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LIG4. This is a phenotypically heterogeneous condition with clinical presentation varying from lymphoreticular malignancies in developmentally normal individuals to significant microcephaly, primordial dwarfism, radiation hypersensitivity, severe combined immunodeficiency and early mortality. Renal defects have only rarely been described as part of the ligase IV disease spectrum.Ligase IV (LIG4) syndrome is a rare disorder of DNA damage repair caused by biallelic, pathogenic variants in LIG4, c.597_600delTCAG, p.(Gln200LysfsTer33), which segregated in the family. LIG4 is encoded by a single exon and so this frameshift variant is predicted to result in a protein truncated by 678 amino acids. This is the shortest predicted LIG4 protein product reported and correlates with the most severe clinical phenotype described to date. We note the clinical overlap with Fanconi anemia and suggest that LIG4 syndrome is considered in the differential diagnosis of this severe developmental disorder.We identified a consanguineous family where three siblings presenting with antenatal growth retardation, microcephaly, severe renal anomalies and skeletal abnormalities, including radial ray defects. Autozygosity mapping and exome sequencing identified a novel homozygous frameshift variant in  The cli3) (LIG4  to micro3)    . She had a termination of her second pregnancy (II-2) at 24 weeks gestation for intrauterine growth retardation, cystic kidneys and anhydramnios, all as visualised on ultrasonography. Post mortem was declined. Her third pregnancy (II- 3) resulted in the birth of a normal female child.During her fourth pregnancy (II-4), ultrasound scan at 15+ weeks gestation showed severe oligohydramnios and the kidneys were not visualised. A termination of pregnancy was carried out at 16 + 3 weeks’ gestation. The post-mortem examination identified symmetrical growth retardation with all growth parameters >2SD below mean, corresponding to a fetus of 14–15 weeks gestation. Examination of the upper limbs showed bilateral proximally inserted hypoplastic thumbs. The left leg was internally rotated with talipes of the left foot. The right kidney weighed 0.07 g and left kidney weighed 0.08 g, each at the 10th centile . HistoloIn the fifth pregnancy (II-5) early ultrasound scans again identified oligohydramnios and abnormal kidneys. On subsequent scans at 17 and 18 weeks the kidneys were not visible. Termination of pregnancy was carried out at 18 + 4 weeks gestation. The post mortem examination identified a male fetus with symmetrical intrauterine growth retardation, with all external growth parameters measuring <5th centile for gestation. The ears were low set and eyelids fused. Upper limb examination showed a small left forearm with the hand connected to the elbow on the left. The left thumb was absent and the right thumb was hypoplastic. There was bilateral positional talipes of the lower limbs, with the lower limb smaller on the left than the right and left 2–3 toe syndactyly. External male genitalia were seen but testes were absent. A skeletal survey confirmed the absent left thumb and radius, hypoplastic and bowed left ulna, hypoplastic right thumb and a hypoplastic sacrum . On direThe mother gave birth to a normal boy following her sixth pregnancy (II-6).The couple provided informed consent for participation in a study approved by South Manchester Ethics committee  and the University of Manchester to determine the cause of their affected children.LIG4 c.597_600delTCAG was predicted to result in a pathogenic frameshift variant p.(Gln200LysfsTer33) and was the most credible variant to account for the phenotype. No homozygous loss of function variants (frameshift or nonsense) in LIG4 are present in the gnomAD database  and the fetal samples from affected fourth and fifth pregnancies (II-4 and II-5). AutoSNPa analysis revealed two shared regions of homozygosity >2 Mb between the two affected individuals, totalling over 9.8 Mb , on chrodatabase  of >140,Sanger sequencing confirmed the presence of the homozygous variant in the affected siblings; both parents (I-1 and I-2) and the unaffected siblings (II-3 and II-6) were heterozygous for this variant.FRAS1  in  to date . Previou to date . The dat in mice .LIG4 presented with dysplastic kidneys, bilateral vesicourethric reflux and urethral valves  have both been previously reported in severe cases of LIG4 syndrome . Howeverl valves , whereasl valves . The sibLIG4 did not correlate with the occurrence of milder kidney or lower urinary tract involvement. It will be informative to document renal involvement in other cases with proximal truncating LIG4 variants. In an individual with biallelic variants in LRIG2, the causal gene for urofacial syndrome (MIM 615112), and a homozygous LIG4 missense variant, the affected child had recurrent episodes of urosepsis, secondary to severe vesicoureteral reflux, leading to left kidney hypoplasia and scarring , a clinically and genetically heterogeneous autosomal recessive disorder which like LIG4 syndrome impacts on the DNA damage repair pathway, and can present with multiple congenital anomalies, including lethal antenatal presentation (Radial ray anomalies have not been reported in individuals with LIG4 syndrome. Previous case reports show other skeletal abnormalities, including hypoplastic ribs, fusion of carpal bones or abnormal vertebrae . The radentation . Approxientation .Table 1CRenal anomalies and limb defects are characteristic of a clinically heterogeneous group of disorders termed acrorenal syndrome . In a seLIG4 is added to the extensive list of genes to be tested when a diagnosis of Fanconi anaemia is considered as part of the differential diagnosis.This report expands the phenotypic spectrum LIG4 syndrome due to premature truncating mutations to include antenatal severe renal phenotype and radial ray skeletal anomalies. The finding in this family facilitates the option for prenatal testing in future pregnancies, and cascade genetic testing in the wider family if married consanguineously to determine their reproductive risks. We recommend that Rajesh Madhu: Methodology, Formal analysis, Investigation, Writing - original draft. Glenda M. Beaman: Methodology, Formal analysis, Investigation, Writing - review & editing. Kate E. Chandler: Resources, Writing - review & editing. James O'Sullivan: Methodology, Formal analysis, Investigation, Writing - review & editing. Jill E. Urquhart: Methodology, Formal analysis, Investigation, Writing - review & editing. Naz Khan: Resources, Writing - review & editing. Elizabeth Martindale: Resources, Writing - review & editing. Tracy A. Briggs: Resources, Writing - review & editing. Jill Clayton-Smith: Resources, Writing - review & editing. Jenny Higgs: Resources, Writing - review & editing. Gauri Batra: Resources, Writing - review & editing. Bronwyn Kerr: Resources, Writing - review & editing. Adrian S. Woolf: Conceptualization, Methodology, Writing - original draft, Supervision, Project administration, Funding acquisition. William G. Newman: Conceptualization, Methodology, Writing - original draft, Supervision, Project administration, Funding acquisition.


