Orthobunyavirus, is a cause of human diseases and less studied worldwide. In this study, a new strain of TAHV was isolated from Aedes sp. mosquitoes collected in Panjin city, Liaoning province. However, the competent vector of TAHV in China is still unknown.Tahyna virus (TAHV), an arbovirus of the genus Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed.The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Ae. albopictus showed positive for the virus in all tested tissues with an extrinsic incubation period of 2 days and a fluctuating increasement of transmission and dissemination rates. Whereas no virus was detected in the saliva of Cx. pipiens pallens. Suckling mice bitten by infectious Ae. albopictus developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, when the virus titer reached 1.70×105 PFU/mL in the brain. Moreover, TAHV was detected in the eggs, larvae and adults of F1 offspring of Ae. albopictus.Phylogenetic analysis revealed that the virus shared high similarity with TAHV and was named the TAHV PJ01 strain. After oral infection with virus blood meals, Ae. albopictus is an efficient vector to transmit TAHV but Cx. pipiens pallens is not. Ae. albopictus is also a reservoir host that transmits the virus vertically, which further increases the risk of outbreaks. This study has important epidemiological implications for the surveillance of pathogenic viruses in China and guiding comprehensive vector control strategies to counteract potential outbreaks in future. Howevern Europe . Neverthnd Culex , and to Culex sp. mosquitoes collected in Xinjiang Uygur Autonomous Region , Dengue virus (DENV), Western equine encephalitis virus (WEEV) and West Nile virus (WNV) (Genera 20 years . Culex pus (WNV) . With glOrthobunyavirus, tentatively named TAHV PJ01 strain, which was isolated from Aedes sp. mosquitoes collected from Panjin city, Liaoning province, China (GPS location: 41°12′ N and 122°07′ E). Here we explore the phylogenetic analysis of TAHV PJ01 strain, and comprehensively evaluate for the first time the vector competence of two dominant mosquitoes in China against it, including replication potential, horizontal transmission and vertical transmission. This study enriched the species diversity of the genus Orthobunyavirus in China, hinting at the potential emerging infectious diseases and their transmission routes, which is of great epidemiological significance for the development of comprehensive vector control strategies for the prevention of arboviral diseases.This study describes a novel member of the genus 2.2.1.Ae. albopictus Guangzhou strain was originally collected as larvae from Guangzhou city, Guangdong Province (GPS location: 23°07′ N and 113°16′ E), in 2019. Cx. pipiens pallens Beijing strain was originally collected as larvae from Beijing city (GPS location: 39°28′ N and 116°28′ E), in 2013. Both mosquito strains were reared under standard insectary conditions at 26 ± 1°C and 75 ± 5% relative humidity (RH) with a photoperiod of 14 h light: 10 h dark cycles. Prior to the infectious feed, adult mosquitoes were provided with 8% sucrose solution.Two mosquito strains were used in this study. 2.2.Ae. albopictus) and BHK-21 (baby hamster kidney) cells were maintained in our laboratory. C6/36 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) at 28°C in an incubator of 5% CO2. BHK-21 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in an incubator of 5% CO2.C6/36  in 2016, and used as a seed stock by passaging once in C6/36 cells and twice in BHK-21 cells. Then, the working stock was generated by inoculation into C6/36 cells, which was subsequently harvested and stored as individual 1.5 mL aliquots in freezing tubes at −80°C. The virus titers were 1.15 × 107 plaque-forming unit (PFU)/mL as detected by plaque assay.The TAHV PJ01 strain isolate was originally isolated from 25–35 g female Kunming mice were used as a blood source and 4-day-old suckling Kunming mice were used as viral infection animal models in this study. All Kunming mice were purchased from Beijing VITAL RIVER Laboratory Animal Technology Co., LTD., and transferred to our laboratory and acclimated to the new environment for 24 h prior to the experiment to minimize the effects of transport.2.3.Viral RNA of TAHV PJ01 strain was firstly extracted from virus stock by QIAamp Viral RNA Mini Kit (QIAGEN), and then reverse transcribed into cDNA using Eastep® RT Master Mix Kit (Promega) according to manufacturer’s instructions. The genome of TAHV consists of 3 segments: L, M and S. The middle region and the ends of segments were amplified by PCR and RACE (Rapid amplification of cDNA ends), respectively. The following reagents were used for PCR reactions: 2 μL of cDNA, 1 μL of forward primer, 1 μL of reverse primer, 10 μL of PrimeSTAR Buffer, 4 μL of dNTP Mixture, 0.5 μL of PrimeSTAR HS DNA Polymerase, and 31.5 μL of nuclease-free water to yield a 50 μL final reaction volume. Amplification reactions were performed in a PCR system (Biometra Tadvanced) programmed as follows: 30 cycles at 98°C for 10 s, 55°C for 5 s and 72°C for 60 s. To obtain the 3′ and 5′ ends sequence, a polyadenylated tail was added to the RNA genome by Poly(A) Polymerase (TaKaRa). Then, first-strand cDNA synthesis, RACE and In-Fusion cloning of RACE products were performed using SMARTer® RACE 5′/3’ Kit (TaKaRa) according to user manual. PCR and RACE products were sequenced at Shenzhen BGI Co., LTD . Genome assembly was performed using SnapGene 2.3.2 . All primers used for amplification were listed in 2.4.Orthobunyavirus together with the representative members of the Bunyamwera serogroup (Bunyamwera virus) were used in the analysis (from GenBank as of 6 June 2022). Accession numbers of all the above strains are listed in The complete sequences of all three segments of 22 strains  belonging to the CSG in the genus 2.5.Ae. albopictus and a saturated sponge for Cx. pipiens pallens. The infected blood meal was kept at 37°C during feeding. After 50 min of feeding, mosquitoes were cold-anesthetized, and blood-engorged mosquitoes were transferred and maintained in the standard conditions.Virus-infected blood meals were prepared containing 1:1 mouse blood and TAHV PJ01 strain suspension supplemented with 3% FBS and 1% heparin sodium. Seven-day-old adult female mosquitoes that had been starved for 12 h were fed with this infected blood meal using a Hemotek membrane feeding system for 2.6.Ae. albopictus and Cx. pipiens pallens, 30 blood-engorged mosquitoes were, respectively, sampled at 2, 4, 6, 8, 10, 12, 16 and 18 day post exposure (dpe). The midguts, ovaries, salivary glands, and saliva of all sampled mosquitoes were dissected and collected carefully with sterile dissecting needles and individually transferred into 1.5-mL microtubes containing 1 mL of RNAiso Plus (TaKaRa). Then, organs were homogenized using 2.5 mm stainless steel grinding balls and an N▪9524R frozen grinder manufactured by Beijing HODER Technology Co., LTD. These samples were then used for RNA extraction and TAHV PJ01 strain detection via RT-qPCR.To evaluate the infection of the TAHV PJ01 strain in 2.7.Ae. albopictus and Cx. pipiens pallens to horizontally transmit TAHV PJ01 strain, at 10 dpe, whole nests of 4-day-old suckling mice were placed in cages containing 50 mosquitoes that previously were fed with the virus-infected blood meals described above. 2 h after exposure, all the mosquitoes were removed from the cage and the whole bodies of newly blood-engorged mosquitoes were detected for virus infection by RT-qPCR. The suckling mice were also removed from the cage, counted red dots of mosquito bites and reared with their mother mouse. The suckling mice were then monitored daily. The healthy mice were sampled on days 4, 7 and 10 (2 mice per day) as routine sampling. The unhealthy mice were sampled immediately once found be sick or dead as additional sampling. For all of the above sampled mice, half of the brain was grinded in the DMEM and filtered to obtain supernatant for plaque assay to determine virus titer; The remaining portion of the brain was used for RNA extraction and TAHV PJ01 strain detection via RT-qPCR.To determine the ability of 2.8.Ae. albopictus to transovarial transmit TAHV PJ01 strain from parents to offspring, the eggs laid by infected mosquitoes during the first and second gonotrophic cycles were collected. In the first gonotrophic cycle, blood-engorged mosquitoes were allowed to oviposit at 6–10 dpe. The eggs were maintained in a desiccator at 26 ± 1°C and 90% RH with a photoperiod of 14 h light:10 h dark cycles for 7 days to permit embryonation, and were subsequently partially hatched. Eggs, hatched larvae, and emerged adults were tested for the presence of TAHV PJ01 strain by RT-qPCR. Then, parent female mosquitoes were fed with normal mouse blood at 10 dpe, and eggs from the second gonotrophic cycle were collected at 13–17 dpe. The eggs, larvae and adults of the second gonotrophic cycle were collected as above for TAHV PJ01 strain detection.To determine the ability of 2.9.Total RNA from midguts, ovaries, salivary glands, saliva, eggs, hatched larvae and emerged adults was isolated using RNAiso Plus (TaKaRa), and from suckling mouse brain using RNeasy® Mini Kit (QIAGEN), according to the manufacturer’s instructions.TAHV PJ01 strain was detected in these samples using GoTaq® Probe 1-Step RT-qPCR System (Promega). A pair of universal primers for CSG virus amplification  and a ne2, virus stocks were discarded and the cells were overlaid with 1:1 of 2% ultra-low gelling agarose (SIGMA) and high sugar-DMEM (2×) (GENOM). Cells were incubated at 37°C of 5% CO2 and observed daily for cytopathic effect (CPE), after CPE emergence they were stained with a 2% crystal violet solution . The number of characteristic plaques was counted to represent the presence of the virus particles. The virus titers were expressed as PFU/mL.Virus titers in mouse brains were detected by plaque assay. The supernatants derived from the mouse brain virus suspensions were serially diluted and inoculated into BHK-21 cells cultured in a 6-well plate with 200 μL volumes. After 1 h incubation at 37°C of 5% CO2.10.Ae. albopictus and Cx. pipiens pallens mosquito populations were characterized via the following indicators: positive rates and vRNA copies of midguts, ovaries, salivary glands and saliva; Infection rate (IR), transmission rate (TR), dissemination rate (DR) and dissemination efficiency (DE); Infection and morbidity of suckling mice; Infection of offspring in the first and second gonotrophic cycle.The vector competence of 2.11.p < 0.05 was considered statistically significant.GraphPad Prism 8.0  was used for data processing and visualization. SPSS 26 software  was used for statistical analysis. Fisher’s exact test was used to compare rates. The Least Significant Difference (LSD) and the Tamhane test were used to compare the means ± standard errors (SEs) of RNA copy across multiple groups when passing and failing the homogeneity of variance test, respectively. An independent sample t-test was used to compare the means ± SEs of RNA copy between the two groups. 