Rabies virus (RABV) has a broad host range and infects multiple cell types throughout the infection cycle. Next-generation sequencing (NGS) and minor variant analysis are powerful tools for studying virus populations within specific hosts and tissues, leading to novel insights into the mechanisms of host-switching and key factors for infecting specific cell types. In this study we investigated RABV populations and minor variants in both original (non-passaged) samples and in vitro-passaged isolates of various CNS regions  of a fatal human rabies case, and of multiple CNS and non-CNS tissues of experimentally infected mice. No differences in virus populations were detected between the human CNS regions, and only one non-synonymous single nucleotide polymorphism (SNP) was detected in the fifth in vitro passage of virus isolated from the spinal cord. However, the appearance of this SNP shows the importance of sequencing newly passaged virus stocks before further use. Similarly, we did not detect apparent differences in virus populations isolated from different CNS and non-CNS tissues of experimentally infected mice. Sequencing of viruses obtained from pharyngeal swab and salivary gland proved difficult, and we propose methods for improving sampling. Lyssavirus, and an estimated 99% of human rabies cases are caused by rabies virus (RABV) transmitted by dogs [Rabies is a viral encephalitis that is caused by viruses of the genus  by dogs . Lyssavi by dogs ,3. Furth by dogs .Lyssaviruses are neurotropic and can infect peripheral nerve endings, after which they travel to the spinal cord via the ventral or dorsal root ganglions. After reaching the brain and infecting local neurons, the virus further spreads to the salivary gland and other organs and is finally secreted through the saliva. Despite their neurotrophic nature, lyssaviruses are capable of infecting muscle cells at the initial infection site, various cell types during the centrifugal spread, and acinar cells in the salivary gland, of which the latter step is essential for virus secretion and infection of the next host ,6.RABV, along with other lyssaviruses, are negative single-stranded RNA viruses (-ssRNA). These RNA viruses are generally known to display high rates of evolutionary change by both natural selection and recombination, enabling them to rapidly adapt to new hosts and cell types ,8,9. IntWhile most viral sequencing is performed on brain tissue specimens, given the presence of high viral loads, there is less information about viral populations in other organs. In this study we investigated RABV populations in three different central nervous system (CNS) regions  of a fatal human rabies case , and thePharyngeal swabs contain secreted virus and are therefore an important specimen for studying virus evolution. However, the low virus loads obtained from pharyngeal swabs severely limit the possibility of investigating virus populations. The parotid salivary gland is easy to sample and contains saliva that is ready to be secreted. Therefore, we also assessed the suitability of using salivary gland biopsies as a read-out for secreted virus.g, after which 200 µL of the supernatant was filtered using a 0.45 µm filter and was incubated on mouse neuroblastoma astrocytes (MNA) for 1 h. After removal of the inoculum the cells were incubated with supplemented DMEM medium at 37 °C in the presence of 5% CO2. Virus was passaged when cytopathic effect was observed; the cell culture supernatant was centrifuged and filtered, and 200 µL was inoculated onto freshly seeded MNA cells. The remainder of the filtered virus was stored at −80 °C.Human CNS biopsies  were obtained from a fatal human rabies case in Qatar described previously . Samples5 or 106 TCID50 of silver-haired bat rabies virus (SHBRV) in the left hind leg. This experiment was designed and performed to validate an in vivo infection model, and the samples used in this study were taken after the initial experiment had finished. Mice were euthanized upon showing neurological symptoms, which appeared between day six and eight. Biopsies of target organs and tissues of interest , as well as a pharyngeal swab were taken, homogenized in 1 mL of virus-transport medium, and stored at −80 °C until further processing. In parallel, organs were fixed in 10% neutral-buffered formalin to verify virus protein expression by immunohistochemistry (IHC). Samples were taken from 10 mice in total, from which the samples of four animals were used to investigate differences in viral populations within the salivary gland and pharyngeal swab isolates, and the samples of the remaining six animals were used to compare virus populations throughout different organs . All animal experiments were performed in compliance with Dutch legislation for the protection of animals used for scientific purposes (implementing EU Directive 2010/63) and other relevant regulations. The research was conducted under a project license (AVD1010020187204) approved by the competent Dutch authority. The specific study protocol (18−7204−01) was approved by the institutional Animal Welfare Body.Six-to-eight-week-old mice (C57BL/6) were intramuscularly inoculated with 10Isolates from original materials from the human RABV patient, the third and fifth passage of these materials on MNA cells, and the samples taken from the infected mice, were included for RNA isolation and sequencing. All samples were centrifuged and filtered through a 0.45 µm filter, after which 100 µL of the filtrate was mixed with lysis buffer (Qiagen). The animal samples were pre-processed with Omnicleave (Lucigen) to cleave present host RNA, and all samples were further processed using the High Pure RNA isolation kit (Roche) following the manufacturers guidelines, including the on-column DNA digestion step. RT-PCR was performed as an initial investigation on the presence of viral RNA, using a RABV genotype 1 RT-PCR  on all icDNA was made using random primers (Thermo Fisher) and SuperScript IV (Thermo Fisher) and dsDNA was made using Klenow (NEBNext). The KAPA Hyper Plus kit (Roche) was used to prepare the library for sequencing with minor adjustments: fragmentation time was reduced to 3 min and the adapters were diluted 1:10. Targeted enrichment was performed using VirCapSeq  and all Consensus sequences were generated using a reference-based alignment against the previously sequenced SHBRV-18 strain (AY705373.1) or the RABV_Nepal_2018 strain (MN534894.1). The reads were re-aligned to the newly generated consensus sequences and minor variants with a 20% frequency cutoff were determined using Geneious version 9.1.8 using default settings.Presence of viral proteins in formalin-fixed paraffin-embedded tissue biopsies was verified by immunohistochemistry (IHC), and presence of virus in the cell culture passages was verified by immunofluorescence (IF). Briefly, 3 µM slides were cut from paraffin-embedded tissues of both human and murine samples, and the RABV nucleoprotein (RABV-N) was detected using the 5DF12 antibody (kindly provided by P. Koraka). Images were acquired using an Olympus BX51 microscope. MNA cultures were fixed with ice-cold 80% acetone and were stained with the FITC-conjugated anti-RABV-N antibody (Fujirebio). A nuclear counterstain with Evans blue was included and images were acquired using a Zeiss AX10 Colibri 7 fluorescence microscope.NGS of RABV isolated from three regions of the CNS of a fatal human case , as well as the following in vitro passages in mouse neuroblastoma (MNA) cells  and non-CNS tissues  of C57BL/6 mice that were experimentally infected with the silver-haired bat rabies virus strain SHBRV, A, a straRABV is predominantly secreted via saliva, making saliva the preferred sample for investigating secreted RABV populations. However, obtaining enough saliva sample to successfully culture or sequence secreted virus is often a challenge, especially when working with small experimental animals. We therefore investigated if the salivary gland tissue is a reliable source for characterizing secreted virus populations by performing pairwise collection and sequencing of viruses isolated from pharyngeal swabs and salivary gland biopsies obtained from mice 1–4. Despite the presence of RABV as detected by PCR, only one of these pairwise comparisons (mouse 3) resulted in successful viral sequences of both samples. In this pairwise sample comparison, we found one SNP and three minor variants in the salivary gland isolate and two different SNPs in the pharyngeal swab isolate, of which one resulted in an amino acid change in the polymerase gene . The secTo compare virus populations in CNS tissues with non CNS tissues, isolates from both CNS  and non-CNS  tissues from mice 5–10 were collected and used for sequencing. Successful sequencing required a Ct value below ~28–29 . Due to A limited number of minor variants were detected in this dataset, similar to the SNPs. However, the majority of them were found in single animals and did not appear to be tissue-specific, as they were observed as SNP in other isolates of the same mouse.In the presented study we investigated virus populations isolated from human CNS tissues, and from several CNS- and non-CNS tissues from experimentally infected mice. Although we did not detect RABV evolution within the analyzed human CNS specimen, multiple SNPs and minor variants were detected within few in vitro passages. Rapid virus adaptation to in vitro culture has been described for RABV previously ,15,23, aWhile the Ct levels in the salivary gland extracts were lower than in pharyngeal swabs of mice, only one of the pairwise comparisons between salivary gland and pharyngeal swab isolates resulted in successful viral sequences of both samples. Given the low number of successful sequences obtained from salivary gland isolates, we cannot definitively conclude if the salivary gland is a reliable source for studying secreted lyssaviruses. Most likely the high level of host RNA in salivary glands resulted in this failure to retrieve complete viral sequences. Furthermore, high levels of RNAses present in the parotid salivary gland are known to drastically reduce the yield of high-quality RNA isolated from its tissue . Given tIn line with a previous study , no diffIn conclusion, this is the first study that performed NGS and minor variant analysis on virus isolated from a broad range of CNS and non-CNS samples of human and mouse origin. The mice experiment was performed independently from the collection and in vitro passaging of the human CNS isolates. While this resulted in insights of inter-host adaptations of both a dog-related RABV strain (human case) and a bat-related RABV strain (mice experiment), more in-depth insights into the dog-related strain could be obtained by performing a specifically designed animal experiment that uses a dog-related RABV isolate. Altogether, better understanding of the evolution and adaptation of RABV in the host will be valuable in increasing the understanding of the spread of RABV and related lyssaviruses within different hosts.