Participants were asked to perform bilateral and unilateral multidirectional jumps, and passive ROM of hip , knee (flexion) and ankle (dorsiflexion) joints was also assessed. Significant between-group differences were observed for hip extension with flexed knee ROM in dominant  and non-dominant  legs; hip flexion with flexed knee ROM in dominant  and non-dominant  legs; and for the ratio of hip abduction . However, no significant between-group differences were observed for bilateral and unilateral jump capacity, or for inter-limb asymmetries (dominant vs. non-dominant leg). Finally, no associations were observed between ROM ratio (dominant vs. non-dominant leg) and bilateral jump performance. These findings lead to the suggestion that differences on passive ROM values in young male athletes may be sport-specific. Additionally, there seems to be need for the implementation of training strategies specifically aimed at improving bilateral or unilateral jump ability, or at diminishing inter limb passive ROM differences in order to improve multidirectional jump performance for neither soccer nor basketball youth male players.This study was performed aimed at comparing multidirectional bilateral and unilateral jump performance and passive range of motion (ROM) of lower limbs between soccer and basketball young players and evaluating associations between inter-limb ROM asymmetry and bilateral jump performance. A total of 67 young male athletes participated in this study, who were classified as soccer (n = 40; 15.55 ± 1.5 y; 1.76 ± 0.12 m; 58.15 ± 10.82 kg; 19.84 ± 2.98 kg·m Team sports athletes need to repeatedly perform high-intensity actions, such as sprinting, change of direction, and jumping , 2, thusBasketball and soccer are among the most popular team sports worldwide. Whereas soccer is practiced by approximately 265 million people worldwide  around 4Jumping ability is crucial for successful performance of several team sports’ fundamental motor skills, in addition to being the most widely used task to improve and indirectly measure lower limb power in multidirectional sports , 13. AltFlexibility is also considered a key component for health and sport performance and is defined by The American College of Sports Medicine  as the cThe assessment of inter-limb asymmetries allows the comparison of the performance of one limb with the contralateral limb . Studies have primarily focused on evaluating the associations between inter-limb asymmetries and injury risk , althougTherefore, the aim of this study was threefold: i) to compare bilateral and unilateral jumping capacity between youth soccer and basketball players, along with inter-limb jumping asymmetries; ii) to compare ROM of lower limbs between youth soccer and basketball players, and ROM ratio between dominant and non-dominant leg; and iii) to explore associations between bilateral jump performance and ROM ratio for each sport modality.This study used a descriptive and correlational design to examine differences in jumping performance and passive ROM between soccer and basketball players, and to determine potential associations between inter-limb asymmetries based on measures of jumping performance and passive ROM. All measurements were executed in a single session, performed in the morning at an indoor private room under standard environmental conditions, with participants wearing the gear normally used during training. All testing was supervised by a group of four researchers accredited in strength and conditioning and ROM. Participants performed a standardized warm-up followed, in counterbalanced order, by jumping performance tests and measures of passive ROM of lower limbs. Jumping tests included countermovement jumps and standing broad jumps, performed both bilaterally and unilaterally, in addition to lateral jumps performed unilaterally. In order to avoid any learning effects for jumping measures, two familiarization sessions took place during participants’ physical training sessions performed during the two weeks preceding testing. Participants were asked to refrain from strenuous exercise 48 hours before each testing session and to adhere to their usual diet.2) and 27 belonged to a basketball academy (age: 15.7 ± 1.66 years. height: 1.76 ± 0.12 cm. body mass: 62.33 ± 16.57 kg. body mass index: 19.84 ± 2.98 kg·m2). All participants trained three times per week (approximately 75 min per session) and competed weekly. Inclusion criteria required that participants completed all the tests, whereas participants were excluded if they had experienced an injury in the preceding 3 months. All participants and their respective parents or guardians were informed about the experimental procedures, along with potential risks and benefits associated with participation in the study. They then signed informed assent and consent forms, respectively. The study was conducted according to the Declaration of Helsinki (2013), and approval was granted by the research ethics committee of University Isabel I (Code: FUi1-PI002).A total of 67 young male athletes participated in this study. Forty belonged to a soccer club academy   x 100) . For eacs . ES of ap > 0.05 for all comparisons).The statistical analysis for the comparison of jump performance and asymmetries between young soccer and basketball players did not reveal significant differences between sports, either for CMJs, or for SBJs and LJs  and non-dominant  legs. Young soccer players showed significantly lower HFFK ROM both for dominant (-4.35%) and non-dominant (-2.94%) legs. Significant differences between sports were also obtained for the HEFK test, both for the dominant  and non-dominant  legs. Young soccer players showed significantly greater HEFK ROM for dominant (24.89%) and non-dominant (24.57%) legs. Moreover, significant differences were observed between young soccer and basketball players for the ratio of HAFK . Whereas soccer players showed higher ROM HEFK values in the dominant leg (ratio = 1.02 ± 0.08), basketball players showed higher values in the non-dominant leg (ratio = 0.97 ± 0.11). No significant differences were observed between young soccer and basketball players for the remaining ROM measures .ROM values and comparisons between soccer and basketball players are shown in p > 0.05 for all associations tested).No significant associations were observed between bilateral CMJ and SBJ performance, and the ratio of ROM observed at lower limb joints (The aim of the present study was threefold: i) to compare bilateral and unilateral jumping capacity between youth soccer and basketball players, along with inter-limb jumping asymmetries; ii) to compare passive ROM of lower limbs between youth soccer and basketball players, and ROM ratio between dominant and non-dominant leg; and iii) to explore the associations between bilateral jump performance and ROM ratio for each sport modality. Significant differences between youth soccer and basketball players were observed for specific passive ROM measured at hips. However, no significant differences between sports were obtained for jumping performance, and the association analysis revealed no significant correlations between ROM ratio and bilateral jump performance, either for soccer, or for basketball youth players.Jumping capacity has been established as one of the most prevalent tests in soccer and basketball due to its inherent role as a principal action of the game, and as an important neuromuscular power output measure , 64. SinScreening tests and exercises aimed at improving flexibility are a crucial part of the injury prevention strategies used by coaches in team sports, based on their capacity to achieve and test for optimal ROM values, respectively . The appOur results revealed smaller HFFK ROM both for the dominant (mean difference: 4.35%) and non-dominant (mean difference: 2.94%) leg for soccer players compared to young basketball players, suggesting lower levels of gluteus flexibility for the former. Interestingly, since gluteus muscle is prone to weakness, leading to adverse changes in movement kinematics, increased risk of injury, and decreased sport performance –87, exerp = 0.98), nearly significant differences were observed for the non-dominant leg . Although no clear explanation has been found in the literature for this observation, and thus further research is needed, it is plausible that differences observed for the HAFK ratio might result from sport-specific development of non-dominant HAFK, due to training and match play demands associated with each sport-modality. In this regard, In this regard, Lopez-Valenciano et al. [As for the values of inter-limb passive ROM ratio (dominant vs non-dominant leg), between-sport branches significant differences were only observed for the HAFK test. Whereas young soccer players showed greater hip passive abduction for the dominant leg, their young basketball counterparts showed greater hip passive abduction for the non-dominant leg. Based on the independent between-sport branches comparison performed for the dominant and non-dominant HAFK values, it is clear inter-limb passive ROM HAFK ratio differences resulted from sport-specific values observed for the non-dominant leg. Whereas similar HAFK values were observed for the dominant leg between soccer and basketball young players  lead to passive ROM differences between these young populations, although there is no need for the implementation of training strategies specifically aimed at increasing ROM values, since inter-limb ROM asymmetries are smaller than thresholds suggested as risk for injury (10–15% difference). In addition, the lack of association between ROM asymmetries and bilateral jump performance suggests training programs focused on minimizing inter-limb ROM differences would not lead to an improvement of multidirectional jump performance.These findings suggest that staffs of both sport disciplines could include similar training models for the improvement of jumping ability  in an effort to achieve athletes’ highest performance levels. Additionally, reducing inter-limb ROM asymmetries does not seem to be a key strategy to increase multidirectional jump performance. Finally, coaches should develop an individualized approach to each player aiming to reduce inter-limb asymmetries and consequently, the risk of injury.S1 Data(XLSX)Click here for additional data file.