3.3.1.Aedes sp. mosquitoes and its genome was sequenced via the next-generation sequencing technology. The complete sequences of the virus have been submitted to the GenBank database . Analysis of the assembled sequences via the BLAST tool revealed that the newly isolated virus was the most correlated to TAHV, which belongs to the CSG of the genus Orthobunyavirus, and it was subsequently named TAHV PJ01 strain in this study.The virus was originally isolated from a pool of Orthobunyavirus, the TAHV PJ01 strain was analyzed the phylogenetic relationship between the CSG based on all three segments, except the MBV, which only had complete CDSs for the S segments in GenBank. Bunyamwera virus, belonging to the Bunyamwera serogroup was used as the outgroup , TAHV strain XJ0625 (82.50%), and TAHV strain XJ0708 and XJ0710 (82.90%), respectively –F.3.2.Ae. albopictus and Cx. pipiens pallens were, respectively, tested at 2, 4, 6, 8, 10, 12, 16 and 18 dpe. For Ae. albopictus, from 2 dpe onwards, TAHV PJ01 strain could be detected in the midguts, ovaries and salivary glands on every sampling day  developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, at day 5 . vRNA copies and virus titer in the brain of all the above mice were, respectively, detected via RT-qPCR and plaque assay. Results showed that nine mice, including those 2 diseased and 1 dead, 9 of the eleven (81.8%) suckling mice had vRNA in their brains within 10 days of observation (p = 0.634). Two diseased mice had an average vRNA copies of 8.64 ± 0.19 lg RNA copies/μL, and their virus titers were 1.70 × 105 PFU/mL and 2.20 × 105 PFU/mL, respectively. The dead mouse had the highest vRNA copies of 9.09 lg RNA copies/μL accompanied by the highest virus titers of 1.10 × 106 PFU/mL  routinely sampled on day 10 had vRNA in its brain at a copy concentration of 2.32 lg RNA copies/μL  were detected to demonstrate vertical transmission. A total of 1,500 F1 progeny in 90 pools in the first gonotrophic cycle and 750 F1 progeny in 48 pools in the second gonotrophic cycle were tested (P<0.001), reaching 30.0% and 1.52 ± 0.05 lg RNA copies/μL, and then steadily decreased to 13.3% and 1.48 ± 0.03 lg RNA copies/μL at the adult stage. In the second gonotrophic cycle, IR, MIR (minimum infection rate) and RNA copy all showed decreasing trends from the egg to the adult stage similar to those in the first gonotrophic cycle. At the adult stage, the IR was 13.3%, the MIR was 1.3%, and the vRNA copy was 1.43 ± 0.04 lg RNA copies/μL. However, there were no significant differences in the same stage between different gonotrophic cycles, except that vRNA copies of positive eggs and larvae in the second gonotrophic cycle were significantly higher than those in the first gonotrophic cycle (P<0.01). The fact that the TAHV PJ01 strain can be detected in adults of F1 progeny demonstrated that the TAHV PJ01 strain can be vertically transmitted from parents to offspring in Ae. albopictus.As e tested . In the 4.Aedes sp. mosquitoes collected from Panjin city, Liaoning province, China (GPS location: 41°12′ N and 122°07′ E). The TAHV PJ01 strain was identified as most closely related to TAHV, belonging to Order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus, CSG. Several kinds of CSG viruses have been shown to cause disease in humans, ranging from mild rashes to severe neuroinvasive diseases, with symptoms including headache, fever, dizziness, vomiting, muscle weakness, stiff neck, seizures, hemiparesis, encephalitis, meningoencephalitis, and meningitis  of 2 days. The EIP refers to the time required for the virus to reach the mosquito saliva after ingesting an infectious blood meal, and is influenced by diverse factors, such as mosquito species and population, virus genetic characteristics, virus-mosquito interactions, and environmental factors . However, 66.7% of the infected suckling mice showed no obvious disease symptoms. This interindividual variation in disease progression may be due to differences in initial viral acquisition via biting and immunity of the suckling mice. In brief, the shorter EIP of TAHV PJ01 strain and its pathogenicity and lethality in mammals force us to pay more attention to its emergence.TAHV has been detected from more than one mosquito species worldwide, suggesting that TAHV PJ01 strain can be transmitted ics etc. . After o factors . Previouf 6 days , and DENf 6 days , 2016. Tic cycle , and a sAedes and Culiseta mosquito species via field observation and/or laboratory infection experiments. TAHV was also confirmed to be capable of transovarial transmission in Culiseta annulata .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1159835/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Arthropod-borne flaviviruses like dengue virus (DENV) and yellow fever virus (YFV) are major human pathogens. In Latin America, YFV is maintained in sylvatic cycles involving non-human primates (NHP) and forest-dwelling mosquitos. YFV supposedly does not circulate north of Panama.Ateles geoffroyi, n = 25), black howler monkeys , and mantled howler monkeys  sampled in 2012 and 2018 were screened by an indirect immunofluorescence assay (IFA) to detected IgG antibodies against DENV, YFV, Zika virus (ZIKV), West Nile virus (WNV), Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Middle East respiratory syndrome coronavirus, and Zaire Ebola virus, and confirmed by plaque reduction neutralization tests (PRNT90) representing all mosquito-borne flavivirus serocomplexes circulating in the Americas.We conducted a serologic study for flaviviruses and other emerging viruses in NHP from southeastern Mexico. A total of thirty sera of black-handed spider monkeys  sampled in 2018 in Tabasco, compared to all other flaviviruses tested. Monotypic endpoint PRNT90 titers of 1:28 for Ilheus virus and 1:22 for WNV in serum of black howler monkeys sampled in 2018 in Tabasco suggested additional flavivirus exposure.A total of 16 sera  showed IFA reactivity to at least one tested flavivirus with end-point titers ranging from 1:100 to 1:1000. No serum reacted with other viruses. Monotypic and high mean PRNTOur findings may suggest unnoticed YFV circulation. Intensification of YFV surveillance in NHP and vectors is warranted in Mexico and potentially other areas considered free of yellow fever. Flaviviruses are widespread in almost all continents and climate zones . In LatiCulex mosquitos at different times and places [Aedes aegypti [In Mexico, dengue fever is endemic, with 25,226 cases reported in 2020 and 6746 in 2021 . WNV wasd places . In cont aegypti . In 2021 aegypti , 11. Typ aegypti .Haemagogus and Sabethes which have been reported in Southeastern Mexico [Aedes mosquitoes, are widely distributed throughout the country, the re-emergence of YFV in urban regions could have devastating consequences. Moreover, because YFV infection is potentially deadly for primates and the three NHP species occurring in Mexico are listed as vulnerable  and endangered (Al. pigra and Ateles geoffroyi), the potential presence of YFV in Mexican NHP may pose a great risk for animal conservation. Here we investigated the presence of flaviviruses in NHP from the southeast of Mexico.With circa 200,000 cases and 30,000 deaths annually, YFV is currently endemic in 47 countries in Africa, Central and South America , 14. Urbn Mexico , 19. BecAteles geoffroyi (black-handed spider monkey), three Alouatta pigra (black howler monkey), and two Al. palliata (mantled howler monkey). The samples from Quintana Roo (2012) were obtained from animals that were part of a wildlife translocation program at a government airport, in which samples were taken from the animals before being relocated and sent to our laboratories for the detection of viral infectious diseases, before their release. The animals were kept in a temporary shelter located in Km 20 + 500 Carretera Federal 307, tramo Bacalar-Carrillo Puerto, Quintana Roo. The samples from Tabasco (2018) belonged to captive monkeys at state zoological facilities La Venta Park-Museum and Yumká Natural Protected Area . Because of the importance of monitoring potentially zoonotic diseases, authorities of both institutions authorized the physical restraint of individuals, which is a technique considered appropriate in captive primates for procedures of short duration. Animal handling was performed by specialized and experienced staff, following protocols of safety, care, and health of each institution. All animals were in good overall conditions at the time of blood sampling  and samples were stored on ice and immediately transported to the laboratory. Serum samples from all NHP that were available at the two locations were tested, without application of additional inclusion or exclusion criteria.We tested a total of thirty NHP sera, twenty-one samples were from Bacalar, Quintana Roo and nine from Villahermosa, Tabasco (both sites in southeastern Mexico); twenty-five Phenuiviridae, Flaviviridae, Coronaviridae, and Filoviridae families, we employed an indirect immunofluorescence test (IFA) termed Biochip Virus-Mosaic Africa-2  following the manufacturer’s instructions. The biochip contains antigens from high-risk pathogens such as DENV, WNV, YFV, ZIKV, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Middle East respiratory syndrome coronavirus, and Zaire Ebola virus. Because of the well-described antibody cross-reactivity among flaviviruses, samples that tested positive for at least one flavivirus were further tested for specific viruses by plaque reduction neutralization test (PRNT). PRNT was performed using ZIKV (strain H/PF/2013), DENV-1 (strain Thailand/16007), DENV-2 (strain 16681), DENV-3 (strain Philippines/H87), DENV-4 (strain Philippines/H241), WNV (strain NY-99), YFV (strain 17D), Bussuquara virus , Ilheus virus . Briefly, sera were inactivated at 56° for 30 min and diluted at 1:80, 1:240, 1:720 and 1:2160 in serum-free DMEM. Subsequently, a mixture of 34 µL of each serum dilution plus 34 µL of virus dilution containing 50 plaque forming units was incubated for 1 h at 37° C with 5% CO2. After this time, 50 µL of the serum dilution and virus mixture plus 250 µL of cell culture media were added to 12-well plates containing Vero FM  and Vero B4 (for DENV1-4) cells seeded one day before (1.4 × 105 cells/well). After one hour of incubation, an overlay containing 2% DMEM and 1.25% carboxymethyl cellulose was added. After three days for WNV, four days for BSQV, five days for ZIKV, six days for ILHV and DENV, and seven days for YFV, the medium was removed, and cells were fixed with 6% paraformaldehyde and plaques visualized using crystal violet solution. A positive result means a serum dilution reduced the number of viral plaques in a given well by at least 50% (PRNT50) or 90% (PRNT90). Additionally, NHP sera were tested for flaviviruses by semi-nested RT-PCR using primers previously reported [Taq DNA polymerase , and Taq DNA polymerase , for the first and second round, respectively, following the manufacturer’s instructions.To detect IgG antibodies against selected members of the Initial IFA screening showed that 53% 16/30) of sera were reactive for at least one of the flavivirus antigens tested , with end-point titers ranging from 1:100 to 1:1000 serum dilution, not allowing unambiguous identification of flavivirus infection histories  in 12.5%  of the captive black-handed spider monkeys sampled in Tabasco in 2018  must be interpreted with caution because of the low number of animals tested. However, absence of antibodies in those animals is consistent with high mortality of yellow fever in NHP belonging to Alouatta species exceeding 60%, in contrast to NHP species of the genus Ateles such as spider monkeys which seem to be more resistant to infection and clinical disease.Because antibody cross-reactivity is very common among flaviviruses, we tested all IFA-positive sera by highly specific PRNT against at least one flavivirus of each serocomplex. Both PRNTera Fig. . High ann Panama  and in sn Panama . The abs90. One black howler monkey serum previously IFA-reactive for all tested flavivirus showed monotypic PRNT90 endpoint titers for WNV , while another serum previously reactive in the IFA at the dilution 1:100 for ZIKV showed monotypic PRNT90 endpoint titers for ILHV , whereas one black-handed spider monkey serum that had previously been IFA-reactive for all tested flaviviruses presented monotypic endpoint titers for DENV-2  (Table 90.No ZIKV, DENV-1, DENV-3, and DENV-4-specific neutralizing antibodies could be confirmed by PRNTOur serological data may support the occurrence of YFV in NHP in Mexico. On the one hand, we cannot exclude that antigenically related flaviviruses could also have elicited cross-reactive antibodies. This hypothesis is compatible with the detection of a monotypic neutralizing antibody response against Wesselsbron virus in a single Brazilian cow during a sero-epidemiological investigation of several thousand sera from livestock and pets . On the The apparent absence of yellow fever northwards of Panama is not entirely understood. In contrast to other Latin American countries, such as Brazil, in which yellow fever outbreaks often occur, wild NHP populations in Mexico are geographically limited and in small numbers. That is probably one of the reasons because, at least so far, no recent human cases have been reported in Mexico. If YFV was indeed present in Mexico, it would be possible that at any time, YFV may emerge in humans or NHPs.Our study is chiefly limited by the small number of NHP that were available for testing and the absence of testing for all potentially co-circulating flaviviruses. However, the latter is most critical for WNV, ILHV and BSQV Fig. A due to Our findings warrant monitoring of potential YFV infections in free-living and captive NHP as well as mosquito surveillance in Mexico and neighboring countries. Additional confirmation, including direct viral detection, is needed to provide definite assessments of the potential occurrence of YFV in areas north of Panama considered free of yellow fever.


High morbidity and mortality of influenza virus infection have made it become one of the most lethal diseases threatening public health; the lack of drugs with strong antiviral activity against virus strains exacerbates the problem.Two independent researchers searched relevant studies using Embase, PubMed, Web of Science, Google Scholar, and MEDLINE databases from its inception to December 2022.Based on the different antiviral mechanisms, current antiviral strategies can be mainly classified into virus‐targeting approaches such as neuraminidase inhibitors, matrix protein 2 ion channel inhibitors, polymerase acidic protein inhibitors and other host‐targeting antivirals. However, highly viral gene mutation has underscored the necessity of novel antiviral drug development. Arbidol (ARB) is a Russian‐made indole‐derivative small molecule licensed in Russia and China for the prevention and treatment of influenza and other respiratory viral infections. ARB also has inhibitory effects on many other viruses such as severe acute respiratory syndrome coronavirus 2, Coxsackie virus, respiratory syncytial virus, Hantaan virus, herpes simplex virus, and hepatitis B and C viruses. ARB is a promising drug which can not only exert activity against virus at different steps of virus replication cycle, but also directly target on hosts before infection to prevent virus invasion.ARB is a broad‐spectrum antiviral drug that inhibits several viruses in vivo and in vitro, with high safety profile and low resistance; the antiviral mechanisms of ARB deserve to be further explored and more high‐quality clinical studies are required to establish the efficacy and safety of ARB. In this study, we review the structure of influenza virus, as well as the etiology, epidemiology, and pathophysiology of influenza virus. We describe the process of virus infection and summarize the current antiviral strategies and the reported antiviral mechanisms of ARB; these might be conducive to the discovery and development of novel antiviral drugs in the days to come. However, sometimes, the attachment of HA to SA receptors does not lead to a successful invasion. NA repeatedly cleaves the SAs by catalyzing the cleavage of α2,6 or α2,3 linkage between the terminal SA residues and adjacent oligo lactose, leading to loose HA‐receptor interactions, encouraging virus crawling and gliding until it meets its appropriate entry receptors, and finally triggering the virus uptake and entry receptors into hosts , virus movement plays a significant role in the virus invasion, depending on the cooperation of HA affinity and NA activity. h, virusAfter HA encounters appropriate entry receptors, the virus particles are taken into hosts. Viruses invade organisms via the clathrin‐mediated endocytosis pathway, during which the cell membrane forms invaginated phagocytic vesicles known as clathrin‐coated pits; the virus particles can penetrate the cytoplasm via the clathrin‐mediated endocytosis pathway.α2,6 or 2,3 glycosidic bonds, preventing the immediate detachment of the influenza virus. NA is responsible for catalyzing the hydrolysis of these important glycosidic bonds; mature virus particles finally detach from the host cells to infect new epithelial cells, spreading influenza viruses in the host.In the late stages of infection (6–10 h), most of the progeny viruses mature via endoplasmic reticulum processing. Viruses are transported through the mucus and destroy the SA receptors on the host cells, encouraging the release of progeny virus particles from the infected hosts. However, mature influenza virus particles still depend on SA residues for connecting with hosts through 2Current antiviral methods are mainly focused on two strategies: directly targeting the virus itself or its hosts. Antiviral drugs targeting viruses mainly affect some stages of the viral replication cycle for protecting against virus invasion, such as interrupting virus adsorption, blocking membrane fusion, inhibiting polymerase and protease, or impairing viral release Figure , whereas2.1The most important step of influenza A virus infection is the attachment of HA to SA receptors on the cell surface; thus, HA inhibition is an attractive and promising antiviral strategy.2.2M2 genesM2 channel blockers, including amantadine and rimantadine, are common antiviral drugs that can inhibit the M2 ion channel activity for preventing the replication of the influenza virus.2.3NA inhibitors are a type of antiviral drugs that can block the activity of viral NA protein.2.4The RNA polymerase complex essential for virus replication and transcription comprises three units: polymerase basic protein 2, polymerase basic protein 1, and PA. Specific residues of PA are highly conversed and mutated, making endonuclease inactive. The antiviral agent baloxavir marboxil  was developed to specifically target PA for inhibiting its activity but not affect polymerase basic protein 1 and 2 activities. Baloxavir has demonstrated broad‐spectrum antiviral effects against many subtypes of influenza A viruses including strains with NA inhibitor‐resistant mutations. Baloxavir has currently been under phase III clinical trials and has shown the desired effect; oral administration of baloxavir had minor side effects and was effective in relieving influenza virus symptoms with a halftime of around 80 h. It was approved for specific treatment of uncomplicated influenza in patients in the United States and Japan in 2018.2.5Considering the emergence of drug‐resistant viruses, it is still important to look for novel targets targeting host cellular factors.3ARB is a small‐molecule indole‐derivative developed in Russia3.13.1.1The broad‐spectrum antiviral activity of ARB specifies that it acts on the important steps of virus–cell attachment. Moreover, ARB plays a direct role in removing the influenza virus, which is similar to the role of other direct‐acting antiviral drugs. The inhibitory effect of ARB on different stages of the virus life cycle, such as cell entry, attachment, internalization, and replication, has also been reported Figure . Therefo‐mediated endocytosis. Virus binding to the plasma membrane is severely inhibited following ARB treatment. Subsequently, ARB can also directly impair clathrin‐mediated endocytosis by preventing the release of vesicles or interrupting dynamin‐2‐induced membrane rupture, leading to the accumulation of clathrin‐coated vesicles or failure of viral transportation in cells, inhibiting virus infection at the entry step.During the early stages of infection, after attaching to HA receptors on the cell membrane, the influenza virus is internalized into clathrin‐coated vesicles and taken into the host cell via clathrin2‐PGC chip quadrupole time‐of‐flight mass spectrometry method, we found that the SA‐linked N‐glycans levels that act as SA receptors significantly decreased in PR8 virus infection. In contrast, the neutral N‐glycans levels increase due to the cleavage of SA‐linked N‐glycans by virus NA protein when the virus rolls and glides on the cell surface at the early step of infection; after that, the virus is successfully uptaken into cells when it meets an appropriate entry receptor. Pretreatment with ARB can remarkably downregulate SA‐linked N‐glycans on the cell surface by obstructing the expressions of sialyltransferases, which is related to the biosynthesis of SA‐linked N‐glycans. This downregulation of SA‐linked N‐glycans by ARB significantly reduces the virus utilization on SA‐linked N‐glycans, and receptor‐dependent rolling and entry capacity of the virus are significantly decreased, thus inhibiting virus rolling, gilding, and entry.Recently, a novel antiviral mechanism of ARB was revealed in our study Figure .68 UsiMechanistically, ARB is a hydrophobic molecule with an indole structure, which is prone to forming