Aquatic bird bornavirus 1 (ABBV-1) is a neurotropic virus that causes persistent infection in the nervous system of wild waterfowl. This study evaluated whether Pekin ducks, the most common waterfowl raised worldwide, are susceptible to ABBV-1 infection and associated disease. Groups of Pekin ducks were inoculated with ABBV-1 through the intracranial , intramuscular , and choanal  routes. Controls  received carrier only. At 1, 12, and 21 weeks postinfection (wpi), 7–14 birds were euthanized to assess virus distribution and lesions. Infection rates in the IC and IM groups were over 70%, while only 4 ducks in the CH group became infected. Neurological signs were observed in 8 ducks only, while over 25% of IC and IM birds had encephalitis and/or myelitis. Seroconversion was highest in the IC and IM groups, and mucosal ABBV-1 RNA shedding was most frequent in the IC group (53%). None of the fertile eggs laid during the experiment tested positive for ABBV-1 RNA. This study shows that Pekin ducks are permissive to ABBV-1 infection and partly susceptible to associated disease. While mucosal shedding may be an important route of transmission, congenital infection appears unlikely. Bornaviridae family, Orthobornavirus genus, and Orthobornavirus avisaquaticae species1. It is a neurotropic virus that circulates in wild waterfowl and is closely related to the congeneric parrot bornaviruses (PaBVs), the causative agents of proventricular dilatation disease (PDD) in psittacine birds5. ABBV-1 was originally detected in Canada geese (Branta canadensis) and trumpeter swans (Cygnus buccinator) from Canada and the United States7. In these species, ABBV-1 has been associated with disease, as illustrated by a retrospective series of postmortem cases from the University of Guelph, which documented that infected birds had a history of neurological signs, and presented with poor body condition, upper gastrointestinal impaction, and variably severe mononuclear inflammation in the central, peripheral, and autonomic nervous systems8. However, since this was not a randomized controlled trial, a definitive causal relationship between infection and disease could not be made. Moreover, while ABBV-1 appears to be widespread in several species of the Anseriformes and Charadriiformes orders10, it has been also detected in distant taxa, such as a bald eagle 11 and an emu (Dromaius novaehollandiae) from a zoological collection12 suggesting the potential for a broad host range.Aquatic bird bornavirus 1 (ABBV-1) belongs to the 13 and used it to inoculate chickens and Muscovy ducks (Cairina moschata)15. In both studies, birds became infected through the intramuscular and intracranial  routes, although Muscovy ducks had a wider virus tissue distribution compared to chickens15. Additionally, despite minimal to no clinical signs, both species developed histologic lesions similar to those seen in naturally infected birds , confirming an association between ABBV-1 infection and inflammation of the nervous tissue15. Lack of neurological signs in experimentally infected birds is consistent with the asymptomatic infections often observed in natural ABBV-1 cases7, corroborating the notion that healthy waterfowl may be carriers of the virus.Recently, our research group isolated ABBV-1 from the brain of a naturally infected Canada gooseAnas platyrhynchos ssp. domesticus) to become infected with ABBV-1 and develop lesions and clinical signs, as well as their ability to shed the virus in the environment, taken as a proxy for transmission potential. Pekin ducks were chosen as an additional waterfowl representative distinct from Muscovy ducks , in order to describe susceptibility of infection among different domesticated anatid species with distinct geographical origin: the Americas (Muscovy duck) and Eurasia (Pekin duck)16. Moreover, Pekin ducks are closely related to mallards (Anas platyrhynchos), a common wild waterfowl from which Pekin ducks have been selected16. Lastly, the Pekin duck was used as it is the most common waterfowl species raised worldwide17, making the question of ABBV-1 susceptibility relevant under a production point of view.In an effort to evaluate ABBV-1 host restriction and pathogenesis in waterfowl species, the goal of the present study was to assess the susceptibility of Pekin ducks (4.16 focus forming units [FFUs]/bird), intramuscular , and choanal  route, with the control  group receiving carrier only through all routes , 6 IM , and 1 CH (#476) bird showed neurological signs , which occurred most frequently (7/8) between 52 and 78 days postinfection (dpi), while only one IM bird (#371) was affected at 124 dpi (1/8). All these birds tested positive for ABBV-1 RNA in the brain and/or spinal cord, with 7/8  presenting also microscopic lesions in the central nervous system (CNS). See below for description of relationship between clinical signs and infection parameters.An additional 3 birds died or had severe clinical signs that warranted humane euthanasia; the cause of disease in these birds was identified as heterophilic bacterial meningitis , severe pulmonary aspergillosis , and septic arthritis . Of these, the CH duck with meningitis tested negative for ABBV-1 RNA in the CNS, while the other two were positive (see below). None of these birds had histologic lesions consistent with ABBV-1 infection, and their demise was considered incidental.No duck in the entire experimental cohort had gross lesions attributable to ABBV-1 infection, such as proventricular dilatation or poor body condition, as assessed during necropsy.In total, 59 ducks underwent histological assessment in multiple tissues (as part of full sampling), and the brain and spinal cord from an additional 31 ducks were processed as part of partial sampling  and were randomly distributed throughout the gray and white matter of the cerebrum, cerebellum, brainstem, and spinal cord Fig. a–c. In tAlthough inflammation was more common and severe in the cerebrum compared to other segments of the CNS in both the IC and IM groups at 12 and 21 wpi , 2 IM , and 1 CH (#476) duck at 12 wpi showed multifocal neuronal chromatolysis in the brainstem , septic arthritis (IC), and heterophilic meningitis (CH) did not present perivascular cuffs in either the CNS or PNS, and did not present neuronal chromatolysis.ABBV-1 RNA was quantified in the brain, spinal cord, proventriculus, kidney, gonads, and choanal and cloacal swabs of all ducks by reverse-transcriptase quantitative PCR (RT-qPCR). The frequency of positive tissues per experimental group and time point is reported in Table In the IC group, virus RNA was first detected at 1 week post-infection (wpi) in the brains of 5/7 (71.4%) birds, and by 12 and 21 wpi, 100.0% of brain and spinal cord samples tested positive. Visceral tissues  began testing positive starting at 12 wpi, and by 21 wpi all organs had ABBV-1 RNA Table . QuantitIn the IM group, no tissues were positive for ABBV-1 RNA at 1 wpi, while by 12 wpi 100.0% of spinal cord and 88.9% of brain samples were positive, with similar high frequencies at 21 wpi. Visceral organs became positive by 12 wpi; however, the frequency of positive rates in these tissues was lower compared to the IC group, ranging between 44.4 to 55.5 and 35.7 to 64.3% at 12 and 21 wpi, respectively ; this bird was euthanized because of pulmonary aspergillosis at 68 dpi. The other (#476) had low-level virus RNA copies in the spinal cord only (101.91) and was euthanized due to progressively worsening ataxia at 63 dpi. The other 2 ducks were sampled at 21 wpi. One (#469) had low virus RNA burden in the brain only (102.32), while the other (#461) was positive in the brain (106.89), spinal cord (106.83), and all visceral organs .In the CH group, 4 birds tested positive for ABBV-1 RNA: 2 at 12 wpi  and 2 at 21 wpi  Table . At 12 wBlood was collected from 115/118 euthanized birds, and consistently tested negative for RNA of the ABBV-1 N gene by RT-qPCR (data not shown). No virus RNA was detected in any tissue of the CO group.Tested by immunohistochemistry (IHC) were 3 ducks that underwent full sampling from the IC, IM, and CH groups at 21 wpi, as well as the one CH duck  that had tested positive in multiple tissues by RT-qPCR, and 1 CO duck from the same time point (negative control). Immunoreactivity was detected in 3/3 IC, 1/3 IM, and 1/4 CH birds. All birds positive by IHC in at least one tissue also showed presence of virus RNA in the CNS, except for one IM duck (#371), which was positive exclusively in the spinal cord by RT-qPCR but showed no immunoreactivity  IC ducks at 12 wpi, and 11/14 (78.6%) and 3/14 (21.4%) IC and IM ducks, respectively, at 21 wpi. The magnitude of virus RNA in the oropharyngeal swabs was highest in the IC group at 21 wpi  values below the threshold. By 12 wpi, 1/8 (12.5%) IM and 2/8 (25.0%) CH ducks has serum samples with OD values marginally above the threshold. At 21 wpi, 9/14 64.3%) IC, 10/14 (71.4%) IM, and 1/14 (7.1%) CH ducks had serum OD values clearly above the threshold  of developing neurological signs compared to those at 21 wpi (p = 0.042).Inflammation of the CNS was also predicted using a multivariable logistic regression analysis, using time postinfection, virus RNA copies in brain and spinal cord, serology, and inoculation route as explanatory variables. Based on the unconditional association results, virus RNA copies in the brain and time postinfection were fed into the multivariable regression model. Results indicated that both variables were significantly correlated with the outcome variable Table . Ducks aEighty-eight, 60, 34, and 61 eggs were collected from the CO, IC, IM, and CH groups between 115 and 148 dpi, of which respectively 44.3, 45.0, 35.3, and 55.7% developed an embryo  led to infection in 4 Pekin ducks, albeit only one of these birds had widespread presence of ABBV-1 in multiple tissues, as shown by RT-qPCR and IHC. This suggests that the upper respiratory tract is a possible, although inefficient, route of infection for ABBV-1, as also shown with PaBVs19. It appears that parenteral  routes of inoculation remain the most effective methods to infection for orthobornaviruses, at least in experimental settings22.This study documents the successful experimental infection of Pekin ducks with ABBV-1. The ability of the virus to infect another waterfowl species (Muscovy ducks) had been previously documented by our research group24. This suggests intra-axonal virus trafficking, as proven with BoDV-1 25, especially in light of the fact that viremia was not detected in our Pekin ducks.The sequence of positive tissues over time indicates both centrifugal  and centripetal  virus spread in both the IC and IM groups, respectively, as also seen for ABBV-1 in chickens and Muscovy