African swine fever (ASF) is a hemorrhagic and often fatal disease occurring in domestic pigs and wild boars. ASF can potentially greatly impact the global trade of pigs and pork products and threaten global food security. Outbreaks of ASF must be notified to the World Organization for Animal Health. In this study, we analyzed the feasibility of applying propidium monoazide (PMA) pretreatment-based infectious virus detection technology to ASF prevention and control and investigated the prospects of applying this technology for epidemic monitoring, disinfection effect evaluation, and drug development. PMA as a nucleic acid dye can enter damaged cells and undergo irreversible covalent crosslinking with nucleic acid under halogen light to prevent its amplification. Although this technology has been widely used for the rapid detection of viable bacteria, its application in viruses is rare. Therefore, we analyzed the theoretical feasibility of applying this technology to the African swine fever virus (ASFV) in terms of gene and cell composition. Rapid infectious ASFV detection technology based on PMA pretreatment would greatly enhance all aspects of ASF prevention and control, such as epidemic monitoring, disinfection treatment, and drug development. The introduction of this technology will also greatly improve the ability to prevent and control ASF. Asfarviridae family, the ASFV is a large, enveloped, double-stranded DNA virus that is directly and indirectly transmitted by soft ticks  is a highly contagious infectious disease in animals caused by the African swine fever virus (ASFV). ASF is characterized by a wide range of clinical symptoms, from subclinical symptoms to sudden death, that often occur within 10 days, but symptoms can occur as early as 4 days after infection . As the ft ticks . The virft ticks . Based oenotypes . The 201enotypes .At present, owing to the lack of effective vaccines and specific therapeutic drugs, ASF prevention and control depends mainly on biosafety, timely detection, culling, and disinfection. In countries without ASF, biosafety and detection are the primary methods of prevention and control, whereas countries where epidemics occur rely mainly on detection, culling, and disinfection. Early virus detection requires highly sensitive and specific diagnostic methods as this enables the rapid implementation of the necessary disease control and eradication measures . BecauseASF was first reported in Kenya in 1921 and has subsequently spread throughout most of sub-Saharan Africa. In 1957 and 1960, it spread across the continent to Spain and Portugal, respectively, and from there to other countries in Europe, South America, and the Caribbean. The 2018 outbreak of ASF is the second transcontinental spread since 2007, with the virus spreading initially to Georgia in the Caucasus and subsequently to neighboring countries and Eastern Europe, possibly from the southeastern region of Africa. To date, ASFV has spread rapidly through China, Mongolia, Vietnam, Cambodia, Laos, the Philippines, Myanmar, South Korea, Indonesia, and other Asia-Pacific countries . Figure Among all animal diseases, ASF is one of the most important viral diseases affecting the pig industry. It has serious social and economic impacts because its detection can often lead to international trade restrictions on pork products, resulting in potentially substantial losses for the pig industry . The difThe disease entered Cuba in the 1980s, resulting in economic losses of $9.4 million . In the Rendleman and Spinelli  estimateOrnithodoros spp. ticks, wild African pigs (mainly warthogs), domestic pigs, and pig-derived products such as pork  is widely used for pathogens . As descFrom a genomic perspective, ASFV is a large double-stranded DNA virus belonging to the family Asfarviridae . DetectiSuidae family  may give reliable results with high sensitivity, and is rapid and convenient. As a nucleic acid dye, PMA can enter the dead virus and bind nucleic acid under halogen lamp irradiation, to inhibit the amplification of nucleic acid from dead virus, which has a good application prospect. However, the pretreatment scheme of PMA will change when the virus structure varies. Based on different viruses, even the length or structure of the target fragments, researchers can optimize the PMA concentration, exposure intensity, illumination time, and dark treatment time to obtain the optimal pretreatment conditions.More significantly, PMA–PCR/qPCR cannot only accurately detect ASFV, but also has high application potential in other fields of ASFV control, such as disinfection effect evaluation, drug research and development. Using effective disinfectants to clean infected sites, trucks, and pollutants is an important step in preventing further spread of ASF. Accurately evaluating both the efficacy of disinfectants and the effectiveness of disinfection is a key step in epidemic prevention and control and will directly affect the success of prevention and control. The efficacy of disinfectant is generally judged by the survival of the virus after disinfectant treatment. Currently, the cell culture method is commonly used. However, this method has some limitations as mentioned above, which seriously affects the efficiency of evaluating the disinfection effect. Both the complicated operation and the requirements for experimental conditions affect its practicability. In ASF epidemic areas, disinfection of pig farms is a necessary part of prevention and control. The long disinfection effect evaluation cycle often puts disinfection workers in a dilemma. Ignorance of the effect of disinfection may lead the staff to take repeated disinfection measures, which will not only result in the wastage of resources but also increase environmental pollution; moreover, incomplete disinfection, wherein the effect of disinfection is not immediately available, will increase the risk of the spread of the disease. Therefore, a method that can quickly identify infectious and non-infectious viruses is urgently needed to interpret the effect of disinfection and provide accurate information for the implementation and adjustment of ASF prevention and control programs. This rapid evaluation method should also help with the development of new disinfection products and schemes.via real-time PCR while reflecting the effect of drugs. Thus, the development of an infectious ASFV detection method based on PMA pretreatment will also benefit the development of anti-ASFV drugs.Drug development is also one of the ways to prevent and control ASF. Although no drugs are currently available for ASF treatment, some compounds with anti-ASFV activity have been reported. Similar to the development of disinfectants, efficacy evaluation is also critical in the development of anti-ASFV drugs. Currently, the absence of cell lines and a long evaluation cycle has hindered the development of drugs. Especially in animal experiments, a direct understanding of the change in viral trend in infected animals after drug administration will undoubtedly enhance progress in the evaluation of drug efficacy and accelerate drug screening and functional analysis. The rapid detection of infectious virus based on PMA pretreatment can be used to monitor the proportion of infectious and non-infectious viruses in animals In summary, rapid infectious virus detection methods are of great significance for the detection, prevention, and treatment of ASF. ASF prevention and control systems urgently need a rapid detection method based on PMA pretreatment. This technology is currently widely used for the detection of viable bacteria but rarely applied to viruses. This may be because the pretreatment of viruses by PMA is more complex, and different viruses need different treatment reminders . The optFX and DZ designed the writing idea of this manuscript. YL, KZ, XY, JS, TZ, MW, XL, and LD reviewed relevant literatures. WP and KL drew the figures. YJ, XW, JL, and JD provided technical support. DZ and BQ wrote the manuscript. FX and XW had primary responsibility for the final content. All authors have read and approved the final manuscript.


Benyviridae to include red algae. Second, our phylogenetic analysis suggests that the evolution of this virus family may have involved cross-kingdom host change events and gene recombination/exchanges between distant taxa. Third, the identification of gene blocks encoding known movement proteins in beny-like RNA viruses infecting non-vascular plants confirms other evidence that plant virus genomic RNAs may have acquired movement proteins simultaneously or even prior to the evolutionary emergence of the plant vascular system. Fourth, novel data on plant virus diversity highlight that molecular evolution gave rise to numerous provisional species of land-plant-infecting viruses, which encode no known potential movement genetic systems.Based on analyses of recent open-source data, this paper describes novel horizons in the diversity and taxonomy of beny-like viruses infecting hosts of the plant kingdom (Plantae or Archaeplastida). First, our data expand the known host range of the family  Benyvirus is the only genus officially recognized by ICTV in the family Benyviridae , with the species Beet necrotic yellow vein virus (BNYVV) being the type benyvirus ..Charavirs orders ,41. It sGoji berry chlorosis virus (GBCV) is positioned rather close to members of the genus Benyvirus in the phylogenetic tree (A. vermiculophyllum (see above). The GBCV RdRp core domain has 52% identity to that of BNYVV. The GBCV CP (ORF2 protein) represents the “tobamo-like” CPL [Martellivirales, particularly the Abisko virus CPL , Xiangshan martelli-like virus 2 CPL  and Bemisia tabaci virga-like virus 2 CPL . Strikingly, the CPL-encoding gene in the GBCV genome is duplicated, and its second copy is represented by ORF3, a protein product of which is most closely related to the Tobacco rattle virus CP (genus Tobravirus) . The ORF4 and ORF5 proteins have no significant similarity to known virus proteins and are marginally related to plant myosin-11 (ORF4 protein) and plant kinesin calmodulin-binding protein (ORF5 protein) (data not shown). ORF6 has been experimentally shown to encode a silencing suppressor protein [Benyviridae and Virgaviridae  [tic tree A. The GBtic tree B. This dike” CPL  with the protein . This gevirales) . Dicranum scoparium and the Antarctic flowering plant Colobanthus quitensis [Benyvirus. Replicases of tecimovirids contain RdRp domains with an obviously close relationship to those of viruses in the genus Benyvirus , in the VLRAs of the moss uitensis . These penyvirus . In parterfamily .Hibiscus green spot virus (HGSV) “accessory” SF-1 helicase as a query [Kitaviridae  [Lathyrus sativus , Quercus castanea  and Litchi chinensis . The RdRp core domain of these viruses has 50–52% identity to that of BNYVV. The BMB helicase sequence is somewhat closer to the tecimovirid “accessory” helicase than to the TGB1 helicase [This group of monopartite capsidless viruses is composed of three plant VLRAs , which w a query . HGSV bevirales) . The HGSvirales) ,13 and sShorea curtisii (accession GJMJ01032282). The unusual organization of this VLRA showed plus and minus chains of the RNA genome assembled in a long hairpin (data not shown). However, despite this probable artifact of sequencing data assembly, the VLRA code for ORF1 replicase with high similarity to the binamovirids Lathyrus sativus, Quercus castanea and Litchi chinensis (RdRp core domain identity 68–75%) and ORF2/3 proteins, which are similar to the BMB1 , Arceuthobium sichuanense virus 3 (BK059270 [Dactylorhiza hatagirea beny-like virus (BK013327) [The largest subcluster of the phylogenetic tree showing benyvirus and beny-like RdRps is represented by numerous plant-specific VLRAs along with three annotated virus sequences, namely, BK059270 ) and DacK013327) )  and Rhyncholacis