supramolecular arrangements by interacting with the aromatic amino acid residues of viral glycoproteins, leading to the interference of membrane fusion. Moreover, ARB can interact with important residues in viral lipid membranes, which may underlie the antiviral effects of ARB.An acidic environment (pH 5.0) is needed for the membrane fusion of the influenza virus and host cells, and changes in pH will hinder the fusion process. ARB is a weak alkaline drug that can increase the pH of the intracellular environment, making the virus unable to complete the process of membrane fusion and release the virus genome, resulting in effective inhibition of virus infection.3.1.2ARB can inhibit influenza virus replication; enveloped RNA viruses are more sensitive to ARB than nonenveloped DNA viruses.3.1.3ARB has immunomodulatory, interference‐inducing, and antioxidant properties.3.23.2.1−1, and that ARB has a comparable or stronger inhibitory effect on virus replication than other anti‐influenza drugs, including amantadine, ribavirin, and OSV.ARB effectively inhibits influenza A ,−1 was orally administered for 10–18 days to a group of workers during an influenza A epidemic and was found to display a significant preventive effect. A Russian study confirmed that prophylactic oral ARB administered to military personnel reduced acute respiratory viral infections and the incidence of mixed bacterial and viral pneumonia.ARB has a good prophylactic effect against influenza virus infection. In a Russian randomized controlled trial,p < .05). Severity of illness, catarrhal symptoms, and intoxication was reduced with ARB compared with placebo. A 2019 study on the safety and efficacy of ARB treatment, including 359 patients with influenza or acute upper respiratory tract infection randomized into treatment group (ARB) and placebo group, showed that influenza symptoms were milder and of shorter duration in the treatment group . Furthermore, the incidence of adverse events was similar in both groups.On the other hand, oral administration of ARB early during the onset of influenza shortens the duration of illness and also reduces symptoms. A study by Kiselev et al.ARBs are effective in preventing and treating influenza in children. A study found that in children, ARB prevented the onset of influenza and other respiratory viral infections, reduced the severity of illness, and decreased the occurrence of other complications.3.2.2SARS‐CoV‐2 is a positive‐sense single‐stranded RNA capsid virus with a high degree of gene sequence homology and belongs to the same genus of betacoronavirus as the SARS‐CoV outbreak in 2003. The main pathogenic mechanism of SARS‐CoV‐2 is to invade host cells by binding to angiotensin‐converting enzyme 2 receptors on the surface of spike glycoprotein,ARB inhibits the fusion of SARS‐CoV‐2 with the cell membrane by dual binding to the receptor‐binding domain and angiotensin‐converting enzyme 2 receptors of SARS‐CoV‐2 spike protein on the host cell membrane; moreover, it interferes with the acidification of endocytic vesicles in the endocytic pathway and inhibits the intracellular release of viral particles. The immunomodulatory effect of ARB may inhibit the excessive release of inflammatory factors, such as IL‐6, in the lung tissues and reduce the chances of inflammatory lung injury.In vitro studies have shown that ARB has very favorable antiviral effects against SARS‐CoV‐2, showing statistically significant decreases in the binding efficiency and inhibition of viral entry and postviral events.p < .01). Moreover, the duration of positive RNA testing was shorter in the ARB group than in the lopinavir/ritonavir group (p < .01).p = .02), less severe involvement on chest computed tomography scan after 30 days (p = .004), and shortened the length of stay (p = .02). However, other parameters such as the need for intubation (p = .6), the need for mechanical ventilation (p = .6), and the time to defervescence or mortality (p = .5) did not show significant differences. Deng et al.p < .05).p = .037), suggesting that the combination of ARB and IFN‐α2b may be beneficial in reducing lung inflammation in patients with mild coronavirus disease 2019 (COVID‐19) but is not effective in accelerating viral clearance (p = .057) or reducing the length of hospital stay (p = .056).ARB alone and in combination with other drugs have good efficacy in reducing the viral load of patients after admission and shortening the time for SARS CoV‐2 nucleic acid to turn negative. In a retrospective study conducted in China comparing 36 patients treated with lopinavir/ritonavir with 16 patients treated with ARB, ARB monotherapy was more effective than lopinavir/ritonavir, as all patients treated with ARB developed undetectable viral load by day 14 postadmission compared with 55.9% of patients treated with lopinavir/ritonavir (n = 45) and control group (n = 36), which were divided according to the use of ARB, ARB failed to improve the prognosis or accelerate SARS‐CoV‐2 clearance in non‐ICU patients; moreover, the clinical outcomes were significantly better in the control group.Conversely, other studies found no benefit of using ARB in patients with COVID‐19. A study of 86 patients with COVID‐19 revealed that ARB monotherapy has a minor advantage in improving the clinical outcome of patients hospitalized with mild/moderate COVID‐19 over supportive care.3.2.3ARB has inhibitory effects on many other viruses such as RSV, human adenovirus, CV, parainfluenza virus, rhinovirus, Hantaan virus, chikungunya virus, Lassa virus (LASV), Ebola virus (EBOV), HSV, and hepatitis B and C viruses. However, most studies on the inhibitory effects of ARB on viruses other than influenza and SARS‐CoV‐2 are animal experiments or at the in vitro stage; thus, clinical trial studies should be supported to promote the application of ARB.Paramyxoviridae and is an enveloped negative‐stranded RNA virus, which can cause upper respiratory tract infections and even severe bronchitis and pneumonia. In a mouse model, it was confirmed that the oral ARB 10 and 50 mg kg−1 d−1 groups had significantly lower titers of RSV than the control group, indicating that ARB has an antiviral effect on RSV in vivo.Adenoviridae that causes pneumonia and upper respiratory tract infections, with human adenovirus types 3 and 7 being the main pathogens of adenovirus pneumonia. Currently, no clinically recommended therapeutic agents are available. In the HEP‐2 cell assay, ARB has an inhibitory effect only when added after human adenovirus type 7; thus, it is concluded that ARB exerts its antihuman adenovirus type 7 effect in vitro by inhibiting a part of virus biosynthesis.−1 and the therapeutic index was 3.5.RSV belongs to the family Picornaviridae, is responsible for several conditions, including respiratory infections, myocarditis, or encephalitis. In a study on the antiviral effects of ARB in mice infected with CV B5, the mice developed interstitial pneumonia and myocarditis; and some mice received oral treatment with ARB for 6 days. At a dose of 50 mg/kg, the drug prolonged survival and reduced the spread of CV in the lungs and heart.ARB has significant inhibitory effects on CVs and HSVs, which cause respiratory symptoms. CV B5, a member of the family −1, respectively.−1). Di Mola et al.ARB also prevents hemorrhagic fever viruses. Chinese studies have demonstrated the antiviral activity of ARB against Hantaan virus.Flaviviridae,−1. ARB obstructed hepatitis C virus entry into cells by affecting lattice‐protein‐mediated endocytosis.As early as 2006, ARB and its derivatives inhibited hepatitis B virus replication in vitro and reduced surface antigen production.ARB also inhibits the foot‐and‐mouth disease virus, Zika virus, and blistering stomatitis virus in in vitro experiments, and the inhibitory effect was more significant when added before or during the infection.3.3−1, respectively,−1 for 4 weeks showed no significant effects on reproductive function and fetal growth and development of pregnant rats.ARB has shown a high safety profile in animal toxicity studies. The 50% lethal doses of ARB in rats and guinea pigs were 3000 and 4000 mg kgARB is well tolerated and safe to use in humans and no serious drug‐related adverse events have been reported over the years of its use in Russia and China. In a controlled study in Russia, 39 healthy volunteers and 22 patients with acute respiratory disease were given an oral placebo and ARB (dose: 200–800 mg) for 10 days with follow‐up for 10–15 days; no significant difference was observed in the results of various examinations between the two groups.3.4ARB has been used for many years in Russia and China for the prevention and treatment of influenza; however, no highly drug‐resistant strains of the virus have been detected. Influenza A/H1N1 and A/H3N2 pandemic strains isolated in Russia in 2008–2009 showed resistance to OSV and amantadine; however, both were sensitive to ARB.However, several influenza strains are resistant to ARB, particularly in the population of influenza B virus strains.4The current clinical options for acute viral respiratory infections are limited, highly viral gene mutation has underscored the necessity of novel antiviral drug development. Unlike other antiviral drugs, ARB is a non‐nucleoside broad‐spectrum antiviral drug that not only prevents viral fusion and replication, but also induces the production of interferon, enhances phagocytosis, and other immunomodulatory effects. ARB has been clinically used for the prevention and treatment of influenza A and B, with a good profile of safety and low drug resistance, and it can be used as a pharmacologic enhancer in combination with other drugs since the mechanism of antiviral action of ARB is different from that of other approved antiviral drugs. Based on aforementioned, ARB is a promising drug for clinical application in the prevention and treatment of respiratory viral infections, and the antiviral mechanisms of ARB deserve to be further explored.However, the research of ARB on many other viruses, such as SARS‐CoV, MERS‐CoV, RSV, CV and Hantaan virus, LASV, etc., mostly not yet in clinical stage, and the conclusions of the current clinical trials on the treatment of COVID‐19 with ARB are still inconsistent. In addition, ARB is currently only produced and used in a few countries, such as China and Russia, and there is a lack of data on the clinical studies on the use of the drug in other countries and populations, so the research on high‐quality clinical trials should be strengthened, especially the global multicenter clinical trial to further confirm its effectiveness and safety and to promote its application.Yue Kang: Conceptualization; data curation; investigation; resources; writing—original draft. Yin Shi: Formal analysis; investigation; resources; visualization; writing—original draft. Silu Xu: Conceptualization; methodology; project administration; supervision; validation; writing—review and editing.The authors declare no conflict of interest.


