host APOBECs and ADARs e.g., . The majSimilar excesses of A-to-G and U-to-C changes were observed in SARS and MERS viruses . A-to-G changes were identified in host microRNA sequences, where levels of editing were observed to be higher in infected than in uninfected samples , thus poThe Caliciviridae family is a family comprised of positive-strand ssRNA viruses and includes noroviruses, major human pathogens that cause acute gastroenteritis, as well as other animal viruses that cause a broad range of diseases across vertebrates, from fish to birds and mammals . Evidenc, P = 0.0291). Specifically, using multiple alignment sites shared among at least 178 HuNoV sequences, we identified 9 resistant and 10 susceptible A’s among conserved A’s, versus 6 resistant and 28 susceptible among variable A’s , with ADAR susceptibility status defined per randomly chosen representative, EF630492.1 (Hu/GII/143/JPN) of GII/4 cluster associated with the largest number of outbreaks . Indeed, ADAR editing plays a key role in the life cycles of these viruses too. In HDV, site-specific editing by ADAR1 p110 in the nucleus facilitates virion formation by editing a stop codon into a tryptophan codon  (The phylogenetic tree presented in /W site) , althoug/W site) . Interes/W site) . However/W site) .We refer the reader to excellent comprehensive reviews by Here we summarize what is known about the evolutionary consequences of ADAR editing across major groups of RNA viruses, highlighting existing gaps in our understanding of the virus–host interactions. Mounting evidence indicates that RNA editing, and ADAR editing in particular, plays a nontrivial role in the molecular evolution of RNA (and other) viruses as a source of novel substitutions. However, for many viral groups, the impact of editing has not been studied at all, whereas for others, nuances of viral interactions with the host immune response—which can act in a pro- or antiviral manner—remain to be elucidated. Some of these gaps in knowledge, such as whether the editing has been studied at the sequence level, are summarized in  and/or chanisms . FurtherThus, understanding the distribution and impact of ADAR editing—as a source of novel mutations, a potential proviral factor, and a cause of changes in host editing resulting in neurological complications—across broad groups of viruses, including in those not currently designated as “pandemic threat,” will serve as an important contribution toward the better handling of future outbreaks and epidemics and will facilitate better diagnostics  of novel diseases. Akin to a person looking for keys under the streetlight, we need to broaden our interests to include areas that are not currently illuminated, to extend the analogy. For example, early in the SARS-CoV-2 pandemic, mild neurological symptoms such as “loss of smell” were mostly ignored e.g.,  until itGenome Biology and Evolution online.evab240_Supplementary_DataClick here for additional data file.


Science, McCarthy et al. promptly observed a pattern of recurrent deletions in four discrete regions of the N-terminal Domain (NTD) of the Spike protein, which provide resistance to antibody neutralization, suggesting convergent evolution due to selective pressure and antigenic drift.1In a recent report in gambits to escape the immune system and to sustain its spread.2 Given that coronaviruses encode an exoribonuclease that provides a proofreading function during the replication, the appearance of variants was at first expected to be marginal. McCarthy’s work anticipated the events, as these deletions were found to be present in some of the lineages causing now a global concern.Evolution is nature’s inexorable imperative. A year into the COVID-19 pandemic, the rollout of vaccines opens a window of hope to end transmission in many countries, yet, recent concerns have arisen due to the SARS-CoV-2’s 3 illustrating SARS-CoV-2 adaptation to selective pressure within a single human being. These findings prompted them to look for similar cases in the GISAID database. Remarkably, they found eight patients with samples from late time points having viruses with deletions within the S gene that were not present at early time points. Tellingly, these viruses could be distinguished from one another by nucleotide differences and appertained to monophyletic clades, suggesting that within each individual, the repeated pattern of deletions was the result of adaptation and not of onward transmission.McCarthy et al. founded their research on the observation of an immunocompromised cancer patient who succumbed to the COVID-19 infection 74 days after diagnosis. They sequenced the S genes from samples obtained 72 days after diagnosis and identified two variants with deletions in the NTD. Avanzato et al. had reported a similar case,McCarthy et al. analyzed a total of 146,795 sequences in GISAID and identified 1108 with deletions in the S gene with 90% of them occupying four discrete sites within the NTD, which they termed “recurrent deletion regions 1–4” (RDRs). Although with a degree of variance, RDR1 typically harbored the Δ69–70 deletion, RDR2 the Δ144/145, RDR3 the Δ220, and RDR4 the Δ243–244 deletion . These lineages are typically defined by substitutions in the receptor-binding domain (RBD), the N501Y in the B.1.1.7 and B.1.351 lineages, and the E484K in the B.1.351. However, these are usually accompanied by other substitutions and deletions. A recent study observed that, far from being a mere companion, the Δ69–70 was key for the increased infectivity of the B.1.1.7 lineage: when reversed and other mutations like the N501Y maintained, the pseudotyped viruses lost substantial infectivity.5Deletions in the NTD have been observed by several groups in immunosuppressed as well as in patients repeatedly treated with convalescent plasma, which would suggest an improvement in fitness by evading the host’s immune response. However, in McCarthy’s work, a virus carrying the deletions was still neutralized by polyclonal antisera. Another study also showed that viruses with Δ69/70 deletion, E484K and N501Y substitutions, were still neutralized by sera from BNT162b2 (Pfizer-BioNTech) vaccinated individuals.Mutations