A new HP-PRRSV strain (SD2020) was isolated from pigs with suspected highly pathogenic porcine reproductive and respiratory syndrome disease in a pig farm in Shandong Province, China, and its genome was sequenced. This pig farm has been using the VR-2332 vaccine strain to immunize pigs for a long time. The phylogenic and single nucleotide polymorphisms (SNPs) analysis of the viruses isolated from dead pigs showed that SD2020 was a natural recombinant virus of the VR-2332 vaccine strain and the JXA1 similar strain, and that two splicing fragments highly homologous to JXA1 in the virus genome were probably derived from the JXA1 wild strain and JXA1-R vaccine strain, respectively. Therefore, the possible recombination events of SD2020 and its mutation site might be related to high pathogenicity. Arteriviridae family with its genotype 1 first identified in Europe (1991) and genetype 2 in America (1992) [Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), brings about huge economic losses to global pig industry. The major manifestation of PRRS is a respiratory syndrome in sows with reproductive disorders. PPRRSV is a worldwide epidemic virus belonging to a (1992) . Of notea (1992) ,5,6.Vaccination is the most effective and commonly used strategy to protect farmed animals from a variety of animal infectious diseases. Since the PRRSV-2 modified-live virus (MLV) vaccine was licensed in the United States in 1994 and PRRSV-1 MLV vaccine in Europe in 2000 , PRRS vaIn this study, we obtained one new full-length PRRSV genome from a pig farm in Shandong province, China. Retrospective data showed that the porcine in this farm was first immunized with the PRRS vaccine, porcine reproductive and respiratory syndrome vaccine live (CH-1R strain) before September 2019, followed by immunization with a different PRRS Vaccine, porcine reproductive and respiratory syndrome vaccine live (strain R98). However, it occurred that there were increasing numbers of nursery pigs with high fever (40.5–42 °C), dyspnea, and depression, with a mortality rate of 60%. We found that the newly identified virus (referred as SD2020) belonged to the PRRSV-2 type, which was naturally recombined from different lineage strains. Of note, the genome of this infectious strain was recombined from two vaccine strains with low pathogenicity. One of the vaccine strains was VR-2332, which had been used in the pig farm for a long time. We also found that the two recombinant regions were highly homologous to the JAX1-R vaccine strain, but some SNPs exhibited the characteristics of wild strains with high pathogenicity. The main regions of the genetic sequence of SD2020 strain are still VR-2332 vaccine strain, and one of the two recombinant regions is also suspected to be a vaccine strain, JXA1P80 vaccine strain. It is well known that vaccine strains in the vaccinated animals do not cause serious illness and death, that are the unique virulence consequences of these highly pathogenic strains. However, the SD2020 strain showed the characteristics of high pathogenicity, and one region of the SD2020 strain was identical to wild type SNP of JXA1 with high pathogenicity. Therefore, we speculated that these SNPs are major contributors to the high pathogenicity of the SD2020 strain. While the amino acid sequences of these genes are currently found to be different compared to those of HP-PRRSV, this strain does not have the typical mutational deletions characteristic of HP-PRRSV found previously before this time. We believe that these SNP alterations provide direction for our search for the cause of elevated pathogenicity of PRRSV. In contrast, these gene sequences deviate from the amino acid sequence of conventional HP-PRRSV and are not characterized by the typical mutational deletions of previous HP-PRRSV. In addition, we presume that the gene fragment that recombines with VR2332 is the key gene fragment responsible for the increased pathogenicity of PRRSV. Analysis of the gene sequence of this newly identified strain provides new directions for our investigation of the key genes responsible for the increased pathogenicity of the virus.2 at 37 °C. The lung samples were added when Marc-145 cells reached a monolayer, after 1 h of adsorption in an incubator with 5% CO2 at 37 °C, the unattached virus was washed off and fresh DMEM with 2% FBS was added. Cells was were split through three blind serial passages until the appearance of cytopathic effects (CPE).The lung samples were collected from diseased pigs and prepared for virus isolation and histopathological studies. In this study, PRRSV was isolated by culturing in MARC-145 as previously described . MARC-14To purify PRRSV, plaque isolation was performed in Marc-145 cells for purification of virus particles. The frozen viral solution was removed and diluted in a gradient with DMEM containing 2% FBS in multiples of 10. When the fusion of Marc-145 cells in the 12-well plate reached 80% confluence, the diluted virus solution was added and adsorbed for 2 h. The virus solution was removed and DMEM mixed 1:1 with low melting point agarose was added. The DMEM contained 0.2% crystalline violet and 2% FBS. The cells were incubated in the incubator for 1 h and then inverted onto 12-well plates. The empty spots were removed and observed after 48 h, and independent empty spots were aspirated with a pipette tip. The viral solution was frozen after two blind passages in Marc-145 cells.TM RT-PCR Kit , with the following three steps: (1) Denaturation, annealing reaction: The reaction solution was prepared as mixture of dNTP Mixture (10 mM each) 1 µL, Random 6 mers (20 µM) 1 µL, Template RNA 3 µL and RNase Free dH2O 5 µL. The reaction was incubated at 65 °C for 5 min and terminated at 4 °C. (2) Reverse transcription reaction: The above denaturing and annealing reaction solution 10 µL, 5×PrimeScript Buffer 4 µL, Inhibitor (40 U/µL) 0.5 µL, Prime Script RTase (for 2 Step) 0.5 µL, RNase Freed H2O 5 µL and Total Volume 20 µL, the reaction was incubated at 30 °C for 10 min, 42 °C for 30 min, 70 °C for 15 min and 4 °C terminated. (3) RT-PCR reaction: 0×PCR BufferⅡ 5 µL, dNTP Mixture (10 mM each) 2 µL, ORF7-1 (10 μM) 0.5 µL, ORF7-2 (10 μM) 0.5 µL, TaKaRa Ex Taq HS (5 U/µL) 0.5 µL, Reverse transcription reaction solution as described above 2.5 µL and H2O 39 µL; the reaction was incubated at 95 °C for 3 min, 30 cycles of 95 °C for 30 s and 55 °C for 30 s and 72 °C for 1 min, 72 °C for 10 min and 4 °C terminated. Primers for PCR were designed to pass through the PRRSV N protein nucleotide sequence .JXA1 (isolated and maintained in our laboratory) was used as a positive control to identify if PRRSV was successfully isolated. Isolated virus, as well as RNA from JXA1, was obtained by MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 , as described in the instructions. The extracted genomic RNA was validated by PrimeScriptViruses diluted 10 times by DMEM containing 2% FBS were inoculated in Marc-145 cells (80% confluence) in 96-well plates. The plates were incubated in the incubator for 96 h. The number of wells in which CPE appeared was observed, and the virus titer was calculated by the Reed–Muench rule.SD2020 Multiplicity of infection (MOI) = 0.1 infected cell when cells were grown to approximately 80% confluency in a cell vial. After incubation at 37 °C for 1 h with rocking every 15 min, the virus suspension was discarded. Cells were cultured with DMEM containing 2% FBS in the incubator. Cells were removed and frozen at −20 °C at 24, 48, 72 and 96 h. Virus suspensions were collected by freeze-thawing the cells three times for the different incubation times.50 was calculated using the Reed–Muench rule and the virus growth curve was plotted.The harvested virus suspension was diluted using DMEM containing 2% FBS, and the diluted virus solution was inoculated in Marc-145 cells (approximately 80%) in 96-well plates. Cells were incubated in the incubator for 96 h followed by evaluation of the number of CPE. The TCID2O2 in methanol for 25 min, and the sections were then washed thrice in in Tris-buffered saline . For antigen retrieval, the sections were placed in citrate buffer , heated in a microwave oven for 40 min, and cooled at room temperature. After washing with TBS, the sections were incubated with 5% bovine serum albumin (BSA) in a humidified chamber at 37 °C for 20 min to block non-specific binding sites in tissues. Sections were then incubated with a PRRSV monoclonal antibody (mouse anti PRRSV-N protein) at a dilution of 1:500 or non-immune mouse serum (control) for overnight at 4 °C. The sections were then thoroughly washed three times in TBS (5 min each), and incubated with HRP-labeled goat anti-mouse IgG (at a dilution of 1:100) at 37 °C for 1 h. After washing three times in TBS, the sections were incubated with diaminobenzidine tetrahydrochloride (DAB) and H2O2 at room temperature for 10 min followed by rinse with distilled water to stop the color reaction. Sections were counterstained with hematoxylin for 30 s and differentiated with 1% hydrochloric acid alcohol for 10 s. After dehydration with gradient alcohol and transparency in xylene, the sections were mounted with gum. Sections obtained from the normal lungs of healthy pig were stained as negative control.Specimens obtained from lung lesions of lung lobe were placed in 10% neutral buffered formalin and embedded in standard paraffin blocks, sectioned at 3 microns and stained with hematoxylin and eosin (HE). Specimens obtained from normal tissues were included as negative control. Immunohistochemistry (IHC) was used to confirm PRRS virus antigen. Tissue sections (3 microns) were dried in the air, dewaxed in xylene and rehydrated in decreasing