When multiple viral populations propagate within the same host environment, they often shape each other’s dynamics. These interactions can be positive or negative and can occur at multiple scales, from coinfection of a cell to co-circulation at a global population level. For influenza A viruses (IAVs), the delivery of multiple viral genomes to a cell substantially increases burst size. However, despite its relevance for IAV evolution through reassortment, the implications of this positive density dependence for coinfection between distinct IAVs has not been explored. Furthermore, the extent to which these interactions within the cell shape viral dynamics at the level of the host remains unclear. Here we show that, within cells, diverse coinfecting IAVs strongly augment the replication of a focal strain, irrespective of their homology to the focal strain. Coinfecting viruses with a low intrinsic reliance on multiple infection offer the greatest benefit. Nevertheless, virus-virus interactions at the level of the whole host are antagonistic. This antagonism is recapitulated in cell culture when the coinfecting virus is introduced several hours prior to the focal strain or under conditions conducive to multiple rounds of viral replication. Together, these data suggest that beneficial virus-virus interactions within cells are counterbalanced by competition for susceptible cells during viral propagation through a tissue. The integration of virus-virus interactions across scales is critical in defining the outcomes of viral coinfection. The co-occurrence of distinct virus populations within a single host occurs commonly, when an individual acquires a second infection before a prior one has been cleared. Under these circumstances, each virus can influence the replication of the other, positively or negatively. These interactions can involve viruses of different species but here we focus on interactions between distinct influenza A viruses (IAVs). For IAVs, coinfection is important to viral evolution and emergence because it can result in the formation of novel, chimeric viruses. We find that two IAVs infecting the same cell often interact synergistically, such that replication is improved compared to a singular infection. Conversely, when monitoring viral dynamics within a mammalian model, we see that virus-virus interactions at the level of the whole host are antagonistic. Our data suggest that, when viruses are propagating through a population of cells, beneficial interactions within cells are counterbalanced by competition for susceptible target cells. This antagonistic interaction is likely a major factor limiting the formation of chimeric IAVs during coinfection. The past three influenza A virus (IAV) pandemics arose through reassortment involving human and non-human IAVs in coinfected hosts –6. Due tWhen viruses coinfect the same host or cell, each can affect the other’s replication. These virus-virus interactions can occur between conspecific viruses (as investigated herein) or taxonomically distinct viruses. They furthermore range from antagonistic to beneficial, depending on the context. At the cellular scale, viruses may compete directly for limited resources within the cell or indirectly through the triggering of antiviral responses. These antagonistic effects are typically potent in the case of defective viral genomes . ConversOur prior work focused on virus-virus interactions between homologous IAVs. We previously reported that coinfection of a cell with multiple IAV particles is often required to initiate a productive infection . FurtherHere, we investigate what viral traits define the extent to which a coinfecting IAV can augment the replication of another. Specifically, we examine the importance of a coinfecting virus’ intrinsic reliance on multiple infection, the host species to which it is adapted, and its homology to the focal virus. To this end, we evaluated the outcomes of coinfection with a set of previously well-characterized IAV strains that vary in host adaptation and reliance on coinfection: influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) (GFHK99), A/mallard/Minnesota/199106/99 (H3N8) (MaMN99), A/Netherlands/602/2009 (H1N1) (NL09), and A/Panama/2007/99 (H3N2) (Pan99) viruses (16). Our results indicate that homology and host adaptation are not critical for defining IAV-IAV interactions within cells. Conversely, IAVs having a low intrinsic reliance on coinfection more effectively augment replication of the focal virus. While these beneficial interactions are readily detected when infection is limited to a single viral generation, under conditions that allow further propagation, antagonistic effects become predominant. Dynamics in vivo and during multiple rounds of viral infection in cell culture suggest that competition for target cells and superinfection exclusion typically limit the opportunity for distinct viral populations to interact within cells.Previously we showed that influenza A virus replication is strongly dependent on viral density. Namely, progeny production is enhanced when population density is sufficient to ensure delivery of many viral genomes to a cell. The extent of this dependence was, however, noted to vary with virus strain. To investigate the importance of this phenotype for interactions between distinct coinfecting strains, we sought to identify a panel of viruses that vary in their reliance on multiple infection. To this end, we evaluated the sensitivity of viral RNA (vRNA) replication to multiplicity of infection (MOI) for a diverse set of IAV strains, including two of avian-origin and two of human-origin. This was done by infecting Madin Darby canine kidney (MDCK) cells with a wild type (wt) virus at a consistent dose of 0.005 genome copies (GC) per cell and infecting simultaneously with increasing doses of a homologous virus, termed var. The var virus carries synonymous mutations to allow differentiation of its genome from that of the corresponding wt virus. Coinfections were limited to a single cycle of infection to ensure that results reflected processes occurring within cells. In line with previous observations, GFHK99wt virus replication is enhanced ~100-fold by co-inoculation with GFHK99var virus . HoweverTo date, our examination of IAV density dependence has centered on homologous interactions that would occur within a given viral population. Here, we sought to test whether the positive effects of increased density would extend to coinfection with distinct IAV strains. Although virus-virus interactions would typically be bi-directional, for the purposes of our experimental design, we measure the impact of coinfection on only one strain in the pairing, referred to here as the focal virus.influenzavirus A is not required for beneficial interactions within the cell  or NL09, MaMN99, or Pan99 virus, then quantified levels of GFHK99wt genomes in the cells. These coinfections were again limited to a single cycle to ensure that results reflected processes occurring within cells. The results show that GFHK99wt genome levels increase with increasing doses of every coinfecting var virus, indicating that phylogenetic relatedness within the genus the cell . Howeverthe cell  while cothe cell . These rTo more rigorously examine how the intrinsic reliance on multiple infection displayed by a coinfecting virus modulates its impact on a focal virus, we used a mutant strain of GFHK99 virus. We previously showed that introduction of a PA K26E mutation into the GFHK99 strain reduces reliance on multiple infection . We confTo test the extent to which the beneficial interaction observed in our cell culture model shapes viral dynamics in vivo, we used a guinea pig model. Mimicking the experiments in cell culture, guinea pigs were inoculated simultaneously with a low dose of GFHK99wt virus and increasing doses of GFHK99var or GFHK99 PA K26E viruses. The titer of GFHK99wt virus was assessed in daily nasal wash samples. Contrary to expectation, GFHK99wt virus replication over the course of the infection was suppressed by the coinfecting virus: relative to GFHK99wt-only controls, GFHK99wt virus vRNA levels were lower during coinfection with either GFHK99var or GFHK99 PA K26E viruses .Unlike in cell culture, multiplicity of infection cannot be controlled in vivo and mixing of coinfecting viruses can be constrained by spatial structure, limiting the potential for cellular coinfection ,19–21. TTaken together, these experiments revealed that the dynamics of coinfecting viruses within an intact animal host are antagonistic. Combined with our cell culture experiments, which indicate that beneficial interactions occur within individual coinfected cells, these data suggest that either the enhancement of focal vRNA replication within cells is not translated into increased viral release or that antagonistic interactions occurring at a higher spatial scale are predominant.To assess whether beneficial virus-virus interactions within cells results in increased virus release, we evaluated the impact of coinfection in cultured cells by quantifying focal viral genomes in both infected cells and released progeny in the supernatant. Similar to the experiments outlined above, a low dose GFHK99wt virus and a high dose of a homologous var or heterologous GFHK99 PA K26E, MaMN99, or NL09 viruses were used. After 24 h, GFHK99wt virus genomes within cells and in the supernatant containing released progeny virions were quantified. Under the conditions of MOI used and at this time point, GFHK99wt virus genome replication in the cells was enhanced during coinfection with MaMN99var and NL09var viruses, but not with GFHK99var or GFHK99 PA K26E viruses . HoweverThe cell culture-based experiments outlined above were all performed under conditions that limited viral replication to a single round, an approach that allows tight control of MOI and detection of intra-cellular virus-virus interactions. Concomitantly, this strategy eliminates the potential for coinfecting viruses to modulate each other’s dynamics during spread through the cellular population. To better understand the dynamics observed in vivo, we therefore adopted a cell culture model in which multiple cycles of viral replication could occur, thereby allowing virus-virus interactions at this higher spatial scale.Thus, cells were infected at low MOIs in medium conducive to multicycle replication. GFHK99wt virus was used at an MOI of 0.005 GC/cell and GFHK99var or GFHK99 PA K26E virus at an MOI of 0.1 GC/cell. GFHK99wt virus replication was monitored up to 48 h post infection. Consistent with minimal interaction between these relatively small viral populations early in the infection, GFHK99wt virus replication was comparable with or without coinfection up to 24 h post infection . HoweverTo further evaluate this concept, we tested the impact of modulating the timing of coinfection on the replication of a focal virus. Here, cells were pre-inoculated at varying times up to 24 h before infection with GFHK99var or GFHK99 PA K26E virus at a high MOI and with GFHK99wt virus at a low MOI. The replication of the focal virus, GFHK99wt, was then assessed. This experimental design was chosen to model a situation in which most cells were infected  by the coinfecting virus prior to introduction of the focal virus. Thus, the potential for cellular coinfection would be high only if cells remain susceptible to infection at the time of GFHK99wt virus introduction. The results show little change to GFHK99wt replication when the coinfecting virus was added up to 4 h before GFHK99wt virus. However, there is a significant decrease in GFHK99wt replication as the interval between infections was increased . These dOur results reveal that, within cells, IAV coinfection can be strongly beneficial. The extent of benefit is not primarily defined by a coinfecting virus’s native host or its homology to the focal strain. Not all coinfecting viruses confer an equal benefit, however, and the magnitude of the benefit is defined by the coinfecting strain’s intrinsic reliance on multiple infection. Despite this clearly cooperative effect within cells, virus-virus interactions at the level of the whole host are largely antagonistic, as competition for susceptible target cells dominates at this scale. Examination of this effect in cell culture suggests that this negative interaction arises at least in part because coinfecting viruses access potential target