ducks, and PaBVs in psittacines14, possibly because of inherent differences between species, or the longer duration  of the current infection trial.While the average magnitude of virus RNA concentration in tissues increased over time in the IC group, in the IM group, there was a slight decrease in the average virus RNA concentration between 12 and 21 wpi in the brain, spinal cord, and gonads. The reasons for this remain unclear. It could indicate variability of inoculation efficiency in the muscle compared to the intracranial route, or a delayed centripetal spread of infection in certain individual birds. Alternatively, it could point to a slow, progressive clearance of the virus, which was not observed in Muscovy ducks14 and in psittacine birds experimentally infected with PaBV26. In the nervous tissue, immunoreactivity was not associated with areas of inflammation, and appeared multifocally and randomly distributed. No immunoreactivity was observed in the kidneys, despite high virus RNA concentrations. This finding is similar to what observed in Muscovy ducks14, and in disagreement with what reported in psittacine birds infected with PaBVs27. Lack of ABBV-1 immunoreactivity in the kidney is consistent with the low number of positive cloacal swabs  seen in our cohort. Presence of virus RNA without ABBV-1 N protein expression may be a reflection of the higher sensitivity of RT-qPCR compared to IHC, or indicate tissue-specific downregulation of virus protein expression.Immunohistochemistry showed presence of ABBV-1 N protein in multiple tissues of IC and IM birds at 21 wpi, and in the single CH duck also at 21 wpi that was positive by RT-qPCR across multiple tissues. Similar wide tissue distribution of the virus has been documented in natural and experimental ABBV-1 infections in waterfowlThe frequency and magnitude of RNA virus concentration in oral swabs was higher in the IC group when compared to the IM group. The reason for such difference could be related to virus trafficking from the brain through the cranial nerves to the oral mucosa or salivary glands, a path that would require longer for the IM group (inoculated in the leg), compared to the IC ducks. Additionally, as only one cloacal swab was positive (with low virus RNA concentration) in the IC group, oral shedding appears to be a more relevant transmission pathway compared to shedding through the urofeces. One CH duck tested weakly positive in the oral swab at 21 wpi, although this bird was negative in all other tested tissues. The reason for this is unclear, it may be due to contamination of the oral cavity of with low-level ABBV-1 RNA present in the enclosure.14 and PaBVs28 can be shed intermittently, we detected viral RNA in the drinking water at all tested time points after 10 wpi, likely as a result of virus RNA accumulation in the trough fitted to the automatic waterer. If the virus could survive short periods of time in the environment, its presence in the water (perhaps nearby areas of congregation) could favor infection through the upper respiratory mucosa, or via skin wounds, a newly proposed route of infection for PaBV29. We attempted to isolate virus from environmental samples, but this was unsuccessful due to the technical difficulty associated with water filtration to allow isolation in cells (data not shown).Low concentrations of viral RNA were present in the drinking water of the IC and IM groups only, with higher titers being recorded in the IC group. This is consistent with virus RNA quantification in the oropharyngeal swabs. Although it has been shown that ABBV-130. Progression of infection is also an important consideration, since it is impossible to determine when a specific duck developed gonadal infection between 12 and 21 wpi, albeit almost 90% of gonads showed presence of ABBV-1 RNA already at 12 wpi in the IC group. Lastly, one cannot confirm whether infected follicles ovulate or undergo regression. Since the ratio of fertile to total laid eggs, as well as the normalized number of eggs did not differ between groups, it appears that ABBV-1 infection did not markedly affect reproductive physiology. In a previous study in free-ranging Canada geese, out of 22 embryos, 23 hatchlings, and 5 non-embryonated eggs, only one non-embryonated egg had evidence of ABBV-1 RNA in its contents31, corroborating the notion that vertical transmission—if possible—is a rare occurrence.All embryos derived from eggs laid during the experiment tested negative for ABBV-1 RNA. When considering the IC group alone, where 100.0% of females and males were positive for ABBV-1 RNA in the gonads, our results suggest that vertical transmission of ABBV-1 is uncommon. Lack of vertical transmission in face of high infection rate of the gonads could be the consequence of ABBV-1 tissue localization, which appears to be predominately in the interstitial, theca, and granulosa cells, while only rarely in the germinal cells. Vertical transmission could be severely hampered by lack of germ-cell infection, as orthobornaviruses establish persistent infection in the nucleus with little extracellular release14 and natural infections in other waterfowl8. These lesions were present with highest frequency in the IC and IM groups, while only 1 infected CH bird presented inflammatory lesions. In both the IC and IM groups, the frequency and severity of encephalitis and/or myelitis peaked at 12 wpi and subsided by 21 wpi. This decline occurred despite birds with inflammatory lesions being still infected , and even showing a statistically significant increase in the amount of virus RNA in the brain and the spinal cord (from 12 to 21 wpi), as seen in the IC group. These data are in agreement with our previous study in Muscovy ducks14 where peak of inflammation was observed at 4 wpi despite virus RNA concentration being highest at 12 wpi in the brain (end of experiment). This could indicate that, while inflammation is eventually cleared, infection of the CNS persists for longer periods of time. Nonetheless, despite this decline in the intensity of inflammation at later time points, regression analysis showed that inflammation was still positively correlated with the burden of virus RNA copies in the brain.Most common microscopic lesions consisted of lymphocytic perivascular cuffs randomly distributed throughout the central nervous system, similar to what observed with ABBV-1 experimental infections in Muscovy ducks32. Possible differentials for this finding include an unexpected background lesion , infection with avian encephalomyelitis virus (AEV) or thiamine deficiency35. Birds with chromatolysis were tested by RT-qPCR and resulted negative for AEV (data not shown). Thiamine deficiency is characterized by degenerative lesions scattered throughout the spinal cord and axonal degeneration35, which was not observed in our case, as lesions were only confined to rare, scattered neurons in the brainstem. Furthermore, all birds were fed a balanced commercial diet, and it would be difficult to justify why only infected birds would be susceptible. At this point, the significance of chromatolysis remains unclear.Neuronal chromatolysis was an unexpected finding; it was present in the brainstem of 4 experimentally inoculated ducks in our study. As chromatolysis was observed only in birds positive for virus RNA, we suspect it might have been caused by ABBV-1 infection, although one CH bird with chromatolysis presented with low levels of virus RNA concentration and lack of IHC reactivity. It is possible that infection might have been cleared and neuronal damage persisted. While neuronal damage is occasionally reported in orthobornavirus infection, lesions are typically characterized by single-cell necrosis, not chromatolysis8, experimental infection of Muscovy ducks and chickens with ABBV-1 failed to reproduce similar signs, except for one chicken with torticollis15. Logistic regression analysis showed that development of clinical signs in Pekin ducks was not correlated with inflammation of the CNS, in agreement with development of perivascular cuffs in numerous asymptomatic birds in the present and other studies15. It is unclear why Pekin ducks developed clinical signs, while Muscovy ducks did not. Possible differences in the adaptive, and possibly innate, immune response could explain this outcome, although both species appear highly permissive to ABBV-1 infection. Lastly, ducks infected intramuscularly had a higher degree of clinical signs compared to the IC and CH groups. While the frequency of clinical sign was too low to confidently assess differences, it is possible that the IM route may have led to more severe damage to the spinal cord and brainstem, as the virus travelled centripetally towards the brain. While inflammation severity in the spinal cord was not different between the IC and IM groups, it should be acknowledged that lesions in the spinal cord can be multifocal, and differences might have been missed due to segmental sampling of this organ. It is also important to recognize that the only CH duck presenting with clinical signs showed incongruous infection parameters, such as minimal amount of viral RNA in the spinal cord and not in the brain, lack of inflammation but presence of chromatolysis, and an ELISA OD value only marginally above the threshold. These conflicting results between indicators of infection and clinical disease pose the questions if the latter may not have been associated with ABBV-1, at least for this one bird. While this is a distinct possibility, there was no evidence of other specific causes for development of clinical signs in our study cohort.Eight ducks presented neurological signs after 12 wpi. This corresponds to 11.6% and 26.1%, respectively, of all inoculated and IM ducks between 12 and 21 wpi. The observed neurological signs included head bobbing, ataxia, and inability to walk. While similar symptoms have been associated with natural ABBV-1 infection in waterfowl37. These differences may be due to species-specific susceptibility to ABBV-1 infection, an intrinsic higher virulence of PaBVs compared to ABBV-1, or age at infection, as shown by a recent study indicating that older cockatiels are more susceptible to development of clinical signs upon PaBV-4 infection (as compared to younger ones), possibly because younger birds may develop immune tolerance38.On the other hand, PaBVs infection appears to yield a much higher occurrence of disease: psittacine birds experimentally inoculated with PaBV-2 or PaBV-4 through the IM route alone or combined with the oral and/or subcutaneous routes presented clinical signs in up to 100% of infected birds Pekin ducks can become infected with ABBV-1 at high rates when inoculated intracranially or intramuscularly. Although