Antricola delacruzi ticks collected in a hot bat cave in the state of Rondônia, Western Amazonia, Brazil. A. delacruzi ticks, are special, compared to many other ticks, in that they feed on both bats  and bat guano  instead of feeding exclusively on vertebrate hosts (blood feeding). Considering this unique life-cycle it is potentially possible that these ticks can pick up/be infected by viruses not only present in the blood of viremic bats but also by virus shed through the bat guano. The viral metagenomic investigation of adult ticks showed that single-stranded negative-sense RNA viruses were the dominant group of viruses identified in the investigated ticks. Out of these, members of the Nairoviridae family were in clear majority constituting 88% of all viral reads in the data set. Genetic and phylogenetic analyses indicate the presence of several different orthonairoviruses in the investigated ticks with only distant relationship to previously described ones. In addition, identification of viral sequences belonging to Orthomyxoviridae, Iflaviridae, Dicistroviridae, Polycipiviridae, Reoviridae and different unclassified RNA viruses showed the presence of viruses with low sequence similarity to previously described viruses.In this study, we describe the viral composition of adult  Flavi-, Bunya- and Rhabdoviridae [Vector-borne diseases are of major concern for many human and animal populations and there are many viruses of great importance that are maintained and spread by blood-sucking arthropods, such as mosquitos and ticks . Many ofoviridae . Bats seoviridae  and can oviridae  and so fRhabdoviridae, Reoviridae, Flaviviridae and to various orders, such as, Bunyavirales and Mononegavirales [Ornithodoros spp. [Hyalomma ticks [Ixodides ticks [After mosquitoes, the second most important arthropod vector for arboviruses of public health concern is ticks and they have been shown to harbour viruses belonging to different families, such as avirales ,6,7. Theavirales . Exampleros spp. , Crimeanma ticks  and tickes ticks . As mentes ticks .Antricola, Nothoaspis, Ornithodoros [Antricola, composed of 17 species, all inhabiting hot caves supporting thousands of insectivorous Mormoopidae bats, especially of the genus Pteronotus [Antricola is hematophagous. The subsequent stages of nymphs and adults are non-parasitic, and are considered to be “guanophagous”; i.e., they are supposed to feed on bat guano, which is very abundant in these hot caves [In the neotropical region, bats are known to sustain a variety of ticks of at least three genera of the Argasidae family (soft ticks): thodoros ,13. Of peronotus . As oppoot caves . Interesot caves ,17.Rhipicephalus microplus in southern Brazil was determined [Antricola ticks collected in a hot bat cave in the state of Rondônia, Brazilian Amazonia, and through this study, we identified a large number of novel viral sequences from various families.Recently, the viral diversity of termined . Apart fAntricola delacruzi ticks from bat guano in a hot cave in Porto Velho Municipality, state of Rondônia, western Amazon, northern Brazil. The cave structure was composed by a hot and humid ecosystem that generates high temperature (33–38 °C) and an atmosphere rich in nitrogen compounds throughout the year. The guano inside the cave was abundant (approximately 10–30 cm in depth), moist, and sticky, as previously described [In July 2017, we collected 180 adult escribed ,18,19. CThe 180 ticks were washed twice with sodium hypochlorite before being washed in sterile water. Thereafter, the ticks were homogenised in pools of five in TRIzol  using Precelly ck14 tubes  prior to RNA extraction using a combined protocol for TRIzol and Genjet RNA extraction kit  as described previously . The RNAThe remaining RNA was used to obtain cDNA and amplified through a Single-primer isothermal amplification (SPIA) approach using the Ovation RNA-seq v2 kit  according to the manufacturer’s instructions. The SPIA products were purified using Genjet PCR purification kit  according to the manufacturer’s instructions before being sequenced at SciLifeLab/Genome Center  using the Ion S5 XL system  and a 530 chip.E-value ≤ 0.0001) using Diamond (version 0.9.10) [The raw HTS data was imported to the CLC genomic workbench (version 11) and trimmed based on quality Q = 20) and length (≥50). The trimmed reads were annotated through blastx ( and leng 0.9.10) . The dathttps://www.ncbi.nlm.nih.gov/orffinder/) and blastp was used to identify related viruses and determine their similarity to each investigated contig. Muscle alignments and the phylogenetic analyses were performed using MEGA7 [In order to be able to analyse longer viral sequences, de novo assembly using CLC genomic workbench (version 11) was performed to create longer contigs. This was done both on reads belonging to, for example, a specific family extracted from MEGAN as well as on the complete trimmed read data set. For the viral contigs studied in more detail, open reading frames were predicted using ORFfinder  remained. The majority of the reads (>70%) remained unclassified after the blastx analysis. Of the classified reads most were eukaryotic, while the remaining mapped to bacteria, archaea and virus  were of viral origin. The reads classified to 47 viral families, however, for most of these families few reads were found. It was only eight viral families that had more than 500 reads assigned to them, shown in Bunyavirales were in clear majority constituting 89.4% of all viral reads in the data set but also the families Rhabdoviridae and Orthomyxoviridae were found. Most Bunyavirales reads classified as being members of the Nairoviridae family, but also potential Peribunyaviridae members were found. However, investigating the reads that, through Diamond and Megan analysis, were classified as Peribunyaviridae members revealed that most of these showed closest similarity to orthonairoviruses and, thus, the focus in the section below will be only on the Nairoviridae family. In addition, a low number of reads (n. 7) were categorised as unclassified Bunyavirales. Considering the abundance of Bunyavirales reads de novo assembly was performed and the analysis focused on contigs (n. 1065) only.As shown in Nairoviridae family (tri-segmented viruses) were in clear majority, constituting 88.2% of all viral reads in the data set. From the 1065 Bunyavirales contigs, 1055 classified as Nairoviridae. All the segments  were represented although at different proportions. The L-segment, encoding the RNA-dependent RNA polymerase (RdRp), was in majority (55.6% of the contigs), while 34.5% and 9% of the contigs represented the M- and S-segments, respectively. The contigs matching to an individual segment together spanned across most of the entire protein coding sequence, however, they did not fully assemble despite some of the contigs overlapping suggesting that they may belong to several different orthonairoviruses.Reads belonging to members of the Five of the S-segment contigs were long enough to cover at least approximately half of the segment and were, thus, analysed more in detail. The longest contig was 2005 nt in length and is believed to contain the complete coding region of a 500 a.a. (amino acid) long nucleocapsid protein (NP). The other four contigs only covered approximate 50% of the coding region, with two of them covering the first half and the other two the second half. Three of the contigs show >99% a.a. similarity to each other and only 70% similarity to the other two suggesting that there are at least two different orthonairoviruses in the data set. Although, there are different orthonairoviruses in the investigated samples, based on the S-segment, they are still genetically closer to each other than to those available in GenBank as the a.a. similarity to the NP of previously described orthonairoviruses, such as CCHFV, Dugbe orthonairovirus and Erve virus, ranged between 35–44%. This was also confirmed by the phylogenetic analysis . Artashat orthonairovirus, Erve virus and Thiafora orthonairovirus, were 30–37%. Phylogenetically, the two glycoprotein sequences from this study grouped together on a clade closest associate with viruses such as Nairobi sheep disease virus, Hazara virus and Erve virus  showed, in the overlapping part, an amino acid similarity of 60%. The similarity to other orthonairoviruses, such as, ve virus .Unfortunately, no contig covered the entire L-segment. The expected size of the L-segment of orthonairoviruses is 7–12 kb, however, the longest contig was only 2720 nt. The protein sequences of the longer contigs were aligned and showed that in many parts of the RdRp they were nearly identical to each other, however, as for the other two segments there were differences observed. Phylogenetic analysis displayed a similar grouping as for the M-segment . The a.aThe genome of rhabdoviruses range between 11 and 15 kb in size, encoding five different proteins. No complete genome was recovered in this study; the longest contig obtained was 6787 nt in length. This sequence contained a larger portion of the L-gene and the predicted protein (RdRp) showed a distant relation to various rhabdoviruses, such as, Tacheng Tick Virus 7, Quarantine head virus and Chimay rhabdovirus, with a.a. identity of around 30%. Despite the distant relationship InterProScan could identify the following domains: Mononegavirales RNA-directed RNA polymerase catalytic domain and Mononegavirales mRNA-capping domain V. In total, there were seven contigs >1000 nt in length and all of them mapped partially to the L gene. In addition, one of the contigs also contained a region (1551 nt) prior to the L gene that we believe is coding for the glycoprotein. Sequence alignment of the overlapping RdRp protein sequences from the tick samples showed a similarity of 80–100%. As expected, the partial RdRp sequences from the Antricola ticks grouped phylogenetically together and it was also observed that they grouped away from lyssaviruses .Orthomyxoviridae reads classified as thogotoviruses, a group of segmented viruses that are known to infect arthropods, such as, ticks as well as in some cases vertebrates. To analyse these reads further de novo assembly was performed and from the 17,553 Orthomyxoviridae reads only 40 reads remained un-matched while the remaining reads were assembled into 29 contigs ranging from 206 to 2369 nt in length. Unfortunately, the complete segments were not recovered through the data analysis but contigs and reads could be found that matched to all the proteins of the six expected segments i.e., to polymerase PB2, polymerase PB1, polymerase PA, glycoprotein (GP), nucleoprotein (NP) and matrix (M). However, for M the complete/near complete coding sequence was recovered, as were parts of the 3′UTR. The obtained M protein (246 a.a.) was divergent showing a protein sequence identity of around 45% to known thogotoviruses available in GenBank, such as, Bourbon-, Oz- and Dhori virus. Comparisons to other thogotoviruses indicate that potentially about 15–30 a.a. are missing at the 5′UTR. In the phylogenetic analysis our sequence grouped on a separate subclade of thogotoviruses  is similar to that of other thogotoviruses, for example that of Dhori virus (AGCAATAACAAGCAGTACTAGA). Investigation of the other thogotovirus-related contigs showed that the protein coverage and a.a. identity varied between the proteins, as shown in Iflaviridae, Dicistroviridae and Polycipiviridae.Although single-stranded negative-sense RNA viruses were found in abundance also positive-sense RNA viruses were found in the ticks. Below follows a description of the viruses detected in the three main viral families found in this group, i.e., in Iflaviridae reads the complete polyprotein was recovered including parts of the UTR. The polyprotein is encoded by 8922 nt and, thus, consist of 2973 a.a. Protein blast analysis showed that the identified Iflaviridae polyprotein was highly divergent to presently known iflaviruses showing around 30–40% a.a. identity across the complete polyprotein. In addition, a near complete polyprotein of a second iflavirus was recovered from the data. The polyprotein is 2882 a.a. in length and is missing the 3′end. This iflavirus showed only a 39% a.a. sequence identity to the first one but was instead more similar to those iflaviruses available at GenBank showing a 73% a.a. sequence identity to Helicoverpa armigera iflavirus (YP_009344960). In the phylogenetic analysis  , were idDicistroviridae family classified to three genera: Aparavirus (n. 382), Cripavirus (n. 2866), Triatovirus (n. 6437). In addition, a portion of the reads were marked as being unclassified Dicistroviridae (n. 539). The genomes of members of the Dicistroviridae are approximately 8–10 kb in length, however, assembling the different genera reads only yielded shorter contigs. The 10,224 reads mapping to the Aparavirus contig was 1010 nt in length and matched with a high nucleotide identity (78–95%) to the capsid gene of various Acute bee paralysis viruses. A contig of 786 nt matched to another region of the capsid protein with similar identities. Also, contigs matching three regions of the replicase gene of the same virus were found. For these the nucleotide identity was around 80%. However, more divergent Aparavirus contigs were also found showing only 30–40% amino acid identity to characterised Aparavirus available in GenBank indicating that several Aparavirus may be present in the investigated ticks. The longest Cripavirus contig yielded a 757 a.a. long protein sequence showing highest protein identity (45–49%) to the non-structural protein of viruses such as, for example, Drosophila C virus and Cricket paralysis virus. Contigs matching the structural/capsid protein of the mentioned viruses to a similar protein identity were also identified. Also, in the Triatovirus contig set both structural and non-structural sequences were identified. The sequences matched to viruses such as, for example, Homalodisca coagulata virus 1, Triatoma virus and Himetobi P virus with the a.a. sequence identity varying from approximate 45–68%.The longest Sopolycivirus, a few to genus Chipolycivirus and a few were marked as unclassified Polycipviridae. The Sopolycivirus reads showed similarity to a number of viruses found in various insects, such as, for example, Lasius neglectus virus 1, Myrmica scabrinodis virus 1 and Solenopsis virus. Only shorter contigs were obtained and the protein sequence identity varied between 25–90%. All of the Chipolycivirus reads classified as Hubei chipolycivirus, through the blastx analysis, and more specifically to Hubei picorna-like virus 81 and 82. These are both picorna-like viruses that were identified and partially genetically characterised from insects through a large metagenomic study in China [Polycipiviridae viruses, most often low ranging from 23–60%.Only 1271 reads classified within this family, majority of these matched to viruses within the genus Partitiviridae, Totiviridae and unclassified double-stranded RNA viruses were identified through the blastx analysis the vast majority of double-stranded RNA reads (94.2%) belonged to Reoviridae and was, thus, analysed in more details. These viruses have 10–12 segments and have been found in a wide range of different hosts.Although Reoviridae reads classified as rotaviruses and, thus, to further analyse the rotavirus reads identified in the data set the reads were de novo assembled and each contig were inspected to identify its protein sequence identity to rotaviruses present in GenBank. In addition, as rotaviruses have 11 segments coding for different proteins it was investigated if all the segments/proteins could be identified. The results are shown in Majority of the Reoviridae viruses further indicating the presence of other rotaviruses from this family.In addition, two of the contigs obtained from the rotavirus de novo assembly did not map to different vertebrate rotaviruses, but instead showed closest similarity (39% and 43% on protein level) to the RNA-dependent RNA-polymerase of Fako virus (isolated from mosquitoes) and Rice gall dwarf virus, respectively. Also, among the reads that remained unmapped after the de novo assembly were those that showed similarity to different plant and insect The 32,371 unclassified viral reads matched, with a varied protein sequence identity (approximate 25–70%), to a vast number of different viruses having mainly an arthropod or plant host. For many of these only a low number of reads matched each respective virus and will therefore not be discussed more in detail. However, for some viruses in the unclassified group a large number of reads were identified and, in some cases, long contigs could be retrieved when de novo assembled.A 11,482 nt long contig yielded a 3770 long a.a. sequence. No stop codon was observed indicating that the contig is missing coding sequence the 3′ end. Through blastp analysis it was shown that the protein had closest similarity to various unclassified viruses such as Hubei picorna-like virus 55, Changjiang crawfish virus 6 and Rosy apple aphid virus. However, it should be noted that only 910 of the 3770 a.a. matched these viruses and then to a very low identity of around 23%. The part matching was in the predicted RNA-dependent RNA polymerase region. Together this indicates that this is a completely novel RNA virus. In addition, other longer contigs included a 6585 nt long sequence coding for a partial RdRp with closest a.a. similarities (30–37%) to viruses such as Ingleside virus and Hubei virga-like virus 14 and a 6342 nt sequence with a 1881 nt long ORF (no stop codon) coding for a protein showing 42% similarity to the first half of a hypothetical protein of Hubei coleoptera virus 2.A. delacruzi ticks collected from a hot bat cave in the Amazonia. These tick are different from most other tick species in that it is only the larvae that is hematophagous, i.e., feeding blood from the bats in the cave, while the other two stages (adults and nymphs) are “guanophagous” i.e., feeding on the bat guano on the ground of the cave [It is estimated, by the World Health Organization, that 17% of all infectious disease are caused by vector-borne pathogens. Ticks have