penicillata Rhyc16 VLRA (ICSC01000014); Dactylorhiza hatagirea beny-like virus with Gymnadenia rhellicani VLRA (GHXH01324014), Ophrys fusca VLRA (GHXI01129489) and Ophrys sphegodes VLRA (GHXJ01414654); and Arceuthobium sichuanense virus 3 with Viscum album VLRA (GJLG01028288), Atriplex prostrata VLRA (EPVF-2046303), Leontopodium alpinum VLRA (DOVJ-2063722), Silene dioica VLRA (GFCG01071918) and Astragalus canadensis VLRA (GGNK01006218)  VLRAs, namely, 1006218) .Red clover RNA virus 1 RNA (7190 bases in length) encodes three proteins (proteins . The 5′-proteins . We presRhyncholacis penicillata Rhyc27837 VLRA (8274 bases in length), despite the close amino acid sequence similarity and domain organization of the encoded RNA polymerase (ORF1 protein) to Red clover RNA virus 1 RNA polymerase (identity 50% for the full-length proteins), codes for two proteins only (R. penicillata Rhyc27837 VLRA (313 residues in length) exhibits no similarity to the Red clover RNA virus 1 ORF2 and 3 proteins. The R. penicillata Rhyc16 VLRA (7871 bases in length) also encodes two proteins, and its ORF1-encoded RNA polymerase has 58% identity of the full-length protein to the R. penicillata Rhyc27837 ORF1 protein. The ORF2 protein has no sequence similarity to R. penicillata Rhyc27837 VLRA-encoded ORF2; nevertheless, both proteins possess potential membrane-bound segments in the N- and C-terminal parts , which represents an isolated group of reclovirids  and can tentatively be regarded as members of another genus of Benyviridae.lovirids , encodeslovirids . ORF1 prlovirids  have simlovirids A. The thGymnadenia rhellicani, Ophrys fusca and Ophrys sphegodes. Moreover, similar proteins are encoded by three additional truncated VLRAs lacking the RdRp-encoding region. All of these proteins contain the characteristic CX(3)CX(10)CX(3)C motif in the N-terminal region  shows ol region  and hydrl region . These cArceuthobium sichuanense virus 3 RNA (7341 bases in length) (Arceuthobium sichuanense virus 3 ORF2 protein has the only counterpart with obvious similarity (identity 38%) found in the Viscum album VLRA (hydrophobic ORF2 protein) , or two ORF2 and ORF3 hydrophobic proteins, with no sequence similarity to the Arceuthobium sichuanense virus 3 ORF2 protein, as in the case of the Atriplex prostrata VLRA (data not shown). length)  encodes  length) , the Arcprotein) . Four otFinally, it should be noted that among other Reclovirids included in Benyviridae to include algae hosts. First, the phylogenetic analysis confirms the placement of monopartite charophyte algae-infecting viruses as a separate branch in the phylogenetic tree of beny-like viruses. These algae viruses are tentatively named “Charavirids”. Second, the presented analyses indicate that benyvirus-like replicases started their evolutionary radiation in red algae (Rhodophyta), which are the most ancient organisms in the kingdom Plantae (Archaeplastida) . Rhodophyta-infecting potentially monopartite beny-like viruses are revealed in two clusters of the phylogenetic tree based on the RdRp core domain. We have found two red algae species carrying potential beny-like viruses, namely, Lithophyllum crustose and Agarophyton vermiculophyllum. The latter virus is positioned in the most basal part of the RdRp phylogenetic tree, and the corresponding VLRA also encodes a tobamo-like CPL, as is known for several members of the genus Benyvirus and some beny-like plant and insect viruses. In particular, the tobamo-like CPL is also encoded by the genomes of Charavirids (see above). Generally, the observed wide distribution of tobamo-like CPL genes [Benyviridae  and family Virgaviridae  strongly suggests that horizontal gene transfer has played a significant role in the evolution of these virus taxa [Hepelivirales) and virga-like RdRp genes  is estimated to be ~900 mya [Hepelivirales and Martellivirales could have occurred in marine red algae species. In support of the proposed role of Rhodophyta as a host for common ancestors of Hepelivirales and Martellivirales, both types of viruses are found in present-day red algae hosts [The data presented in this paper expand the known host range of the multipartite viruses in the family PL genes  in monoprus taxa . The divrus taxa . On the ~900 mya . Therefo~900 mya . It shou~900 mya ,46,47. Tae hosts . Dicranum scoparium) and the TGB in Polypodiopsida (Fern benyvirus) [The analysis presented in this paper also highlights important clues to the evolution of plant cell-to-cell movement systems. First, the available data suggest that MP evolution started at least in lower land plants, as in these hosts, we have found typical movement gene blocks, such as the TCMB in Bryophyta (VLRA of the moss nyvirus) ,11.Endornaviridae and Partitiviridae [Snake River alfalfa virus, Carrot flavi-like virus 1 and Gentian Kobu-sho-associated virus [Novel data on plant virus diversity suggest that molecular evolution may have given rise to numerous potential plant-infecting virus species, which encode no known movement genetic systems. Previously, this fact has been a point of discussion. In fact, it is known that persistent plant viruses lack cell-to-cell movement systems and do not cause visible symptoms; accordingly, they are transmitted only vertically via gametes. Persistent plant viruses have been found in a few virus families, such as iviridae ,50,51,52ed virus ,54,55,56Similar to many persistent plant viruses, all Reclovirid replicating RNAs have been identified by high-throughput sequencing ,44. TherIn this work, we compared the genome sequences  of some novel plus-RNA viruses from red algae, a marine euglenozoan protist and land plants. Despite the concerns related to the potential risks of contig misassembly and contamination and the existence of unknown genome segments, in general, the reported sequence analyses of beny-like genomes confirm previously reported evolutionary relationships between viruses of non-flowering plants, flowering plants, arthropods and fungi, as well as suggest novel evolutionary links between viruses of red algae and land plants. We proposed several viruses as founding members of new potential taxa (subfamilies or genera) in the virus family Benyviridae. Additionally, the identification of gene blocks coding for movement proteins in viruses of non-vascular plants indicates that beny-like viruses may have acquired movement proteins prior to the emergence of the plant vascular system.


Methods: A prospective cohort study of twenty CrossFit® athletes aged 20–36 years was conducted. Data were collected regarding age, anthropometrics, CrossFit® training experience and upper-limb ROM. The ROM was measured using the ROM-SPORT method. After 7 months, athletes performed a clean movement with a load of 80% one repetition maximum. A Bayesian Student’s t-analysis, binary logistic regression analysis and Receiver Operating Characteristic analysis were performed. Results: The optimal upper-limb ROM profile that predicted correct CPCM performance was 78° in shoulder extension, 173° in shoulder flexion, 107° in shoulder external rotation, 89° in shoulder internal rotation, 153° in elbow flexion, 99° in elbow pronation and 92° in wrist extension . Shoulder external rotation, elbow pronation and wrist extension were found to be the most important ROMs for the efficient and safe performance of CPCM . Conclusion: The upper-limb ROM profile is associated with proper clean performance. Further studies are warranted to determine whether improving flexibility on upper-limb ROM may improve proper clean movement performance.Background: The aim of this study was to determine the optimal upper-limb range of motion (ROM) profile for the catch phase of the clean movement (CPCM) and to identify the key ROMs for performing the CPCM in CrossFit The tra® practitioners reported that the average prevalence of injury during CrossFit® training over the past 6–12 months ranged from 5% to 74%, with incidence rates ranging from 0.74 to 18.9 per 1000 h of training; the most commonly injured regions were the shoulder (22.6–26%), spine (14.3–24.0%) and knee (12.9–18.0%) [® practitioners. In addition, their findings highlight that the most common injuries in both male and female CrossFit® participants involve the elbows (12%) and wrists (11%) [Two recent systematic reviews involving 15,386 CrossFit® athletes. The survey typically asked about risk factors associated with the number of years of participation [Generally, these survey-based research studies identify risk factors for shoulder injuries by comparing data between injured and uninjured CrossFitcipation ,6, athlecipation ,6, weeklcipation ,5,6, precipation ,7, compecipation , anthropcipation ,6 and gecipation ,6.Shoulder injuries are the result of the complexity and extreme demands of the WOD, which is often performed with high loads and improper technique ,7,8,9,10® and other sports [® athletes require adequate upper-limb flexibility for the phases of the movement that require a high ROM [Weightlifting movements often predispose the athlete to upper-limb injuries, especially shoulder and wrist injuries, in CrossFitr sports . The cler sports . These er sports . In addihigh ROM ,14,15. Ihigh ROM ,17,18,19® athletes. This contribution to strength and conditioning and health training can provide a complete understanding of what ROM is required in each joint to successfully perform these weightlifting movements [® efficiently and safely has not yet been investigated [Strength and conditioning coaches and athletic trainers should know the reference values (ROM cut-points) for each upper-limb movement  required in weightlifting exercises in order to implement a flexibility program that allows for correct technical movement and reduces the risk of injury. Achieving the ROM cut-point values required to achieve optimal technique in weightlifting movements should be the goal of flexibility training for CrossFitovements . Howeverstigated . ® athletes. Therefore, the aim of this study was to determine the optimal upper-limb ROM profile for the catch phase of the clean movement and to identify the key ROMs for performing the catch phase of the clean movement in CrossFit® athletes who correctly execute the catch phase of the clean movement will have a greater ROM in the shoulder, elbow and wrist than athletes who do not execute the catch phase of the clean movement correctly. It was also hypothesised that external rotation of the shoulder, pronation of the elbow and extension of the wrist would predict correct execution of the catch phase of the clean movement.The hypothesis of the study was that CrossFit® athletes who properly applied the catch phase of the clean movement (CPCM) technique. This study followed the Strengthening the Reporting of Observational Studies in Injury and Illness in Sports Guidelines and Checklist (STROBE-SIIS) [A case-control study embedded in a prospective cohort study was conducted to determine the optimal upper-limb training ROM for CrossFitBE-SIIS) . The stu® athletes aged 20–36 years was recruited from a CrossFit® gym. A sample of twenty-six CrossFit® gym. A warm-up and dynamic flexibility were performed before the ROM and CPCM tests.Instruction in the testing procedure of the ROM and 80% 1RM of the CPCM test (familiarisation session) took place one week prior to the testing procedure at the CrossFitn = 4), elbow (n = 3) and wrist (n = 2) ROMs. Three measurements were taken for each ROM test and the median was used for the subsequent statistical analysis. Information was also collected regarding confounding variables, such as age, anthropometric measures, CrossFit® experience and the athlete’s level of competition. For seven months (events), the athletes performed their usual CrossFit® training and competitions  and no shoes. Testing was conducted in an independent room of the CrossFit® gym athletes were invited to participate in the