strengths of alcohols (100% to 0%). After deparaffinization and rehydration, endogenous peroxidase activity was blocked with 3% HTM RT-PCR Kit . The complete virus genome was amplified using 10 pair primers  according to the manufacturer’s instructions, followed by amplification using Prime Script primers  and sequBLASTn was used to search related genes of the SD2020 virus strain, and the reference virus sequences were obtained from Genbank. Whole genome sequences of SD2020 and reference viruses were aligned using CLUSTALW . To deteWith JXA1 (EF112445) and JXA1 P80 (FJ548853) as reference sequences, we analyzed B and D region sequence SNPs of SD2020, whole genome sequence SNPs of CH-YY (MK450365) and 15SC2 (KX815427), and whole genome sequence SNPs of vaccine rJXA1-R (MT163314) in the market.Gross pathological observation results showed serious edema and hemorrhage in the lung A. The hi50 of SD2020 was log106.66/mL and the TCID50 of JXA1 was log107.1 TCID50/mL  share the same genomic sequence. Therefore, we concluded that this PRRS outbreak was caused by a single virus strain infection. The virus strain was designated as SD2020, and its sequence full-length (GenBank accession no. MW408254) was 15386 bp.Alignment of SD2020 and VR-2332 sequences revealed that the overall homology was 97.53%. Meanwhile, SD2020 shared 66.54% sequence identity with the Lelystad virus, indicating that SD2020 belongs to genotype 2 PRRSV. Furthermore, we compared the obtained genomes with the sequences of multiple virus genomes of the PRRSV-2 lineage that had been detected, including NADC30, MN184C, JA142, CH-1a, P129, HB-1, HB-2, JXA1, etc. The main sequence of SD2020 virus genome is highly homologous to the vaccine strain VR-2332 used in pig farms. Simultaneously, the partial sequence of the SD2020 virus genome had significantly higher homology with the JXA1 virus strain, than VR-2332. Based on these results, we speculated that SD2020 was a recombinant virus from the VR-2332 vaccine strain and JXA1-like strain.Compared with the homologous region of the SD2020 strain and the VR2332 strain, there was a deletion of three amino acid residues in NSP2 protein . The same pattern of NSP2 deletion has been found in both Chinese and American strains [The recombination analysis (similarity plot in SIMPLOT v3.5.1) supported that the SD2020 has a recombinant genome A. The RDTo further analyze the evolutionary characteristics of SD2020 gene sequence, phylogenetic tree  was constructed for each region by MEGA X to locate SD2020′s evolutionary branch. According to recombination sites, we divided the whole SD2020 virus gene sequence into five regions, namely region A/B/C/D/E. Obviously, the phylogenetic analyses with separate regions show that regions A, C, and E were derived from the VR-2332 virus strain, whereas regions B and D were highly similar to the JXA1-like virus strains D. Given Interestingly, RDP analysis indicated that regions B and D were, respectively, from JXA1P80 (vaccine strain) and JXA1 (wild-type strain). Therefore, we believe that more in-depth analysis is necessary to trace the specific sources of different JXA1-like fragments in the SD2020 viral genome.We searched for similar sequences with the B and D regions of SD2020 by BLASTn and found that CH-YY strains were highly homologous to SD2020 in these two regions, In addition, 15SC2 was highly homologous to the B region of SD2020. Further analysis and a literature review showed that both CH-YY and 15SC2 belonged to the JXA1 family without recombination to other sublineages [With JXA1 (EF112445) and JXA1 P80 (FJ548853) as reference sequences, we analyzed B and D region sequence SNPs of SD2020, whole genome sequence SNPs of CH-YY (MK450365),15SC2(KX815427), and whole genome sequence SNPs of vaccine rJXA1-R(MT163314) in the market . The resOf the 97 SNPs that can distinguish JXA1 from JXA1P80, only 10 SNPs (scattered in the whole virus genome) of 15SC2 were the same as those of JXA1, the rest of the 87 SNPs belonged to the JXA1P80 type. Therefore, we speculated that 15SC2 was derived from the JXA1-P80 vaccine strain merely through point mutation, which was consistent with the previous study [In addition, a single-base insertion/deletion combination (involving three amino acid changes) and a 12-base insertion (involving four amino acid changes) could distinguish JXA1 from JXA1P80. Unlike JXA1, a G was inserted at bit 13,664 and a C was missing at bit 13,673 in JXA1P80. Similar mutations were observed in SD2020 and CH-YY, except that T was inserted at site 13664. In JXA1 P80, there was a 12-nucleotide insertion between C13693 and A13704. This 12-base insertion can be used as a genetic marker to distinguish vaccine strains from the wild-type strains. Of note, this 12-nucleotide insertion was not observed in SD2020 and CH-YY.From August to October in 2019, PRRS broke out in a pig farm in Shandong Province, China. Retrospective data have showed that the surveyed farm has been using VR-2332 vaccine to prevent PRRS for five years, without an outbreak of related disease. Unexpectedly, after the latest vaccination , all of the nursery pigs were diagnosed with high fever (41–42 °C), dyspnea, depression, and even death. The mortality rate was as high as 100%. The major symptoms of sick pigs included difficult breathing, high fever, abdominal respiration, and sitting like a dog, which consequently led to death from respiratory failure. The above-mentioned symptoms were evaluated to have the maximum gross-lung-lesion scores according to the previously reported standard scoring system. The analysis of PRRS pathogenesis in pig farm and pathological changes of dead pigs indicated that the PRRSV strain that caused the current PRRS outbreak was highly pathogenic.After obtaining the full-length sequence of the virus genome, we found that the virus characterized by highly pathogenic PRRS has some very interesting characteristics. The sequence of the SD2020 virus genome is almost identical to the sequence of vaccine strains (VR-2332) used in the farm, and two regions  of SD2020 are highly similar to JXA1-like virus strains. Therefore, the SD2020 virus strain obviously experienced the natural recombination of the vaccine strain and the wild strain, which makes it intriguing to identify the wild virus strain which was involved in the recombination with VR-2332 vaccine strain.JXA1 virus strain was firstly isolated in Jiangxi Province, China in 2006 . Recent By searching for the virus strain with the highest homology to the B and D region sequence, we identified CH-YY strain (highly homologous to these two regions) and 15SC2 strain (highly homologous to B region). Both CH-YY and 15SC2, as naturally infected strains, have been isolated in Chinese mainland in recent years , In addiC. Similar SNP analysis techniques had also been used by Wu Bin et al. to trace the evolutionary path of human H5N1 influenza virus and speculate on the SNP sites that change the virulence of the virus [SNP analysis technology has been widely used in genetic typing and base site mutation analysis related to the biological characteristics of many kinds of viruses, including influenza virus ,26,27,28he virus . Tomasz Further SNP analysis revealed the more specific sources of the two viruses. Specifically, we analyzed all SNP between the JXA1 wild-type strain and JXA1P80 vaccine strain, and determined all SNP types of CH-YY and 15SC2 strains which were most genetically similar to SD2020 strain, and further conducted a parallel control analysis of the rJXA1-R strain genome . The resRelated research was found that the pathogenicity of wild strain and vaccine strain was significantly enhanced after recombination ,31. TherCombining SNP analysis with sequence recombination analysis results, we found that the main genetic marker features of region B and D were highly similar to those of CH-YY, indicating that they had a very close common ancestor. Based on this, the common ancestor virus strain was speculated to have the characteristics of different JXA1 virus families in different regions and to be a recombinant strain of JXA1 and JXA1-R virus strains. Furthermore, this common ancestor recombined with VR-2332 under natural conditions to generate the SD2020 strain with high morbidity and mortality. Regions B and D of SD2020 strains were derived from the JXA1-like highly pathogenic wild-type strain and safe vaccine strain, respectively. Region A/C/E, highly homologous to VR-2332, are typical vaccine sequences with low pathogenicity and high safety. Based on the above-mentioned analysis results, we speculated that the D region might be directly related to high pathogenicity of SD2020. The mutation sites in D region, which were same as those of wild-type but different from those of the vaccine strain, were likely to be the highly pathogenic characteristic sites of SD2020. Only 10725G, 13344A and 13418T belonged to this type of mutation site. These three mutation sites are suggested to be further investigated in order to better understand the biological mechanism of PRRSV pathogenicity.In this research, we determined that SD2020 is a new natural recombinant virus strain through a variety of sequence analysis methods, and reasonably speculated the evolutionary origin and pathway of SD2020. At the same time, we speculate that three SNP sites may be related to the virulence of the virus, according to the biological characteristics and SNP characteristics of the virus. Our work uncovered some key gene or protein characteristics of highly pathogenic PRRSV. However, further experimental validation of this speculation is needed. In addition, the SNP site related to viral virulence requires further exploration.