been somewhat overlooked as important viral arthropod vectors, as few tick-borne viruses of human and animal concern have been known. However, in the last decade several pathogenic tick-borne viruses have been detected showing their importance . In thisthe cave ,19. ConsNairoviridae belonging to the order Bunyavirales. Approximately, 88% of all viral reads in our data set were categorised as belonging to this family and among these sequences matching to all three segments  were identified. Most viruses in this family are tick-borne and are transmitted to different mammals (including bats), birds, fish and reptiles through the blood meal [The results show that a vast number of viruses, from different families and orders, are present in this special ecosystem of ticks and bats. One viral family did, however, stand out and that was the ood meal . In addiRhabdoviridae. These are non-segmented negative-sense RNA viruses known to infect a wide range of hosts, including, vertebrates, invertebrates and plants [One other interesting finding, especially considering the association of the tick and bats, was the presence of viral sequences belonging to the family d plants . Of majod plants . The rhad plants .Orthomyxoviridae and are known to be transmitted to vertebrates by ticks [Orthomyxoviridae members. Depending on the segment investigated the a.a. identity varied between 47–97% to Dhori thogoto virus indicating that the thogotovirus in these ticks most likely belong to its own species within the thogotovirus genus.Sequences matching to all segments of thogotoviruses were also identified. These are segmented viruses belonging to the by ticks . A numbeReoviridae and particularly within the rotavirus genus. Rotaviruses are globally spread viruses that cause gastroenteritis and in development countries they are a major cause of death among young children [An additional finding that could be of interest from a veterinary and public health perspective is the presence of sequences being classified as members of the children . These achildren . The cloIn addition, to the above discussed viruses a number of other highly divergent viruses were identified in the samples. For many of the viruses identified the similarity to other viruses was low, even on protein level, indicating that these might represent novel viruses within previously uncharacterised genera.A. delacruzi ticks have both similarities and differences to those reported from other tick species and from other parts of the world. Vandergrift, K. and Kapoor A. (2019) investigated eight viral metagenomic articles and compared the virome of ticks collected from five different countries  and concluded that flaviviruses were the most common positive-sense RNA virus [Rhipicephalus microplus [Bunyavirales and Mononegavirales and more specifically to the families Chuviridae, Rhabdoviridae, Phenuviridae, Nairoviridae and Orthomyxoviridae. These results are similar to ours as the main negative-sense RNA viruses in our study belong to the families Nairoviridae, Orthomyxoviridae and Rhabdoviridae. However, we could not identify any member belonging to the Chuviridae but it is known that viruses from this family are present in Brazil as it has been identified in Rhipicephalus microplus ticks collected in the southern part of the country [Colitvirus of the family Reoviridae, however, in our study members of the genus Rotavirus were identified. But as discussed previously this finding may reflect the special feeding properties of these ticks.Taken together, the virome of the NA virus . Unlike icroplus  no flavi country . The onlA. delacruzi ticks and the bats it is interesting to speculate whether the bats could act as a reservoir for tick-borne viruses and potentially also being directly involved in spreading these viruses to other mammals. In addition, as the A. delacruzi ticks in the adult and larvae stage feed on bat guano rather than taking a blood meal it would mean that they could potentially ingest and be infected by viruses being shed through the faeces and is, thus, not dependent on a viremic bat. Attempting viral isolation as well as simultaneous viral investigations of ticks (adult and larvae), of bat guano and of blood samples from bats in the same cave would be a possible approach to further investigate the origin, host specificity and ecology of the identified viruses. Further studies are needed to determine the host specificity and possible transmission routes of these different viruses between the ticks and bats. Considering the shared ecological niche of the


Studies to ascertain a possible relationship between herpesviruses and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) have relied heavily on classical approaches, specifically, serological examination for antibodies against virus proteins, primarily structural, and/or increases in viral load –21. ThesBlomberg et al.  used a sAlong these lines, measuring the response in patients' sera using purified virus lysate would only detect antibodies to proteins that are components of the virion  but not proteins expressed during virus lytic/abortive-lytic replication . FurtherIntroduction and Discussion of their manuscript that a recent study (p < 0.857).Lastly, Blomberg et al.  indicatent study  implicatIn conclusion, although the suspension multiplex immunoassay seems appropriate, it may lack the sensitivity of the luciferase immunoprecipitation system  and VirSThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Infection with hepatitis E virus (HEV) represents the most common source of viral hepatitis globally. Although infecting over 20 million people annually in endemic regions, with major outbreaks described since the 1950s, hepatitis E remains an underestimated disease. This review gives a current view of the global circulation and epidemiology of this emerging virus. The history of HEV, from the first reported enteric non-A non-B hepatitis outbreaks, to the discovery of the viral agent and the molecular characterization of the different human pathogenic genotypes, is discussed. Furthermore, the current state of research regarding the virology of HEV is critically assessed, and the challenges towards prevention and diagnosis, as well as clinical risks of the disease described. Together, these points aim to underline the significant impact of hepatitis E on global health and the need for further in-depth research to better understand the pathophysiology and its role in the complex disease manifestations of HEV infection. Hepeviridae [Hepatitis E virus (HEV) is a quasi-enveloped, positive strand RNA virus belonging to the family eviridae . HEV is eviridae . Howevereviridae . Furthereviridae ,5,6,7. Aeviridae . Accordieviridae . Orthohepevirus, containing four species, namely Orthohepevirus A, B, C, and D with A containing the genotypes HEV-1 to HEV-8 [Orthohepevirus C, casually termed rat HEV [HEV belongs to the genus to HEV-8 . The gen rat HEV ,11,12,13 rat HEV . HEV-3 a rat HEV ,15. HEV- rat HEV ,16. HEV- rat HEV . In cont rat HEV ,16. HepaAlthough usually a self-limiting disease in immunocompetent persons, hepatitis E can cause serious complications in at-risk populations such as pregnant women  and orgaThis review focuses on the history and discovery of the hepatitis E virus and discusses reported outbreaks in order to share new aspects and insights into its epidemiology and global circulation. Furthermore, it explores studies on the virology of HEV to portray a detailed picture of the known stages of the viral life cycle. Finally, it briefly summarizes the clinical aspects to underline the significant need for further research into the viral pathophysiology contributing to this underrated emerging infectious disease.An epidemic of HEV was reported in 1955 in Delhi, India, with about 29,000 cases of icteric hepatitis . After t7G), followed by three open reading frames . The 3′ UTR ends with a poly(A) tail (An) like the eukaryotic mRNA structure. Viral replication starts with the translation of the ORF1-encoded non-structural polyprotein. An RNA-dependent RNA polymerase (RdRp) subsequently transcribes the full-length negative-sense RNA. This RNA serves as a template for the synthesis of two viral positive-sense RNAs in infected cells, a full-length genomic RNA, and a subgenomic RNA containing the capsid protein-encoding ORF2 and polyfunctional protein (PP)-encoding ORF3 [HEV particles have an icosahedral shape, are non-enveloped, and form virions with a diameter of about 27–34 nm . The HEVing ORF3 . The genORF1 encodes a non-structural polyprotein of varying length that consists of seven domains comprising a methyl transferase (Met), X and Y domains, a papain-like cysteine protease (PCP), a proline-rich hypervariable region (HVR), RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) . Of thesN-glycosylation sites [® p239, which is currently only approved in China.The ORF2 encodes for the capsid protein. Its N-terminal signal peptide shuttles it to the extracellular compartment. The ORF2 protein contains three potential on sites ,42,43, aon sites ,44. The on sites ,46. ORF2The ORF3 protein