study on a voluntary basis. Twenty-six CrossFit® athletes  voluntarily participated in this study. General inclusion criteria that applied to both samples were that they were actively training for CrossFit® and had at least two years of CrossFit® training experience. Those athletes who had upper limb or back pain or injury during the study or six months prior to the start of the study were excluded. All CrossFit® Open. A double-blind method was developed in which the athletes and examiners did not know which participants were assigned to each cohort (CT group (cases) or IT group (control)). All athletes were informed of the benefits and risks of this study and signed a written informed consent form prior to testing, which was approved by the institution. The power of the sample size for this study was analysed as described in the statistical analysis section. Specific criteria for cases were the correct performance of the CPCM test and participation in at least one Spanish CrossFit® experience  and injury history.First, weights and heights were measured with a mobile stadiometer  with accuracies of 0.1 cm and 0.5 kg, respectively. The body mass index was calculated from the body mass and height by dividing the body mass (kg) by height (m) squared. A correction of 0.5 kg was made for the clothing weight. Next, the athletes were asked about their age, CrossFithttp://www.randomizer.org. The inclinometer was calibrated to 0° with either the vertical (shoulder extension and flexion) or horizontal  before the start of the test session. Elbow flexion and wrist extension were calibrated with the position of the arm and forearm, respectively (Aerobic and dynamic stretching exercises were performed before the ROM test. The main passive ranges of motion of the shoulder (extension (SE), flexion (SF), external rotation (SER) and internal rotation (SIR)), elbow (flexion (EF) with shoulder flexion at 180°, pronation (EP) and supination (ES)) and wrist (flexion (WF) and extension (WE) with elbow extension at 0°) were measured with an inclinometer . The ROM tests were performed in a random order that was decided using the software ectively . ® athletes in the clean movement. Of the phases of this movement, the catch phase requires a greater range in the upper limbs. The ROM was measured using the ROM-SPORT method [95%), a third measurement was taken if a difference of more than 6° was detected between the two measurements [The movements measured were chosen because they are used by CrossFitT method ,24. Two urements . Each meurements . 95%. The intraexaminer reliability of ROM measurements at the shoulder, elbow and wrist using an inclinometer  was established previously ,26. The Each athlete was asked to perform a general (aerobic exercise and dynamic stretching exercises) and specific warm-up for the clean movement before approaching the target load (80% 1RM) of the test. This load is considered optimal for stabilising the technical gesture of an Olympic movement ,28. After a 5 min break, the clean movement was recorded in the frontal and sagittal planes by an Olympic movement expert using a SONY FDRA-X33 4K Ultra HD digital video camera . Later, the CPCM technique was analysed using the Kinovea Version 0.9.5 software . The athletes were divided by the expert into a CT group and an IT group according to the technique used for the CPCM. The expert, who was a graduate of physical activity and sport sciences with a weightlifting coach level 1 qualification, focused their examination on the catch position of the clean, which is the phase with greater technical complexity and greater demands on the upper limbs’ ROM. The expert  considerThe sample size was calculated post hoc with input parameters  to provide a difference between two independent (two groups) tests ). The effect size was calculated from the average of the ROM variables . p-values) for hypothesis testing. This method is based on quantifying the relative degree of evidence for two competing hypotheses [01) versus the alternative hypothesis , using the Bayes factor (BF01–BF10). Hypotheses H1 and H0 refer to the probability that the comparison of the evaluated variables is different and equal, respectively.Due to the final sample size, it was decided to use Bayesian statistics instead of frequentist statistics. Bayesian inference was recently proposed as an alternative that is more robust than traditional frequentist statistics  analyses were performed using the open-source statistical software Jamovi version 1.6.23  to determine the optimal cut-off values for the ROMs assessed . The preThe correlation between the ROM  and the execution of the CPCM was determined using Pearson’s chi-square statistic.Six athletes were excluded from the preliminary sample because they showed shoulder pain during the assessment session. Finally, a sample of 20 athletes was selected for the study. The power of the sample size was 0.83 .10 ≥ 20.55, 95% credibility interval ≥ 1.39 (large)).The characteristics of the athletes who met the inclusion and exclusion criteria of this study are shown in 10 ≥ 12.74, 95% credibility interval ≥ 1.27 (large)) between the CT and IT groups in terms of training experience, training frequency, training duration, SER ROM and WE ROM.There were differences  = 12,857, p = 0.000, ղ2 = 0.802), EP ROM ≥ 112° (χ2(20) = 12,535, p = 0.000, ղ2 = 0.792) and WF ROM ≥ 90° (χ2(20) = 12,857, p = 0.000, ղ2 = 0.802) was associated with an adequate CPCM technique.The chi-square test revealed that with a SER ROM ≥ 123°  with the exception of SF ROM . A possible explanation for this finding could be that CrossFit® athletes perform overhead exercises more frequently than powerlifters, bodybuilders, and weight and machine fitness practitioners. In addition, CrossFit® functional exercises are performed with a complete ROM of the technical gesture in order to be counted by the competition judges. However, bodybuilders and weight fitness practitioners do not usually use overhead and wide ROM exercises , as their main goal is aesthetics and muscle hypertrophy [®, 94.8° and 156.2° in powerlifting, 160.3 and 183.6° in bodybuilding; SIR ROM: 43.1° and 57.4° in powerlifting vs. 89° in CrossFit®, 57.4° in powerlifting, 60.5° in bodybuilding; SER ROM: 78.2° and 77.9° in powerlifting vs. 107° in CrossFit®, 104.7° in bodybuilding; SE ROM: 10.5° in powerlifting vs. 78° in CrossFit®, 40.8° in bodybuilding) were likely due to the angle assessment procedure used in each study. Fixation and control of the scapula may be the methodological variable that has the greatest influence on the ROM [To our knowledge, only five previous cross-sectional studies investigated ROMs in strength sports ,65,66,67rlifting ,65, bodyrlifting . In the ertrophy ,66. Likeertrophy . The resertrophy ; the useertrophy  of warmiertrophy . However the ROM .As for the second objective of this study, SER, EP and WE were the most important ROMs for the correct execution of the CPCM with a probability of success of more than 85%. These facts were previously observed in the difference of the ROM means between the two groups (CT group vs. IT group) according to the CPCM, where differences were found in these three ROMs. The kinematic description of the clean movement remarkably shows these three ROMs ,30.The increased flexibility of the shoulder internal rotators, the elbow supinators and the wrist flexors could be due to the fact that these muscles are trained using a comparatively large ROM during the CPCM. Eccentric training is an effective method of increasing muscle flexibility . However® athletes differed according to the CPCM. Athletes who had more years of training experience and training volume (training frequency per week and training duration) were found with strong robustness to correctly execute the CPCM. Furthermore, body mass and body mass index also have a moderate influence on performance in the CPCM. It is possible that low values of both body composition variables are associated with higher athletic performance [® and bodybuilding. The greater flexibility of female athletes predisposes them to the correct execution of the CPCM. With regard to ROM, moderately higher values (B10 = 3 to 10) were found in female athletes than in male athletes for SER and EP ROMs. Therefore, the differences in ROM by sex were not sufficiently robust to achieve the study’s aim of separating male and female athletes. However, these moderate differences in the SER and EP ROMs in favour of the female athletes and even the higher values in the rest of the ROMs can be considered as an important factor for the correct performance of the CPCM. In this sense, the percentage of success observed in this study regarding the correct execution of the CPCM was 80% female athletes versus 25% male athletes. Previous work investigating the upper-limb ROM in strength athletes did not develop this study objective [On the other hand, the age, anthropometric data and sport experience of the CrossFitformance ,78. Withformance , body maformance ,80,81 anformance ,81 in stbjective ,65,66,67® promotes a progressive training program and safe exercise execution technique, improving body composition and physical fitness, including flexibility training  may improve the performance of the clean and reduce injury in CrossFit® athletes.Although CrossFit® athletes should improve the upper-limb ROM profile, technical training (refining clean movement patterns) and a minimum amount of training experience prior to increasing loads on the cleans. Further studies are warranted to determine whether improving flexibility for upper-limb ROMs may improve the clean technique and performance.The optimal upper-limb profile that predicted correct execution of the catch phase of the clean movement was 78° in shoulder extension, 173° in shoulder flexion, 107° in shoulder external rotation, 89° in shoulder internal rotation, 153° in elbow flexion, 99° in elbow pronation and 92° in wrist extension. It was found that three ROM for the SER, EP and WE ROMs are associated with the proper execution of the catch phase of the clean movement with a probability of success of more than 85%. CrossFit


Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe. Direct heart infection, not systemic inflammation, drives influenza virus–associated cardiac pathology in IFITM3 KO mice. Seasonal influenza virus remains a major contributor to human mortality, and the potential for emergence of new pandemic strains is an ever-present worldwide concern  miR133b and miR206 into the viral genome in a manner similar to previous engineering of a virus with blunted replication specifically in hematopoietic cells (7 median tissue culture infectious dose (TCID50)/ml] in embryonated chicken eggs, indicating that relative replicative capacities of the engineered viruses were unaffected in the absence of specific miRNA targeting.To disentangle systemic lung inflammation from cardiac infection in IFITM3 KO mice, we sought to selectively attenuate influenza virus in cardiomyocytes. Influenza virus strain A/Puerto Rico/8/1934 (H1N1) (PR8) is a pathogenic mouse-adapted virus that we previously showed disseminates from the lungs to the hearts of WT and IFITM3 KO mice  (38–44).To validate that PR8-miR133b/206 is attenuated in cells expressing the relevant miRNAs, we infected a mouse myoblast cell line known as C2C12. Compared to the control virus, PR8-miR133b/206 was significantly attenuated in C2C12 cells, suggesting that targeting by miRNAs 133b and 206 potently restricts infection of myoblasts . As a coTo measure the overall pathogenicity of PR8-miR133b/206 compared to control virus, we infected WT and IFITM3 KO mice and tracked their weight loss and survival. Consistent with enhanced disease severity in IFITM3 KO mice, the KO animals lost significantly more weight than WT mice in infections with both viruses . CompariSimilarly, all WT mice recovered from infections with either virus strain, while both infections were lethal in IFITM3 KO mice . DespiteTo further validate the utility of our engineered viruses in dissecting determinants for cardiac dysfunction, we first examined viral loads and lung inflammation after infection by PR8-miR133b/206 and PR8-miRctrl. As shown in Given the inclusion of muscle/cardiomyocyte-specific miRNA-targeting sequences, we predicted that PR8-miR133b/206 infections would be attenuated in cardiac tissues. To test this prediction, we quantified virus titers in the same experimental mice used to derive the data from lungs (5 and 10 days after infection). As expected for PR8-miRctrl, we observed primarily low or undetectable viral titers in WT mouse hearts and significantly higher titers in the cardiac tissue from IFITM3 KO mice at both time points . In PR8-Overall, we have established a controlled experimental system to interrogate roles of lung inflammation versus direct cardiac infection in influenza-associated cardiac dysfunction. Namely, IFITM3 KO mice infected with PR8-miRctrl or PR8-miR133b/206 allow direct comparison of heart phenotypes in animals with equivalently severe lung infections, but with or without high virus replication in the heart.Fibrosis is a broadly observed consequence of severe infectious insults to cardiac tissue in humans. Because IFITM3 KO mice exhibit significant cardiac fibrosis following infection with influenza virus . FibrotiGiven that cardiac fibrosis is a well-established risk factor for cardiac electrical conduction irregularities , we testCardiac manifestations of influenza virus infection are widely attributed to severe lung inflammation, which contributes to systemic tissue damage and exacerbates preexisting heart conditions  to the heart and how productive infection in the heart is achieved thereafter. Single-cell analyses may prove useful for identifying cell subsets in the heart that are infected initially by influenza virus. In addition, virus strain–specific differences influencing not only overall virulence but also tissue tropism may influence cardiac infection and pathology. Certain strains of influenza virus have been shown to preferentially infect upper versus lower respiratory tract, and other sites of extrapulmonary tropism are noted for particular virus strains (PR8-miRctrl and PR8-miR133b/206 were generated as previously described (50) in sterile saline. Mice were monitored daily for weight loss and morbidity and sacrificed if weight loss exceeded 30% of starting body mass or other end-point criteria (severe hunched posture and lack of ambulation) were met. All procedures were approved by the Ohio State University (OSU) Institutional Animal Care and Use Committee (IACUC).Mice 8 to12 weeks of age were anesthetized with isoflurane  and intranasally infected with PR8-miRctrl or PR8-miR133b/206 (50 TCID2 in a humidified incubator. C2C12 and HEK-293T cells were infected with PR8-miRctrl or PR8-miR133b/206 at a multiplicity of infection of 1.0 for 24 hours. For determination of influenza A virus (IAV) infection percentages via flow cytometry, cells were stained with anti-H1N1 IAV NP  and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific). Flow cytometry was performed on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software.C2C12, HEK-293T, and MDCK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% EqualFETAL bovine serum  at 37°C with 5% COIFNβ and IL-6 concentrations in organ homogenates were analyzed using mouse DuoSet ELISA kits (R&D Systems). Quantification of serum IL-6, IL-8, IL-1β, and TNFα was performed using a mouse V-PLEX Proinflammatory Panel 1 kit  and was analyzed by the OSU Center for Clinical and Translational Science. Creatine kinase quantification was performed using the Mouse Creatine Kinase MB ELISA Kit .For subsurface ECG recordings, anesthesia was provided by isoflurane in oxygen at a flow rate of 1.0 liter/min. Mice were placed in a prone position on a heated pad to maintain body temperature, and subcutaneous electrodes were placed under the skin (lead II configuration). ECGs were recorded for 5 min on a PowerLab 4/30 (AD Instruments). Anesthesia was maintained for the duration of the reading. Manual visual inspection of ECG tracings for artifacts or abnormalities was performed before data analysis. ECG traces were then analyzed using LabChart 8 Pro software (AD Instruments) wherein the analysis software performed automatic detection of P, Q, R, S, and T wave onsets, amplitudes, and intervals following data recording. The selections generated by the software were then manually optimized, and individual values and averages of 10-s intervals were recorded.For immunohistochemistry, lungs and hearts were fixed in 10% formalin and maintained at 4°C until embedded in paraffin. Organs were sectioned by the OSU Comparative Pathology and Mouse Phenotyping Shared Resource. Masson’s trichrome staining was used to identify fibrotic replacement of cardiac tissue. Lungs were subjected to hematoxylin and eosin staining to measure gross pathology. Digital images of heart sections were generated using Aperio ImageScope software (Leica Biosystems). Fibrosis images were analyzed via ImageJ (version 2.0.0) as previously described (


We report human monkeypox in a man who returned to the United States from the United Kingdom and reported no sexual contact. He had vesicular and pustular skin lesions but no anogenital involvement. The potential modes of transmission may have implications for the risk of spread and for epidemic control.  The 2022 multicountry monkeypox outbreak has been linked primarily to intimate contact among men who have sex with men . The palmar vesicle was unroofed; a swab of the expressed clear fluid tested positive for nonvariola orthopoxvirus DNA by quantitative PCR (qPCR) and was confirmed as monkeypox virus DNA by qPCR specific for clade 2/3 (West Africa) monkeypox  to clarify viral shedding. We detected virus DNA in a saliva sample, as well as from patient-collected conjunctival and rectal swabs using both the non-variola orthopoxvirus and clade 2/3 monkeypox virus qPCRs. Lesions resolved by day 26 after symptom onset.This patient tested positive for monkeypox virus DNA from several nonlesion samples. The nasopharyngeal and saliva findings are noteworthy because the patient did not report respiratory symptoms. In addition, the detectable viral DNA in the rectal swab specimen in the absence of visible anal lesions or pain indicates a potential for sustained sexual transmission, although the viral DNA levels were low; contamination during self-collection cannot be ruled out. We were unable to assess whether internal rectal lesions were present. This case highlighted the distinctiveness of clinical manifestations as they indicated potential routes of transmission during the 2022 multicountry outbreak of monkeypox. This patient did not report recent sexual contact, did not have evidence of genital lesions or inguinal lymphadenopathy (This case also demonstrates the importance of local monkeypox virus testing, rather than centralized testing in public health or commercial reference laboratories. Local testing enabled diagnosis in <12 hours and immediate notification to local and state public health authorities for isolation and contact tracing. Additional information about monkeypox in a patient without viral prodrome or sexual exposure, California, USA.


Campylobacter species using the polymerase chain reaction (PCR). Based on the PCR results, the animal-level prevalence was 25.96% (range 0–73.98%) and all the positive samples came from four (57.14%) of the 7 herds tested. The current study shows that Campylobacter spp. could be a causative agent in infertility observed in a number of herds in Zimbabwe. Strategies for treatment and control of campylobacteriosis should be enhanced in the country. More research and surveillance are needed to determine the epidemiology of Campylobacter species in Zimbabwean cattle herds.Infertility remains a challenge in breeding herds in most developing countries. In the current study, 104 penile sheath washes were collected from bulls of different breeds and ages from different cattle breeding farms in Zimbabwe. The samples were submitted to the Central Veterinary Laboratory, Zimbabwe, for screening of  Campylobacter spp. and is associated with a number of disease conditions in cattle, primarily related to poor fertility in breeding cows [Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis are most commonly associated with early embryonic death and abortion [Campylobacteriosis is an infection caused by the bacteria ing cows . Campyloabortion , the latabortion . Financiabortion . In counabortion , BGC emeabortion .Campylobacter spp. in breeding bulls was investigated in samples submitted to the Central Veterinary Laboratory for diagnostic testing.In this study, the prevalence of Campylobacter spp. One hundred and four bulls of different ages and breeds were sampled from extensive beef production farms between September 2021 and February 2022. The bulls came from a total of seven farms. The farms are located in different provinces of Zimbabwe, that is, Mashonaland East, Harare, and Matabeleland North. Veterinarians submitted samples for different reasons including prebreeding examinations, surveillance, low pregnancy rates in cows, and abortions.The Central Veterinary Laboratory (CVL), Zimbabwe, provided veterinary diagnostic testing services to farmers and public and private veterinary practitioners. The diagnostic tests carried out include tests for bovine sexually transmitted infections (STIs) such as Skilled veterinarians carried out preputial scraping of the bulls to obtain samples for diagnostic testing. The collected washes were put in a 10 ml container with phosphate-buffered saline (PBS-pH 7.0) and sent to the CVL in cooler boxes for analysis within 24 hours.DNA was extracted from the samples using the Zymo Quick DNA Miniprep Plus kit  according to the manufacturer's instructions. The concentration and purity of extracted DNA were checked using a Nanodrop spectrophotometer .μl reaction containing 9.3 μl of RNA-free water, 8.7 μl of the master mix , 1 μl each of Campylobacter spp. specific primers (primer sequence F-5′ GGC TGA TCT ACG ATT ACT AGC GAT 3′ and R-3′GCG CGC ATT AGA TAC CCT AGT AGT CC 5′), and 5 μl of the DNA template was used in each PCR tube. Nuclease-free water was used as a negative control. The presence of Campylobacter spp. DNA was determined using PCR reaction undertaken as previously described [25 The PCR test was carried out using a thermocycler .μl PCR products alongside a 100 kb ladder on 2% agarose gel containing a fluorescent dye in 1X TAE buffer at 90 volts for 1 hour. Visualisation of the gel was performed using a gel documentation system (Kodak Gel Logic System 100). The expected band size for Campylobacter spp. was 600 base pairs (bp).Gel electrophoresis was carried out using 10 Campylobacter spp., divided by the total number of herds tested.Data were entered into MS Excel , and animal prevalence was determined by calculating the number of positive bulls divided by the total number of sampled bulls. Herd prevalence was calculated by the number of herds, which had at least one bull testing positive for Campylobacter spp. was 25.96%, and all the positive samples came from four (57.14%) of the 7 herds. Only three (42.9%) herds out of 7 herds had all the bulls testing negative for Campylobacter spp. . In recent years, farmers have lost cattle due to drought and diseases. The current thrust of the Zimbabwean government is to replenish and increase the national herd . One of e losses . The litCampylobacter fetus subspecies venerealis was performed in 1989 [The current prevalence and impact of campylobacteriosis in Zimbabwe are unknown. The last known study on prevalence of  in 1989 , where 1 in 1989 . PremoveCampylobacter spp. The high prevalence of Campylobacter spp. infection noted in this study warrants further research on campylobacteriosis in Zimbabwe. It is important to note that the positive Campylobacter spp. samples identified in the CVL laboratory were the first in over ten years . In the past decade, farmers and animal movement regulatory authorities did not focus much on bovine sexually transmitted infections, and this may have resulted in the introduction of the infection into herds unknowingly through cattle movement, loaning, and sharing of bulls [Campylobacter spp. is affected by climate change, locations, animal breeds, and age.In this study, only 3 herds out of 7 had all bulls testing negative for of bulls . The difCampylobacter spp. with the aim of developing intervention to reduce such losses.As STIs decrease productivity of cattle by inducing reproductive losses, reduced conception rates, extended calving seasons, and increased costs, replacing bulls and preventing loss of genetic potential due to culling the disease are of great economic importance. A comprehensive study should be carried out in Zimbabwe to determine the epidemiology of Campylobacter spp. in bull herds in Zimbabwe. There is a need for further surveillance of Campylobacter spp. in the country using advanced molecular techniques, which will be able to elucidate the subspecies responsible. Veterinary authorities should enforce strategies which can minimize the spread of cattle STIs in the country.The study reports a high prevalence of