will still appear as long as the virus continues to replicate within hosts, as a matter of mere probabilities. So far, selective pressures toward the virus might have given rise to variants with a fitness advantage based on infection, replication, or transmission efficiency, although potential evasion from antibodies—like the one observed in McCarthy’s paper—might result as a by-product or due to immune pressure within an individual during chronic COVID-19. As the population becomes less naive to the virus through natural infection or vaccination, the selective pressure to escape acquired immune responses will be higher and will force the virus to find a way. Variants with antigenic drift would introduce antigenic novelty and enable reinfection, as happens with influenza A, whose hemagglutinin protein requires a very small number of mutations to evade pre-existing immunity.H.G. Wells famously wrote “Adapt or perish, now as ever, is nature’s inexorable imperative”. This quote very elegantly summarizes evolution itself. In the midst of the COVID-19 pandemic, the virus never ceases to adapt, but neither do we. Thus, the best way forward to address variants of concern is to intensely monitor their emergence to be able to act proactively. Even if identification is limited by the lack of broadly established sequencing systems, the appearance of immune escape variants would probably be self-explanatory at the clinical level with vaccinated individuals recontracting the disease. In addition, one perk of mRNA-based vaccines is that they can rapidly be tweaked, or new sequences added for booster doses. Additional reasons for optimism are (a) T-cell responses may not be affected by the deletions and mutations of new variants as they recognize multiple antigens and epitopes conserved in the variants of concern and may provide protection beyond neutralizing antibodies, (b) even if vaccines fail to protect against infection or mild/moderate disease, they may still be effective against severe cases, and (c) reinfection with new variants may not lead to severe COVID-19. In the long run, although SARS-CoV-2 keeps evolving and adapting, so evolves our understanding of the virus and our tools to reduce its impact. Inexorably, the adapted virus must perish. We shall prevail.


Plant viruses cause many of the most important diseases threatening crops worldwide. Over the last quarter of a century, an increasing number of plant viruses have emerged in various parts of the world, especially in the tropics and subtropics. As is generally observed for plant viruses, most of the emerging viruses are transmitted horizontally by biological vectors, mainly insects. Reverse genetics using infectious clones—available for many plant viruses—have been used for the identification of viral determinants involved in virus–host and virus–vector interactions. Although many studies have identified a number of factors involved in disease development and transmission, the precise mechanisms are unknown for most of the virus–plant–vector combinations. In most cases, the diverse outcomes resulting from virus–virus interactions are poorly understood. Although significant advances have been made towards understanding the mechanisms involved in plant resistance to viruses, we are far from being able to apply this knowledge to protect cultivated plants from all viral threats.The aim of this Special Issue was to provide a platform for researchers interested in plant viruses to share their recent results related to the various aspects of plant virology: ecology, virus–plant host interactions, virus–vector interactions, virus–virus interactions, and control strategies. A total of 15 papers have been contributed by 96 authors from 18 countries to the issue, comprising ten research articles, one short communication, and four reviews .Nicotiana clevelandii to Chenopodium foetidum, can be boosted by adaptation to a bridge plant species, Arabidopsis thaliana, using mutants of the potyvirus plum pox virus. Kim et al. [Arabidopsis thaliana genotypes infected by the cucumovirus cucumber mosaic virus that were linked to virus seed transmission. On a more applied aspect, Nancarrow et al. [Plant virus ecology looks at virus–host-environment interactions and, in a broad sense, includes studies on biodiversity and evolution. Several papers in this Special Issue focused on this topic. Martínez-Turiño et al.  describem et al.  also anam et al.  studied m et al. , using aw et al.  estimateHigh-throughput sequencing (HTS) technologies have become indispensable tools to characterize plant virus diversity thanks to their ability to detect virtually any virus without prior sequence knowledge. Three papers published in this Special Issue deal with HTS. Kutnjak et al.  present The study of the precise interactions established between viruses and plant hosts to cause a productive infection is a fundamental subject that has received the attention of three research groups contributing to this Special Issue. Teixeira et al.  reviewedBemisia tabaci complex, have been mostly conducted with tomato yellow leaf curl virus. In this Special Issue, Chi et al. [Deciphering virus–vector interactions is an emerging research subject in plant virology. Thus far, studies concerning begomoviruses, an important group of emerging pathogens transmitted by the whiteflies of the i et al.  showed tMixed-infections with two or more plant viruses are frequent in the field, with viruses being able to interact in multiple and intricate ways. These interactions are generally categorized as synergistic, antagonistic, or neutral. In this Special Issue, Elvira González et al.  experimeAlthough a number of biotechnological approaches have been developed to produce virus-resistant plants in recent years, the use of classical genetic resistance remains the strategy of choice for practical control of plant viruses. In this Special Issue, Yan et al.  reviewedOverall, the papers in this Special Issue reveal different perspectives of current research on plant viruses, from applied field studies to investigations into the intricate mechanisms involved in the tripartite interactions between viruses, plants, and vectors.