For more than 70 years, the countries of South America have been attempting to eliminate foot-and-mouth disease (FMD), but a regional strategy had not been established by all the affected countries until 1988. The Action Plan 1988–2009 of the Hemispheric Program for the Eradication of Foot-and-Mouth Disease (PHEFA 1988–2009) resulted in an FMD-free status in 88.4% of the bovine population of South America. However, countries of the Andean sub-region maintained an FMD endemic. In addition, sporadic outbreaks in vaccinated cattle populations have been reported in countries of the Southern Cone, endangering the disease-free status in these countries. Within this context, the PHEFA 2011–2020 was approved to eliminate FMD from the subcontinent, and this review describes the most important milestones during its execution. FMD in Ecuador and sporadic outbreaks in the Southern Cone sub-region were effectively eliminated. The outbreaks that occurred in Colombia in 2017 and 2018 were successfully controlled. The type C virus was removed from the vaccines in use in most countries, based on a risk assessment. This review also describes the progress made by the countries advancing toward official recognition as FMD-free in all their territories, with Bolivia, Brazil, and Peru leading the progressive suspension of vaccination to achieve FMD-free status without vaccination. Consequently, at the end of PHEFA 2011–2020, Venezuela was, and still is, the only country in the region whose control program has suffered setbacks, and no evidence has suggested that the transmission and infection of the bovine population have been eliminated. At the end of 2020, a new PHEFA Action Plan 2021–2025 was approved with a five-year horizon, to complete the eradication of the disease in the Americas. Foot-and-mouth disease (FMD) was introduced in South and North America in 1879, with two different outcomes. While in North America the sporadic occurrence of outbreaks resulting from imports of animals and products was confronted with an elimination strategy by stamping-out and quarantining, in South America the infection spread to bovine populations in all affected countries   was established as the high-level technical and political mechanism to coordinate national plans for the control of FMD in South American countries . Bovine The first PHEFA Action Plan extended from 1988 to 2009, and although it did not eradicate the disease, it made significant progress: approximately 85% of the South American bovine population was recognized as free from FMD, with or without vaccination. Nevertheless, some areas still experienced FMD endemics /World Organization for Animal Health (WOAH) launched in 2012 , 5. The This review aims to describe the key milestones that characterized the implementation of the second PHEFA action plan between 2011 and 2020, namely, FMD elimination in Ecuador, FMD elimination in Paraguay following the 2011 outbreaks, FMD elimination in Colombia after the 2017 and 2018 outbreaks, the epidemiological situation in Venezuela, the evidence to not having experienced a type C outbreak since 2004 allowing the suspension of vaccination against this serotype, progress toward WOAH recognition of FMD-free status in more zones within the continent, and transitioning toward more zones achieving FMD-free without vaccination status. By the end of the PHEFA Action Plan 1988–2009, Ecuador was experiencing epidemic outbreaks of FMD throughout the country, caused by type O virus, due to the poor implementation of vaccination campaigns and animal movement control .In their phylogenetic analysis of the type O FMD viruses circulating in the Andean sub-region of South America in 2002 and 2008, Malirat et al. , suggested a loss of protective response, which was supported by in vivo studies using the Protection against Podal Generalization test in cattle.A controversy emerged with Duque et al.  observedIn August 2011, the last case of FMD was documented, and the continental region of Ecuador achieved in 2015 disease-free status with vaccination, according to the WOAH, upon completion of serological studies showing the absence of virus transmission, while the Galapagos Islands achieved recognition as FMD-free without vaccination. Since then, Ecuador has been implementing serological surveys annually to check the immune status of vaccinated populations and confirm the absence of virus circulation to maintain its FMD-free status .At the start of PHEFA 2011–2020, the countries of the Southern Cone sub-region  had systematic vaccination programs in place and were recognized as FMD-free  30 days post-vaccination in estimates from the EPP tests. However, the Podal Generalization tests performed 79 days after vaccination and 79 days after revaccination, showed an estimated protection of 75.0 and 87.5%, respectively. Furthermore, the Reference Laboratory of PANAFTOSA-PAHO/WHO estimated the immune coverage of the O1 Campos vaccine strain against the strain isolated in Paraguay in 2011 in the EPP test using ELISA-CFL to be 78.99% 30 days after vaccination and 99.70% 30 days after revaccination , 5 to 6 months after the last vaccination cycle, small herds of some departments included in the containment zone showed a level of protection significantly lower than expected, which may have led to the establishment of niches facilitating virus transmission after reintroduction. Vaccine matching tests against isolates obtained in 2018 with EPP using the VNT and ELISA-CFL tests performed by the WOAH Reference Laboratory of PANAFTOSA-PAHO/WHO showed that the vaccine provided protection equivalent to 99.90% in a panel of sera obtained 30 days after vaccination and 99.99% in a panel of sera obtained 30 days after revaccination . After tIn March and April 2019, four cross-sectional serological surveys were conducted on the bovine population. Three of them covered the entire vaccinated bovine population in the national territory with its official FMD-free status suspended, and one covered the protection zone. Tests to detect virus transmission and assess the apparent prevalence of FMD protective antibodies were performed. The selection of sampling units was carried out in two phases, and samples were randomly distributed and stratified according to the size of the herd. The three surveys conducted in the territory with official FMD-free status suspended estimated levels of protection against the virus between 83.7 and 95.7% for type O virus and between 80.1 and 94.2% for type A; similar results were observed, regardless of the size of the herd. No evidence of virus transmission was observed .In February 2020, the WOAH Scientific Commission recommended the reestablishment of FMD-free status with vaccination with a subdivision of the area in four FMD-free areas to reduce the impact of new potential virus incursions from Venezuela . One booVenezuela, Colombia, and Ecuador reported their first FMD outbreaks in 1950, 1951, and 1956, respectively and were affected by type O and type A viruses . BetweenHowever, in 2016, Venezuela reported to COSALFA that the provision of vaccines, particularly those for the social plan, would be restricted due to the critical economic situation of the country that year . In 2017The deterioration of the vaccination program can be observed by comparing the average number of vaccinated bovines by year. In 2015, 15,448,097 bovines  were vaccinated during the vaccination cycles, corresponding to a coverage >90% of the bovine population , whereasThe occurrence of FMD caused by the type C virus in South America was described by Saraiva and López , and morvia 3 routes: the environment, wild animals, and carrier cattle or endemic niches. Additionally, the risk of non-detection by surveillance activities carried out by veterinary services was evaluated , compelling these countries to confirm the elimination of the virus in vaccinated populations by suspending vaccination campaigns.In 2020, at the request of the 13 countries of COSALFA, the representatives of the six sub-regions of the Americas in COHEFA approved the third Action Plan of PHEFA covering the 2021–2025, with the overall purpose of completing the eradication of FMD in South America and strengthening the prevention and response capacity of the veterinary services of the countries in this continent. Such a goal can be achieved with actions aimed at three specific objectives: (a) eradicating the FMD virus in the territory of Venezuela and mitigating the risk in the Northern Andean sub-region, (b) making the transition to the official status of FMD-free without vaccination in the FMD-free countries still using vaccines, and (c) maintaining the status of the FMD-free territories without vaccination .In its more than 33 years of execution, PHEFA has provided valuable insights in the efforts to eliminate FMD in South America.First, PHEFA has had a regional governance mechanism made up of COHEFA and COSALFA, which has allowed not only coordinated actions under a master program that has guided national programs, but also permanent monitoring of progress in the elimination of the disease and a space for discussion and genuine collaboration between the public and private sectors with the support of international cooperation. As a result, the definition and implementation of regional strategies to solve problems and delays found during the execution of the program have been made possible.Second, the adoption of oil-type vaccines accompanied by quality control of all the series in use, complying with regional and international standards, has allowed national programs to rapidly control outbreaks and eliminate infection, since the vaccination programs reached high coverage, both at the herd and population level.Third, a pattern of sporadic outbreaks in bovine populations that reached FMD-free status with vaccination revealed the persistence of FMD virus transmission and the presence of endemic niches in vaccinated populations. These niches corresponded to sub-populations with low immunity due to lower coverage and/or bad practices in vaccination campaigns.Fourth, the introduction of studies to measure post-vaccination immunity, not only in a generalized manner, but also to characterize it through simple indicators such as the size of the farm, the age of the animals, and the identification of geographic clusters, served to identify the failures in vaccination and introduce corrections, both in the frequency of vaccination and in its application. The review and strengthening of vaccination campaigns, including additional cycles for young animals, as was done in Colombia, Ecuador, and Paraguay, was a decisive strategy for mitigating the risk of transmission and eliminating the infection.Fifth, the isolation of the active viruses in each outbreak of the disease has allowed a phylogenetic characterization of the different lineages of viruses present in the region, linking them to their area of occurrence, and has revealed that the transmission patterns among the sub-regions of South America  have been limited, demonstrating a particular segregation of risk in the region.Finally, the movement of animals, conditional on compliance with the vaccination program, has been strengthened with the improvement of the cadaster and identification of herds supported by computer tools, which ensure centralized and effective control of the movement of each batch of animals, without requiring the individual identification of the animals, except in the case of batches intended for the export of livestock products.The territories that have taken the step toward withdrawing the vaccine and being recognized by the WOAH as FMD-free without vaccination are contributing to the absence of virus transmission, confirming the elimination of FMD in those areas.AR led the article conception, writing, and review of this article. MS-V has shared the article conception task with AR and has contributed to the writing and review. EP and LB have contributed to the writing and review. MM and OC has contributed to the review. All authors contributed to the article and approved the submitted version.