cells asynchronously, leading to competition for a limited supply of susceptible cells.Our data suggest a model in which dominant virus-virus interactions differ at the within-cell and within-host scales . Within For IAVs, cellular coinfection is beneficial in large part because it allows complementation of (very common) incomplete viral genomes, increasing the likelihood of productive infection ,15,24. IAt the within-host scale, our data reveal a competitive effect wherein the virus introduced at a higher dose or an earlier time point suppresses the replication of the other. Similar dynamics have been reported previously. In particular, superinfection exclusion, in which the replication of the secondary virus diminished if it is introduced outside of a somewhat narrow time window, is well documented. The duration of the window observed here (4–8 h) is in line with previous work ,22,23,25in vivo  as injected into the bottom of the tube and the sample was centrifuged (Beckman Coulter Optima XL-100K Ultracentrifuge) at 25,000 rpm for 2 h at 4°C. The resultant viral pellet was resuspended in PBS and RNA extracted using the Qiagen Viral RNA mini kit and protocol instructions.MDCK cells were seeded into T75 flasks at 5 x 10Female Hartley strain guinea pigs weighing 300–350 g were obtained from Charles River Laboratories  and housed by Emory University’s Department of Animal resources. Prior to intranasal inoculation, nasal lavage and euthanasia, guinea pigs were anesthetized by intramuscular injection with 30 mg/kg ketamine and 4 mg/kg xylazine. Guinea pigs were inoculated intranasally with IAV in 300 μl PBS. Daily nasal washes were performed up to 6 d post GFHK99wt inoculation. Briefly, with the animal’s nose suspended in a downward orientation over a Petri dish, 1 mL PBS was instilled into the nares in 200 μl increments and allowed to drop back out. Liquid was pooled into a 1.5 mL tube, aliquoted and stored at -80°C.RNA was extracted from cells and viral samples using the Qiagen RNeasy mini kit and the Qiagen Viral RNA mini kit, respectively, using included protocol instructions. Extracted vRNA was reverse transcribed using a 1:1 ratio of universal influenza primers  S2 Tabl and MaxiAll statistical analyses were performed in R (version 1.3.959). Log transformed values for genome copy numbers and the mean of technical replicates were used for statistical analyses.S1 Fig(Related to (TIF)Click here for additional data file.S2 Fig3 GC and increasing doses of GFHK99var (A) or GFHK99 PA K26E (B) virus. The viral titer in nasal washes collected from each guinea pig is plotted and the limit of is indicated by the dashed line. Guinea pig ID numbers are shown in grey boxes appended to each facet.(Related to (TIF)Click here for additional data file.S3 Fig4 GC of either GFHK99var (A) or GFHK99 PA K26E (B) virus either 12 or 24 h prior to a dose of 104 GC of GFHK99wt virus. The viral titer in nasal washes is plotted and the limit of detection is indicated by the dashed line. Guinea pig ID numbers are shown in grey boxes appended to each facet.(Related to (TIF)Click here for additional data file.S4 FigLevels of defective viral genomes (DVGs) were quantified by ddPCR using primers targeting the terminal and internal regions of the PB2, PB1, PA, and M gene segments. Ratios of terminal to internal copies that are <2.0 indicate low DVG content. Virus stocks tested are named above each facet. Two different stocks of GFHK99wt virus were used.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


In health economics, costs can be divided into both direct and indirect categories. Direct costs tend to consist of medical costs, which are those directly attributed to health care interventions , and non-medical direct costs such as monitoring and professional caregiving. Indirect costs tend to comprise those related to lost productivity due to illness (or treatment), burden on systems outside of the healthcare domain, and other costs that can sometimes outweigh the entire sum of direct healthcare costs.The most common life-threatening complication of lung and hematopoietic stem-cell transplantation (HSCT) is bronchiolitis obliterans syndrome (BOS). BOS is currently diagnosed as a 20% decline in the forced expiratory volume in one second (FEV1) from the best (baseline) post-transplantation value, and is a major cause of morbidity and mortality amongst lung and stem cell transplant patients. BOS affects half of all lung transplant patients within the first 5 years post-transplant, rising to the majority of patients (~80%) within the first decade following transplant.We estimated both direct and indirect costs for the first 10 years following BOS diagnosis, a viewpoint that highlights a tremendous imbalance between healthcare and non-healthcare costs. The lost workforce resulting from BOS-related infirmity will cost society more than $3.7 Billion over the next decade, a figure that is more than double the estimated 10-year cost of treating BOS ($1.4B), including diagnostics, immunosuppressives, and additional complications. As such, BOS is estimated to present a burden of cost that must be evaluated in a new light to include the wider societal perspective.


The prevalence of irritable bowel syndrome with constipation (IBS-C) is estimated to be between 4.3% and 5.2% among adults in the United States. Little is known about the health care resource utilization and costs associated with IBS-C.To (a) evaluate the annual total all-cause, gastrointestinal (GI)-related, and IBS-C-related health care costs among IBS-C patients seeking medical care in a commercially insured population and (b) estimate the incremental all-cause health care costs among IBS-C patients relative to matched controls.Patients aged ≥ 18 years with continuous medical and pharmacy benefit eligibility in 2010 were identified from the HealthCore Integrated Research Database, which consists of administrative claims from 14 geographically dispersed U.S. health plans representing 45 million lives. IBS-C patients were defined as those with ≥ 1 medical claim with an ICD-9-CM diagnosis code in any position for IBS (ICD-9-CM 564.1x) and either ≥ 2 medical claims for constipation (ICD-9-CM 564.0x) on different service dates or ≥ 1 medical claim for constipation plus ≥ 1 pharmacy claim for a constipation-related prescription on different dates of service during the study period. Controls were defined as patients without any medical claims for IBS, constipation, abdominal pain, or bloating or pharmacy claims for constipation-related prescriptions. Controls were randomly selected and matched with IBS-C patients in a 1:1 ratio based on age (± 4 years), gender, health plan region, and health plan type. Patients with diagnoses or prescriptions suggesting mixed IBS, IBS with diarrhea, chronic diarrhea, or drug-induced constipation were excluded. Total health care costs in 2010 U.S. dollars were defined as the sum of health plan and patient paid costs for prescriptions and medical services, including inpatient visits, emergency room (ER) visits, physician office visits, and other outpatient services. The total cost approach was used to assess total all-cause or disease-specific health care costs for patients with IBS-C, while the incremental cost approach was used to examine the excess all-cause costs of IBS-C by comparing IBS-C patients with matched controls. Generalized linear models with bootstrapping were used to assess the incremental all-cause costs attributable solely to IBS-C after adjusting for demographics, Elixhauser Comorbidity Index (ECI) score, and other general and GI-related comorbidities not included in the ECI score.A total of 7,652 patients  were included in the analysis. The mean (± SD) age was 48 (± 17) years, and 83.6% were female. The mean annual all-cause health care costs for IBS-C patients were $11,182, with over half (53.7%) of the costs attributable to outpatient services, including physician office visits and other outpatient services . Remaining total all-cause costs were attributable to hospitalizations (21.8%), prescriptions (19.1%), and ER visits (5.4%). GI-related costs  comprised 39.8% of total all-cause costs, while IBS-C-related costs  accounted for 11.9% and were primarily driven by costs of other outpatient services (50.3%). After adjusting for demographics and comorbidities, the incremental annual all-cause health care costs associated with IBS-C were $3,856  per patient per year, of which 78.1% of the incremental costs were due to medical services, and 21.9% were due to prescription fills.IBS-C imposes a substantial economic burden in terms of direct health care costs in a commercially insured population. Compared with matched controls, IBS-C patients incurred significantly higher total annual all-cause health care costs even after controlling for general and GI-related comorbidities. Incremental all-cause costs associated with IBS-C were mainly driven by costs related to more frequent use of medical services as opposed to prescriptions.


Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17–26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections. Vector-borne diseases are bacterial, viral, or parasitic infections transmitted to the human host through the bite of infected arthropod species, such as mosquitoes, ticks, midges, and flies . In 2020Approved antiviral drugs target viral proteins involved in key viral steps, from viral entry to viral gene expression and genome replication. Direct targeting of nucleic acid is very sporadic, as achieving selective targeting has always been extremely challenging. Nucleic acids have been shown to fold into structures alternative to the classical double helix, which do not obey the Watson-Crick hybridization canon and are therefore defined as non-canonical nucleic acid structures. Among these non-canonical structures, G-quadruplexes (G4s) have been shown to play key biological roles both at the human and viral level ,13,14,15Several algorithms have been validated to predict the presence and distribution of putative quadruplex (G4)-forming sequences (PQSs) in genomes . The difThis work shows that arboviruses embed both canonical and bulged PQSs and that the different viral families show different patterns of PQSs enrichment or depletion. The conservation of each predicted PQS among virus isolates was also analysed to correlate the presence of highly conserved putative G4 sequences with their possible biological role. Our data provide new information on the evolutionary conserved PQSs among human arboviruses, provide insights into unexplored aspects of arbovirus biology and reveal innovative anti-arbovirus targets.Arboviruses were grouped according to the Expasy ViralZone and NCBI taxonomy classifications ,30. A toFirst, for each virus, the GC content of the reference genome alone and of all the sequenced virus isolates was calculated and expressed as an average value . Taken tNext, the pqsfinder algorithm was run on all reference genomes. The algorithm was set up to recognize sequences with G-runs containing at least two G residues. Each PQSs could have loops with a maximum length of 12 nucleotides and a maximum of one loop with a length of zero nucleotides. The pqsfinder algorithm was used to identify canonical PQSs and PQSs harboring a single bulge . PQSs coThe analysis that considered canonical and bulged PQSs showed that the reference genomes of positive-strand ssRNA viruses, Flavivirus and Alphavirus, are particularly enriched in PQSs. In particular, the Japanese encephalitis, the Langat, the Louping ill, the Tick-borne encephalitis and Zika viruses were predicted to embed more than one hundred PQSs in their genomes. In general, the members of the Flavivirus family embed an average of 94 PQSs per genome. The second group of positive-strand RNA viruses, the Alphaviruses have an average of 67 PQSs per genome, with the Semliki Forest virus topping the list with 95 PQSs. Negative-strand ssRNA viruses, with the genome consisting of a single linear RNA strand, showed an average of 37 PQSs, with the Chandipura virus and the Australian bat lyssavirus showing the highest number of PQSs  and the non-Indiana Vesicular stomatitis virus strains showing the fewest . Segmented negative-strand ssRNA viruses and the dsRNA Banna virus, although not so different in GC content from the other viruses, were predicted to have very few PQSs . A closer look at the PQSs strand location  showed tThe