Alternaria leaf blotch caused by Alternaria alternata apple pathotype  is an important fungal disease that affects the production of apples worldwide. Mycoviruses harbored in plant pathogenic fungi can confer hypovirulence in their hosts and have attracted widespread attention as potential biocontrol tools. In this study, the coinfection of two mycoviruses, named A. alternata chrysovirus 1 strain QY2 (AaCV1-QY2) and A. alternata magoulivirus 1 (AaMV1), respectively, were isolated from A. alternata f. sp. mali strain QY21. Sequence analyses revealed that AaCV1-QY2 virus belonged to the genus Betachrysovirus and AaMV1 virus belonged to the genus Magoulvirus. These two mycoviruses were found to be associated with hypovirulence in A. alternata, among which AaCV1-QY2 might play a relatively leading role. Because the elimination of AaMV1 from the strain QY21 does not affect the hypovirulence trait, which indicates that the virus AaCV1-QY2 can independently induce slow growth and reduce host virulence. Moreover, the presence of viruses decreased the accumulation of the mycotoxin alternariol (AOH) in A. alternata strains. Intriguingly, AaCV1-QY2/AaMV1 mycoviruses can be horizontally transmitted to other A. alternata strains, and this coinfection can promote the interspecific transmission efficiency of AaCV1-QY2. To our knowledge, this study reports the first description of the member of Chrysovirus is related to hypovirulence in Alternaria spp. that facilitates the development of biocontrol measures of A. mali Roberts. The majority of mycoviruses have double-stranded RNA (dsRNA) or positive-sense single-stranded RNA (+ssRNA) genomes, and they can or cannot have a capsid . Due to oduction . Most vioduction . From anCryphonectria hypovirus 1 (CHV1) is used to control chestnut blight caused by Cryphonectria parasitica, and is effectively commercialized in Europe ] (Botourmiaviridae [Fusarium oxysporum ourmia-like virus 1 (FoOuLV1)] (Partitiviridae [Colletotrichum liriopes partitivirus 1 (ClPV1)] (Endornaviridae [Rhizoctonia solani endornavirus 1 (RsEV1)] (Mitoviridae [Sclerotinia sclerotiorum mitovirus 1 (SsMV1)] (Reoviridae [Rosellinia necatrix mycoreovirus 3 (RnMYRV-3)] (Genomoviridae [S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1)] ] , BotourmoOuLV1)] , Partiti(ClPV1)] , Endorna(RsEV1)] , Mitovir(SsMV1)] , ReoviriMYRV-3)] , and GenHADV-1)]  familiesHADV-1)] . For exaHADV-1)] . The recAlternaria species are very successful pathogenic genera that cause a wide range of plant diseases  that induce phytotoxic effects on a broad range of plant species, generally as virulence factors, aggravating the severity of disease symptoms , causing severe leaf blotch disease all over the world       , belonging to the family Hypoviridae, was identified from an apple orchard in China. And AaHV1 is the first mycovirus reported to slow the growth and attenuate the pathogenicity of Alternaria spp.  , A. alte(AaPmV1) , A. alte , A. alte1 (AAV1) , A. alte (AaCV1) , and A. (AaBRV1) , classifmination . Moreoveria spp. . HypovirA. alternata f. sp. mali strain QY-2 isolated from an orchard in North China, which cause apple leaf blotch disease, and two hypovirulence-inducing mycoviruses, designated as A. alternata chrysovirus 1 strain QY2 (AaCV1-QY2) and A. alternata ourmia-like virus 1 (AaMV1) was identified. Meanwhile, we performed genome characterization to clarify the molecular properties of AaMV1 and AaCV1-QY2, investigate their influence on phenotypic alteration, pathogenicity, transmission efficiency, and nHST production in A. alternata. We also preliminarily explored the interplay between these two mycoviruses in the induction of hypovirulence during coinfection in the laboratory. This is the first description of the coinfection of mycoviruses associated with impaired growth and attenuation of virulence in A. alternata.In this study, we screened the dsRNAs in Alternaria alternata f. sp. mali strain QY-2 was isolated from an Alternaria blotch-diseased leaf of Malus domestica cv. Tengmu 1 in Hebei province of China in 2018. All collected and derived strains were cultured on potato dextrose agar (PDA) plate at 25°C in the dark. The hypovirulence strain QY21 is a single-conidium isolate progeny of QY-2. Mycelial plugs and asexual spores were preserved in glycerol (15%) at −80°C in the Lab of Plant Disease Epidemiology and IPM of Hebei Agricultural University (AUH), Baoding, Hebei province, China, all of which were verified by Koch’s rule and identified by sequencing of the translation elongation factor-1 α (TEF-1α) gene.Malus micromalus) leaf using the TRIzol reagent  as described by the manufacturer. Total RNA quality was evaluated by agarose gel electrophoresis.The 5 mm mycelial plug of the strain QY21 was cultured on PDA medium overlaid with sterile cellophane for 4 days, then fungal mycelia were harvested and ground in liquid nitrogen. Viral dsRNA was extracted from 400 mg of mycelia or virus particles using cellulose  column chromatography as previously described . Separatde novo using MEGAHIT version 1.0  was performed using the SMART TM RACE cDNA Amplification kit  according to the manufacturer’s protocol.Virome sequencing was performed on the Illumina HiSeq 2000 sequencing platform by SHBIO Biotechnology Corporation . Total RNA of the strain QY21 was used as a template for deep sequencing analysis. Preparation of the cDNA library was performed with TruSeq RNA Sample Prep kit v2 . Raw reads were cleaned by removing adapter sequences, and low quality reads were trimmed with FASTP version 1.5.6 before assembly. All clean reads were assemble sion 1.0 . The assSequence assembly of each region of genome was performed with the SeqMan software v7.1.0 . The open reading frames (ORFs) and conserved domains of the mycoviruses were predicted from the National Center for Biotechnology Information (NCBI) website, and their homologous amino acid (AA) sequences were searched using BLASTp programs, respectively. Multiple sequence alignment was performed using the CLUSTAL X v2.0 . A maximFor reverse transcription-polymerase chain reaction (RT-PCR) detection of the different mycoviruses in fungal strains, first-strand cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit  using total RNA as template. Then, the segmental sequences were amplified using Taq DNA polymerase  with the specific primers  to identify the presence of the corresponding mycoviruses. All primers used in this study are listed in g for 15 min to remove cell debris. The resulting supernatant was further ultracentrifuged at 100,000 × g for 1 h  to collect pellets. The precipitates were resuspended in 0.5 ml of PB buffer. Followed by centrifugation at 10,000 × g for 1 min, the final supernatants were overlaid and ultracentrifuged at 70,000 × g for 3 h in sucrose density gradients (10–60%). Virus particle fractions were further centrifuged at 100,000 × g for 3 h, and the resulting pellets were re-suspended in 50 μl of 0.05 M PB buffer. The purified virus particle was visualized with the transmission electron microscope (TEM) Hitachi H-7650  after staining with 2% (wt/vol) phosphotungstic acid solution (pH 7.4).Mycoviruses-infected QY21 strain was used for virus particle purification. Approximately 20 g of mycelia cultured on PDB for 7 days were harvested and ground into fine power in liquid nitrogen. The obtained powder was homogenized with 0.1 M phosphate buffer (pH 7.0) containing 0.1% β-mercaptoethanol and shaken at 150 rpm for 30 min at 4°C, and then centrifuged two times at 10,000 × We performed cycloheximide treatment to eliminate the virus from the strain QY21 as previously described with minor modifications . BrieflyAfter removal of the mycelium cultured on PDA plates, 1 g of agar plugs were cut from the center of 7-day-old fungal colonies and placed in a 5-ml microcentrifuge tube. Extraction solvent [ethyl acetate/dichloromethane/methanol ] containing 2% formic acid was added to each tube, and the plugs were extracted by ultrasonic treatment for 60 min. Then the supernatant was transferred to a clean tube after centrifugation at 4,000 × g for 2 min and freeze-drying the samples for 12–24 h to remove all water. Thereafter, the samples were redissolved in 500 μl of methanol-water (50:50 v/v) with 1% formic acid and filtered through a 0.22-μm pore size nylon membrane . Mycotoxins in the extract were then quantitatively analyzed by high-performance liquid chromatography (HPLC) Agilent 1290 system  using an Agilent Eclipse Plus C18 (50 nm × 2.1 mm × 1.8 μm).The sample injection volume was 2 μl with a flow rate of 0.25 ml/min at 40°C. The mobile phase was composed of phases A (water) and B (methanol), both with 1% formic acid. The elution program was set as follows: 10% B , 10–90% B (0–6 min), 90% B (6–7 min), 90–10% B (7–7.1 min), 10% B (7.1–9 min), and held for further 3 min for re-equilibration . The mycotoxin alternariol (AOH) stock standard was diluted with methanol-water (50:50 v/v) solution containing 1% formic acid to six concentrations  to construct a calibration curve, so as to determine the concentration of mycotoxin in the samples. The toxigenic potential of the virus-free and virus-infected strains was evaluated by testing the pathogenicity of 10 μl of mycotoxins extracted from the different strains inoculated in Begonia leaves.Alternaria strains. Virus-carrying strains QY21 or QY21-C1 were used as virus donors; the virus-free isogenic strain QY21-C2 or other 99 virus-free allogenetic strains were used as virus recipients, respectively. First, a mycelial plug of the fungal strains QY21 or QY21-C1, respectively, was co-cultured for 14 days on a fresh PDA plate with mycelial plug of each recipient, which was kept close to each other (10–15 mm). Then, the mycelial plugs, which were close to the recipient strain but far away from the strain QY21, are transferred to a new PDA plate and cultured in the dark at 25°C for 7 days. Virus transport in these derivative strains was evaluated based on visual observation of distinctive mycovirus-associated colony morphologies and virus detection by RT-PCR with virus-specific primers.The co-culture experiment was chosen to evaluate the potential horizontal transmission of viruses harbored in To determine the transmission trait of the mycovirus of virus-infected strain QY21 to Begonia leaves. Plugs of virus-carrying