Chikungunya virus (CHIKV) is currently an urgent public health problem as high morbidity from the virus leaves populations with negative physical, social, and economic impacts. CHIKV has the potential to affect every organ of an individual, leaving patients with lifelong impairments which negatively affect their quality of life. In this review, we show the importance of CHIKV in research and public health by demonstrating the immunopathology of CHIKV as it presents in different organ systems. Papers used in this review were found on PubMed, using “chikungunya and [relevant organ system]”. There is a significant inflammatory response during CHIKV infection which affects several organ systems, such as the brain, heart, lungs, kidneys, skin, and joints, and the immune response to CHIKV in each organ system is unique. Whilst there is clinical evidence to suggest that serious complications can occur, there is ultimately a lack of understanding of how CHIKV can affect different organ systems. It is important for clinicians to understand the risks to their patients. Togaviridae family and is transmitted by Aedes aegypti mosquitoes in tropical and subtropical regions, and by Aedes albopictus mosquitoes in tropical, subtropical, and temperate regions, which are currently the main vector [The emerging disease, Chikungunya (CHIKV), is currently an urgent problem in public health as high morbidity from the virus leaves populations with negative social and economic impacts ,2,3. CHIn vector ,5,6. Whin vector . CHIKV hn vector ,8.Most recently in 2022, as of 5 May, 50,459 cases and seven deaths have been reported in just four months . These cAedes aegypti mosquito was identified as its mode of transmission into humans [Aedes albopictus mosquitoes, due to a mutation allowing for enhanced viral infectivity [Aedes albopictus mosquitoes had already successfully occupied much of the globe and could be found in tropical, subtropical, and temperate regions due to high propagule pressure driven by human activities, this mutation allowed for a rapid invasion of the virus, leading to outbreaks in Southeast Asia, North America, South America, and Central America [Aedes albopictus mosquitoes, occurred in Italy in 2007 and in France in 2010, where they raised concerns about the emergence of the virus in temperate regions and how it could potentially affect public health, since these are highly populated and affluent areas of the world [Evidence suggests that Chikungunya-like cases were recorded as early as 1823 in Tanzania, and 1827 and 1828 in the Caribbean, but clinical descriptions also suggest the possibility of outbreaks as far back as the 1600s ,12. Howeo humans . CHIKV r America ,14. The he world ,16.Even with the lengthy epidemiological history of CHIKV, data suggest that outbreaks may have been missed due to limitations in diagnosis, surveillance, and non-symptomatic infections ,17. DiagCHIKV has the potential to affect every organ of an individual, leaving patients with lifelong impairments which negatively affect their quality of life ,18,20. TThe general immunology of CHIKV has been summarized in a review by Petitdemange et al. . BrieflyThe full virion replication cycle is typically complete in 8 h . Once reFollowing the release of proinflammatory cytokines and chemokines, adaptive immunity is activated . CD8 T lOverall, there is a significant inflammatory response during CHIKV infection, which may result in the clinical pathogenesis observed in diseased patients affecting several organ systems, such as the brain, heart, lungs, kidneys, skin, and joints . Organ-sChikungunya causes numerous neurological syndromes and disorders, including encephalitis, meningoencephalitis, optic neuritis, Guillain-Barré syndrome, paralysis, acute disseminated encephalomyelitis, and neonatal hypotonia ,45,46,47The immunology of a CHIKV infection in the brain and how complications such as encephalitis arise is understudied, but research is ongoing, and many studies have provided insight. In a zebrafish model, a real-time CHIKV infection was observed, revealing that the virus infected the brain parenchyma and persisted longer than in other tissues . CHIKV wCynomolgus macaques showed activation of astrocytes, the most common cell type in the central nervous system [Consistent with the zebrafish model, cytokine profiling of individuals with neurological disease during CHIKV infection showed an increase in TNF-α, IFN-α, and IL-6 in CSF samples, characteristic of a type I IFN response ,55,56. Ts system ,58. Heres system . In a gls system . While ts system .Other important immune cells, in addition to astrocytes, are microglia, which are the macrophages of the brain . In CHIKThere are many reports that detail a wide range of cardiac symptoms involved during CHIKV infection, including arrythmias, palpitations, abnormal electrocardiograms and echocardiograms, myocardial infarctions, heart failure, cardiac arrest, cardiomegaly, and myocarditis among others ,65. WhilOne interesting consequence of CHIKV infection which is found across nearly all age groups is myocarditis, which results when the virus directly infects the cardiac tissues and causes damage ,67,70. KInterestingly, overactivation of TLR3 is more closely associated with pathogenesis of myocarditis . FurtherWhile rare, CHIKV seems to be capable of infecting the lungs, causing acute respiratory distress syndrome (ARDS) and some cases of respiratory failure, which have resulted in the death of at least one patient ,86,87,88When it comes to clinical presentations and differential diagnosis, the absence of a respiratory illness or respiratory symptoms is an indication of an arboviral disease, such as CHIKV . TherefoThere is not much data which reveal how CHIKV infection affects the kidneys, but what little clinical evidence exists shows that CHIKV can cause acute kidney injury (AKI), usually if rhabdomyolysis, acute interstitial nephritis, thrombotic microangiopathy, or a renal lesion is already present ,98,99. FThe kidneys are one of the most common organs to be transplanted, which presents an interesting concern for CHIKV infection . In addiThere are several different types of cutaneous or mucocutaneous presentations of CHIKV, reported in all age groups and of varying severity ,121,122.While many case reports only describe the symptoms involved, some also give results of Tzanch smears, a diagnostic tool to corroborate cutaneous diagnoses, which showed lymphocytes, acantholytic cells, necrotic keratinocytes, and a small number of neutrophils in bullae ,125. AddCHIKV is transmitted through mosquito bites, and after going through the dermis and epidermis layers of the skin, the virus then attaches to fibroblasts, starting the replication process . When CHFrom a clinical perspective, arthralgia is a common symptom of CHIKV infection, with approximately 87% of all cases having this presentation as part of the acute stage of infection . This arThere are other alphaviruses which cause persistent joint pain, including Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), and o’nyoung’nyoung virus (ONNV), which CHIKV is closely genetically related to ,135. In The Th17 response is implicated in both autoimmune diseases as well as in the removal of extracellular bacteria, and is marked by a subset of CD4 T cells which produce IL-17, IL-6, and TNF, and seem to require IL-23 for pathogenicity ,140. In Outside of a potential Th17 response, it is known that chronic chikungunya arthritis syndrome is also accompanied by increased levels of CD4 T cells, IL-6, IL-12, and macrophage inflammatory protein . HoweverIn mouse models, joint pain is associated with the increased level of CD4 T cells and viral clearance, rather than viral titer . Teo et In the past several decades, chikungunya has become an ever-growing concern due to increasing cases, abnormal and deadly presentations of disease, and a lack of treatment or vaccine ,144. CHI


Liming contributes to the alleviation of acidity in highly weathered soils. For sugarcane, the use of green harvest methods and new soil tillage systems requires an adjustment of lime application rates. In the present study, the effects of different lime rates and tillage systems on sugarcane performance and soil chemical fertility parameters were assessed. Three experiments were conducted in two locations between April 2015 and October 2019. The study design was a randomized block field study with four replicates. Four lime rates were applied once at sugarcane establishments in each soil tillage system and location: no liming (control); lime recommended rate (LRR); two times LRR (2× LRR); and three times LRR (3× LRR). The three soil tillage systems were conventional (CT), deep-strip (DT), and modified deep-strip tillage (MDT). Soil chemical fertility, leaf nutrient concentrations, and sugarcane yield components were analyzed, and correlations were identified by principal component analysis (PCA). The soil acidity was adequately alleviated in all tillage systems. Increasing the lime rate improved the lime distribution and soil fertility parameters. Applying lime at rates higher than LRR improved stalk and sugar yields, longevity, agronomic efficiency index (AEI), and correlated with a longer residual effect of liming, mainly in the last ratoon. Saccharum spp.) in the world  × 100, where Y1 = sugarcane stalks or sugar yield in the treatment control; Y2 = sugarcane stalks or sugar yield using 2 or 3× LRR; and Y3 = sugarcane stalks or sugar yield using LRR. The AEI determined the increments of stalk and sugar yields compared with the control (increased yields) and considered the ratio of the increment under 2 or 3× LRR to the increment under LRR, expressed as a percentage. Accordingly, values above 100% indicate that the higher lime rate was more efficient than LRR, while values below 100% indicate that LRR was more efficient than the higher lime rate. Values near or equal to 100% indicate no difference in efficiency between the lime rates.Data were submitted for analysis of variance (ANOVA) with subsequent comparison of means. Fisher’s test, supported to the least significant difference (LSD), was performed comparing soil chemical and fertility attributes for each soil depth, foliar diagnosis, and yield components in the same growing season. The 0.1 level of probability was used at all tests.p ≤ 0.1) and determined through the Kaiser rule, considering eigenvalues >1 or explaining over 85% of the total variance [To identify that the major soil attributes and the sampled depths correlated with sugarcane stalks and sugar yield, the principal component analysis (PCA) technique was constructed separately for each tillage system and location using Pearson’s correlation coefficients (variance . Correlavariance . The avevariance .The present study assessed the effects of increasing lime rates on chemical fertility parameters throughout the soil profile and sugarcane performance during the complete sugarcane growing cycle under different tillage systems. In general, regardless of the tillage system, soil acidity was adequately alleviated, and increasing the lime rate improved the distribution of lime in the soil profile and soil fertility. The highest lime rates provided more pronounced residual effects.Because micronutrient fertilization was adequate, higher-than-recommended lime rates did not negatively impact the nutritional status of the plant. Stalk and sugar yields and the AEI were positively impacted by higher-than-recommended rates, but sucrose concentration and TRS were less responsive to the lime rate, regardless of the tillage system.2+ and Mg2+ concentrations and BS were observed in PCA, mainly at depths ≤ 0.6 m under DT and at all soil depths under CT and MDT. Conversely, AS was negatively correlated with stalk and sugar yields at all depths, regardless of the tillage system. In addition, higher sugarcane yield and longevity were associated with a higher residual effect of liming, mainly in the last ratoon.Positive correlations of the yield parameters with soil CaThe stalk and sugar yields were highest at the higher-than-recommended lime rates, regardless of the tillage system and location. When all growing seasons were averaged, the AEI was highest under CT and DT in Macatuba and under MDT in Piraju.This study provides solid information for chemical assessments of clayey soils, but similar studies in medium-textured and sandy soils are also necessary due to the widespread cultivation of such soils with sugarcane in Brazil.