number of canonical PQSs was then calculated, excluding the bulged sequences from the first prediction . The maxt-test and p-values were calculated [t-test was used to determine whether the average PQSs number of the shuffled genomes (one hundred per virus) was significantly different from the PQSs number predicted on the relative viral reference genome. p-values lower than 0.001 were considered significant. Significance analysis and generation of corresponding p-values indicated that 52% of the considered viral genomes/segments were significantly enriched in PQSs, while 37% showed significant depletion in PQSs compared to the presence of G-runs on shuffled genomes/segments. The remaining 11% of viral genomes/segments showed no significant enrichment or depletion in PQSs. The PQS prediction was highly significant for positive-strand RNA viruses, with the exception of the Eastern equine encephalitis virus and the Ross River virus. Flaviviruses are all enriched in PQSs, whereas Alphaviruses, with the exception of Barmah Forest and Semliki Forest viruses, are depleted in PQSs. When considering negative-strand RNA genomes, viruses with single linear RNA genomes are all statistically enriched in PQSs, whereas segmented viruses display segments with PQSs enrichment, PQSs depletion and segments that are not significantly enriched in PQSs . The Banna virus (dsRNA) has segments 10 and 11 with non-significant p-values, the other 10 segments have a statistically significant PQS prediction, despite the low number of predicted PQSs.Next, the significance of the predicted PQSs was then calculated. To assess whether the predicted PQSs in arboviruses were statistically relevant or random, the results from viral genomes were compared with those obtained by viral genome simulation. Shuffled genomes (one hundred per virus), with the same nucleotide composition but different order with respect to the references, were generated. The presence of PQSs was predicted using the same parameters as in the first analyses . To estilculated . The oneOnce the presence of PQSs in arboviruses had been predicted and their statistical relevance had been assessed, their distribution within the genomes was examined  bulged and canonical PQSs were conserved to the same extent, with viruses such as the Sagiyama and the Semliki Forest viruses having more than 87% of fully conserved canonical and bulged PQSs. The pattern of conservation among negative polarity RNA viruses is much more diverse. Segmented RNA viruses do not appear to conserve bulged PQSs, nor do they have a high conservation rate of canonical PQSs. Single linear negative polarity RNA viruses tend to conserve both canonic and bulged PQSs, with viruses such as the Australian bat Lyssavirus with no conservation potential and viruses such as the Isfahan virus and the Vesicular stomatitis (non-Indiana strains) having high conservation rates of both canonical and bulged PQSs. The Banna virus (dsRNA) showed no strong conservation of either type of PQSs . The genomic location of all PQSs with more than 85% conservation was then determined . Genome The presence and possible key roles played by nucleic acid secondary structures, in particular G4s, during the viral cycle of major human pathogens, such as HIV-1, HSV-1 and many others , has begArboviruses are a major threat to humans with no specific pharmacological treatment and few prevention strategies . Here weOur data show that frequency of PQSs is not related to the GC content of viral genomes, confirm that the clustering of G-runs is not random, and suggest a specific biological role for G4 structures at the arboviral level. Viruses, especially those with an RNA genome, such as that of arboviruses, mutate with high frequency ,38,41. TThe conservation of PQSs in viruses is one of the strongest indications of the biological relevance of G4s. The utmost conservation of G-tracts in a G4-forming pattern indicates that they are required for infection/replication/transmission of the virus. Our data show that, depending on the virus class, both canonical and bulged PQSs are conserved among virus isolates, suggesting that also bulged G4s play a role in the biology of arboviruses. In addition, our analysis suggests that PQSs in the coding sequences and 3’UTRs of positive-strand ssRNA viruses, especially Flaviviruses, could play an essential role during viral infection.The role of G4s in regulating transcription and translation when embedded in coding regions has begun to be elucidated at the human level : our datOur data also show that G4s are not particularly abundant or conserved in negative-strand ssRNA or dsRNA arboviruses. Negative-strand and dsRNA viruses have more complex viral cycles than positive-polarity ssRNA viruses: under these conditions, G4-mediated slowing of viral transcription and replication may be more likely to be avoided .Accessible viral genomic sequences  belonging to the 40 arboviruses infecting humans were downloaded from the genome database of the NCBI in January 2023. Genomes were grouped in FASTA-format files. Dengue viruses were clustered according to the four reported serotypes (Dengue virus 1–4) . VesiculAll the analyses were performed using R (version 4.2.2). The FASTA files containing the reference genomes and the FASTA files containing the multiple alignments were loaded onto the R platform and GC content was calculated using Biostrings (2.66) ,49. PQS t-test was performed comparing the PQS predictions of the shuffled genomes with the PQS prediction of the reference genome. Significance was expressed as a p-value.The R universalmotif (version 1.16.0) was used to shuffle the reference genome sequences . Each reTo calculate the percentage of conservation of each PQS the vmatchPattern function of Biostrings (version 2.66) was used, setting the parameter with.indels = TRUE, to count PQS with longer loops as conserved. The multiple aligned genomes were loaded onto the R platform and the number of times each predicted PQS was present in the aligned genomes was counted. The percentage of conservation was also calculated, taking into account the number of aligned genomes analysed. The conservation values were then plotted together with the density pattern and the PQS scores using Gviz (version 1.42.0) .Only PQSs with more than 80% conservation were annotated on viral genomes. GTF files were uploaded using rtracklayer (version 1.58.0) . AnnotatArboviruses are a heterogeneous family of viruses that are transmitted to humans by arthropod vectors. This work has shown that many members of the family, mainly belonging to the Flavivirus subgroup, embed highly conserved PQSs in their genomes. The conserved PQSs are located in coding regions and at genome ends, reinforcing the critical role of G4s in the regulation of viral cycles. These findings pave the way for a broader understanding of the mechanisms regulating arbovirus infections and suggest that highly conserved PQSs may be novel and innovative antiviral targets against arbovirus infections.


Monkeypox virus has recently been detected in multiple countries. Two cases of monkeypox virus were reported in Egypt as part of an ongoing international outbreak. We report the whole-genome sequence of a monkeypox virus that was retrieved from the first confirmed case in Egypt. The virus was fully sequenced on the Illumina platform, and phylogenetic analysis demonstrated that the current monkeypox strain is closely related to clade IIb, which caused recent multicountry outbreaks. Orthopoxvirus genus of the Poxviridae family of enveloped viruses, harboring a linear double-stranded DNA genome of around 200 kb. Mpox virus is an emerging zoonotic pathogen that was first isolated from monkey in 1958. Mpox symptoms are similar to those of smallpox infection, including fever, headache, muscle ache, lymphadenopathy, and a characteristic rash that develops into papules, vesicles, and pustules, which eventually scab over and heal (Monkeypox (Mpox) virus is a member of the As of 10 October 2022, 71,237 laboratory-confirmed cases and 1,097 probable cases, including 26 deaths, had been reported to the World Health Organization (WHO) from 107 countries. According to the WHO nomenclature, Mpox is genetically divided into two clades; clade 1 Mpox virus, formerly known as the Congo Basin  clade, is associated with high mortality rates, and clade 2 Mpox virus, formerly known as the West African clade, is associated with milder infection and lower mortality rates . On 8 SeMT903345.1) through the tools Merge overlapping pairs, Trim reads, Map reads to reference, and Extract consensus sequence using CLC Genomics Workbench v21 .Viral DNA was extracted using a QIAmp DNA minikit . To amplify the entire Mpox virus genome, we used two pools of the primalSeq v2 primer sets . MultiplOP597769. To determine the viral lineages, we used Nextclade v2.9.1 (https://clades.nextstrain.org). Results showed that the Mpox strain MOH-NRC-0002/2022 belongs to lineage A.2.1 in clade IIb. The full genome sequence of the MOH-NRC-0002/2022 strain was compared to all other complete Mpox virus sequences available in the NCBI database on 15 January 2023 using BLAST (MT903345.1) and MPXV_USA_2021_TX (GenBank accession number ON676707.1), respectively. Gigante et al. showed that lineage A.2 had several mutations, compared to the dominant lineage B.1, in 2022 , with 192.1× coverage, and has been submitted to GenBank under accession number  in 2022 . Mpox viThis work was approved by the Medical Research Ethics Committee of the National Research Centre (Egypt) (protocol number 1-4-6).OP597769.1. The accession numbers for the Illumina MiSeq sequence raw reads in the NCBI Sequence Read Archive (SRA) are PRJNA887588 (BioProject), SAMN31178476 (BioSample), and SRR21841757 (SRA) (hMpxV/Egypt/MOH-NRC-0002/2022).The genome sequence of the Egyptian Mpox virus strain was submitted to GenBank and is available under accession number


Neurodynamic techniques have yielded good clinical results in the treatment of various pathologies. The objective of this study is to examine the short‐term effects of neurodynamic techniques of the sciatic nerve on hip ROM (range of motion) and on the amplitude and latency of the soleus H‐reflex and M‐waves, in young asymptomatic subjects. In a double‐blind controlled trial design, 60 young asymptomatic participants were randomly assigned into six groups with different levels of manipulation of the sciatic nerve. The passive straight leg raise test was used to evaluate the hip ROM amplitude. All evaluations were performed before, 1 min after, and 30 min after intervention. For each time‐point, spinal and muscle excitability were also tested. ROM increased in all groups, but none of the treatment groups had superior effects than the group with no treatment. This means that ROM testing maneuvers increased ROM amplitude, with no add‐on effect of the proposed neurodynamic techniques. Neurophysiological responses changed similarly in all groups, showing that the aftereffects were not intervention‐specific. We observed a significant negative association between the change in limb temperature and the change in latencies of all potentials. ROM‐testing procedures performed repeatedly increase ROM amplitude. This observation should be considered when evaluating the aftereffects of therapeutic interventions on ROM amplitude. None of the explored neurodynamic techniques produced acute aftereffects on hip ROM amplitude, spinal or muscle excitability different to the induced by the ROM testing maneuver. One session of neural mobilization does not produce modification of the ROM or the spinal excitability.ROM‐testing procedures performed repeatedly increase ROM amplitude.There was an association between changes in limb temperature and latencies. The first involves alternating movements of adjacent joints, which are thought to produce nerve sliding  Butler, comprisesHowever, a note of caution has also been added by Ginanneschi et al. , as neur22.1In a double‐blind controlled design, we tested the effects of a NT single session on hip ROM and H‐reflex excitability in asymptomatic young subjects. Based on previous reports, our hypothesis establishes that NT will increase ROM and H‐reflex amplitude .Investigators (blind to the group assignment) carried out the neurophysiological evaluations and ROM, in this order, at three different time‐points: Interventions and evaluations were always performed on the subject's