possible, infection through the upper respiratory tract is inefficient. Infected ducks developed inflammatory lesions  exclusively in the nervous system, where highest virus RNA concentration was also observed. Of the virus-inoculated ducks, approximately 9% displayed neurological signs; however, neurological symptoms were not correlated with inflammation or magnitude of virus RNA concentration in the nervous tissue. Shedding occurred mainly through the oral mucosa, and allowed for accumulation of viral RNA in the drinking water. Despite the presence of virus RNA in male and female gonads, vertical transmission did not occur. When compared with the results of a similar study done in Muscovy ducks, Pekin ducks appear to be similarly susceptible to infection, although more likely to shed the virus and to develop clinical signs. Overall, this study described for the first-time infection of Pekin ducks with ABBV-1, and suggests that this species is not only susceptible to ABBV-1 infection, but can also promote its environmental dispersal.13. The infectious and control inocula were prepared following the same methods used by our research group in previous studies15. Briefly, for the infectious inoculum, a stock of confluent 100–150 mm dishes of cells persistently infected with CCL-141 was lysed by osmotic shock followed by one cycle of freeze/thaw in an ultra-cold freezer (− 80 °C), cleared from cell debris by centrifugation, precipitated with polyethylene glycol (PEG)-8000, and resuspended in phosphate-buffered saline (PBS) with 1% fetal bovine serum (FBS) to obtain a ratio of supernatant to final resuspended volume of approximately 100:114. The virus stock was titrated in CCL-141 cells by limiting dilution in a 96-well-plate format. As the virus does not cause cytopathic effect, positive wells were detected by immunofluorescence (IFA) using a monospecific antibody directed against the ABBV-1 N protein (Pacific Immunology). The titer was calculated using the Karber’s method, reported as 50% tissue culture infectious dose (TCID50)/mL, and converted to focus forming units (FFUs). The control inoculum (carrier) consisted of PBS precipitated with 30% PEG, centrifuged, and then resuspended in PBS with 1% FBS to obtain a ratio of initial PBS to final resuspended volume of approximately 100:1.The ABBV-1 strain for this experiment was initially isolated from the brain of a naturally infected Canada goose (GenBank number MK966418), and propagated in immortalized duck embryo fibroblasts (American Type Culture Collection CCL-141)Anas platyrhynchos domesticus) were purchased from a local hatchery  and delivered to the Research Isolation Unit of the Central Animal Facility of the University of Guelph. Upon arrival, ducklings were neck tagged and divided into four groups: control , intracranial , intramuscular , and choanal . Experimental groups were kept in separate, approximately 6 m2 pens with negative pressure, and 12 h of light and 12 h of dark. Ducklings were placed on the floor with shavings, and food and water were offered ad libitum. The water bowls were flushed daily and re-filled automatically. Infrared lamps were provided for optimal temperature until 3 weeks of age.A layout of the experimental plan and sampling is provided in Supplementary Fig. 14. For the intracranial (IC) group, each duck was inoculated with 50 μL of inoculum containing 104.16 FFUs of ABBV-1 directly into the subdural space of the right cerebral hemisphere. Each duck in the intramuscular (IM) and choanal (CH) groups was inoculated with 100 μL of virus inoculum containing 104.46 FFUs of ABBV-1 in the right gastrocnemius muscle or directly into the choanal slit (delivered through the mouth), respectively. All ducks in the control (CO) group were inoculated with carrier only using the same 3 inoculation routes and volumes employed for the infectious inocula.After 24 h from arrival, ducklings were inoculated with ABBV-1 or control carrier, as described previouslyFollowing inoculation, ducks were monitored daily for the presence of clinical signs. Ducks with neurological signs, or unable to reach food and water were euthanized regardless of sampling schedule, and were grouped with the subsequent sampling point.2) inhalation. Older ducks were euthanized by pentobarbital overdose delivered intravenously (100 mg/kg), after being sedated with intramuscular medetomidine (0.02 mg/kg) and ketamine (30 mg/kg).From each group, seven ducks at 1 and 12 weeks postinfection (wpi), and 15–18 ducks at 21 wpi were scheduled to be randomly selected and euthanized for sample collection . For 3–6 ducks from each group at each time point, additional tissues were sampled to increase the granularity of the histology assessment . For tissues collected for both RNA extraction and histology, two samples were collected separately; for the brain, after removal of the entire brain from the skull, a portion of approximately one third of the frontal cerebrum (~ 50 mg) was sampled for RNA extraction, while the rest was fixed in formalin. Ducks that died unexpectedly or were euthanized outside of sampling schedule underwent full sampling, and test results were tallied with the next sampling time point for analytical purposes.Oropharyngeal and cloacal swabs for RNA extraction, and 2 mL of blood (jugular vein) for both RNA extraction and serology were collected from every duck immediately after euthanasia. For each bird, a full postmortem was carried out to assess any macroscopic abnormalities, and select tissues  were sampled for both RNA extraction and histology  at 37.7 °C with 80% humidity, and candled daily. Unfertile eggs were discarded, while embryonated eggs were incubated up to 18 to 21 days, and then transferred to 4 °C overnight. Embryos were then extracted and dissected to collect separately the brain and a pool of internal organs , which underwent RNA extraction.Presence of virus RNA in the environment was tested in the drinking water starting at 4 wpi, and then every 2 weeks until the end of the experiment. From each pen, a 1.5 mL volume of drinking water was collected from an automatic waterer fitted with a trough. A sterile disposable pipette was used to collect the water, which was transferred to a sterile 1.5 mL screw cap tube and transported to the laboratory for RNA extraction.All experimental procedures were approved by the Animal Care and Use Committee of the University of Guelph  and conducted in accordance with the relevant regulations set out by the Canadian Council on Animal Care.All tissues sampled during postmortem examination were fixed in 10% neutral buffered formalin and routinely processed for histology . For partial sampling, brain, lumbar spinal cord, proventriculus, ventriculus, kidneys, and gonads were collected. For full sampling, tissues included brain, spinal cord , ischiatic nerves, brachial plexus, right gastrocnemius muscle (only for ducks in the CO and IM groups), trachea, lungs, heart, esophagus, proventriculus, ventriculus, small intestine (at the level of the Meckel’s diverticulum), colon, liver, pancreas, spleen, thymus, cloacal bursa, thyroid glands, adrenal glands, kidneys, gonads, and nasal passages (only for ducks in the CO and CH groups). The nasal passages  were decalcified 24–48 h post fixation using Cal-Ex II Fixative Decalcifier (Fisher Scientific), and sectioned coronally. Coronal sections of the cerebrum, optic lobe, brainstem, and cerebellum, and at least two coronal and/or transverse sections of each segment of the spinal cord were included to examine the central nervous system (CNS).15, with minor adaptations to better reflect the range of lesions observed in Pekin ducks.Histology slides stained with H&E were examined and scored in a nonblinded fashion by one member of the investigative team (F.A.). Presence of microscopic lesions was tallied nominally for each evaluated tissue. Since mononuclear encephalitis and myelitis were exceedingly the most common lesion, this type of inflammation was evaluated using a semi-quantitative scoring system developed for a similar study in chickens and Muscovy ducks2). Each region received a subscore resulting by adding the distribution score  and intensity score . The final score for the brain was calculated as the simple average between all assessed brain regions . For the spinal cord, the final score corresponded to the calculated subscore. For descriptive purposes, lesions were ranked into mild (scores 1–2), moderate (3–4), and severe (5–6).Three different regions of the brain  and the spinal cord  were scored separately , by assessing the average thickness of the perivascular cuffs  and the average number of vessels affected (distribution of inflammation) in 10, randomly selected 100 × fields . Lastly, for ducks that developed neurological signs outside specific time points, IHC was carried out on both the brain and the spinal cord. The positive control used for the IHC technique consisted of brain tissue from a Canada goose naturally infected with ABBV-1, as identified in a previous study8. Only cells that displayed nuclear, or nuclear and cytoplasmic immunoreactivity were considered positive.Virus distribution in tissues and cells was evaluated by immunohistochemistry (IHC), using a rabbit monospecific antibody against the ABBV-1 N protein at 1:6000 dilution, and coupled with a Nova Red chromogen (Vector Laboratories), as previously described13. For tissues, copy numbers of virus RNA  were calculated by interpolating the threshold (Ct) value of the samples against a standard curve made of serial dilutions of a 500 bp gBlock gene fragment of the ABBV-1 N gene, which was run with each plate14. For swabs and water, the standard curve was made by extracting RNA of a serially diluted stock of known amount of virus in water, and the titre was expressed as focus forming units (FFUs) RNA equivalent per μL. All samples were run in triplicates wells using the Roche LightCycler® System; samples were considered positive if the cycle threshold (Ct) was < 35.Tissues samples from birds , embryos (brain and pooled tissues), and swabs were placed into separate sterile screw cap tubes containing 0.7 mL of preserving solution  ammonium sulfate with a pH of 5.2) and stored at − 80 °C until processing. Drinking water samples were directly placed into the ultracold freezer, without preservatives. Total RNA was extracted from approximately 300 μg of tissue and 150 μL of swab liquid or drinking water, using the E.Z.N.A® RNA Kit II or the Viral RNA Kit, respectively (both from Omega Bio-Tek). The purified RNA was used to amplify the target region of the ABBV-1 N gene using primers and fluorescent probes coupled with the Luna® Universal Probe One-Step reverse transcription quantitative polymerase chain reaction (RT-qPCR) kit (New England Biolabs), as described previouslyg for 10 min) and used to assess presence of antibodies by enzyme-linked immunosorbent assay (ELISA) across all groups and time points, as previously described15. Briefly, wells of 96-well microtiter ELISA plates (ThermoFisher) were coated using a recombinant full-length ABBV-1 N protein (Biomatik) at 25 ng/well at 4 °C, subsequently blocked with 5% fish gelatin in PBS overnight at 4 °C, and then incubated for 2 h at 37 °C with 50 μL of duck sera diluted 1:200 in PBS-T with 1% fish gelatin. After abundant washes (3 times with PBS-T and 2 times with water), a secondary HRP-conjugated goat anti-avian IgY  was applied at 1:10,000 dilution for 2 h at 37 °C. Following additional abundant washes, binding was detected by adding 50 μL/well of chromogenic substrate . The optical density (OD) of the reaction was read at a wavelength of 450 nm, using an EnSpire multimode plate reader (Perkin Elmer).Following the euthanasia of each duck, blood was immediately collected from the jugular vein and allowed to coagulate for up to 8 h. Sera were separated by centrifugation . Calibration was carried out by dividing the raw OD values of each sample on a plate by the OD of its calibrator and multiplying by the average of all calibrators across plates. The cut-off for positive sera was determined as the average of all the CO sera + 3 times the standard deviation (SD). All samples and calibrators were run in triplicate, and calculations were conducted on the averages of the 3 wells.Differences in the frequency of inflammation between different areas of the CNS within each experimental group  was evaluated by pairwise comparison for proportions and Holm’s tests correction for multiple comparisons. Differences in the average severity of CNS inflammation scores, as well differences between average OD ELISA values were tested by Kruskal–Wallis test with Dunn’s test for multiple comparisons. A two-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used to evaluate differences between the average concentration of virus RNA copy number in the tissues of infected ducks, with inoculation route and time point as explanatory variables (main effects model). The daily number of eggs laid across experimental groups was compared with a Kruskal–Wallis test, after normalizing by the number of females (as assessed during necropsy) in each group.A multivariable logistic regression model was used to predict the occurrence of neurological signs , using as explanatory variables time postinfection , severity of inflammation , virus RNA copies in the brain and spinal cord (continuous), serology (OD) values (continuous), and inoculation route . A second multivariable logistic regression model was used to predict inflammation of the CNS  using time postinfection , virus RNA copies in brain and spinal cord (continuous), serology (OD) values (continuous), and inoculation route  as explanatory variables. Presence of chromatolysis was not included due to the low frequency of this variable in the cohort.–5. Only day from 12 and 21 wpi were included, and for each univariable logistic model, 82 observations were present.Independent variables were included into the multivariable models if they were shown to be significantly associated with the dependent variable using univariable logistic models with a relaxed p-value (p ≤ 0.2). Collinearity between explanatory variables was tested by pairwise Spearman’s correlation tests: when two variables were highly correlated , only the one with the smallest p-value was considered for inclusion in the multivariable models. The virus RNA concentration was expressed as a log10 copy number per 150 ng of total RNA, and any zero values were changed to 1.0 × 10p < 0.05.Differences in the proportion between fertile over total eggs laid in each group were tested by a chi-square test. Statistical analysis was carried out using R software  for comparison of proportions and regression analyses; GraphPad Prism for iOS, version 9 , was used for all the other tests. Significance for each test was set at https://arriveguidelines.org).Animal use and experimental procedures involving animals were approved by the University of Guelph Animal Care and Use Committee . The study is reported in accordance with ARRIVE guidelines (Supplementary Information.


Despite epidemiological evidence associating gallstone disease (GSD) with cardiovascular disease (CVD), a dilemma remains on the role of cholecystectomy in modifying the risk of CVD. We aimed to characterize the phenotypic and genetic relationships between GSD and two CVD events – stroke and coronary artery disease (CAD).We first performed a meta-analysis of cohort studies to quantify an overall phenotypic association between GSD and CVD. We then investigated the genetic relationship leveraging the largest genome-wide genetic summary statistics. We finally examined the phenotypic association using the comprehensive data from UK Biobank (UKB).P = 6.00 × 10–4) and CAD (P = 2.27 × 10–15), corroborated by local signals. The shared genetic architecture was largely explained by the multiple pleiotropic loci identified in cross-phenotype association study and the shared gene-tissue pairs detected by transcriptome-wide association study, but not a causal relationship (GSD to CVD) examined through Mendelian randomization (MR) . After a careful adjustment of confounders or considering lag time using UKB data, no significant phenotypic effect of GSD on CVD was detected , further supporting MR findings.An overall significant effect of GSD on CVD was found in meta-analysis . Genetically, a positive shared genetic basis was observed for GSD with stroke (Our work demonstrates a phenotypic and genetic relationship between GSD and CVD, highlighting a shared biological mechanism rather than a direct causal effect. These findings may provide insight into clinical and public health applications.The online version contains supplementary material available at 10.1186/s12916-023-03072-6. Gallstone disease (GSD) and cardiovascular disease (CVD) are both common and costly global public health issues, of which comorbidity has long been documented –4. In a N = 62; stroke: N = 23; coronary artery disease (CAD): N = 241) elucidated by recent genome-wide association studies (GWAS) [CYP7A1, NPC1L1, ABCG5/8, APOE, and FABP2) affecting both traits have also been identified, suggesting a potential intrinsic link underlying GSD and CVD [Both GSD and CVD are known to under genetic influences, with SNP (single-nucleotide polymorphisms) -heritability estimates of 25% and 40% –13, as ws (GWAS) –16. Mult and CVD –20.To better elucidate the relationship between GSD and CVD, we first meta-analyzed currently available epidemiological investigations of prospective design on GSD (or cholecystectomy) and incident CVD to quantify a crude overall phenotypic association. We then leveraged large-scale genome-wide genetic data as well as a complied analytical strategy – genome-wide cross-trait analysis – to determine shared and distinct genetic architecture. Such analysis features in several analytic aspects: genetic correlation analysis to estimate overall and local genetic correlation, cross-trait meta-analysis and transcriptome-wide association study (TWAS) to identify potential pleiotropic loci, and Mendelian randomizations (MR) to make causal inferences . FinallyP < 5 × 10–8) after removing SNPs in LD (r2 > 0.25 across a 250 kb window) were identified. We extracted relevant information of 62 GSD-associated index SNPs for instrumental variables (IVs) as well as downloaded full set summary statistics. The effect size and relevant information of GSD IVs are shown in Additional file The hitherto largest GWAS of GSD was obtained from meta-analyzing data of UK Biobank (UKB) and FinnGen, comprising 550,437 European individuals  . IndepenP-threshold of 5 × 10–8. GWAS summary data of CAD was conducted meta-analyzing data across nine studies, totaling 181,522 cases and 984,168 controls of predominantly European ancestry (> 95%) [P < 5 × 10–8). For both GWASs, we extracted relevant information of these trait-associated index SNPs (23 stroke-associated index SNPs and 241 CAD-associated index SNPs) for reverse Mendelian randomization (MR) analysis as well as downloaded full set summary statistics for genome-wide cross traits analysis.Two most common CVD events, stroke and CAD, were included in our study. GWAS summary data of stroke was obtained from the multi-ancestry meta-analyzing data of GIGASTROKE Consortium, among which 1,308,460 were European individuals  . This meA table detailing relevant information of each included dataset is presented , numbers of GSD, GSD diagnosis, CVD definition, years of follow-up, confounders adjustment, effect size, and quality assessment. The Newcastle–Ottawa Scale was used to evaluate the quality of included studies , ranging studies . We pool studies . To expl studies . All anaP-value  was used to define statistical significance.We performed pairwise genetic correlation analysis using cross-trait linkage disequilibrium score regression (LDSC)  and geneP-value  was used to define statistical significance.We estimated the pairwise local genetic correlation using SUPERGNOVA . This alHom and SHet, which effectively combine summary statistics across traits controlling for population structure and cryptic relatedness. While SHom is the most powerful when genetic effect sizes are homogeneous, this assumption is often violated in the context of meta-analyzing multiple traits. As an extension of SHom, SHet maintains statistical power even in the presence of heterogeneity by assigning greater weights to larger trait-specific effect sizes. Therefore, we adopted the SHet estimate in our analysis. We applied PLINK clumping function to obtain independent SNPs (parameters: –clump-p1 5e-8 –clump-p2 1e-5 –clump-r2 0.2 –clump-kb 500) [PCPASSOC < 5 × 10–8 and Psingle-trait < 1 × 10–3 (for both traits). Particularly, a significant pleiotropic SNP satisfies the following conditions was further considered as a novel shared SNP: (1) was not driven by any single trait (5 × 10–8 < Psingle-trait < 1 × 10–3); and (2) was not in LD (r2 < 0.1) with any previously reported genome-wide significant SNPs of single-trait, and none of their neighboring SNPs (± 500 kb) reached P < 5 × 10–8 in single-trait GWASs.We next conducted a Cross-Phenotype Association (CPASSOC) analysis to identify potential pleiotropic loci . CPASSOC-kb 500) . Among eWe used Ensemble Variant Effect Predictor (VEP)  for detahttps://github.com/hailianghuang/FMsummary) to identify a credible set of SNPs that were 99% likely to contain the causal SNP at each of the significant shared SNPs obtained from CPASSOC. This algorithm maps