To investigate respiratory virus infections in diarrhea cases and identify the risk of respiratory virus transmission through feces.Fecal specimens were collected from diarrhea cases in enteric disease clinics in Beijing, China, from 2019 to 2020. Cases that tested negative for norovirus, rotavirus, sapovirus, astrovirus, and enteric adenovirus were included in the study. Real-time RT-PCR was used to detect 16 groups of respiratory viruses, and the major viruses were genotyped. Viruses isolation and digestion of clinical specimens and nucleic acid by artificial gastric acid or artificial bile/pancreatic juice were used to evaluate the risk of respiratory virus transmission through feces.A total of 558 specimens were collected and 47 (8.42%) specimens were detected positive, 40  in 2019, and 7  in 2020, including 20 (3.58%) for human rhinovirus (HRV), 13 (2.32%) for Bocavirus (BoV), 6 (1.08%) for parainfluenza virus I (PIV), 4 (0.72%) for coronavirus (CoV) OC43, 3 (0.54%) for respiratory syncytial virus (RSV) A, and 1 (0.18%) for both BoV and CoV OC43. Syndrome coronavirus 2 (SARS-CoV-2) and other viruses were not detected in this study. Eight genotypes were identified in the 13 HRV specimens. BoVs 1 and 2 were identified in nine BoV specimens. HRV infectious virions were successfully isolated from 2 clinical specimens and clinical specimens of HRV, RSV, PIV, and CoV could not be detected after 4 h of digestion and their nucleic acid could not be detected after 2 h of digestion by artificial gastric acid or artificial bile/pancreatic juice.There may be a risk of respiratory virus transmission from diarrhea cases, and interventions against SARS-COV-2 epidemics are also effective for other respiratory viruses. Diarrhea is the main symptom caused by enteric pathogen infection; however, several respiratory virus infections also present with this symptom. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in the final months of 2019, causes severe acute respiratory diseases, multiple organ injuries, and fatal outcomes . SARS-CoA prospective observational study found that among diarrhea cases, the RNA of human influenza A virus (IAV) and human influenza B virus (IBV) became undetectable in sputum on days 7 and 10 after infection, respectively, although it could be detected in feces 24 days after infection. In addition, their RNA and antigens were detected in intestinal tissues, and diarrhea symptoms were observed in patients whose feces tested positive . These fBocavirus (BoV) was first identified in Swedish children with acute respiratory infections (ARTIs) in 2005 and divided into four genotypes (BoVs 1 to 4) . After tHuman rhinovirus (HRV) has been associated with lower respiratory tract (LRT) illness and more severe clinical outcomes in pediatric and other vulnerable populations. At present it has the most well-defined role in causing the “common cold” . HRV is Several respiratory viruses, including CoV, IAV, IBV, BoV, RSV, MPV, and HRV, can be transmitted by the respiratory tract and cause diarrhea or have the same cellular receptor as the gut virus. Therefore, this study aimed to investigate the epidemiological distribution and clinical characteristics of respiratory viruses among outpatients with diarrhea in Beijing to further understand the causes of diarrheal diseases and provide information on the risk of respiratory virus transmission through feces.Three enteric disease clinics located in the Chaoyang district of the urban area, Tongzhou district of the suburbs, and Huairou district of the outer suburbs were enrolled in this study. Diarrhea cases were defined as patients with three or more loose fecal samples within a 24 h period. Fecal samples of children aged 0–10 years with diarrhea were collected in clinics in Chaoyang district and Tongzhou district, and samples of the whole age group were collected in Huairou district. Thereafter, 10–15 samples per month were collected from each district. These cases were first detected for norovirus, rotavirus, sapovirus, astrovirus, and enteric adenovirus, and negative cases were included in the study.Viral RNA was extracted from 140 μL of a 10% (w/v) fecal suspension in phosphate-buffered saline using the QIAamp Viral RNA Mini Kit . Approximately 60 μl of the total nucleic acid eluate was recovered in nuclease-free tubes. RNA was stored at −20°C until further use.A panel of respiratory viruses, including influenza virus A  and B, RSV, parainfluenza virus I to IV (PIV I to IV), HRV, MPV, coronavirus , and BoV, was detected in these specimens using commercial real-time RT-PCR kits . Commercial real-time PCR kits  was used to detect severe acute respiratory SARS-CoV-2.Escherichia , Vibrio parahaemolyticus, Yersinia enterocolitica, Bacillus atrophaeus, Aeromonas hydrophila, Campylobacter jejuni, Plesiomonas shigelloides, Aeromonas hydrophila, Campylobacter jejuni, Campylobacter coil, Clostridium difficile, Vibrio cholera, Vibrio vulnificus, Blastocystis hominis, Dientamoeba fragilis, Entameba histolytica, Giardia lamblia, and Cryptosporidium, were detected using the commercial TaqMan™ Microbial Array Specialty Card . Escherichia  were also identified by the real-time PCR method  with 1,000 bootstrap replicates.The genotypes of the HRVs and BoVs were identified using the Basic Local Alignment Search ToolHi-hela cells were selected for HRV isolation. The cytopathic effect was detected by inverted microscope and identified by real-time PCR . Besides, the positive specimens and nucleic acids were incubated for 1, 2, 3, or 4 h with phosphate-buffered saline (PBS), artificial gastric acid or artificial bile/pancreatic juice . Artificp-value of less than 0.05 is considered statistically significant. All analyses were performed using SPSS software version 20.0.The differences in the detection rates of respiratory viruses between 2019 and 2020 were analyzed using the chi-square test and Fisher’s exact test. A two-sided 2 = 16.648, p = 0.00).From 2019 to 2020, 558 fecal specimens from diarrhea cases, including 300 fecal specimens in 2019 and 258 fecal specimens in 2020, were collected in enteric disease clinics, and 16 groups of respiratory viruses were identified, as shown in Single virus infection was found in 46 cases, and one case was a mixture of BoV and CoV OC43. Among 46 cases of single infection, HRV was the most prevalent , followed by BoV , PIV I , CoV OC43 , and RSV A . No cases were infected with SARS-CoV-2, IAV, IBV, PIV , CoV , RSVB, or MPV. All these viruses were mostly detected in 2019, and the differences in the detection rates of HRV, PIV I, and BoV between 2019 and 2020 were statistically significant, as shown in V. parahaemolyticus, Y. enterocolitica, B. atrophaeus, A. hydrophila, C. jejuni, P. shigelloides, A. hydrophila, C. jejuni, C. coil, C. difficile, V. cholera, V. vulnificus, B. hominis, D. fragilis, E. histolytica, G. lamblia, Cryptosporidium, and Escherichia  positive was detected among these cases.Among the 47 respiratory virus-positive cases, an HRV-positive case and a BoV-positive case were detected with EPEC infection, with Ct values of 30 and 34.5, respectively. In addition, no The clinical characteristics of the cases infected with a single respiratory virus are shown in The temporal distribution of cases of infection with a single respiratory virus is shown in p = 0.038). HRV, PIV I, BoV, CoV OC43, and RSV A were detected in Chaoyang in the urban area and Tongzhou in the suburb, while only HRV, BoV, and PIV I were detected in Huairou in the outer suburb, as shown in These detection rates of respiratory viruses in Chaoyang, Tongzhou, and Huairou districts were 12.50% (19/152), 8.17% (26/318), and 2.27% (2/88). The detection rates in these districts in 2019 were 16.19% (17/105), 16.92% (22/130), 3.08% (2/65), 4.65% (2/43), 2.22% (4/180), and 0% (0/35) in 2020, respectively. The difference in the detection rates among the three districts in Beijing was statistically significant  showed cytopathic effect and were identified as positive by real-time PCR .5, after 4 h of treatment and digestion by artificial gastric acid or artificial bile/pancreatic juice, both clinical specimens and nucleic acid of BoV could be detected and Ct value of the nucleic acid was slightly higher than that of clinical specimens. The clinical specimens of HRV and RSV could not be detected after 4 h of treatment, while their nucleic acids were not detected after 2 h of treatment. For PIV and CoV with Ct value of 28 and ΔRn ranging from 1.0 to 1.2 × 105, both clinical specimens and nucleic acid could not be detected after an hour of treatment. All these clinical specimens and nucleic acid were detected with slightly decreased ΔRn values and slightly increased Ct values after 4 h of treatment by PBS  for IVA (H3N2) to 14.42%  for IVB  since its first isolation in the 1950s, and HRV also causes pneumonia hospitalization in vulnerable people, such as children, the elderly, and those with underlying diseases . HRV-assCI: 5.7–6.1) for AGE cases and 6.3% (95% CI: 6.2–6.4) for ARTI cases , respectively, which are similar with that of EV and cause similar clinical symptoms as well  can be found below: 第(02)总, Beijing Center for Disease Control and Prevention. Informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.The studies involving human participants were reviewed and approved by 2021 QW, ZG, and LS conceived and designed the experiments. LS, HY, and WL performed the experiments. LS and CL analyzed the data. QW, PY, DZ, and LJ contributed to reagents and materials. YT, BL, and YW collected the clinical data. ZG and LS contributed to the writing of the manuscript. All authors reviewed the manuscript.