High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated  to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data. Plant viruses and virus-like diseases are ever-emerging threats to agricultural and horticultural production . To ensuTotal RNA (totRNA) was used as input for HTS. However, a potential limitation of this approach can be the low amount of viral RNA within the background of plant RNA. Virus reads generated by totRNA sequencing from plant material might be low in number and difficult to analyse due to the high amount of plant rRNA in the total RNA . RibodepTo apply HTS as a routine tool for plant viruses’ detection, there are technical and biological challenges . It is iSo far, no guidelines or standards on how to use HTS in a diagnostic setting are available. We therefore decided within the framework of the Euphresco project “The application of Next-Generation Sequencing technology for the detection and diagnosis of non-culturable organism: Viruses and viroids (NGS-detect)” to evaluate one viral enrichment method for its suitability for viral diagnostics. In this interlaboratory comparison, plant leaf material infected with viruses representing different genomic organisations, i.e., single stranded DNA and RNA (positive and negative single-stranded), was analysed using HTS of ribodepleted totRNA. The design of this part of the study had the characteristics of a test performance study (TPS) as all participants were required to follow the same protocol and use the same commercial sequencing provider. Participating laboratories were also assessed in terms of their capacity to analyse the sequencing data using their own bioinformatic pipeline, which has the characteristic of a proficiency test (PT). In addition, the data generated by each laboratory were analysed using Virtool to assess its performance.Twenty laboratories were invited to participate in the study and nine laboratories agreed to participate in this study, of which eight performed the study and reported their results.All laboratories were able to extract sufficient RNA for further processing . None ofEach laboratory used their own bioinformatic pipeline , except At a later step, the raw data were reanalysed using Geneious to generate virus consensus sequences. Consensus sequences generated from TPS_08 were used as references to map the reads from other participants, followed by alignment to confirm their similarity. BLASTn search revealed that ACMV, AaNV, CGMMV, CMoMV, CMV, PEMV2, PEMVsatRNA, PepMV, PhCMoV, and ToMV genomic sequences share high similarity to GenBank sequences, between 95.7 and 99.9% nt identity . In contThe datasets provided by the participant laboratories were reanalysed using Virtool. Twelve out of the fourteen viruses included in this study were detected by the PathoscopeBowtie approach. The percentages of mapped reads to the organisation taxonomic units (OTU) database of plant viruses were between 4.9 and 18.6% for Pool 1, and between 15.2 and 31.3% for Pool 2 . Both AaIn order to compare the datasets, the raw reads were normalised to subsets of two million reads randomly extracted. Three replicates of the normalised reads were then reanalysed using Virtool. The calculated proportion of reads mapping to each virus (weight) are shown in (a)the practical performance of each laboratory to prepare the samples for HTS;(b)their ability to correctly identify the viruses using their own bioinformatic strategies;(c)the checking of the variation in sequencing quality of one commercial provider; and(d)the performance of Virtool for data analysis as an alternative standard analysis option.High-throughput RNA sequencing is a robust virus diagnostic tool for plant virus identification and whole genome sequencing without prior knowledge of the pathogen(s) in the infected plant. In order to adopt it in routine plant virus diagnostics, this study was designed to assess:We opted to provide the infected material in the form of freeze-dried leaf material as, in general, freeze-dried material is more stable in transportation in contrast to fresh material. We expected delays in the delivery of the material, in particular to non-European countries, which proved correct in one case. Even in this case, all virus sequences could be recovered successfully. The participants were provided with individual vials of samples that had to be pooled by the participants themselves before extraction, as it was virtually impossible to harvest all viruses at the same time considering the infection dynamics were very different for the individual viruses (ranging from a few days to a few weeks for symptom development).Host-sequence extraction using bioinformatic means is also a possible option to analyse HTS data. However, we opted for the sequencing of ribodepleted totRNA because host-sequence extraction requires host reference sequences which are not always available and higher bioinformatics power/infrastructure would have been necessary. Furthermore, the number of virus reads generated from totRNA HTS can be low due to the high background of host RNAs and can therefore lead to false-negative results. Increasing the sequencing depth could overcome these obstacles, although this comes at a higher cost. Ribodepleted totRNA is a suitable alternative approach that reduces the amount of extracted host and host-associated microbial rRNAs, thus enhancing the threshold of virus detection . DespiteIn this study, we were able to successfully detect ssDNA and ssRNA plant viruses. Other studies also showed that it is possible to detect dsDNA and dsRNA plant viruses using a ribodepleted totRNA approach . This suAppropriate analysis of the data is a bottleneck for disease causal agent identification . The proCoronaviridae: Coronavirinae: Gammacoronavirus) is the causative agent of infectious bronchitis in several avian species, leading to multisystemic disease and economic loss in the poultry industry [Interestingly, false-positive results were also reported. AvCoV  in the contigs. The translated ORFs are scanned for viral protein motifs. The contigs can then be used for BLAST search directly on the NCBI and the results require a virology-expert interpretation. In the Virtool analysis, the viruses’ weight or the calculated proportion of reads mapping to each virus were