is a polyfunctional 13-kDa protein of 113 (genotype 3) or 114aa . Computational homology scans did not reveal any domains comparable to other known viral proteins. It has been shown to bind to microtubules and be involved in particle assembly and egress by interaction with the tumor susceptibility gene 101 protein (TSG101), a key protein involved in the endosomal sorting complexes of the ESCRT  transport pathway and involved in the budding of the viruses ,48,49,50HEV exists as quasi-enveloped viral particles in blood and cell culture supernatant and as non-enveloped virions in bile and feces . When thNaked HEV particles possibly attach to target cells via heparin sulfate proteoglycans (HSGPs)  and heatThe subsequent steps are viral assembly and release. ORF2 and ORF3 and positive-sense genomic HEV RNA are known to form a complex in the ER–Golgi intermediate compartment and produce viral progeny particles ,66. The HEV outbreaks occur mainly in resource poor countries, due to waterborne infections caused by HEV-1 and possibly HEV-2 , often dThe largest outbreaks involved 79,000 ENANBH cases in Kanpur, India, between 1990 and 1991, and 119,000 cases in China between 1986 and 1988 ,94,95. SHepeviridae is divided into the genus Orthohepevirus with the species Orthohepevirus A, B, C, and D, and the genus Piscihepevirus. The species Orthohepevirus A contains eight distinct genotypes that infect humans and other mammals [Orthohepevirus A including host range, transmission routes, and global distribution are summarized in Taxonomically, the family  mammals . It is k mammals ,143. Det(Orthohepevirus C) have been reported [Orthohepevirus A genotypes [Moreover, sporadic human infections with rat HEV reported ,13,144. enotypes   have also been reported [HEV-3 and HEV-4 are zoonotically transmitted from pigs, wild boar, deer, and rabbits in developed countries , where preported ,13. HEV reported ,175, cliAs a large percentage of HEV infections do not present with symptoms, the disease often goes undiagnosed. In individuals presenting with hepatitis, symptoms are indistinguishable from those of hepatitis A. Standard diagnostic tests for hepatitis E include the detection of HEV-specific antibodies, including both anti-HEV IgM and IgG, as well as amplification of the viral genome using conventional PCR or real-time reverse transcription PCR . During L. mauritiana [L. platyphylla [®, is thus far only approved in China [®, only two candidates have entered clinical trials [Although the main site of replication for HEV is the liver, non-hepatic replication has been shown in the brain, kidney, placenta, and others . This mauritiana  and L. ptyphylla , as welltyphylla , and thetyphylla  as potentyphylla . Target-typhylla  and prottyphylla ,202 in Htyphylla , severaltyphylla , Brequintyphylla , and thetyphylla . Howevertyphylla  and the typhylla  advancedtyphylla . Finallyin China , prokaryin China , expressin China ,211, vecin China , and chiin China ,214. So l trials ,216. An Infection with hepatitis E virus is still an under-reported disease worldwide. However, awareness of the prevalence of this virus has grown exponentially in the last decades. Before and since its discovery, there had been numerous large-scale outbreaks in the developing world with tens of thousands of cases, demonstrating that HEV is the most common cause of viral hepatitis worldwide. While HEV-1 and HEV-2 occur mainly in waterborne epidemics in areas with insufficient hygienic conditions, sporadic cases of HEV-3 and HEV-4, which are transmitted zoonotically or via contaminated blood products in industrialized countries, have confirmed that HEV is not a health burden solely of resource poor regions. Although much effort has been put into elucidating the mechanisms of infection, replication, and pathology of the virus, many questions are still left unanswered, underlining the importance of further research on its virology. This is especially important as hepatitis E infections—depending on the genotype—can cause severe liver disease in risk groups such as pregnant women and immunocompromised persons. With these aspects in mind, it is clear that further research is vital to develop more effective treatment options and approved vaccination strategies.


Belgian artist Pierre-Joseph Redouté is the “most celebrated flower painter of quite possibly the entire history of botanical art” and “remembered as one of the greatest botanical illustrators in history,” notes Grace Costantino, from the Biodiversity Heritage Library. Redouté, whose patrons included members of French royalty and other luminaries interested in botany, published more than 2,100 plates depicting more than 1,800 species of flowers, many of which had never been illustrated before. Born in 1759 at St. Hubert in the Belgian province that is now Luxembourg, Redouté was descended from a family of Belgian painters. He trained in his father’s studio, and by the time he was 13 years old, Redouté was earning his living as an artist. In 1782, he teamed with his elder brother, Antoine-Ferdinand, to design the stage scenery for the Théâtre-Italien in the rue de Louvois, Paris. There Redouté started painting flowers during his free time and studied with Dutch artist Gerard van Spaendonck, a professor of flower painting at the Muséum National d’Histoire Naturelle. Spaendonck taught the young Redouté engraving and watercolor techniques, which he continued to use and refine. Artist and author Wilfrid Blunt asserts that Redouté owes much of his success to those discoveries and describes Redouté’s technique as using “pure water colour, gradated with infinite subtlety and very occasionally touched with body-colour to suggest sheen.” Iris germanica featured on this month’s cover comes from Les liliacées, a misleading title for a volume of prints that also features many other plants. This collection was published while the artist was under patronage from the Empress Joséphine, first wife of Napoleon, and is considered to be Redouté’s masterpiece. The image The plant’s gentle intricacy and distinctive three-fold symmetry are apparent in the two indigo blooms, each shown from a different perspective, paired with a third stalk poised to bloom. Faintly striated sword-shaped leaves fan out around the stalks and petals, tapering into finely pointed tips. The upright petals, or standards, and the pendant petals, or falls, are the same color. Author and gardener Steve Bender notes that “Standards and falls may be the same color or radically different colors. Dream up any color combination, and you can probably find it.”Iris comprises more than 300 species and perhaps 50,000 varieties of flowers. Flowering irises fill gardens with an assortment of eye-catching colors in the spring and summer and have been the subject of many well-known paintings by renowned artists.Named for the Greek goddess Iris, who traveled along rainbows to deliver messages between the gods and the Underworld, the genus Iris germanica—which is not actually native to Germany but rather to the Mediterranean and central Asia—has become established throughout the world and naturalized throughout much of Europe and the United States. Irises are diverse in their ecology, flourish under myriad conditions, and have increased in variety and geographic range largely because of human activity. The Missouri Botanical Garden notes that “the presumed father of most modern bearded iris cultivars,” Although generally hardy, irises are susceptible to a number of viruses, including Iris mild mosaic virus, iris severe mosaic virus, iris fulva mosaic virus, bean yellow mosaic virus, narcissus latent virus, and the more recently documented cucumber mosaic virus, broad bean wilt virus, and tobacco ringspot virus. Irises can be affected by multiple viruses at the same time, and the viruses are typically spread by aphids. contagium vivum fluidum.”During the late 19th century, about 50 years after Redouté died, Adolf Eduard Mayer showed that tobacco mosaic disease could be transferred among plants. Dmitry Iosifovich Ivanovsky discovered a microscopic infectious agent that could permeate porcelain Chamberland filters (bacteria could not). And Martinus Beijerinck replicated Ivanovsky’s discovery and called this new pathogen “Since that time, rapid transit, global trade, urbanization and destruction of natural habitats, and modern agricultural practices have accelerated the emergence and spread of viruses among humans, animals, and plants. As the landmark 1992 Institute of Medicine report on Emerging Infections reminds us “. . . in the context of infectious diseases, there is nowhere in the world from which we are remote and no one from whom we are disconnected.” The emergence of SARS-CoV-2 underscores how connected the modern world is, and shows that, like the artist’s iris, emerging viruses continue to expand their diversity and range. Spending a few quiet moments enjoying Redouté’s striking botanical illustration may offer a brief respite to public health professionals, clinicians, and researchers engaged in detecting, preventing, and responding to emerging viruses.