Human pegivirus (HPgV) is best known for persistent, presumably non-pathogenic, infection and a propensity to co-infect with human immunodeficiency virus or hepatitis C virus. However, unique attributes, such as the increased risk of malignancy or immune modulation, have been recently recognized for HPgV. We have identified a unique case of a woman with high levels HPgV infection in two pregnancies, which occurred 4 years apart and without evidence of human immunodeficiency virus or hepatitis C virus infection. The second pregnancy was complicated by congenital heart disease. A high level of HPgV infection was detected in the maternal blood from different trimesters by RT-PCR and identified as HPgV type 1 genotype 2 in both pregnancies. In the second pregnancy, the decidua and intervillous tissue of the placenta were positive for HPgV by PCR but not the chorion or cord blood (from both pregnancies), suggesting no vertical transmission despite high levels of viremia. The HPgV genome sequence was remarkably conserved over the 4 years. Using VirScan, sera antibodies for HPgV were detected in the first trimester of both pregnancies. We observed the same anti-HPgV antibodies against the non-structural NS5 protein in both pregnancies, suggesting a similar non-E2 protein humoral immune response over time. To the best of our knowledge, this is the first report of persistent HPgV infection involving placental tissues with no clear indication of vertical transmission. Our results reveal a more elaborate viral-host interaction than previously reported, expand our knowledge about tropism, and opens avenues for exploring the replication sites of this virus. Pegivirus genus within the Flaviviridae family [Flaviviridae family. HPgV has two structural proteins (E1 and E2), two predicted proteins of undetermined function (X and p* protein), and six non-structural (NS) proteins [Pegivirus C, formerly GBV-C) have been classified, along with many variants [Human pegivirus (HPgV) is a member of the e family . It is aroteins E and E2, proteins ,4. To daroteins E and E2, variants ,6, whichKnowledge regarding the biology and behavior of this virus remains scarce. HPgV-1 is a lymphotropic virus with a positive association between viremia with a risk of adult lymphomas ,8. The pIn pregnant women, the prevalence of HPgV infection is reported to be 1.1–6%, with a presumed rate of vertical transmission of up to ~65%, without HCV or HIV co-infection. These reports, however, were all from small cohorts and the route of transmission was not determined and only presumed to have been trans-placental based on detection of the virus in the newborn infant  ViroCap to detect and genotype the virus ,31, and We obtained samples from pregnant women who provided informed consent for the collection of biological samples and clinical data . ViroCap, VirScan and RT-PCR assays were performed on 1st-trimester plasma samples corresponding to week 12 and 11 for the first and second pregnancies, respectively. Additional plasma samples were collected at week 16 and 39 for both pregnancies, as well as cord blood at both births and the placental/fetal tissues at post-partum for the second pregnancy, were used for end-point RT-PCR assays as shown in Figure 1.www.viprbrc.org (last accessed 7 January 2022) [Nucleic acid isolated from samples with Quick DNA/RNA Viral Kit  was used was used as input for the ViroCap assay . Viral sry 2022) . Phylogery 2022) .The presence of HPgV was analyzed by nested-PCR  and one-10 pfu for complex formation at 4 °C overnight. Immunoprecipitation (IP) was then performed at 4 °C for 4 h using 1:1 ratio of Protein A and Protein G Dynabeads mix (Invitrogen). As described previously [VirScan assay was performed as previously described . In brieeviously , beads wwww.uniprot.org/align/; accessed on 15 September 2022) to determine each peptide’s starting and ending position. Then, we manually aligned the peptides with the polyprotein sequence.Sequences were aligned to the Vir3 reference genome (obtained from Dr. Steve Elledge) using Bowtie2 (version 2.2.6) with the option –very-sensitive-local. Samtools (version 1.6) was used to index and sort the BAM files and raw counts generated using the option -idxstats. All counts were normalized to 1 million reads prior to additional downstream analyses. The VirScan analysis pipeline was performed as previously described . In brieThe patient is a Caucasian woman (not Hispanic or Latino) living in the United States of America at the time of recruitment and sample collection. The first pregnancy  of the individual resulted in a normal spontaneous vaginal delivery and the infant and postpartum course were normal. The second pregnancy 4 years later  was complicated by congenital heart disease (CHD) . Maternal body-mass index was 20.4 and 21.76 for the first and second pregnancies, respectively. Prior and during both pregnancies, the mother had no history of gestational diabetes, hypoglycemia, hyperglycemia, alcohol use, or tobacco use. The newborn of the first pregnancy had a gestational age of 39 weeks and 5 days, a birth weight of 3972 g, and is a male. The second pregnancy’s newborn had a gestational age of 39 weeks, a birth weight of 3434 g, and is a female. The mother had a history of human papillomavirus (HPV) infection. Prior to her second pregnancy, the mother developed an eye melanoma. She also had 3 liver lesions (hemangioma/benign cysts) during her second pregnancy which was also complicated by iron-deficiency anemia. The mother was negative for HIV and hepatitis B surface antigen by serology testing in the clinic. There are no other covariates known to affect HPgV infection. A summary of the samples obtained, and assays performed is shown in By viral sequence analysis of first trimester maternal plasma from both pregnancies, we identified the complete coding sequences of the HPgV genomes. The consensus sequences from both genomes were identical and without mutations. The sequence was ~91% identical to the best match in the NCBI nucleotide database (Sequence ID MN551063.1), and the virus was determined to be most similar to HPgV-1 genotype 2 based on comparison with representative 5′-untranslated region (UTR) sequences from known genotypes .2 EBS of 5.013 and 5.757 in the first (P1) and second (P2) pregnancies, respectively.Using VirScan, we observed evidence of antibody responses against several viral species, including commonly observed group of viruses such as human herpesviruses, rhinoviruses, and adenoviruses, which corroborates findings from other groups ,45. ReleFrom both pregnancies, the maternal plasma samples consistently tested positive for HPgV by end-point RT-PCR, but the cord blood samples were negative . Taking Prolonged persistence of the HPgV have been noted ,13,46, iTo put our findings into perspective, a small study from Brazil found that among 63 pregnant women under treatment for HIV, 25% had HPgV infection. Of those with HPgV, five had HPgV-positive newborns, corresponding to a vertical transmission of 31% . Pre-natSeveral viral infections have a documented history of causing birth defects, including congenital heart disease. In the 1970, immunologists created ToRCH as a group of viruses associated congenital malformations . The rec−38; score = 324, UniProtKB: A0A895ZPP4_9FLAV). The molecular function of the NS5 protein includes RNA binding, and RNA-directed 5′-3′ RNA polymerase activity and is involved in viral RNA genome replication [Detection of antibodies targeting the HPgV E2 protein is associated with viral clearance. There have been no reported antibody responses to the NS proteins in HPgV-1 infections [lication . Intereslication . Perhapslication . FurtherSeveral genotypes of HPgV-1 have been identified world-wide by genome sequencing ,12,56,57In summary, we have identified a unique case of persistent HPgV-1 in a woman with two pregnancies in 4 years. Although the mother had HPV infections, the etiology of the HPgV-1 infection in our patient is not known and did not coincide with common co-infections (HIV and HCV). To the best of our knowledge, we are the first group to directly assess vertical transmission by testing fetal and placental tissues, and umbilical cord blood for the presence of HPgV. We saw no evidence of vertical transmission of the virus in two consecutive pregnancies. Our observation of this long-term persistent HPgV-1 infection and humoral response to NS5 epitopes during pregnancies highlights a complex interplay between virus and host and suggests putative effects on maternal health and fetal development to be explored in future studies.


To investigate the proliferation cycle of a virus, virus-host interaction, and pathogenesis of a virus, virion particles must be concentrated from the media of virus cell culture or the sera of virus-infected patients. Ultracentrifugation of the culture media is a standard method for concentrating virion particles. However, this method is time-consuming and requires special equipment (ultracentrifuge). Moreover, a large number of infectious viruses are lost during enrichment. We developed a new method of hepatitis C virus (HCV) concentration to overcome the issues associated with traditional methods of virus concentration. We used an aqueous two-phase system (ATPS) to concentrate the virus. HCV, which causes various liver diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, was used as a model virus to test the efficacy and reliability of the ATPS. The efficiency of HCV concentration by the ATPS was approximately three times higher than that by ultracentrifugation. Moreover, the infectivity of the concentrated HCV, which is a labile virus, remained the same after concentration of the virus by the ATPS. Considering the simplicity and effectiveness of the ATPS, it is the method of choice for concentrating viruses. The concentration and/or purification of viruses is essential to investigate the basic aspects related to the viral life cycle, viral morphology, virus-host interaction, pathogenesis, immunity, and functions of viral proteins, as well as application aspects in the production of vaccines and the development of anti-viral drugs . For insHepacivirus genus in the Flaviviridae family -5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate . An Alexa 555-labeled anti-mouse antibody  was used to visualize the HCV core protein. Fluorescence microscopy was performed on a Zeiss Axio Scope A1 microscope.Renilla Luciferase Assay System .Huh-7.5.1 cells were inoculated with virus samples (JFH-5a Rluc). After 4 h of viral inoculation, the culture medium was replaced with fresh medium. The cells were further cultivated for 3 days. Cells were harvested and lysed in passive lysis buffer , and luciferase activity in cell lysates was measured using the The recovery efficiencies of virion particles and viral RNAs after the concentration process by either ultracentrifugation or ATPS were calculated as follows:We performed concentration of HCV using either an ATPS or a conventional ultracentrifugation method . To deteThe sizes and amounts of nanoparticles enriched by the ATPS and ultracentrifugation were analyzed using NTA and TEM. The amounts of nanoparticles in the solutions before and after enrichment varied, but the sizes of the nanoparticles in the peaks were similar (approximately 100 nm) among the solutions c. The siThe nanoparticles in the unenriched and enriched samples were visualized using TEM d. SeeminAccording to the NTA, three times more nanoparticles were enriched by the ATPS than by ultracentrifugation . HoweverThe relative amounts of HCV RNAs in each fraction, quantified by qRT-PCR, are shown in fugation b.Renilla luciferase) in the NS5A region, which allows easy monitoring of viral infectivity by measuring Renilla luciferase activity in cells [Renilla luciferase activity in the cell lysates was measured to monitor the infectivity of the virus. As shown in We monitored the infectivity of the concentrated HCV using HCV strain JFH5a-Rluc-ad34 containing a reporter gene . For instance, adenovirus was enriched from the crude lysate of HEK293 cells using a PEG 300-phosphate system . Howeverg) applied during sedimentation and the dissolving process of viral pellets is a likely reason for the further reduction of virus infectivity after ultracentrifugation. On the contrary, the recovery rate of HCV RNA by the ATPS (~40%) was 2.7 times higher than that by ultracentrifugation  and EVs  which co