low, which can be roughly proportional to low titre. In this case, the low weights reported in the analysis may not be attributed to the virus titre but to the reference database, as the majority of the OTUs used in this version of Virtool were not closely related to our viruses and only a few virus-isolate sequences were used by Virtool as references to establish the weight values. AV-1, CNDV, CLRV, ClYVV, and LNYV sequences generated in this study were very divergent isolates that shared low nucleotide identities to the most similar sequences in GenBank . This deOne major advantage of Virtool is the use of curated databases that are checked for correct entries, thus limiting the potential of misidentifying viral reads. This means that the wrong detection of the citrus exocortis Yucatan viroid could have been avoided using the Virtool approach. Currently, a new version of Virtool is available with an updated OTUs database. However, this advantage is also a disadvantage as manpower is needed to update the databases regularly to include new virus and viroid sequences. This requires not only bioinformatics expertise but also expertise in virology.The percentages of reads assigned to each virus varied among the different labs. Nevertheless, there were high percentages for the tobamoviruses, i.e., CGMMV and TMV, followed by CMV, a cucumovirus . This isHTS is a powerful tool for diagnostics that can identify unknown pathogens. As it can be seen from our study, bioinformatics expertise is required for the correct interpretation and diagnostics. Virtool provides a powerful open-source bioinformatics platform that can be implemented in a diagnostic laboratory. In addition, the data analysis with Virtool is reasonably straightforward in comparison to other bioinformatics approaches. Even though HTS costs have dropped, implementation of HTS for routine diagnostics is still cost-prohibitive, thus this method will probably be reserved for critical samples rather than routine diagnostics. It is worth noting that often the customer has no influence on the turnaround time at the sequencing supplier, thus this time needs to be taken into consideration. Currently, novel long-read sequencing technologies such as MinION from Oxford Nanopore Technologies may change this as the sequencing can be carried out relatively inexpensively in-house ,21.cox or nad5 [Diagnostic tests require sufficient controls in order to minimise the risk of false-negative or false-positive results . These c or nad5 ,24. Howe or nad5 ,25. It i or nad5 . This isThe study was organised by the Julius Kühn Institute  and assisted by the Netherlands Food and Consumer Product Safety Authority (NVWA).Nicotiana benthamiana plants were inoculated mechanically with 14 different plant virus species . Fifteen days post-inoculation, the non-inoculated leaves were tested using ELISA, PCR, or RT-PCR to confirm virus infection  in order to carry out totRNA extraction according to the manufacturer’s instructions. In addition, aliquots of the RiboMinus™ Plant Kit for the RNA-Seq (Invitrogen) kit were also provided to each participant. The chemicals were shipped by fast courier (cool pack) and arrived at their destination within three days .1.Preparation of pooled samples:The lyophilised plant material for the HTS_TPS was mixed into two pooled samples (Pool 1 and Pool 2) and each laboratory was asked to combine the vials as in 2.Total RNA extraction:The RNeasy Plant Mini Kit was used for the isolation of total RNA from the pooled samples following 3.Ribosomal RNA depletion:The RiboMinus™ Plant Kit for the RNA-Seq kit was used for depleting the ribosomal RNA following 4.High-throughput sequencing:Ribodepleted RNA was sent for library construction  and HTS was performed on the Illumina HiSeq 4000 platform (2 × 150 bp) at Eurofins Genomics GmbH, except for one laboratory in which the pools were sequenced at GenomeScan  on the Illumina NextSeq 500 platform after library preparation using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).5.In-house bioinformatic analysis:Each laboratory used their own bioinformatic pipeline to analyse the HTS data .6.Reporting the results:A form was provided to each lab for collecting information on their procedures and data analysis, and for reporting the viruses detected.7.Assessing Virtool:Raw reads were uploaded to a JKI cloud server by each laboratory and then analysed on a single Virtool pipeline at JKI.The above steps of the interlaboratory comparison are summarised in N. benthamiana was used ).Each sample was analysed for viruses using Virtool (v3.9.8), which is a sample manager that can run multiple diagnostic analysis workflows . For rapTo normalise the reads from each laboratory, three subsamples of each of the two million reads were randomly extracted from the raw data sets provided by each laboratory using Geneious Prime (version 2021.1.1). The subsamples were then reanalysed with Virtool.The statistical analysis of the generated raw data sets and the subsamples  was performed using R version 4.0.3 . The datThis study showed that the participants’ technical (wet lab) performance is sufficient to detect viruses in infected plants. The ribodepletion approach for HTS was found to be adequate for the detection of the plant viruses used in this study (ssDNA and ssRNA positive and negative senses). This strategy provides a good tool for plant virus diagnostics but the bottleneck concerns bioinformatic competence. The interlaboratory comparison underscores the range of bioinformatic pipelines used by the different participants for data analysis and the level of expertise. The inability to correctly identify new viruses or viruses not found in databases suggests that plant virology expertise is required. Virtool also allowed for the identification of all viruses, specifically twelve viruses (plus a satellite RNA) with PathoscopeBowtie and two viruses with the NuVs approach. Thus, Virtool can offer an alternative open-source platform for plant virus detection. This interlaboratory comparison was a very good exercise as all laboratories could re-check their data on their own and identify why they deviated in their analyses from the “correct” findings. This helped to improve and optimise their bioinformatic pipelines for plant virus diagnostics.