the primary signal and uses a flat prior with the steepest descent approximation, assuming at least one causal variant exists within a given region [We conducted a fine-mapping analysis using FM-summary  based on 49 Genotype-Tissue Expression (GTEx) version 8 tissue expression weights [To identify associations with regard to transcriptome gene expression in specific tissues, we conducted a transcriptome-wide association study (TWAS) using FUSION ( weights . We firsP-threshold of 0.05/2 (number of CVD phenotypes) was defined as statistical significance.We next conducted two-sample MR analysis to detect the putative causal relationship between GSD and CVD, and reported study design according to STROBE-MR  the exclusion of palindromic IVs ; (ii) thP-value of less than 0.05 was considered statistically significant.Descriptive statistics were conducted to characterize the baseline UKB participants. Continuous variables were summarized as means and standard deviations, and categorical variables as frequencies and percentages. We constructed Cox proportional hazard regression models to estimate the HR and 95%CI among individuals with GSD compared to those without GSD for the time of follow-up. We used three sets of adjustments. Estimates in model 1 were adjusted only for sex, age, assessment center, and the top 40 genetic principal components. Estimates in model 2 were further adjusted for income, Townsend deprivation index, physical activity, smoking, drinking, sleep duration, hypertension, dyslipidemia, and waist-to-hip ratio (WHR). Estimates in model 3 were adjusted for, on top of model 2, type 2 diabetes mellitus (T2DM), and body mass index (BMI). In addition, a sensitivity analysis without the exclusion of less than two years of follow-up or a diagnosis of CVD within two years after developing GSD was performed. Finally, we repeated all these in following analyses including (i) limiting the exposure to cholecystectomy alone; (ii) investigating the sex-specific relationship between GSD and CVD; (iii) ruling out the reverse-direction association between CVD and the risk of subsequent GSD. All analyses were conducted using SAS version 9.4 . A two-sided n = 4), the United States (n = 4), and Europe (n = 2). The follow-up periods for these studies varied from 5 to 32 years. Of the 10 included studies, seven were prospective cohort studies, while the remaining three were retrospective cohort studies. The diagnosis of GSD was established using self-report questionnaires and medical records. The quality assessment scores for these studies ranged between 7 and 9, indicating high quality  were included in the analysis –8, 41–44P < 0.01, I2 = 88%)  (data not shown). Specific to a particular CVD event, participants from 3 cohorts with a history of GSD had a 25% increased risk of stroke , whereas participants from 8 cohorts with a history of GSD had a 21% increased risk of CAD   and CAD (P = 2.27 × 10–15) showed a positive genetic correlation with GSD using LDSC, which remained consistent in GNOVA , all withstood Bonferroni correction . With regard to CAD, two significant local regions were found, including 19q13.33  and 4p16.3  [P < 0.05/2,353)  . All estPCPASSOC = 4.78 × 10–8) located near ABHD17C, a locus involved in protein depalmitoylation by enabling palmitoyl-(protein) hydrolase activity [In total, we identified five significant pleiotropic SNPs shared between GSD and stroke  located near HGFAC, encodes a member of the peptidase S1 protein family, an enzyme activating hepatocyte growth factor (HGF), which further influence metabolic diseases and CVD [CPASSOC = 4.52 × 10–9), mapped to TERT, a gene known to encode for telomerase reverse transcriptase, which was closely related to CAD [PCPASSOC = 5.33 × 10–9) was located near ABCA1, a gene regulating cholesterol transporting [For GSD and CAD, we found a total of 49 significant pleiotropic SNPs among which nine were novel , SNRPD2 associated with CAD, and RAC1 associated with CAD and lipid metabolism. Notably, all of these genes were located at pleiotropic loci identified in cross-trait meta-analysis, including RAC1, FAM220A, and DAGLB at 7p22.1; SPPL3, C12orf43, and UNC119B at 12q24.31; as well as SNRPD2, DM1-AS and DMPK at 19q13.32.To investigate specific expression-trait associations that are likely shared by both traits, we further performed TWAS using gene expression data available at 49 tissues. Numbers of 48 shared gene-tissue pairs were identified for GSD and CAD, but none was identified for GSD and stroke . This estimate did not alter in IVW , MR-Egger regression  or weighted median approach . Sensitivity analysis removing pleiotropic SNPs or palindromic SNPs revealed null findings. As for CAD, a consistent null result was observed . In the reverse-direction MR, genetic predisposition to stroke or CAD did not seem to affect GSD risk , during which 1,254 GSD patients and 24,709 GSD-free individuals developed CAD . From a genetic perspective, our work demonstrated biological links underlying these complex traits, highlighting pleiotropy rather than causality. From a phenotypic perspective, the absence of associations between GSD and CVD risk further corroborates MR findings. Our work advances understanding to the complicated relationship underlying GSD and CVD, providing important implications for the prevention and treatment of the two common conditions.SULT2B1 and FAM83E, situated in the region 19q13.33; RGS12 and HGFAC, located in the region 4p16.3). These findings shed light on the shared biological mechanisms underlying GSD and CVD, encompassing cholesterol metabolism [GSD and CVD shared a moderate global genetic basis in our study. Extending to local level, we further identified two significant local signals (19q13.33 and 4p16.3) for GSD and CVD. Interestingly, the findings of SUPERGNOVA and CPASSOC analyses simultaneously discovered four pleiotropic genes , which was further corroborated by the findings of local genetic correlation  and fine mapping analysis (having a posterior probability of 1.00 in the 99% credible set). It encodes a member of the peptidase S1 protein family. Initially, the protein is synthesized as an inactive single-chain precursor and subsequently undergoes endoproteolytic processing to be activated in a heterodimeric configuration. It acts as serine protease that converts hepatocyte growth factor to the active form. The increased circulation of HGF has been reported to be related to a wide variety of CVDs and metabolic disease [Contrary to the limited evidence observed for causal relationships, we identified multiple pleiotropic loci, suggesting that the previously reported phenotypic links could be largely explained by common biological mechanisms. Here, we highlight two interesting examples.  disease , 54. Fur and CVD . HGFAC w disease , 56.On a gene-tissue pair level, our TWAS analysis further revealed shared biological hypotheses between GSD and CAD. The three loci  identified in both CPASSOC and TWAS analysis implicate common biological mechanisms in GSD and CVD, involving lipid metabolism , 58, infTaken together, our findings deliver important clinical and public health implications. First, we provide evidence suggesting that GSD is very unlikely to pose a direct effect on the risk of CVD, implying that the treatment of GSD may not confer additional substantial cardiovascular health benefits, which partly supports the previous findings on that individuals with gallbladder removal did not benefit from a lowered CVD risk . From a N < 10,000) [Several limitations need to be acknowledged. First, the characteristics of GSD vary across races . However 10,000) . Future In conclusion, leveraging large-scale population-based prospective observational studies and summary statistics from the hitherto largest GWAS, our study demonstrates an intrinsic link underlying GSD and CVD (stroke and CAD). While GSD is not likely to elevate the risk of stroke and CAD, it shares biological mechanisms with these conditions. Clinically, the treatment of GSD may not directly confer additional substantial cardiovascular health benefits. Further studies are needed to confirm our findings and to clarify potential mechanistic pathways.Additional file 1: Table S1. Characteristics of genetic instruments of gallstone disease. Table S2. Data sources, sample sizes, number of IVs. Table S3. The detailed search strategy for meta-analysis. Table S4. STROBE-MR checklist of recommended items to address in reports of Mendelian randomization studies. Table S5. Characteristics of included studies in meta-analysis. Table S6. Subgroup analyses for relationship between GSD and CVD. Table S7. Significant local regions after correcting for multiple testing . Table S8. Genome-wide significant SNPs identified by cross-trait meta-analysis between gallstone disease and stroke . Table S9. Genome-wide significant SNPs identified by cross-trait meta-analysis between gallstone disease and coronary artery disease . Table S10. Linear closest genes of each cross-trait analyses-identified SNP for gallstone disease and stroke. Table S11. Linear closest genes of each cross-trait analyses-identified SNP for gallstone disease and coronary artery disease. Table S12. List of SNPs in the 99% credible set identified from fine-mapping analysis for each CPASSOC-identified locus shared between gallstone disease and stroke. Table S13. List of SNPs in the 99% credible set identified from fine-mapping analysis for each CPASSOC-identified locus shared between gallstone disease and coronary artery disease. Table S14. Results from colocalization analysis for each pleiotropic locus identified from CPASSOC between gallstone disease and stroke. Table S15. Results from colocalization analysis for each pleiotropic locus identified from CPASSOC between gallstone disease and coronary artery disease. Table S16. Significant shared transcriptome-wide association analysis results between gallstone disease and coronary artery disease. Table S17. Baseline characteristics of UK Biobank participants by gallstone disease status during follow-up for stroke. Table S18. Baseline characteristics of UK Biobank participants by gallstone disease status during follow-up for coronary artery disease. Table S19. Observational associations between cholecystectomy and cardiovascular diseases. Table S20. Sex-specific observational associations between gallstone disease and cardiovascular diseases. Table S21. Observational associations between cardiovascular diseases and gallstone disease.Additional file 2: Figure S1. Forest plot of pooled relative risk of incident stroke and CAD in participants with GSD. Figure S2. Estimated causal association between cardiovascular diseases and gallstone disease using two-sample Mendelian randomization.