Host population size, density, immune status, age structure, and contact rates are critical elements of virus epidemiology. Slum populations stand out from other settings and may present differences in the epidemiology of acute viral infections. We collected nasopharyngeal specimens from 282 children aged ≤5 years with acute respiratory tract infection (ARI) during 2005 to 2006 in one of the largest Brazilian slums. We conducted real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) for 16 respiratory viruses, nested RT‐PCR‐based typing of rhinoviruses (HRVs), and collected clinical symptoms. Viruses were common causes of respiratory disease; with ≥1 virus being detected in 65.2% of patients. We detected 15 different viruses during 1 year with a predominance of HRV (33.0%) and human respiratory syncytial virus  infections, and a high rate of viral coinfections (28.3%). We observed seasonality of hRSV, HRV and human coronavirus infections, more severe symptoms in hRSV and influenza virus (FLU) infections and prolonged circulation of seven HRV clusters likely representing distinct serotypes according to genomic sequence distances. Potentially unusual findings included the absence of human metapneumovirus detections and lack of typical FLU seasonal patterns, which may be linked to the population size and density of the slum. Nonetheless, most epidemiological patterns were similar to other studies globally, suggesting surprising similarities of virus‐associated ARI across highly diverse settings and a complex impact of population characteristics on respiratory virus epidemiology. Large epidemiological study of respiratory viruses in children from one of the biggest Brazilian slums using sensitive molecular PCR assaysViruses are common causes of respiratory disease in pediatric patients from a Brazilian slum15 distinct viruses circulate in the Brazilian slum, with highest prevalence of rhinoviruses and human respiratory syncytial virusThe epidemiology of virus‐associated respiratory infections presents surprising similarities worldwide despite drastic differences in socioeconomic status and population characteristics Data on virus‐associated ARI from slum communities, particularly from Brazil, are scarce. In a study based on clinical symptoms, ARI in children inhabiting a Brazilian slum were very frequent, representing 50% of child infections events through a 1‐year period.In Brazil, 11.4 million people, nearly 6% of the country's population live in slums  and climatic patterns.2 area (approximately 59.300 inhabitants/km2) and distributed in 21 thousand dwellings including shacks, masonry buildings, townhouses and older and solid buildingsHere, we investigated 16 respiratory viruses by real‐time (reverse transcription)‐polymerase chain reaction (RT‐PCR)–based methods in one of the biggest slums of Latin America, namely Paraisópolis. This community is located within the urban area of São Paulo city, inhabited by 89.000 residents in a 1.5 km2Nasopharyngeal aspirates were collected longitudinally from February 2005 to February 2006 from 282 infants and children. Inclusion criteria were patient age equal or below 5 years and presence of at least 2 symptoms of ARI including cough, coryza, sneezing, dyspnea, wheezing and/or fever. From 282 patients, 12 presented with 2 symptoms (4%), 37 presented with 3 symptoms (13%), 52 presented with 4 symptoms (18%), 84 presented with 5 symptoms (30%) and 95 patients presented with all 6 symptoms that were recorded (34%); for 2 patients we lacked clinical data. From the 282 included patients, 22 presented underlying disease (8%). More specifically, a total of 21 patients had heterogeneous symptoms of heart disease, 16 of which tested positive for at least one virus and 5 of which tested negative. One patient that tested negative for all respiratory viruses was infected with HIV, none presented cystic fibrosis or tuberculosis. The underlying disease did not lead to exclusion from this study. Samples were collected from the pediatric hospital Darcy Vargas (n = 157) and the Paraisópolis Basic Health Unit (n = 125), in the Paraisópolis area, São Paulo, Brazil. The gender‐balanced cohort comprised 136 males and 146 females at a median age of 14 months . Samples per season were: Summer, n = 29 ; autumn, n = 56 ; winter, n = 125 ; spring, n = 72 . Ethical approval was obtained from the Hospital Israelita Albert Einstein research ethics committee (protocols 10/1291 and 191/3). Informed consent was obtained from parents or guardians of children. Samples, symptoms and demographic data were collected by medical personnel upon medical consultation.Laboratory analyses were done using real‐time RT‐PCR assays for 16 respiratory viruses including enteroviruses (EVs), human rhinoviruses (HRVs), human parainfluenzaviruses 1 to 4 (hPiV1‐4), human coronaviruses (HCoV) 229E, NL63, OC43 and HKU1, human metapneumovirus (hMPV), human respiratory syncytial virus (hRSV), human parechoviruses (hPeVs), adenoviruses (AdVs) and influenza A and B viruses (FLU) as described previously.www.megasoftware.net/) on a data set comprising 434 nucleotides from the viral VP2/VP4 domains after deletion of 5′‐untranslated region sequence portions. Recombination in the data set was discarded using RDP V4.95. Only HRV was typed due to frequent detection and rapid evolution allowing sufficient phylogenomic resolution.The HRV evolutionary history was inferred using a Neighbor‐Joining algorithm, a p‐distance substitution model and a complete deletion option in MEGA6  using the Wilson method.Statistical analysis was performed by using SPSS 13.0 (IBM) with 3Many epidemiological features of respiratory viruses in our study were similar to those from other reports worldwide. The first similarity was a high overall virus detection rate of 65.2% (184 of 282 patients) together with the genetic diversity of the respiratory pathogens, including all viruses that were tested except hMPV (Table The second similarity was the predominance of HRV (33.0%) and hRSV 12.1%) detections , whereas HCoV, AdV, hPiV, EV, and hPeV were significantly more frequently detected as coinfections   Figure B which wP = .004), a pattern already observed in surveillance studies carried out in two distinct São Paulo city hospitals.P = .011; Figure The fourth similarity was the seasonal variation of hRSV and HCoV detections. Particularly, hRSVs were more frequently detected during autumn  and of hRSV infections with dyspnea    Figure E. The asP = .001 and P = .01, respectively)  sampling in an area of difficult access, (b) sensitive methodology targeting all major respiratory viruses, (c) contiguous sample collection over 1 year, and (d) the combination of clinical and virological data.Our study has several limitations, including (a) a relatively small sample size, (b) lack of a control group of children with ARI sampled simultaneously in non‐slum areas, (c) sampling over only 1 year, since respiratory virus epidemiological patterns may greatly vary over time, (d) absence of information regarding daycare attendance and influenza vaccination status, (e) sampling of only one slum community, preventing generalization of results and (f) the absence of screening for bacterial pathogens, such as Our data indicate that the epidemiology of virus‐associated ARI is not consistently affected by SES, population density, and climatic factors. Multi‐centric epidemiological studies conducted over several consecutive years from multiple areas representing replicates of climatic, SES‐ and population‐associated conditions, analyzed with identical quality‐controlled methodology would be desirable to better understand respiratory virus infection patterns and potential effect modifiers. Meta‐analyses of the vast literature describing viral ARI epidemiology worldwide may provide cost‐efficient alternative approaches, yet studies focusing on respiratory virus epidemiology in slum populations are scarce compared to studies from affluent settings.


Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a “hot-spot”-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed.in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep.Each Gc-protein with amino acid deletions within the “hot spot” of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations. in vitro growth of such a variant virus isolate was severely impaired. Furthermore, the deletion mutant was attenuated for adult sheep. This study provides important new insights into the mechanisms of virus persistence within chronically infected malformed fetuses and explains the mystery of the “hot-spot” of sequence variations not only observed in SBV, but also in related viruses. The model proposed can represent an example of virus mutations resulting in antibody induced immune escape.Schmallenberg virus (SBV) is a pathogen of veterinary importance and is used as a model virus for studying peribunyaviruses, a complex and highly divergent family of RNA viruses. An SBV-infection of naïve dams during pregnancy may lead to the induction of severe malformation in the fetus. In the medium (M) genomic segment of SBV and related viruses, a region of high sequence variability was detected, affecting the N-terminal major immunogenic domain of envelope protein Gc in malformed fetuses. Variation of this mutation hot spot in fetuses was demonstrated to result in immune-evasion from neutralizing activity in the infected fetus. Accordingly, the neutralization capacity of SBV-specific antisera collected from SBV-infected animals on the  Schmallenberg virus (SBV), which emerged in 2011 in Central Europe, is transmitted by Culicoides biting midges and causes no or only mild non-specific and short-lived clinical signs in adult ruminants .Like other orthobunyaviruses, Schmallenberg virions contain three segments of negative-stranded RNA genome. The large (L) genome segment encodes the RNA-dependent RNA polymerase (RdRp); the medium (M) segment encodes the viral glycoproteins Gn and Gc, as well as a non-structural protein (NSm); and the small (S) segment the nucleocapsid protein (N) and the non-structural protein NSs –6.Within the insect vector season, in which the virus was detected for the first time year 2011), SBV spread very rapidly across the European ruminant population. In the center of the epizootic in northwestern Germany, the Netherlands, and Belgium more than 90% of the tested cattle were seropositive. During the following years, SBV circulated further in Germany, albeit at a much lower level. However, in summer and autumn 2014, the virus circulated again widely in continental Europe  and in t, SBV sprIn malformed calves and lambs, very high SBV genome copy numbers have been demonstrated, mainly in the central nervous system (CNS) ,13, and In January 2015, different tissue samples  of a malformed lamb were submitted to the Friedrich-Loeffler-Institut to be analyzed for the presence of SBV-genome (submission number BH04/15). For the detection of viral RNA, independent real-time RT-PCR assays targeting either on the S-, the M-, or the L-segment  were useSurprisingly, using the M-segment-based PCR, only the lung sample was positive, every other tissue sample tested negative indicating nucleotide variations in the primer and/or probe binding sites or the presence of an M-segment of a related Simbu serogroup virus instead of an SBV-M. As in other viruses with segmented genomes, natural genetic reassortment occurs also in orthobunyaviruses –27. SequIn order to investigate the potential mechanisms further, the reactivity of SBV-specific neutralizing antibodies with various M-segments of SBV was analyzed. The first SBV isolate BH80/11), which was initially obtained from a blood sample of a viremic cow in 2011 1, which , one strSince it was not possible to isolate virus from the diagnostic samples BH04/15 and BH19/14 or to generate infectious virus containing the large deletions identified in these samples by a previously described reverse genetics system , BSR-T7/The original SBV isolate BH80/11 reacted with each of the tested SBV-specific anti-Gc monoclonal antibodies while the M-segment of the virus strains BH148/12 and BH248/12-1 which display only a few aa substitutions across the M-segment could be stained by five out of six monoclonal antibodies . The onlin vitro growth of this very unique virus isolate and the SBV reference strain BH80/11 were compared to each other; the strain 697/2012 showed impaired growth characteristics (4.75 tissue culture infectious dose 50% per ml (TCID50/ml) on mammalian cells in the presence of either monoclonal anti-Gc antibodies [Despite all efforts to isolate virus from highly positive Indel virus containing samples or generate recombinant viruses using reverse genetics, only strain 697/2012 could be isolated on baby hamster kidney-21 (BHK-21) cells. Subsequently, the eristics , a phenotibodies , sera oftibodies  or sheeptibodies  or sera tibodies .2.75 TCID50/ml despite the presence of sera from animals infected with wild-type SBV. The effect of anti-Gc antibodies was equally impaired, and the sera collected from cattle immunized with subunit vaccines had hardly any effect on the in vitro growth of this virus , and in January 2015, different tissue samples  of a malformed lamb (submission number BH04/15) were submitted to the Friedrich-Loeffler-Institut to be analyzed for the presence of SBV-genome.In addition, the first SBV isolate  according to the manufacturer’s recommendation and analyzed for the presence of SBV-genome by independent real-time RT-PCR assays based either on the S-, the M-, or the L-segment ,23. SequThe M-segment plasmids with mutations, deletions and insertions in the hypervariable region according to the wild-type sequences of strains BH148/12, BH248/12-1, BH174/12-1, BH77/12-1, BH04/15, BH19/14 and 697/2012 were generated by fusion PCR on the basis of the cDNA construct pT7ribo_SBV_M which displays the SBV strain BH80/11 sequence downstream of a T7 promoter . Primer Culicoides variipennis larvae (KC) and baby hamster kidney (BHK) cells as described previously [The samples of the diagnostic submissions BH 19/14 and 04/15 were homogenized in phosphate-buffered saline (PBS) and inoculated onto 2 the inoculum was removed and replaced with Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS). After two cell passages a cytopathic effect (CPE) was visible and the presence of SBV was confirmed by real-time RT-PCR and sequencing of the S segment.Isolation of the ovine SBV strain 697/2012 was carried out from brain material of a neonate sheep with severe malformation, delivered dead at parturition. Approximately 2g of brain was homogenized in PBS. 300μl supernatant of this homogenate was inoculated onto a sub-confluent monolayer of BHK cells. Following an incubation of 3 hours at 37°C/5% COIn vitro growth kinetic experiments were performed using BHK cells which were inoculated with either SBV isolate 697/2012 or BH80/11 with a multiplicity of infection (MOI) of 0.1; experiments were performed in duplicates. Supernatants were collected at 0, 8, 24, 48, and 72 hours post infection and titers were calculated by counting CPE-positive wells of BHK cells and displayed as TCID50/ml.4.75 TCID50/ml as determined by endpoint titration using BHK cells. The medium was supplemented with 10% of either FCS (negative control), monoclonal anti-Gc antibodies [2 on BHK cells, and subsequently assessed for CPE. The titre reduction was calculated in relation to the final virus titres of the respective virus grown in the presence of FCS.10-fold dilution series of the SBV reference strain BH80/11 and the newly isolated Gc deletion variant 697/2010 were prepared in MEM starting with a titer of 10tibodies , sera oftibodies  or sheeptibodies  experimetibodies . The dil5 plaque forming units (PFU) in 1ml of SBV isolate 697/2012. The animals were monitored daily to assess clinical disease. Whole blood samples were taken on day 0 and each day thereafter for a period of 14 days. Viral RNA was extracted as described above and analyzed by an S-segment based real-time RT-PCR [Six Cheviot lambs (<1 year old) were inoculated subcutaneously with 8x10e RT-PCR . FollowiS1 Data(XLSX)Click here for additional data file.