Begomovirus has a wide host range and threatens a significant amount of economic damage to many important crops such as tomatoes, beans, cassava, squash and cotton. There are many efforts directed at controlling this disease including the use of insecticides to control the insect vector as well as screening the resistant varieties. The use of synthetic virus or infectious clones approaches has allowed plant virologists to characterize and exploit the genome virus at the molecular and biological levels. By exploiting the DNA of the virus using the infectious clones strategy, the viral genome can be manipulated at specific regions to study functional genes for host–virus interactions. Thus, this review will provide an overview of the strategy to construct infectious clones of Begomovirus. The significance of established infectious clones in Begomovirus study will also be discussed.Begomovirus. Also, this technique’s impacts and benefits in controlling and understanding the Begomovirus infection will be discussed.Begomovirus has become a potential threat to the agriculture sector. It causes significant losses to several economically important crops. Given this considerable loss, the development of tools to study viral genomes and function is needed. Infectious clones approaches and applications have allowed the direct exploitation of virus genomes. Infectious clones of DNA viruses are the critical instrument for functional characterization of the notable and newly discovered virus. Understanding of structure and composition of viruses has contributed to the evolution of molecular plant pathology. Therefore, this review provides extensive guidelines on the strategy to construct infectious clones of  The first infectious clones of bacteriophage QB was reported in 1978 and this was achieved by inserting the cDNA genome into a plasmid vector  and was Begomovirus is the largest member of the group, which has about 322 species. Begomoviruses have either monopartite or bipartite genome. It can only be infecting dicots plants through whitefly (Bemisia tabaci) as the vector. This virus is present in both Old World (both genomes) and the New World  .Replication-initiator protein (Rep) encoded by the AC1 gene is vital for virus replication. In the fections . Throughfections ,106. A pBhendi yellow mosaic virus (BYMV) has been conducted through the construction of C2 mutants in BYMV. Two stop codons were inserted into the ORF that encodes the C2 protein. The host plant inoculated with BYMV bearing a mutated C2 protein displays the suppression of symptoms and a decrease in viral load. This suggests that BYMV C2 protein is vital in infection as it involves symptom determination and the viral replication process [Begomovirus, C2 protein may play a different role. Stanley et al. [Beet curly top virus (BCTV) does not play any role in infectivity. In contrast, a study conducted by Baliji et al. [Spinach curly top virus (SCTV) has suggested that C2 mutant of SCTV produces a milder symptom of infection, which in turn indicates that C2 protein is required for infectivity.Elucidation of the role of C2 protein in symptom determination and replication in the  process . Howevery et al.  reportedi et al.  in SpinaBegomovirus function in the development of symptoms. The loss of C4 function leads to the attenuation of the symptom and reduced infectivity [Begomovirus, the function of AC4 protein is less conserved as compared to monopartite Begomovirus. This is shown by the alignment of the AC4 region sequence of bipartite Begomovirus, indicating that the sequence is less conserved. Bipartite African cassava mosaic virus with AC4 mutants (ACMV) expressed symptoms and infection [Tomato golden mosaic virus (TGMV), mutants of AC4 show no effect on the virus’s symptom development and infectivity [Begomovirus may function as a suppressor of PTGS by binding to miRNAs and impeding plant growth [In general, ORF encodes for AC4 protein in ectivity ,112,113.nfection ,115. Whiectivity . On the t growth ,118. ThiBegomovirus genome mimics the DNA-A from bipartite Begomovirus [Begomovirus [Tomato leaf curl China virus (ToLCCNV) by Yang et al. [Nicotiana benthamiana and no symptoms were observed. However, the plant begins to show a downward leaf curling symptom when transfected with ToLCCNV and ToLCCNB. This infectivity test showed that ToLCCNB is needed for the symptoms to appear. Also, the DNAβ with a mutated βC1 retains its capability for silencing suppressor, no typical symptom in Nicotiana benthamiana is observed when it is coinfected with ToLCCNV [Begomovirus genome.Monopartite omovirus . In receomovirus ,120. DNAomovirus . A studyg et al.  found th ToLCCNV . Table 2Begomovirus-causative diseases is based on the viral vector’s control, which is the whitefly (Bemisia tabaci) even this approach is known to be laborious [African cassava mosaic virus (ACMV) clones into the cassava plant [One of the approaches to managing and controlling aborious . Withoutva plant . This inva plant . Additiova plant . To deplva plant ,127.Cassava mosaic virus (Begomovirus) is more rapid in drawing out the infection symptom, which is as early as 10 days post-inoculation. Moreover, successfully infected plants produced are more significant in number. Hence, this indicates that using biolistic mediated transmission is more efficient than the whitefly-transmission technique [As for early screening for resistant variety, biolistic inoculation was used to deliver cloned virus into cassava plant infection, speeding up evaluating cassava cultivars’ tolerance in the breeding program. The usage of biolistic has been reported to help determine the resistance level to cassava mosaic disease in cassava breeding lines . The bioechnique ,128.Bean golden mosaic virus was demonstrated using the PCR–Gibson Assembly technique [Begomovirus infectious clones, and the single-step GA was successfully used to assemble the genome of the virus in an isothermal reaction. Golden Gate cloning and yeast homologous recombination has not yet been reported in the development of infectious clones in begomoviruses, and this might be due to their small genomes and cloning of the genome being relatively easy.For decades, the assembly of DNA constructs and viral clones has been conducted using the use of restriction endonucleases to create compatible ends. The presence or absence of restriction sites in the viral genome and vector sequences prevented this approach from being successful. Plant virologists have adopted several cloning methods to overcome these limitations such as overlap-based methods, Gibson assembly, Golden