The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system. They arThe core structure of ESCRT is illustrated in ESCRT-I, structurally forming an elongated heterotetramer, was the first discovered ESCRT structure. It consists of VPS23/TSG101, VPS28, VPS37, and MVB12/UBAP1 (Ubiquitin Associated Protein 1) in a 1:1:1:1 ratio. On one hand, the UEV domain of VPS23/TSG101 interacts with ESCRT-0 and ubiquitinated membrane proteins. ESCRT-I can bind two ubiquitin moieties on the endosome due to the association of UBAP1 and yeast MVB12 with ubiquitin. On the other hand, VPS28 interacts with the GLUE domain of ESCRT-II protein EAP45, thereby interacting with the ESCRT-II complex. Additionally, ESCRT-I and Alix can recruit ESCRT-III components, which mediate the final scission step during cell division. Moreover, ESCRT-I is involved in regulating various cellular processes such as viral infection  and apopAs a key factor in intracellular membrane transport and degradation pathways, ESCRT-II participates in forming and dissociating intracellular vesicles in the endolysosomal system. In yeast cells, this complex consists of VPS36, VPS25, and VPS22, while in mammals, it is composed of the endosomal sorting complex required for transport-associated proteins (EAP). The Y-shaped structure of ESCRT-II is formed by EAP30, EAP45, and EA20 in a 1:1:2 ratio . SimilarIn contrast to early ESCRT complexes  that form stable protein complexes in the cytoplasm, the ESCRT-III complex assembles only transiently on endosomes. ESCRT-III belongs to the CHMP family, soluble monomers that are widely present in eukaryotes and participate in intracellular membrane transport and degradation pathways. It also acts as a membrane remodeling machinery, providing the driving force for membrane constriction at the neck and subsequent scission . The ESCThe AAA-ATPase complex Vacuolar protein sorting 4 (VPS4) consists of the type I AAA-ATPase Vps4 and its cofactor Vta1. By harnessing the energy provided by ATP hydrolysis, it disassembles the ESCRT-III complex on endosomal membranes, enabling recycling. Upon completion of ESCRT-III disassembly, the Vps4 complex dissociates into its inactive protomers. Therefore, the Vps4 complex terminates each round of MVB cargo sorting and vesicle formation . Two typALIX protein is a multifunctional cellular protein with a structure depicted in 2.The molecular mechanism underlying membrane invagination is illustrated in 2.1.Degradation and recycling of intracellular substances are fundamental mechanisms for maintaining cellular homeostasis and responding to adverse external conditions. Most cells employ several distinct degradation systems: the ubiquitin-proteasome degradation system, endocytosis, autophagy, and lysosomal degradation system . Among tThe formation of autophagosomes requires the induction, transport, and fusion of membrane-bound organelles. Previous studies have shown that disruption of the ESCRT machinery in mammals leads to autophagosome accumulation, indicating the important role of ESCRT in regulating autophagy . The rel2.2.ESCRT is an evolutionarily conserved membrane remodeling complex with the unique ability to catalyze membrane constriction within necks. Among them, ESCRT-III, in collaboration with the AAA ATPase VPS4, plays a central role in the main membrane remodeling and scission functions of the ESCRT machinery . ESCRT-INot all organisms require the complete ESCRT complex, but ESCRT-III and VPS4 appear to be indispensable, with CHMP2, CHMP3, CHMP4, and VPS4 considered essential components in membrane remodeling. However, even in the presence of ESCRT-III proteins, membrane fission is halted when AAA ATPase VPS4 is absent. Furthermore, ESCRT demonstrates critical activity in many other cellular membrane remodeling events, such as enveloped virus budding, cytokinesis abscission, exosome biogenesis , plasma 2.3.The ESCRT machinery was initially discovered through genetic studies in yeast, where mutations led to improper sorting of proteins into vesicles, functionally equivalent to lysosomes . These mESCRT complexes play crucial roles in this process, with the ubiquitination of cargoes providing a critical signal for initial cargo binding by ESCRT-0 . The ESC2.4.in vitro  domains. These motifs mediate the recruitment and interaction of class E proteins to facilitate virus budding, envelopment of retroviruses , and other RNA viruses , redirecting cellular ESCRT proteins to the cytoplasmic membrane for viral particle release and egress from infected cells.Many enveloped viruses utilize the ESCRT pathway to catalyze membrane scission, leading to the release or budding of viral particles . This pr3.1.Some envelope RNA viruses utilize the ESCRT pathway to facilitate budding and release from host cells. By interacting with ESCRT proteins, these viruses can redirect intracellular proteins to the cell membrane, promoting viral particle budding and separation. The involvement of ESCRT plays a critical regulatory role in the infection, replication, and spread of enveloped RNA viruses.3.1.1.Avian paramyxoviruses, including Newcastle disease virus (NDV), are single-stranded RNA viruses that can cause highly contagious diseases in various avian species. The M protein of paramyxoviruses has been identified as a nucleocapsid-associated protein that plays a crucial role in the virus life cycle . During in vitro and are indispensable for the in vivo multiplication and pathogenesis of the infection they cause (Sendai virus (SeV), belonging to the Paramyxoviridae family and Respirovirus genus, is a pneumotropic virus in rodents and one of the most extensively studied members of the Respirovirus genus . Its strey cause . It is tey cause . Oda et ey cause . Furtherey cause . While tHuman parainfluenza virus type 1 (HPIV1) is a common respiratory pathogen and a single-stranded, negative-sense, non-segmented RNA virus belonging to the Paramyxoviridae family. The viral genome consists of 15,600 nucleotides and encodes six proteins from six genes. Among them, the C protein serves as an important virulence factor in HPIV1. It can inhibit apoptosis, influence host gene transcription, and regulate the production and signaling of type I interferon . The C pNipah virus (NIV) is a deadly zoonotic virus belonging to the Paramyxoviridae family, capable of causing severe respiratory and neurological diseases in humans and animals. The M protein of NIV is considered to play a central role in virus assembly and budding . When ex3.1.2.Human Immunodeficiency Virus (HIV) is a single-stranded RNA virus belonging to the Retroviridae family. It primarily infects human immune cells, leading to immune system impairment. HIV-1 Gag is synthesized in the cytoplasm and subsequently directed to the plasma membrane, where virus assembly takes place . The forFollowing this, 3.1.3.The Flaviviridae family comprises enveloped, single-stranded positive-sense RNA viruses, including the Hepatitis C virus (HCV), which is the causative agent of chronic hepatitis. It encodes a large polyprotein precursor of approximately 3,000 amino acid residues, which is processed by viral proteases and cellular signalizes to generate three structural proteins  and seven non-structural proteins  and its binding to HCV assembly, thus compensating for the apparent deficiency of the late domain. On the other hand, HCV proteins involved in virus assembly, including the core and NS2, are ubiquitinated, and the recognition of ubiquitinated NS2 and potentially other viral proteins by HRS UIM might play a role in ESCRT recruitment and HCV assembly, providing a potential mechanism by which HCV proteins could be recruited to ESCRT complexes . Subsequvia exosome secretion by Tamai et al.  is an acute, contact-transmitted infectious disease that can cause abortion, stillbirth, weak-born piglets, mummified fetuses, and acute respiratory distress in pigs of all ages. The causative agent, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an enveloped, non-segmented, positive-sense RNA virus belonging to the Arteriviridae family and the genus Arterivirus . PRRSV iAmong the ESCRT proteins that are crucial for PRRSV infection, TSG101 has been identified as the most upstream factor. It has been reported that TSG101 is involved in the replication of classical swine fever virus and the assembly and release of various viruses . As a noAlthough significant progress has been made in elucidating the PRRSV lifecycle, a comprehensive understanding of PRRSV assembly is still lacking. The