and virus-free fungal strains were inoculated onto Begonia leaves, respectively. After 7 days of culture, total RNA was extracted from leaves . The presence of the virus in leaves was confirmed by RT-PCR detection.Conidia collected from the cultures of the infected strains QY21 and QY21 were used to prepare the subculture of single spore to analyze mycovirus vertical transmission rates. A total of 100 single-spore cultures were carried out for each virus-carrying strain. The number of single-spore isolates containing the virus AaCV1-QY2 was recorded. The presence of the virus in these isolates was confirmed by RT-PCR detection.To assess morphology and growth rates, 4-day-old mycelial plugs of the strains QY21, QY21-C1, and QY21-C2 were transferred to a PDA plate and cultured at 25°C for 3–7 days in the dark. The morphology of colonies was photographed, and the morphology of hyphae and spores was observed with a light microscope. And the diameters of each colony were measured using the crisscross method at 7 days to calculate mycelial growth rates, with each strain having five replicates.5 spores/ml with sterile distilled water. The prepared spore suspension was dropped onto a concave glass slide and placed on a culture dish containing wet filter paper for 18 h. The germination rate of spores was calculated by counting the number of germinated spores. Spores were considered to have germinated when bud tube length is equal to or greater than the diameter of spores. Approximately 100 spores of A. alternata per strain were observed within each replicate, and all experiments were repeated three times.The potential effects of mycoviruses on spore production and germination were investigated using the following method. Fresh 5 mm fresh mycelial plugs of each strain were inoculated on PDA medium under near-UV light (12 h light/12 h dark) for 7 days. Spores were collected by flushing the culture with sterile distilled water containing 0.05% (v/v) Tween and residual mycelia were removed by filtration through a glass fiber filter. The spore production of mycelia per square centimeter was measured with a hemocytometer. For the spore germination assay, the concentration of spore suspensions of each strain was adjusted to 1 × 10For virulence assessment, mycelial plugs or mycotoxin of each strain were inoculated on detached apple fruit and Begonia leaves. Diameters of lesions developed after 7 days of inoculation were photographed and measured. All tests were maintained at 25°C in a humid container. Each of the above tests was conducted three times, and each strain had five replicates.post hoc t-tests to determine significant differences at a significant level of p < 0.05 with SPSS 22.0 software. Unless otherwise stated, all experiments were repeated at least three times independently.Statistical analyses were performed using a one-way analysis of variance (ANOVA) followed by A. alternata strain QY21, and it was treated with DNase I and S1 nuclease before 1% agarose gel. Multiple dsRNAs were detected, especially three distinct dsRNA bands with the size of approximately 0.8, 3.0, and 3.5 kbp, respectively, indicating the presence of viral infection in the strain QY21 .The full-length cDNA sequences of the dsRNA segments were determined by the combination of RT-PCR with specific primers  and the LC350277.1, identity: 93%). The length of ORF1 is 3,351 bp, which has the potential to encode 1,117 AA residues contained a putative RdRp domain, starting at position 205 nt with an AUG codon and terminating with a UAA codon at positions 3,556–3,558 bp . No known domains were predicted in ORF3, ORF4, or ORF5; however, ORF3-, ORF4-, and ORF5-encoded proteins showed the highest sequence identities with proteins deduced from dsRNA3, dsRNA4, and dsRNA5 of AaCV1, respectively. The 5′-untranslated regions (UTRs) are relatively conserved among the five RNA segments of AaCV1-QY2 , so it was temporary named as A. alternata ourmia-like virus 1 (AaMV1).The putative genomic organization of AaMV1 and AaCV1-QY2 were shown in 3,558 bp . ORF2-enaCV1-QY2 . With thaCV1-QY2 , and simaCV1-QY2 . OFR6-enBetachrysovirus in the family Chrysoviridae  was significantly lower than that of the virus-free strain QY21-C2 (1.6 × 106 spores/cm2)  .In addition to the influence of virus infection on the morphology of fungal host, the mycelial growth and spore germination rate of virus-infected strains was significantly inhibited compared with the virus-free strain . The resTo verify and evaluate the hypovirulence-inducing effect of the mycoviruses AaMV1/AaCV1-QY2 on the fungal host, virulence assays were performed on detached apple fruits and Begonia leaves. After 7 days of inoculation, the average diameter of diseased spots on Begonia leaves caused by the virus-infected strains QY21 (0.18 cm) and QY21-C1 (0.21 cm) were significantly smaller than that caused by the virus-free strain QY21-C2 (1.51 cm) . MeanwhiA. alternata. In this study, the virulence assay of toxins extracted from the three A. alternata strains in this study showed that total mycotoxins of the strains QY21-C1 and QY21-C2 had a remarkably stronger pathogenicity in Begonia leaves than in the strain QY21 ssRNA with a size of 2.3–3.6 kb and includes a single ORF encoding RdRp. In addition, ourmia-like mycoviruses frequently lack coat protein and movement protein, hence the genomes are commonly found in the lipid vesicles of cells as an RNA/RdRp nucleoprotein complex rather than as virus particles , Magnaporthe oryzae chrysovirus 1-A (MoCV1-A), F. oxysporum f. sp. dianthi mycovirus 1 (FodV1), and BdCV1 induce hypovirulence in the their hosts  suppresses non-self-recognition of the fungal host and facilitate coinfection through the horizontal transmission of mycoviruses between different strains  related to members of the genera https://www.ncbi.nlm.nih.gov/genbank/, MK672910; https://www.ncbi.nlm.nih.gov/genbank/, MK672913; https://www.ncbi.nlm.nih.gov/genbank/, MK672912; https://www.ncbi.nlm.nih.gov/genbank/, MK672911; https://www.ncbi.nlm.nih.gov/genbank/, MK836314; https://www.ncbi.nlm.nih.gov/genbank/, MW492 539; and https://www.ncbi.nlm.nih.gov/, PRJNA832420.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: YW and KC designed the study and revised this manuscript. BL analyzed the data and wrote the manuscript. YC, JZ, PD, and ZJ performed the experimental work. XM, TH, and SW helped to revise this manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The International Virus Bioinformatics Meeting 2022 took place online, on 23–25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus–host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting. The International Virus Bioinformatics Meeting (ViBioM) was the fifth edition of the virus bioinformatics meeting organized by the European Virus Bioinformatics Center (EVBC). The EVBC was founded in 2017 to bring together experts in virology and virus bioinformatics in Europe ) from 14ViBioM 2022  was planned to take place in Valencia, Spain in March 2022. As the number of Omicron cases in Europe were rapidly increasing in January 2022, we decided to again switch to an online format to avoid creating a transimission hotspot for SARS-CoV-2 and to make the conference planning less complicated (in terms of safety regulations and travel restrictions).Virtual meetings have several advantages and disadvantages. The flexibility of listening to selected talks and not being compelled to travel  makes online meetings accessible to a broader range of scientists. Therefore, we had an incredibly high amount of registered participants. Even during the ongoing conference there were registrations up to the last day. There was a lot of fluctuation among the 380 registered participants and not everyone attended all talks. However, we had a solid base between 100–150 participants during each of the talks. From all registered participants, ∼19% are EVBC members; thus, ViBioM is attracting scientists far beyond the EVBC community. As in 2020, the participants made it possible to create a highly interactive scientific environment even without physical face-to-face interactions. Breakout rooms of the speakers for continued discussion during the coffee breaks were extensively used.Another benefit of the virtual format was the flexibility we had when putting the program together. Due to the high amount of submissions on SARS-CoV-2-related research, we decided to add an additional conference day solely focusing on SARS-CoV-2. The extended meeting took place 23–25 March 2022. In total, the meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions.A number of high-quality presentations were given by leading experts and junior scientists on several different topics in virus bioinformatics. From over 50 submissions (a ∼25% increase compared to 2020 ), we selDuring three virtual poster sessions, 52 posters were presented. In order to imitate onsite poster sessions, we set up a breakout room for each poster. Participants were free to visit and switch between the breakout rooms at all times during the poster session. The posters were made available beforehand. Three presenters were selected for the Best Poster Award: Gabriel Lencioni Lovate  and Fernando González-Candelas , one of the ViBioM 2022 organizers. Two keynote speakers were invited on this topic: Francois Balloux  opened the conference with a talk about the changing landscape of SARS-CoV-2 genetic diversity; and Philippe Lemey  opened the afternoon session and spoke about SARS-CoV-2 genomic epidemiology see . From thAs the COVID-19 pandemic unfolded, viral genomic data was produced at an unprecedented scale, allowing us to track the SARS-CoV-2 evolutionary and epidemiological dynamics and providing important insights for intervention strategies. Here, I will highlight a number of developments in a Bayesian statistical framework in support of SARS-CoV-2 phylodynamic reconstructions, including the integration of individual travel history and mobility data  and its The ongoing SARS-CoV-2 pandemic has observed an unprecedented amount of rapidly generated genome data. These data have revealed the emergence of lineages with mutations associated to transmissibility and antigenicity, known as variants of concern (VOCs). A striking aspect of VOCs is that many of them involve an unusually large number of defining mutations. Current phylogenetic estimates of the substitution rate of SARS-CoV-2 suggest that its genome accrues around 2 mutations per month. However, VOCs can have 15 or more defining mutations, and it is hypothesised that they emerged over the course of a few months, implying that they must have evolved faster for a period of time.In this talk I will present detailed molecular clock analyses of genome sequence data from the GISAID database to assess whether the emergence of VOCs can be attributed to changes in the substitution rate of the virus.Our results indicate that the emergence of VOCs is driven by an episodic increase in the substitution rate of around 4-fold of the background phylogenetic rate estimate that may have lasted several weeks or months. This outcome stands in contrast with the notion that the virus has overall increased its mutation rate. In sum, this study underscores the importance of monitoring the molecular evolution of the virus as a means of understanding the circumstances under which VOCs may emerge.Kunaphas Kongkitimanon, Martin Hölzer, and Hugues Richard contributed to this work.The evolution of the SARS-CoV-2 virus has demonstrated the emergence of waves of variants that reveal more worrying phenotypes, e.g., resulting in higher antibody escape or transmissibility . Many nehttps://github.com/rki-mf1/covsonar (accessed on 1 May 2022)). Based on the mutation profiles, VOCAL then detects the potentially concerning samples and ranks them according to three tiers of alert level: high, moderate, and low impact. Finally, VOCAL