Gate cloning and yeast homologous recombination. Overlap-based methods are versatile, flexible and overcome the major constraints associated with the use of restriction enzymes. Overlap-based cloning allows for the rapid and efficient assembly of infectious clones. A simple approach to obtain infectious clones of the echnique . The dimBegomovirus, the presence of conserved sequences allows the Begomovirus to utilize its degenerate “universal” primers to amplify the entire genome, even from total plant DNA. Genotypic sequences of begomoviruses were generated through a viral metagenomic approach using RCA–NGS (rolling circle amplification–next generation sequencing). The method was described to enable the analysis of the diversity of begomoviruses in North America, and 19 complete genome begomoviruses and one alphasatellite were assembled [The potential of synthetic biology has been demonstrated through the development of functional and artificial genomes. In particular, the assembly of synthetic replicons from virus genomes has been shown to be an effective method for de novo synthesis. Several infectious clones from plant virus’s genomes have been reported to have been synthesized entirely using de novo synthesis and assembly approach ,131,132.ssembled . With thCell-free cloning techniques have been used to obtain infectious clones from plant viruses ,135. UncAgrobacterium tumefaciens and its binary vectors is a cost-effective and highly efficient method for analyzing the Begomovirus’s infectivity. Thus, it is widely used by virologists. To date, the processes for producing a full-length infectious clone construct have been simplified and continuous developments of kits and methodologies have been made available to allow a better understanding of viral infectivity.The availability of infectious clones for begomoviruses has proven to be a powerful molecular tool. This technique allows the functional study of a viral gene and its biological properties, and facilitates the genetic screening of germplasm for resistance to viruses. The agroinoculation of viral clones using


The COVID-19 pandemic has catastrophically impacted the world. Before the success in vaccination, this virus shows no sign of stop spreading. Nearly all the countries have implemented stringent approaches to slow down the transmission of the virus, but the virus still caused over 2 million deaths and the number is increasing. Therefore, preventing the virus spreading is still necessary to protect most people, especially the ones with pre-conditions. Mainland China has successfully eradicated the COVID-19 virus infection in Wuhan in 2020. After that, several small-scale outbreaks occurred in many cities in China, but none of these COVID-19 virus infections caused the widespread. In this review, we would like to give a detailed presentation of the approaches that were implemented by the China government to suppress the virus spreading by considering the unique characteristics of this virus and the paths of the virus transmission. Both the pros and cons of these strategies will also be analyzed. The experiences and lessons learned during the virus-fighting in China, expectedly, will be a useful source of reference for other regions in overcoming the threat caused by the COVID-19 virus. COVID-19 has swept through the globe within a brief time and after one year; it is still continuously extending its territory. Millions of people are newly infected daily in the world. Several factors are important to contribute to the widespread of this virus. Fundamentally this virus is highly contagious. In some extreme cases, one patient could transmit this virus to over one hundred other people. To most of the victims, however, this virus only causes mild symptoms such as fever, cough, dizziness, etc. just like a regular cold without threatening life The pandemic caused by COVID-19 across the whole globe indicates that preventing the spread of this virus is nearly an impossible task. The reason is complicated because the virus could infect people in diverse ways. Also, the virus survives for a long time and there are too many non-symptomatic virus carriers too. Even whether wearing a mask will prohibit the dispersal of the virus is still an unsolved puzzle because the virus could be spread via a non-respiratory route. However, the outbreaks in China, including the Wuhan outbreak, have been contained and eradicated within few months. Now the Wuhan city is virus-free and the people regain the freedom to live a normal life, except wearing the mask as a precautious approach. Other cities such as Beijing and Shanghai have also experienced more than one wave of outbreaks of COVID-19 infection. Quickly, these infection events were under control and the infected individuals were quarantined on time. Frankly, the cost to suppress the spreading of the virus is not cheap. However, the benefits gained from the elimination of the virus are also valuable. Nevertheless, some valuable experiences were accumulated during the prevention of the virus spreading A common measurement for virus infection capacity is “R0”, or “R naught”, which shows how contagious a virus is by measuring the average number of people who contract the virus from one infected person. An R0 larger than 1 means the infection is exponentially expanding and restrictive measurements must be applied to contain the spreading of the virus. The estimated R0 for Covid-19 is diverse given by different studies with a range of 1.1 to 2 The long-live ability of the COVID-19 virus has been recognized since the beginning of the pandemic. When the first outbreak of this virus in Wuhan in 2020, a study reported that the virus has unusual survival ability in the environment of the hospitals The initial outbreak of COVID-19 was in winter. People expected that when the summer comes, the pandemic might get less severe owing to that the virus might lose resistance to the high temperature, high humidity, and high UV, just like many infectious viruses such as SARS and influenza which are seasonal and become less transmissible as the climate warms. However, some studies showed that the transmission ability of COVID-19 was not sensitive to climate change. Data collected from 244 cities in China suggested that the basic R0 was not significantly associated with temperature changes and UV intensity Disinfection of the environment and preventive hand sterilization was based on the fact that the virus can't survive the disinfectants or alcohol-based sanitizer. To test the effects of different disinfectants against the virus, the survival of the virus