dominant limb, defined as: “the leg used to kick a ball”  lower limb surgery in the previous year, (ii) low back pain (iii) positive diagnosis of any neurological disorder.Sixty subjects (27 women) matched the criteria and were enrolled .Subjects received a detailed description of the experimental procedure and the associated risks  and signed consent forms. All procedures were approved by our Local Ethics Committee, complying with the Declaration of Helsinki.2.3Figure 2.3.1Subject remained seated on a gurney with hips and knees flexed at 90°, with back support. A footstool (adaptable in height) was on the floor to give feet support. Self‐adhesive electrodes were used for recording and applying stimulation.After skin preparation, the soleus EMG was acquired by means of surface electrodes arranged in a belly–tendon manner, with the reference electrode on the Aquilles tendon and the active electrode on the soleus muscle; the ground electrode was placed ≈4 cm above the active electrode. EMG signals were recorded by means of D360 amplifiers (Digitimer), amplified (×250–1000) and bandwidth filtered between 3 and 3000 Hz. EMG was sampled at 10 kHz and stored in a computer by means of a CED 1401 mkII Power A–D converter (Cambridge Electronic Design), which also triggered a Digitimer DS7A stimulator. Skin temperature was monitored  by means of a fine temperature probe attached just at the side of the active EMG recording electrode, the probe was connected to a Cibertec Citer which sampled temperature at 0.1 Hz and delivered the signal to the CED1401; therefore, temperature recording was synchronized to EMG recording.We stimulated the tibial nerve (1 ms pulses) with the cathode on the popliteal fossa and the anode over the patella. Stimulation pulses were delivered at 0.1 Hz to avoid any induction of post‐activation depression. Different stimulation intensities were used to record the H‐reflex (at two points of the ascending‐leg of the recruitment curve) and the compound muscle action potentials (CMAP). Ten responses were obtained for each intensity.CMAP acquired at stimulation intensity 20% above the supramaximal.MAX: acquired at the stimulation intensity producing the largest H‐wave amplitude, we also recorded its corresponding M‐wave.H50: at pre testing, it was acquired at the stimulation intensity inducing H‐wave amplitudes in the ascending leg of the recruitment curve equivalent to the 50% of the HMAX. We also recorded its M‐wave. However, for post and post‐2 the stimulation intensity was adjusted to have M‐waves equivalent in amplitude to those obtained at pre; this was done to have similar levels of antidromic volleys in the axons of the motoneurons during H‐reflex recording  was analyzed in Celsius grades; skin temperature is a valid parameter to estimate muscle temperature as shown previously , peak to peak amplitude and latency of the following variables were studied (in this order):2.3.2pre, post, post‐2), the ROM was tested three times (1 min rest) and the average was calculated.The passive straight leg raise test was used to evaluate the ROM amplitude, this is a valid and reliable test : This group underwent a slide sciatic nerve maneuver. Subjects adopted the “slump test” position, characterized by trunk flexion with the sacrum in touch with a vertical back‐support Figure No neural mobilization (nNM): This protocol was included as a way of controlling the specific maneuvers of the NM group : A similar starting position as for NM was used. However, participants maintained cervical flexion and knee extension (to the point of maximum muscle‐tension) for 30 s (without movement), with assistance from the researcher. The intervention included five sets, 1 min rest.ROM testing control: Participants remained at rest for the same period of time as the other groups (7 min). The inclusion of this group controls if ROM testing maneuvers modify ROM amplitude along the protocol. It serves as control for the three groups above presented.Control group without ROM testing: Participants remained at rest as the previous group but without ROM testing. The inclusion of this group controls the effects of ROM testing on spinal excitability.Temperature‐control group without ROM testing: The same as above, but skin temperature was monitored, it serves to control for putative effects of temperature on the neurophysiological tests. This last group was included after performing experiments and data analyses in the other five groups.2.5For each subject, H and M waves amplitudes were normalized with reference to the amplitude of the CMAP at the corresponding evaluation time‐point. The median of the 10 responses acquired at each time‐point was the score introduced in the analyses. CMAP and ROM were considered in absolute values.50 was evaluated with a repeated measures analysis of variance (3×6 ANOVARM) with factor TIME (pre‐post‐post2) and GROUP (six groups). Note that the intensity of stimulation for acquiring the M‐wave of the H50 was adjusted at post and post‐2 to match the amplitude of the responses obtained at pre (see above). For this reason this variable was analyzed in isolation. The same approach was used to evaluate the M‐wave of the HMAX, CMAP, and ROM.First, we tested the assumption of normality of distributions for each variable and group (KS for one sample test).Then, results at the three testing points for M‐wave amplitude of the H50 and HMAX; therefore, if factor RECRUITMENT interacted significantly with any other factor, the estimated effect would be different for H50 and HMAX. We proceeded the same way to analyze all wave‐latencies, but in the case of M‐waves latencies RECRUITMENT had three levels .For the amplitudes of the two H‐waves, the same statistical model was used but adding factor RECRUITMENT. This way, the analysis allowed us to estimate if the effects were differently expressed for HFor the ANOVA, the degrees of freedom were corrected with Greenhouse–Geisser coefficients if the sphericity was not assumed.temperature‐control group without ROM testing, we performed correlation analyses to look for the relation between putative changes in H and M waves scores (in latency and amplitude) with changes in temperature; for this purpose we used Pearson's or Sperman's correlation analyses, depending on the violation of normality of the variables.With data derived from the p < 0.05. Data in the graphs are means and standard deviation (SD).Significance was set at a 33.150 acquisition was stable along the three testing time‐points  in the six groups ; this was expected as it was imposed by the experimental condition to control the level of antidromic volleys in the axons at the different testing times during H50 acquisition. Figure M‐wave amplitude during H3.2MAX) did not change along the three testing points  and this was not different for the six groups   Figure .3.3F2,108 = 4.2Ԑ = 0.6p = 0.039TIME). No significant interactions by group was detected indicating that the effect was not different in the six groups. The effect was small and post hoc analyses between pairs did not show significant differences between any pair  , the H‐waves (H50 and HMAX) were introduced in the same ANOVA to know if the protocol had a different impact on both waves.Contrary to the case of the M‐waves . Second, amplitudes were modified along the three testing points . No significant interactions between them or with factor Group were detected, which means that the effect was not different for all groups (including the Control groups). Subsequent post hoc analyses indicated an increase in the amplitudes (HMAX and H50) from pre to post (Bonferroni p = 0.015), which remained at post‐2 (Bonferroni p = 0.028). The mean increase from pre to post was small in size . As expected, post hoc analyses showed that latency was shorter for the Mmax  compared to M‐wave of H50 and M‐wave of HMAX . Wave latencies were significantly delayed (≈0.4 ms) along the three testing points . The lack of significant interactions of factor TIME with any other factor indicated that latencies increased not differently in all M waves explored, along the three testing times and in all groups , the effects were observed in all groups.The latency of the H‐waves was different for the two recruitment points tested3.7The ROM was tested in four out of the six groups. Three groups underwent intervention techniques  and a fourth group only executed the ROM testing technique; the other two groups, without ROM testing, served to control the putative effect of ROM testing and superficial leg temperature on excitability scores.F2,72 = 4.4ε  = 0.8p = 0.015TIME.). The increased was not different in the four groups evaluated . Post hoc analyses indicated an increment from pre to post (Bonferroni p = 0.051) which remained at post‐2 ; the size of the effect was small, about two degrees. Therefore, the testing protocol itself increases the ROM and NM, nNM and ST interventions had no effect on increasing ROM beyond the explained by ROM testing itself  from pre to post and pre to post‐2 versus changes in skin/muscle temperature (pre to post and pre to post‐2). Analyses were performed pooling M‐waves and H‐waves together. These analyses were aimed at understanding the putative effect of limb temperature change on H and M waves parameters.Thus far, the results indicated that the effects observed on M and H waves amplitudes and latencies where not different in the six groups tested in our experiment. A same pattern had been observed initially before including in the analyses of the MAX (an its corresponding M‐wave) and H50 (an its corresponding M‐wave); the effect was very small but consistently expressed in the subjects F2,18 = 17.7 p < 0.001POTENTIAL‐TYPE. The same was observed along the three testing time‐points F2,18 = 29.6ε  = 0.5p < 0.001TIME. There was not interaction between these two factors . This effect was observed within each testing‐time‐point, which means there is a drop in temperature during sequential recording of CMAP, Hp = 0.147, Spearman Rho, Figure p < 0.001, Pearson), with a negative coefficient (r = −0.46), thus a drop in temperature caused latency to increase, Figure The association between the changes in the amplitude of the potentials and the changes in limb temperature along the testing time‐points was not significant  for M‐waves that for H‐waves (Dewhurst et al., First, the latencies of all recorded neurophysiological signals showed small but significant increments. A decrease in muscle temperature, which is reported to increase H and M waves latencies and amplitudes in young healthy subjects (Dewhurst et al., Other works have also reported and effect of the NT techniques on the latencies of H‐reflex. Regrettably, the lack of a proper control group and the fact that in some of them a consistent location of the stimulation electrode cannot be guaranteed, hinders any comparison Adel, . This ma4.3Despite the fact that 60 subjects were enrolled, the groups included were not very large. Subjects were split in groups of 10. This can always have an impact on the power of observations, but the number of subjects in our study were similar to the used in other studies with positive results.5One session of neural mobilization techniques does not produce modification of the ROM or spinal excitability in healthy people. However, based on literature, it appears possible a positive aftereffect of NT after multi‐session protocols or single sessions with different application times. This possibility deserves to be studied; however, the use of proper control conditions should be carefully considered, including the control of the aftereffects of ROM testing maneuvers on ROM amplitude.Álvaro Cancela: conceptualization, data curation, formal analysis, investigation, software, writing—review and editing. Pablo Arias: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, validation, writing—original draft. Beatriz Rodríguez‐Romero: conceptualization, data curation, investigation, methodology, visualization, writing—review and editing. Marcelo Chouza‐Insua: conceptualization, formal analysis, methodology, resources, supervision, writing—review and editing. Javier Cudeiro: conceptualization, project administration, supervision, writing—review and editing.No conflicts of interest, financial or otherwise, are declared by the authors.The participants gave their informed consent prior to their inclusion in the study.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.