combines and summarizes all results in an HTML report to help users to investigate the raised alerts quickly ) during two scenarios of emerging VOCs in 2021: the Delta variant (April) and the recent Omicron (December). Our testing set demonstrated that all of the VOC samples were correctly detected as a high concern (Delta: 30/30 (100%); Omicron: 3372/3446 (97%)). For Delta, we detected an additional set of 21 samples, which were mainly assigned to lineages B.1.617.1 and B.1.617.3 and have also been reported as concerning during that time. In conclusion, VOCAL is a specialized tool for the early detection of potentially concerning variants from enormous collections of SARS-CoV-2 genomes. The tool is freely available as stand-alone annotation and visualization or as a comprehensive workflow for molecular surveillance ).We retrospectively assessed the prediction power of VOCAL by considering all German sequences .BREAKFAST is freely available at This session was chaired by EVBC member Magda Bletsa  Two keynote speakers were invited on this topic: Emma Hodcroft  opened the session with a talk on phylogenetics, pandemics, and what comes next see ; followeSince the announcement of the first variant of concern (VoC) in December 2020, the COVID-19 pandemic has been increasingly shaped not only by viral spread, restrictions, and immunity, but also by variants with increased transmission and immune evasion. Detecting and tracking these emerging variants—and deciding how to react to them—has been no small challenge. With over 7 million publicly available sequences, and millions of unique clusters of sequences, identifying those with mutations of interest and determining if they might be the next VoC is far from straightforward. As the pandemic progresses, heterogeneity in immune history, through infections, vaccinations, and boosters, also means increasing heterogeneity in how ‘concerning’ a VoC may be: the impact of Omicron varied widely across countries. In turn, future variants on the ‘road to endemicity’ may pose different risks to different populations.Though it is impossible to predict what future variants may mean for how much SARS-CoV-2 continues to impact society, the return of pathogens that were suppressed during the restrictions of 2020 and early 2021 are a reminder of the common disparity in data and understanding between SARS-CoV-2 and the world of viruses we live in. How do we pivot our real-time test of the role that sequencing, modeling, and immunity panels can play in public health to a sustainable real-life integration of research and healthcare for a better understanding of human viruses overall?John Juma, Vagner Fonseca, Samson Limbaso, Peter van Heusden, Kristina Roesel, Rosemary Sang, Bernard Bett, Alan Christoffels, Tulio de Oliveira, and Samuel Oyola contributed to this work.https://www.genomedetective.com/app/typingtool/rvfv/ (accessed on 1 May 2022). A user can provide up to 4000 multi-FASTA sequences. Validation of the tool has been performed on a large dataset comprising of partial and whole genome sequences obtained from public database. The Rift Valley Virus typing tool was able to correctly classify all 129 RVFV sequences at species level with 100% specificity, sensitivity and accuracy. All the sequences in lineages A  in Africa has been shaped mainly by the environmental changes such as abnormal rainfall patterns, climate change, and land subsidence that occurred over the last few decades. These gradual environmental changes are believed to have effected gene migration from macro  to micro (reassortment) levels. Presently, 15 lineages of RVFV have been identified to be circulating within the sub-Saharan Africa see . InternaThis session was chaired by EVBC member Kevin Lamkiewicz . Friedemann Weber  was invited for a keynote talk to speak about diverse anti-interferon strategies by members of the genus phlebovirus see . From thThe genus Phlebovirus  contains species covering a wide spectrum of virulence. Rift Valley fever virus (RVFV), for example, is highly pathogenic, whereas the Sandfly fever Sicilian virus (SFSV) displays an intermediate level of virulence. Although the importance of the mosquito-borne phleboviruses is increasingly recognized, we are only beginning to understand their mechanisms of pathogenicity. A key virulence factor of phleboviruses is the non-structural protein NSs, an inhibitor of the antiviral type I interferon (IFN) system . Our groThus, our investigations have demonstrated two surprisingly different IFN escape strategies by these related phleboviruses. While the highly virulent RVFV destroys key host factors of innate immunity, the more benign SFSV only sequesters them.https://github.com/gruber-sciencelab/SMEAGOL (accessed on 1 May 2022)). In order to provide the community with a comprehensive overview of potential single-stranded RNA (ssRNA) virus-RBP interactions, we have applied SMEAGOL to 197 ssRNA virus genomes  phages. Community-type CrM demonstrated a high alpha-diversity and a high relative abundance of Caudoviricetes [CrAss] and Malgrandaviricetes phages. Distance-based redundancy analysis (dbRDA) allowed us to determine the metadata affecting the virome composition (Inflammatory bowel diseases (IBD) are a group of chronic inflammatory diseases of the gut. It is commonly divided into two major variants, ulcerative colitis (UC) and Crohn’s disease (CD). The pathophysiology is unknown; however, it is thought to result from an aberrant immune reaction to the commensal gut microbiota. Community-typing is a common practice in bacteriome analysis allowing for the stratification of individuals based on their gut microbiome  ,66. Simiposition B, left.  samples B, right.type CrM C. We conLuca Nishimura was selected for Best Poster Award.Ancient DNA has been extracted from historical samples such as bones and teeth. Recently, ancient DNA studies have shed light on ancient people’s genomes and have elucidated the population structures and migration histories at ancient times. Additionally, we can discover the ancient microbial or viral genomic information from the ancient DNA of human remains. Some viruses that existed in ancient people were pathogenic and valuable to understanding the pandemic in ancient times. On the other hand, most were non-pathogenic and related to ancient people’s health. Therefore, it is crucial to analyze those ancient viruses to comprehend viral evolutions and ancient people’s health conditions.Here, we utilized whole genomic sequencing data of the “Jomon” people, who lived in the Japanese archipelago more than 3000 years ago, to analyze the ancient viral genomes. As a result, we obtained several ancient viral genomic information related to oral commensal bacteria and marine habitats. They might be related to ancient Jomon people’s diet. Moreover, we successfully reconstructed an ancient Siphovirus contig89 phage genome from 3800-year-old specimens and utilized it to construct phylogenetic trees . Our resThe EVBC was founded in 2017 to bring together experts in virology and virus bioinformatics , 66 new EVBC is offering several services to our members and the virus bioinformatics community. We are publishing a monthly newsletter, informing about recent research results, upcoming events, job vacancies and further announcements. Moreover, we are curating a list of specific bioinformatics tools to be applied in virology.At the last meeting in 2020, we experienced an increase in registrations after announcing the online event. This has made the meeting accessible to a broader range of scientists, in particular younger researchers and researchers newly entering the field. This brought us to the idea to set up a monthly online lecture series ‘viruses in silico’ to keep scientists up to date with the latest developments in virus bioinformatics, especially new tools. This lectures is attended by 20–80 participants each month. In addition, we are organising a monthly ‘Viromics Webinar Series’ for early career researchers studying viruses in complex communities (together with the Center of Microbiome Science at OSU and EMERGE). For the future, we are also planning to set up a workshop program.In a survey among EVBC members, we were asked to introduce our members. We are now posting two member profiles each month to help you to get to know each other and possibly find interesting collaboration opportunities.http://evbc.uni-jena.de/ (accessed on 1 May 2022).To learn more about our work or to become an EVBC member, please have a look at our website As in previous years, in 2022 members of the community met at the International Virus Bioinformatics Meeting to discuss current research of the field. This report summarizes the presented work and we hope that it will allow the wider community to profit from the meeting by gaining interesting insights into the field of virus bioinformatics and its current state-of-the-art research.evbc@uni-jena.de.We encourage interested researchers to join us at the next International Virus Bioinformatics Meeting to be held in 2023 in Valencia. For more information, do not hesitate to contact us via


Ligase IV (LIG4) dificiency is a very rare clinical syndrome with around 50 cases reported to date. This syndrome is caused by biallelic pathogenic variants in the LIG4 gene, which cause DNA damage repair disorders, mainly manifesting as severe immunodeficiency.We report the case of a 15-month-old male child with pancytopenia, growth retardation, microcephaly, history of vaccine-related rubella, elevated immunoglobulin G, and decreased T- and B lymphocytes. Next-generation sequencing revealed LIG4 pathogenic genes and compound heterozygous mutations, namely the missense mutation c.833G > T (p.Arg278Leu) and deletion mutation c.1271_1275del (p.Lys424Argfs*20).This case suggests that LIG4 dificiency can manifest not only as immunodeficiency but also with increased serum IgG levels and pancytopenia, which constitutes an additional clinical phenotype. Furthermore, this case suggests that LIG4 deficiency should be considered upon differential diagnosis of myelodysplastic syndrome in children. LIG4 dificiency is a very rare genetic disease that is caused by mutations in the LIG4 gene 13q22-q34). It is associated with impaired DNA double-strand break repair mechanisms, and its incidence is currently unknown 3q22-q34., 6. The Pancytopenia is common in hematological diseases and secondary to other diseases. The most common causes are suppression of bone marrow hematopoiesis, or malignant hematological diseases of the bone marrow hematopoiesis (MDS), or immunological disease. MDS is a disorder characterised by ineffective haematopoiesis, which has a tendency to evolve into acute myeloid leukaemia (AML) . PrimaryThe patient was a 15-month-old boy who was admitted to our hospital due to failure to growth and poor appetite. He was a full-term gestational child delivered at 40 + 3 weeks of pregnancy. His birth weight was 2.3 kg, head circumference was 31 cm, and body length was 50 cm. There was no history of neonatal asphyxia. He resistance to complementary foods. At 6 months of age, he underwent an operation for polydactylism. He had repeated eczema-like rashes and scattered bleeding points all over the body. He did not have repeated respiratory or digestive tract infections after birth. Moreover, he had no family history of malignant tumours. Regarding his vaccination, he completed the BCG dose, third dose of hepatitis B vaccine, third dose of polio vaccine, third dose of DPT vaccine, second dose of meningococcal polysaccharide vaccine (Group A), and first dose of measles vaccine. The patient had not received any vaccination in the last 5 months before admission.9), neutrophil count (NE) (1.40 × 109/L), haemoglobin (HGB) (87 g/L), and platelet (PLT) count (7 × 109/L).His physical examination showed a weight of 7.4 kg (− 3 SD), height of 72 cm (− 