Hysteroscopy is one of the commonest performed procedures in contemporary gynaecology and is considered the gold standard endoscopic procedure for the evaluation and treatment of women with intrauterine pathology . HysteroThe structure and delivery of health care services vary across national health care systems, with differing credentialing requirements, funding, mechanisms for reimbursement and laws and regulations. These differences have hampered a clear understanding of how contemporary hysteroscopic services are currently being delivered globally and how best to do this. Specifically, there is no consensus regarding the terminology used to describe the different hysteroscopic procedures, including the setting in which they are performed and the model of care  of the patient undergoing hysteroscopic procedures. Terms such as “in-office”, “outpatient”, “ambulatory”, “day-case”, “in-patient”, “operating room” are used frequently interchangeably without standardised definition or common understanding.There is, thus, a pressing need for the implementation of a common terminology to describe hysteroscopic procedures that can be used uniformly in clinical practice and research. The creation and adoption of a standard nomenclature will be helpful for clinicians and patients by allowing the quality and outcomes of clinical practice to be more robustly compared. Furthermore, research studies can be reliably compared, aiding the interpretation and generalisability of findings, as well as facilitating data syntheses .Therefore, the American Association of Gynecologic Laparoscopists (AAGL), the European Society for Gynaecological Endoscopy (ESGE) and the Global Community of Hysteroscopy (GCH) formed an international working group of experts in hysteroscopy to develop a consensus statement of recommended terminology to use for describing different aspects of hysteroscopic procedures: (i) pain management, (ii) the setting where procedures are conducted, (iii) the model of care relating to the length of stay and need for admission, (iv) the type of procedure and (v) the approach to hysteroscopy.In June 2018, concerns about the lack of a standardised nomenclature to describe hysteroscopic procedures was identified by a group comprising of five expert hysteroscopists from Europe and the United States. The need to create a nomenclature that objectively and uniformly defined hysteroscopic procedures prompted this group to create a larger working group of leading international hysteroscopists to identify areas in which a standard nomenclature was lacking.In December 2018, seventy gynaecologists with extensive experience in hysteroscopic procedures were selected to represent the international community. An email was distributed among the members of this group enquiring about identified areas of current hysteroscopic practice that, in their opinion, needed a common terminology. On January 31, 2019, a preliminary proposal regarding areas of hysteroscopic practice where standard nomenclature was needed, namely pain management, procedural setting, the model of care, and approaches to hysteroscopy, was drafted. This broad, preliminary consensus was presented and discussed at the Global Hysteroscopy Congress in Barcelona, Spain in June 2019, at the ESGE Annual Scientific Meeting in Thessaloniki, Greece in October 2019 and at the Annual Global Congress of the AAGL in Vancouver, Canada in November 2019.Following this wide consultation, the AAGL, the ESGE and the GCH created an international working group of 15 experts in hysteroscopy with the objective of revising the preliminary draft and prepare a consensus statement for terminology to be used for hysteroscopic procedures. Each scientific organisation contributed with five members in the group including practicing clinicians and researchers who had demonstrated leadership and expertise hysteroscopic procedures.A total of three online video meetings took place from April to June 2021 in which a final agreement for standardised nomenclature was obtained. The process for arriving at a consensus was as follows. Where there was consensus about terminology for a particular area of hysteroscopic practice, the statements were adopted and revised only for editorial reasons. Where consensus could not be achieved, the proposed statements were removed, and members of the working group had the opportunity to provide verbal and written comments, suggestions, and to propose changes. These statements were revised accordingly to encompass the views of the working group members. The revised statements including alternative statement options where applicable, were disseminated in advance of the next online meeting and subsequently discussed in that forum. If voting failed to reach a consensus, the same process was followed before and during the third and final online working group meeting. Where members could not attend the online meetings, their written opinions were sought in advance to allow formulation of consensus of the whole group.The adoption and implementation of a common terminology to standardise reporting of hysteroscopic procedures was proposed to cover five domains; pain management, healthcare setting, model of care, type of hysteroscopic procedure and the hysteroscopic approach to the uterine cavity. A summary of the terminology is given in Technological advances have led to the miniaturisation of hysteroscopes and ancillary instrumentation, which has facilitated the conduct of procedures without the need for anaesthesia or with the use of local genital tract anaesthesia alone. The feasibility of conducting procedures without the need for conventional general or regional anaesthesia is dependent upon several factors both clinical and non-clinical and these include the type of procedure, patient preferences, clinician expertise, the available instrumentation and infrastructure and how health services are reimbursed and regulated.Thus, the management of pain is a key consideration when undertaking hysteroscopic procedures and needs to be clearly and consistently reported. A hierarchical description of pain management, consisting of five levels, is recommended. .There is currently no common understanding on the best terminology to define the setting in which hysteroscopic procedures are carried out. Terms such as “in-office”, “outpatient”, “ambulatory”, “day-case”, “in-patient”, “operating room” are used without standardised definition and often incorporate both the place (facility) where procedures are conducted and whether patients are admitted and how long they stay for.In order for a clearer understanding of the procedural situation, it is recommended that the setting is defined in alignment with the “International Association for Ambulatory (Day) Surgery (IAAS) Suggested International Terminology and Definitions”  and accoTo enable clarity over the definition of setting for hysteroscopy and ensure consistency with the IAAS definitions , it is rHysteroscopic procedures can be diagnostic and / or therapeutic (operative). Diagnostic procedures aim to visualise the uterine cavity to detect or exclude endometrial and structural abnormalities  with or without tissue sampling (blind or directed biopsy). Operative procedures remove endometrial and myometrial uterine pathologies with the aim of providing a therapeutic benefit by alleviating gynaecological symptoms as well as allowing histological analysis of removed tissue. The suggested terminology recommended to distinguish the type of hysteroscopic procedure is shown in The traditional approach conducting hysteroscopy to access the uterine cavity consists of inserting a vaginal speculum to visualise the cervix, which is then grasped with a toothed forceps (tenaculum/vulsellum) to provide counter traction when dilatating the cervical canal and subsequently introducing the hysteroscope through the cervical canal and into the uterine cavity under direct visualisation. This technical approach was consolidated after the introduction of hysteroscopy to routine gynaecological practice in the 1990’s when procedures were generally conducted exclusively under general or regional anaesthesia. Advances in surgical technology, and in particular miniaturisation of endoscopes and ancillary instrumentation, has allowed hysteroscopy to be performed without the need for vaginal instrumentation because cervical dilatation is not routinely required. This progression allowed hysteroscopy to be undertaken without the requirement for an anaesthetist or a formal hospital operating room (theatre) and hysteroscopy conducted in more convenient and accessible office and outpatient clinic settings became established.Once office/outpatient clinic hysteroscopy became established, some practitioners began using techniques which did not require conventional vaginal instrumentation with specula and forceps . The hysIt is recommended that the approach to hysteroscopy, namely how the cervix is visualized and accessed to enable entry into the cervical canal and uterine cavity, is defined according to whether vaginal instrumentation is used or not .A proforma to report hysteroscopic procedures according to this nomenclature is presented in Hysteroscopy is considered the gold standard procedure for the diagnosis and management of women with intrauterine pathology and is one of the most common interventions in contemporary gynaecological practice . DespiteContemporary hysteroscopy is performed in a variety of health care settings using different methods of pain control. Hysteroscopic procedures using smaller diameter endoscopes and improved operative technologies, are increasingly being performed without the use of general anaesthesia in variety of facilities. Avoidance of regional or general anaesthesia is associated with a decrease rate of complications, faster recovery times, high rates of acceptability, equivalent effectiveness and greater cost-effectiveness . HoweverThe working group felt that the type of hysteroscopic procedure should be dichotomised into diagnostic procedures and those where the hysteroscope was directly used to treat uterine conditions or remove intrauterine pathologies. Finally, the group believed that the approach to hysteroscopy, namely the technique to visualize and traverse the cervical canal to access the uterine cavity should be reported in a standard fashion. This was in recognition of the miniaturisation of endoscopes and ancillary equipment over time that has facilitated hysteroscopic procedures being performed without the need for any other vaginal instrumentation, namely vaginal specula or forceps applied to the intravaginal cervix . These, The strengths of this consensus statement include the initial wide, international consultation to identify key areas where common definitions or hysteroscopic practice were needed and the subsequent formation of a working group of practicing expert clinicians and researchers by the three leading organisations in the field. This group produced and finalised the standard nomenclature by consensus following several iterations of the proposed statements and classifications until overall consensus was reached. Weaknesses of our approach include that we did not conduct a systematic literature search to identify published literature pertaining to standardised reporting of hysteroscopy. However, our literature searches did not find such papers and our international panel of leading experts in hysteroscopy were unaware of any standard nomenclature. Despite the international make-up of the working group, not all geographic areas were represented. Furthermore, our consensus was not reached using a highly structured approach, such as adoption of a formal Delphi process . These mWe anticipate that wide, international adoption of this standard terminology will vastly enhance communication in the field of hysteroscopy. In turn this will facilitate better understanding of clinical practice, the conduct and feasibility of techniques, and the cost-effectiveness of hysteroscopic interventions. Clinical and research collaborations will be facilitated, and data syntheses supported to robustly inform clinicians and patients.Hysteroscopy is the gold standard technique for the evaluation and management of uterine disorders and is widely used in modern gynaecological practice. However, a clear definition and understanding of the terminology used to describe hysteroscopic procedures is lacking. The production of this international consensus statement for terminology to describe hysteroscopic procedures, covering pain management, setting, model of care, type of procedure and hysteroscopic approach, has the potential to enable more effective communication for both clinical and research purposes with the ultimate aim of improving patient care and clinical outcomes.A summary of the standard terminology describing hysteroscopic procedures is provided in the appendix.