in different disinfectants such as bleach, hand sanitizer, ethanol, povidone-iodine, chlorophenol, chlorhexidine, benzalkonium chloride, etc. were analyzed. No infectious virus could be detected after 5 min treatment, except for hand sanitizers, which took a long time to kill the entire virus at room temperature. The team also found that the virus was very stable even at a variety of pH levels (pH3-10) and can still be detected in infectious concentrations for 60 minutes The efficiency of human cell infection by the virus is critical for the spreading of the virus. ACE2 is a widely expressed protein distributing on the cell membrane and was identified as the major mediator of COVID-19 virus invasion. The mechanism of COVID-19 virus invasion of the human cell via binding to ACE2 has been studied in detail. After binding to ACE2, the spike (S) glycoprotein of the COVID-19 virus is cleaved into S1 and S2 subunit by Transmembrane protease serine 2 (TMPRSS2) and FURIN and then entry into the host cell. A single-cell mRNA sequencing from major adult organs revealed that ACE2 shows more expression in the kidneys and digestive system than in the lungs and trachea Compared with the SARS virus and influenza virus, COVID-19 is more contagious and infects more organs. Some other important receptors or co-receptors that bind to the S protein of COVID-19 in addition to ACE2 may exist. For example, a recent study found that Tyrosine-protein kinase receptor UFO (AXL) binds to the N-terminal domain of the S protein and co-localizes to the cell membrane. Knocking out AXL significantly reduces COVID-19 infection in lung cells. The expression level of AXL is closely correlated with S protein abundance in bronchoalveolar lavage fluid cells from COVID-19 patients The abundance of the non-symptomatic carrier of the COVID-19 virus causes more trouble to prevent the spreading of the virus. Asymptomatic infection refers to those who have no relevant clinical manifestations, such as fever, cough, sore throat, and other self-perceived or clinically recognizable symptoms and signs, but the respiratory tract and other samples tested positive for the virus. As early as February 2020, half of the people infected with the COVID-19 virus on the Diamond Princess cruise ship in Japan were found to be asymptomatic The recognition of the multiple ways for the COVID-19 transmission was based on the experiences accumulated over a long time of battle against this virus. The strategies to trace the virus transmission and to interrupt the virus spreading are also based on the detailed understanding of the virus transmission mechanisms.Similar to other coronaviruses such as SARS-CoV and MERS-CoV, the COVID-19 viral particles mainly infect the human respiratory tract Fomite transmission can occur by touching the COVID-19 virus-contaminated objects unintentionally and therefore is believed to be another potential route to spread the virus. Clinical reports discovered that environmental samples of COVID-19 wards were identified with viral RNA To increase the accuracy in deciding if a COVID-19 patient is no longer infectious, and to find the virus carrier in the close contacts of the confirmed COVID-19 cases, the anal swab was also implemented in addition to the oropharyngeal swab in controlling the recent outbreaks in China. The reason is that the COVID-19 could infect the gastrointestinal (GI) epithelial cells. The virus RNA was detected in patients' stool Since the SARS outbreak in 2003, it has been believed that coronavirus can transmit via direct or indirect contact with the mucosa of the eyes The risk of a pregnant woman getting COVID-19 is not higher than others but it draws public attention to COVID-19 virus transmission from infected mothers to the fetus. A few clinical cases reported that vertical transmission is rare (2 of 31 COVID-19-positive pregnant women) but possible The reservoir hosts of COVID-19 are generally considered as bats since sequence analysis showed it shared high nucleotide homology with SARS-like coronavirus isolated from Chinese horseshoe bats While the spreading of COVID-19 virus shows no sign of slow down before the widely vaccination globally, the Mainland China has achieved zero local new case for nearly a half year after the Wuhan outbreak. Although the new cases still continuously appear in several different places later, China has developed the strategies to eradicate these infections locally. The experiences accumulated during these fights against COVID-19 are valuable but may not be applicable to other places owing to complicated reasons.With the largest population in the world and with the first and a large-scale outbreak of the COVID-19 pandemic in Wuhan, China quickly suppressed the spreading of the virus and achieved the zero-local new case on March 19 of 2020. Afterward, some small-scale outbreaks appeared in several cities especially the ones close to the border to other countries. Nearly all these outbreaks were under control and achieved “zero” new case finally. The benefit of the “zero” policy is that once there is any new case reported, the path of the virus transmission can be determined through the epidemiological investigation. In addition, when there is no new case, the community becomes safe and the normal economic activities can be resumed.However, such a goal can only be achieved when there is a border to control the flow of people. China has enforced border control when the pandemic becoming severe across the globe rd wave of virus spreading sustained much longer and become more devastating. Immediately after the Chinese New Year, the Hong Kong government issued the restriction releasing policy again in anticipating that the forthcoming vaccination may reduce the risk of community transmission of the virus. After all, the stringent restriction on social distancing in Hong Kong lasted for around 2 months in total and results in over 190 deaths during the COVID-19 pandemic. If the policy was more stringent and follows the rule of 14 days no new case before releasing the restriction, Hong Kong may have the chance to eradicate this virus at the first hand , when the pandemic is still at its peak in 2020, most people chose to give up visiting each other. In fact, the government extended the CNY holidays to encourage people to stay home. In addition, all the public areas were closed including the restaurant, theaters, parks, etc. Even the public transportation was canceled for a brief period in cities heavily hit by COVID-19. Since the virus takes less than 2 weeks to cause symptoms in most people, the sharp and stringent