2 SD), head circumference of 40.2 cm , six primary teeth, and normal facies , but normal IgA and IgM values. Upon re-checking 2 months later, IgG was still elevated , whereas IgG4 protein quantification was normal (0.477 g/L，0.08-1.4). Lymphocyte immunophenotyping was performed twice, which showed that the counts of T lymphocytes and especially CD4 + T cells were significantly diminished , PLT 2 U), dexamethasone (0.2 mg/kg for 4 days), and oral prednisone acetate  to suppress the autoimmune reaction. Despite this, the patient’s PLT count did not increase . PLT-specific and histocompatibility (HLA) antibodies were also positive. Furthermore, his thyroid-stimulating hormone level was high, and thyroxine was normal.Whole exome sequencing revealed two heterozygous variants of the LIG4 gene: c.833G > T (p.R278L) and c.1271_1275del (p.K424Rfs*20). Family verification results showed that these two variants were inherited from the mother and father respectively (both heterozygous)  and c.2440C > T (p.Arg814*). Dard et al. . These vNotably, our patient had a rubella virus infection and was vaccinated at 8 months of age. His mother was not found to have rubella virus infection during pragnancy. This is shares similarities with another case reported by Japanese researchers was vaccine-derived rubella virus infection in a Japanese patient with DNA ligase IV deficiency, whose successful bone marrow transplantation and immune reconstitution led to the eradication of the rubella virus . The vacElevated IgG levels in a PID case warrant further serological work up, mainly regarding oligoclonality and antigen specificity. Oligoclonal increases of serum IgG levels may be encountered in SCID. The patient lacked IgG subclasses, and only found that elevated IgG levels and the IgG4 level was normal. There may be abnormal distribution of IgG subclasses in autoimmune diseases and tumor diseases. It was reliable that information about IgG levels and responses to CMV and rubella,and the same time which is indicative of the ability to develop antigen specific humoral responses. However, there was no information on the patient’s ability to mount antibody responses to a simple vaccination, such as tetanus toxoid, and to weaker antigens, including those of bacterial polysaccharides such as anti-pneumococcal and anti-haemophilus influenza type b antibodies.9/L) even after treatment with high-dose intravenous infusion of gamma globulin, PLT transfusion, and glucocorticoids. This suggests that conventional immunotherapy was ineffective in our case for the treatment of thrombocytopenia,sendering haematopoietic stem cell transplantation may be the successful treatment [Previous studies have also reported improvement of PLT counts after intravenous immunoglobulin injection . Howeverreatment , 15–17.Our patient had an initial MDS diagnosis based on persistent pancytopenia. However, there were no obvious signs of ineffective haematopoiesis in bone marrow smear of the patient. The inclusion criteria of suspected paediatric MDS are as follows, including any of the following cytopenias: low WBC, low HGB, low PLT < 150 × 109/L, hypocellular bone marrow . PaediatIn conclusion,we report a LIG4 deficiency which reveals the complexity of this syndrome. It can present with increased IgG levels and pancytopenia, misleading diagnosis towards MDS.DNA LIG4 dificiency is a rare and complex disease. It can present as immunodeficiency, with increased IgG levels and pancytopenia. As conventional immunotherapy for thrombocytopenia may be ineffective. We should be taken to not exclude LIG4 dificiency in patients with MDS, especially in the presence of malformations or signs of immune deficiency.


Mycoplasma pneumoniae (MP) pneumonia has been increasing. However, few studies have investigated the impact of respiratory virus coinfection in patients with MP pneumonia, and their results have been inconclusive. This study aimed to investigate the impact of respiratory virus coinfection in children hospitalized with MP pneumonia.The prevalence of refractory This study enrolled 145 children hospitalized with MP pneumonia between May 2019 and March 2020. The patients were divided into two groups: the respiratory virus coinfection and non-coinfection groups. All the children underwent polymerase chain reaction testing for respiratory virus infection. Information on clinical, laboratory, and radiologic findings were obtained retrospectively via medical chart reviews.), severe pneumonia , and poor response to the stepwise approach for MP pneumonia . In addition, higher levels of liver enzymes and lactate dehydrogenase at admission were associated with respiratory virus coinfection in children with MP pneumonia.Children in the respiratory virus coinfection group were younger than those in the non-coinfection group. Respiratory virus coinfection in children hospitalized with MP pneumonia was significantly associated with persistence of fever more than 6 days (adjusted odds ratio [aOR], 2.394; 95% confidence interval [95% CI], 1.172–4.892The results of this study suggest that respiratory virus coinfection in children hospitalized with MP pneumonia may be associated with refractory MP pneumonia. Mycoplasma pneumoniae (MP) is one of the most common causes of community-acquired pneumonia requiring hospitalization in children [The hospitalization due to community-acquired pneumonia has been a global big social burden, especially in children –3. Amongchildren , 4, accochildren , 2, 5. Mchildren . Howeverchildren . Furtherchildren , 8, has children .Previous studies have identified predictive biomarkers of refractory MP pneumonia and severe MP pneumonia, such as serum lactate dehydrogenase (LDH) and interleukin 17 A –11. AlthThis study enrolled 145 children  hospitalized with MP pneumonia who underwent polymerase chain reaction (PCR) for respiratory virus coinfection, which is routinely performed for all patients hospitalized due to pneumonia in our hospital, from May 2019 to March 2020. The patients were divided into two groups according to the presence of respiratory virus coinfection: the respiratory coinfection and non-coinfection groups. Information on clinical, laboratory, and radiologic findings were obtained retrospectively via medical chart reviews. Our Institutional Review Board (IRB) approved this study and waived the need for informed consent (IRB no. CNUH-2019-261).M. pneumoniae Real-Time PCR kit, Slan; Biocore, Seoul, South Korea) from sputum or nasopharyngeal swab samples and MP serologic tests . All the 145 children showed positive PCR results for MP and 108 showed a highly positive MP-specific IgM titer at the time of admission. The remaining 37 children, who were an initially negative for MP-specific IgM, were identified as undergoing seroconversion of MP-specific IgM within one week of hospitalization.MP infection was confirmed using both PCR . The presence of 16 respiratory viral pathogens, including adenovirus, respiratory syncytial viruses A and B, rhinovirus, influenza viruses A and B, parainfluenza viruses 1–4, bocavirus, metapneumovirus, enterovirus, and corona viruses OC43, 229E, and NL63, was detected in nasopharyngeal swab samples by means of a PCR assay using the Anyplex II RV16 Detection kit . In some of the patients with available sputum samples, the Pneumobacter PCR  assay was performed, which can detect Streptococcus pneumoniae, Haemophilus influenza, MP, Chlamydophila pneumoniae, Bordetella pertussis, and Legionella pneumophila.The cut-off values for interpreting an MP infection status based on the MP-specific IgM titers were assessed in accordance with the manufacturer’s instructions, as positive  and negative . Macrolide resistance was evaluated by identifying the point mutations at both positions 2063 and 2064 in domain V of the 23 S rRNA of MP by the GENECUBE system and GENECUBE P value < 0.05 was considered significant.Comparisons of demographic characteristics, clinical courses, laboratory findings, microbiological features, and radiologic findings between the non-coinfection and coinfection groups were performed using chi-square tests as appropriate for the variables. Logistic regression analysis was performed to identify the factors associated with respiratory virus coinfection in patients with MP pneumonia, after adjustment for age and sex. To exclude the effect of bacterial coinfection, adjustment for age, sex, and bacterial coinfection was also performed. All statistical analyses were performed using IBM SPSS Statistics for Version 24.0 . A P = 0.008; Table P = 0.060). The most commonly co-infected respiratory virus was rhinovirus  and hospitalization  were longer in the respiratory virus coinfection group than in the non-coinfection group , alanine aminotransferase (ALT), and MP-specific IgM titer were significantly higher in the respiratory virus coinfection group than in the non-coinfection group Table .Table 3Streptococcus pneumoniae was found in 8 cases; Haemophilus influenzae, in 11 cases; and both S. pneumoniae and H. influenzae in 8 cases. No significant differences were observed in the proportion of macrolide resistance in MP pneumonia and bacterial coinfection between the respiratory virus coinfection and non-coinfection groups , persistence of fever more than 6 days , severe pneumonia based on chest radiography findings , and poor response to the stepwise treatment for MP pneumonia . Previous studies have shown that the respiratory virus coinfection rates in children with MP pneumonia ranged from 27.3 to 56.1% , 14, 21.3.4%. PreRespiratory syncytial virus is the most common cause of community-acquired pneumonia in children across all ages, followed by rhinovirus . In thisIn the present study, we observed significant associations of respiratory virus coinfections with severe clinical manifestations of MP pneumonia and poor response to the stepwise treatment for MP pneumonia. These findings suggest that a respiratory virus coinfection in MP pneumonia may be associated with more severe clinical outcomes than those in pneumonia caused by a viral infection alone. Studies on the mechanisms underlying the effects of respiratory virus coinfections in MP pneumonia are currently lacking. The altered immune function during the illness of MP pneumonia or immunomodulatory drugs, such as systemic corticosteroids, commonly used for the treatment of MP pneumonia, may play a role in the association of poor clinical outcomes with respiratory virus coinfections in MP pneumonia , 24. AltA previous study showed that the duration of fever was longer in children with MP pneumonia and respiratory viral coinfection than in those without respiratory virus coinfection. However, that study showed no significant differences in clinical features and severity of pneumonia between the two groups . AnotherThe asymptomatic elevation of liver enzyme levels is frequently encountered in MP infection . The preMP infection can be confirmed using PCR and/or serology tests, although both tests have limitations in the early diagnosis of MP infection . A negatThis study has several limitations. Most of the patients were referred, and therefore, the treatment strategies for MP pneumonia before referral to our hospital were different and might have affected the clinical course of MP pneumonia. To address this issue, we treated all patients hospitalized with MP pneumonia using the stepwise treatment approach for MP pneumonia after considering the treatment received at previous hospitals . The genIn conclusion, we identified the clinical significance of respiratory virus coinfection in children with MP pneumonia. Our results suggest that respiratory virus coinfection in children hospitalized with MP pneumonia may be associated with refractory MP pneumonia. Respiratory virus coinfection should be considered in children with MP pneumonia showing poor or no response to the stepwise treatment approach.