lockdown breaks the transmission of the virus and gives people a time window to identify and quarantine the infected ones. In comparison, most other countries rely on law and self-obedience to keep the social distance. For example, the UK policy allows people to leave their homes with a necessary excuse Tracing the travel route of individuals is illegal in most countries including China. However, when the vaccine is not available and the COVID-19 virus is highly contagious and sometimes lethal, it is necessary to prevent the potential risk as early as possible. For example, when no new COVID-19 case was reported starting from March 19, 2020, after the Wuhan outbreak, many areas in China have started to resume the normal. To reduce the risk, both the central and the local governments implemented the health declaration QR code to identify the individuals with potential risk. The QR code is based on the recording of the travel route of the cell phones carried by nearly everybody China has applied restrictions to cross-border activities since the COVID-19 pandemic outbreak. Initially, all the international flights were canceled. When the domestic pandemic was under control, some international flights were resumed. However, all the routine flights facing a potential risk of one-week suspension if there are COVID-19 positive passengers on that flight When the infection accelerates, more victims are found. However, many of these infected individuals show no symptoms, or the symptoms are relatively unharmful. These people are advised to stay home for self-quarantine in many countries. This has resulted in infections among family members. Inevitably, these infections also leak to the community later. Starting from the Wuhan outbreak, all the infected individuals, no matter with or without symptoms, will be quarantined in the facilities provided by the government in China To deal with the recent outbreaks in several cities of China, the cities chose to lock down the whole city to screen for the positive virus carrier or COVID-19 patients. The lockdown often associates with the “freezing” of all the people movement. This strategy has been efficient to make the Wuhan virus outbreak under control because there was a large number of infections and active transmission of the virus. However, when only a limited number of cases were identified in a city, implementation of the lockdown causes inconvenience to all the residents if the government can't support the least needs of the people efficiently. Also, the tedious virus test for all the people is not economical. Therefore, if a city has equipped with a good epidemiological investigation team, implementation of PEI to find the people potentially infected by the virus is a cost-effective way. For example, the small-scale outbreaks in Shanghai, a city with a nearly 20 million population, were managed via PEI COVID-19 is so far the most devastating infectious disease on a scale similar to the Spanish flu in human history. However, the challenge brought by this disease is even worse because of the modern interconnected economic activities globally. Thus, the destruction caused by this virus is also severe in human history. In addition to the over 2 million deaths caused by this virus, the global economy was impaired fundamentally. Most of the countries have their economies shrink except the mainland China with a slowdown of economic growth. The reason is simply that China earned more than half a year of virus-free time which allows the restart of the economic activities. In addition, the rest of the world has been deeply involved in the wrestling with the virus and has increased demand for the products from China, even more than the amount they used to have because many countries lose the normal ability to be as productive as before under the virus defending circumstances. Since China has been one of the major manufacturers for the global market before the “COVID-19” crisis, the successful elimination of the virus in early 2020 gave China a significant opportunity to further increase its productivity and expand its share of the international market.The COVID-19 firstly broke out in Wuhan China. Afterward, the virus quickly swept most of the other countries. Owing to insufficient knowledge about this virus, most of the countries did not take immediate action to prevent the virus invasion. China has learned from the Wuhan disaster quickly and applied strict isolation and quarantine approaches to contain the virus from spreading. However, the people who live in those cities that were locked down experienced some challenging difficulties. In few cases, people suffered the sacrifice of life that is not directly caused by the virus but by the lockdown. But the payback is cutting the pain of suffering to be short. With the price of fewer than 100,000 cases of infection and 5000 deaths, China has eradicated the virus within 2 months. Importantly, several smaller-scale outbreaks were also successfully eliminated without causing any deaths.The total cost for the COVID-19 pandemic was 25 billion USD in China till 31th May 2020 Conclusively, mainland China has become a unique example of fighting the COVID-19 virus. Some of the strategies are applicable to other countries too. For example, the rigorous virus test is an cost-effective and easy-way to identify the risky non-symptomatic virus carriers. The establishment of the “Fang Cang hospital” is also an applicable approach to prevent the community spreading of virus. However, this approach may not be easily acceptable because some people may consider the hospitalization is not deserved with minor symptoms. Some of the strategies implemented by China may not be suitable for other countries because of the different infrastructure of the government, clinical organizations, and culture, etc. For example, the tracing of the cell phone is forbidden by many countries because it may violate the human rights. The implementation of such an approach has to be protected by the central government as China is able to do. Similarly, the PEI approach may violate the personal rights as well. In fact, several people infected by COVID-19 have become victims owing to the leaking of personal information during PEI, although such information leaking is strictly prohibited by law.After all, it is difficult to tell whether the approaches implemented by China are better than the other approaches implemented by other countries. But if simply analyze the impact on the economy, the change of the life of the people and the deaths caused by COVID-19, China has won more and lose less.