mechanisms of other identified ESCRT components involved in PRRSV infection remain unclear. Further research is needed to explore the interaction mechanisms between PRRSV and the ESCRT system to better understand the PRRSV lifecycle and viral proliferation process. Moreover, considering the importance of TSG101 in various viral infections, it holds promise as a broad-spectrum antiviral target for development.3.1.5.Ebola virus (EBOV) is a single-stranded negative-sense enveloped RNA virus belonging to the family Filoviridae, which can cause a severe hemorrhagic fever syndrome with a high fatality rate. The viral matrix protein VP40 is a major component of EBOV virions and is essential for filamentous particle formation . OligomeMarburg virus (MARV) is a filamentous virus capable of inducing severe hemorrhagic fever in both humans and non-human primates, with a mortality rate reaching up to 90%. Comprising seven distinct proteins, the filamentous MARV particles manifest a complex architecture. Among these constituents, the nucleoprotein (NP) encases the non-segmented negative-sense RNA genome. Coordinating with viral proteins VP35, L, VP30, and VP24, NP assembles into an elongated helical nucleocapsid . A surro3.1.6.The Arenaviridae family stands as a dynamically evolving and diverse collective of viruses. It encompasses an array of enveloped viruses characterized by a bipartite negative-sense RNA genome, encoding four distinct viral proteins, and marked by a non-lytic life cycle. This genome comprises two individual single-stranded RNA entities: a larger segment encoding the viral polymerase (L) and a petite zinc finger motif protein (Z), as well as a minor segment encoding both the viral nucleoprotein (NP) and the glycoprotein precursor GPC; . The Z pNotwithstanding the existence of diverse late-domain motifs within certain arenaviruses, the extent to which virus budding hinges on each motif appears to diverge among different viruses. For instance, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) exclusively features a sole PPxY motif within its Z protein, a critical determinant driving the budding process . In cont3.1.7.Rabies virus (RABV) is a well-established zoonotic pathogen belonging to the Lyssavirus genus within the Rhabdoviridae family. Characterized by a single-stranded negative-sense RNA genome, this virus encodes a set of five essential viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein L; . In the Infectious hematopoietic necrosis virus (IHNV), much like RABV, resides within the Rhabdoviridae family and boasts a negative-sense single-stranded RNA genome. This genomic structure encodes a cluster of six distinctive proteins: nucleoprotein (N), polymerase-associated phosphoprotein (P), matrix protein (M), unique glycoprotein (G), large RNA-dependent RNA polymerase (L), and a concise intergenic region nestled between the G and L genes . Through3.1.8.Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality . CSFV isLiu et al. investigated the Tsg101, VPS25, CHMP4B, and CHMP7 proteins that play essential roles in viral trafficking and Clathrin-mediated viral entry. CHMP4B and CHMP7 interact with Rab5 to transport CSFV virions from early endosomes to late endosomes. They first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II), and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Tsg101 and CHMP4B are associated with LAMP-1 in the lysosomes, probably leading to uncoating and release of nucleic acid for genome replication in the endoplasmic reticulum area .3.2.In addition to enveloped RNA viruses, the ESCRT complex is also involved in the assembly and release processes of enveloped DNA viruses. For instance, Hepatitis B virus (HBV) and Herpes simplex virus type 1 (HSV-1).3.2.1.HBV is the major pathogen responsible for human liver disease. It is an enveloped, DNA-containing pararetrovirus that replicates primarily through reverse transcription of its pregenomic RNA pgRNA; . DespiteDuring HBV infection, all components of ESCRT-0 are essential for HBV replication. The core antigen of HBV (HBcAg) interacts with ESCRT-0, facilitating the assembly and release of HBV particles. Specifically, the HBcAg protein forms a complex with the HRS subunit of ESCRT-0, which promotes the intracellular aggregation and assembly of HBV core proteins, ultimately leading to the release of HBV particles. Additionally, the surface antigen of HBV (HBsAg) can interact with the ESCRT complex, facilitating the release of HBV particles. Studies by Chou et al.  have shoDue to the absence of the P (S/T)AP-like late-domain motif in HBV structural proteins, which is known to interact with TSG101, it was previously believed that TSG101 was dispensable for HBV export. However, recent research findings have demonstrated that the knockdown of TSG101 reduces extracellular HBV DNA levels and results in the accumulation of viral capsids within cells. This supports the essential role of TSG101 in the process of HBV extracellular capsid release . Althoug3.2.2.HSV-1 is a structurally complex enveloped double-stranded DNA virus belonging to the Herpesviridae family. It is a human pathogen that can cause diseases such as oral herpes, keratitis, and encephalitis. Its capsid can package together with the DNA genome in the cell nucleus . The virGenerally, viruses recruit one or more early ESCRT components to access ESCRT-III, with the most common being ALIX and the ESCRT-I complex . HoweverConsidering the aforementioned findings, it is reasonable to hypothesize that ESCRT-III may be directly recruited by proteins present in the capsid or envelope of HSV-1. HSV-1 appears to redundantly utilize multiple cellular ESCRT components for ESCRT-III recruitment, or the virus may completely bypass the requirement for Bro1 domain proteins, ESCRT-I, and ESCRT-II. In the latter scenario, the structural proteins within HSV-1 particles, whether internal or external to the capsid, might have evolved to directly recruit and trigger the assembly of ESCRT-III subunits at sites of envelope constriction and scission . The int3.2.3.Autographa California Multiple Nuclear Polyhedrovirus (AcMNPV) is the most intensively studied baculovirus and is the type species of the virus family Baculoviridae. Baculoviruses are enveloped, insect-specific double-stranded DNA (dsDNA) viruses that replicate in the nuclei of infected cells. During the infection cycle, baculoviruses produce two phenotypes of enveloped virions: occlusion-derived virions (ODV) and budded virions (BV). The ESCRT pathway is conserved in sequenced insect genomes and the expression levels of certain components of ESCRT-I, -II, -III, Vps4, and Alix were significantly upregulated upon AcMNPV infection .Prior studies revealed that efficient entry and egress of AcMNPV BV are dependent on functional Vps4 . Since V4.ESCRT plays a crucial role in various cellular processes, including the budding of enveloped viruses, regulation of innate immune responses, and degradation of damaged or unwanted cellular components. Although significant progress has been made in understanding the molecular mechanisms of ESCRT-mediated virus budding, much remains to be explored. For instance, the molecular mechanisms underlying the regulation of ESCRT components’ expression and activity require further investigation. Moreover, the precise roles of ESCRT in virus replication and pathogenesis need to be elucidated. An enhanced understanding of how the ESCRT system is hijacked by viruses can greatly contribute to our knowledge of normal cellular physiological functions.In the future, research on ESCRT function in virus-host interactions will provide new insights into the development of antiviral therapies. A better understanding of the ESCRT complex’s role in innate immune response regulation will lead to the development of novel approaches to treating viral infections. Moreover, the identification and characterization of novel ESCRT-interacting viral proteins and their interaction mechanisms will expand our knowledge of virus-host interactions. Finally, the development of new technologies, such as single-particle cryo-electron microscopy, will allow us to determine the three-dimensional structure of ESCRT and provide a more detailed understanding of its molecular mechanisms. Overall, further research on ESCRT and its interactions with viruses will help to develop new strategies to combat viral infections.CW: Writing – original draft. YC: Writing – review & editing. SH: Writing – review & editing. XL: Writing – review & editing.


Nirmatrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro), is clinically useful against infection with SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to many monoclonal antibody therapies, potential SARS-CoV-2 resistance to nirmatrelvir is a major public health concern. Several amino acid substitutions have been identified as being responsible for reduced susceptibility to nirmatrelvir. Among them, we selected L50F/E166V and L50F/E166A/L167F in the 3CLpro because these combinations of substitutions are unlikely to affect virus fitness. We prepared and characterized delta variants possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F. Both mutant viruses showed decreased susceptibility to nirmatrelvir and their growth in VeroE6/TMPRSS2 cells was delayed. Both mutant viruses showed attenuated phenotypes in a male hamster infection model, maintained airborne transmissibility, and were outcompeted by wild-type virus in co-infection experiments in the absence of nirmatrelvir, but less so in the presence of the drug. These results suggest that viruses possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F do not become dominant in nature. However, it is important to closely monitor the emergence of nirmatrelvir-resistant SARS-CoV-2 variants because resistant viruses with additional compensatory mutations could emerge, outcompete the wild-type virus, and become dominant. Resistance to nirmatrelvir, an oral antiviral agent that targets SARS-CoV-2 and is clinically useful against infection with Omicron variants, is currently not well understood. In this study, the authors characterize mutant viruses with reduced sensitivity to nirmatrelvir in vitro and in vivo. Omicron variants possess more than 30 amino acid substitutions in the spike protein, and its subvariants BA.4/5, BA.4.6, and BA.2.75 are less susceptible to therapeutic monoclonal antibodies including REGEN-COV [casirivimab (REGN10933) plus imdevimab (REGN10987)], bamlanivimab (LY-CoV555) plus etesevimab (LY-CoV016), evusheld [tixagevimab (COV2-2196) plus cilgavimab (COV2-2130)], and xevudy [sotrovimab (S309)]6. BA.4/5, BA.4.6, and BA.2.75 are susceptible to bebtelovimab (LY-CoV1404) but the latest subvariants BQ.1.1 and XBB escape from it7. The antiviral compounds nirmatrelvir, remdesivir, molnupiravir, and ensitrelvir, which target virus proteins other than the spike protein, are effective against such variants and subvariants.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, is still prevalent three years since its emergence and continues to spread around the world. It accumulates amino acid substitutions frequently and many variants of concern (VOCs) have appeared and caused several waves of infection. Among these variants, omicron (lineage B.1.1.529) was identified at the end of 2021 and became globally dominant9 and nirmatrelvir11.Several antivirals have been widely used for to treat COVID-19 including GS-441524 (remdesivir), EIDD-1931 (molnupiravir), PF-07321332 (nirmatrelvir), and S-217622 (ensitrelvir). Remdesivir and molnupiravir inhibit virus RNA-dependent RNA polymerase, whereas nirmatrelvir and ensitrelvir interfere with 3CL protease . As these drugs are increasingly used in clinical settings, there is concern about the emergence of viruses with reduced sensitivity or resistance. Amino acid substitutions associated with resistance were reported for remdesivir12. When treatment is initiated within three days of onset, approximately 90% protection against severe COVID-19 and hospitalization has been reported13. Amino acid substitutions that are responsible for resistance to nirmatrelvir have been reported in 3CLpro (nsp5). In silico analysis showed that the N142L, E166M, Q189E, Q189I, and Q192T substitutions reduced the potency of nirmatrelvir in vitro14. A screen using a VSV-based system revealed that the Y54C, G138S, L167F, and Q192R substitutions confer resistance to nirmatrelvir15. Naturally occurring amino acid substitutions such as S144A, A173V, and E166D/G also contribute to resistance to nirmatrelvir16. Deep mutational analysis has shown that the E166V, P252L, and T304I substitutions reduce the IC50 value to nirmatrelvir18. In another study, resistant viruses possessing the E166V and T304I substitutions in combination with other amino acid substitutions in nsp5 were selected through virus passaging in the presence of nirmatrelvir in cell culture19. The E166V and T304I substitutions conferred strong resistance but resulted in a loss of viral fitness; viral fitness was restored by compensatory substitutions such as L50F and T21I18. The L50F and T21I substitutions had little effect on the sensitivity to nirmatrelvir18. Another resistant virus selection experiment in vitro identified the combination of the L50F, E166A, and L167F substitutions as being associated with resistance to nirmatrelvir20. Each E166A and L167F substitution conferred reduced sensitivity to nirmatrelvir20. Recent studies have reported that the virus fitness of viruses possessing the L50F and E166V substitutions and the L50F, E166A, and L167F substitutions is comparable to that of wild-type virus in cell culture20. Another preprint reported that a recombinant virus possessing the L50F, E166A, and L167F substitutions showed similar pathogenicity in hamsters and transmitted to co-housed hamsters21. Therefore, among the many resistant mutations reported, we chose to study Nsp5-L50F/E166V19 and Nsp5-L50F/E166A/L167F20 because they appeared unlikely to affect virus fitness in vitro. By using reverse-genetics based on the delta variant, we prepared mutant viruses possessing these substitution combinations and characterized their replication in vitro, their pathogenicity in hamsters, and their airborne transmissibility in hamsters. We also assessed their replication in co-infection experiments with wild-type virus in the presence or absence of nirmatrelvir in the hamster model.Nirmatrelvir (PF-07321332) is co-formulated with the pharmacokinetic enhancer ritonavir 50 value of PF-07321332 (nirmatrelvir) against wild-type virus was 8.08 μM , whereas those against Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F were greater than 100 μM. Although EIDD-1931 (molnupiravir) and GS-441524 (remdesivir) showed similar IC50 values against all three viruses tested, the sensitivity of Nsp5-L50F/E166V to S-217622 (ensitrelvir) was moderately reduced and that of Nsp5-L50F/E166A/L167F was appreciably reduced; the IC50 values were 1.41 μM  and 19.34 μM . These results indicate that both mutant viruses have reduced sensitivity to PF-07321332 (nirmatrelvir) and show reduced sensitivity to S-217622 (ensitrelvir) as well.To characterize mutant viruses possessing amino acids mutations that are responsible for reduced sensitivity to nirmatrelvir, we prepared a wild-type delta B.1.617.2) variant and its mutant viruses possessing the L50F and E166V substitutions in Nsp5 (Nsp5-L50F/E166V) or the L50F, E166A, and L167F substitutions (Nsp5-L50F/E166A/L167F) by using a BAC-based reverse genetics system. No nucleotide mixtures of more than 10% were identified in the three stock viruses. These three viruses were tested for sensitivity to the antivirals PF-07321332 (nirmatrelvir), EIDD-1931 (molnupiravir), GS-441524 (remdesivir), and S-217622 (ensitrelvir) in a focus reduction assay  and lungs ((p = 0.00794 or p = 0.0476) of the treated hamsters compared to the untreated animals, the wild-type virus was predominant in most of the hamster nasal turbinates. However, in the lungs of three hamsters, Nsp5-L50F/E166V became dominant, and in the lungs of one hamster, Nsp5-L50F/E166A/L167F became dominant. These results demonstrate that although Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F are less fit than the wild-type virus, Nsp5-L50F/E166V could be problematic by dominating the wild-type virus in lungs in the presence of nirmatrelvir.We next examined the competitive growth capability of each virus by co-infecting the wild-type virus with a mutant virus at a ratio of 3:7 based on virus infectious titers. The ratios of each mutant virus to wild-type virus in the inoculum based on deep sequence analysis were 48:52 (wild-type: Nsp5-L50F/E166V) and 42:58 (wild-type: Nsp5-L50F/E166A/L167F) , since it is the antiviral drug used most often to treat COVID-1924. Some of these drug-resistant viruses are less fit than the wild-type virus25, but others are not28. Regardless of whether viral fitness is reduced, such resistant viruses rarely spread worldwide. If a resistant virus does spread globally, either the selective pressure is extremely high due to the widespread use of the antiviral (which is unlikely), or the resistant virus acquires greater fitness through the acquisition of additional amino acid substitutions29. Combination therapy is thought to be a better strategy to treat viral infections and reduce the likelihood of emergence of resistant viruses30, but it is possible for viruses resistant to both antivirals to emerge32.Viruses, especially RNA viruses, generate variants. Although SARS-CoV-2 has a proofreading mechanism, many people have been infected and each individual producing a large amount of virus could lead to the emergence of viruses resistant to monoclonal antibodies or antivirals when used as monotherapy. In influenza patients, viruses resistant to neuraminidase (NA) or PA inhibitors have been detected after treatment with the antiviralsIn conclusion, Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F are unlikely to dominate in nature. However, secondary adaptive mutations may make these mutant viruses dominant to wild-type virus. Moreover, mutant viruses possessing other resistance-conferring substitutions could emerge and dominate in the human population. Therefore, we need to closely monitor the amino acid mutations that confer resistance to nirmatrelvir and secondary adaptive mutations by undertaking active virus surveillance to detect such viruses as soon as possible after emergence.All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan.2. The cells were regularly tested for mycoplasma contamination by using PCR and confirmed to be mycoplasma free.VeroE6/TMPRSS2 (JCRB 1819) cells were propagated in the presence of 1 mg/ml geneticin  and 5 μg/ml plasmocin prophylactic (Invivogen) in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. VeroE6-TMPRSS2-T2A-ACE2 cells were cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin–streptomycin, and 10 µg/mL puromycin. HEK293T cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS. All cells were maintained at 37 °C with 5% COActive components of remdesivir and molnupiravir , and nirmatrelvir (PF-07321332) were purchased from MedChemExpress. S-217622 (ensitrelvir) was kindly provided by Shionogi Co., Ltd. Compounds were dissolved in dimethyl sulfoxide (in vitro) or 0.5% methylcellulose (in vivo) prior to use.34. The mutations responsible for the L50F/E166V and L50F/E166A/L167F substitutions in 3CLpro were introduced during the PCR step. To rescue these viruses, pBeloBAC11 encoding the wild-type virus , Nsp5-L50F/E166V (Accession No. OR116092), or Nsp5-L50F/E166A/L167F (Accession No. OR116093) was transfected into HEK293T cells. At 3 days post-transfection, the supernatant containing the viruses was collected and inoculated onto VeroE6/TMPRSS2 at 37 °C to prepare virus stocks. The stock viruses were deep sequenced to confirm the absence of unwanted mutations, and no position contained unwanted nucleotides that exceeded 10% of the population in all stock viruses.The full-genome nucleotide sequence of SARS-CoV-2  was assembled into the pBeloBAC11 vector to generate infectious cDNA clones under the control of a cytomegalovirus (CMV) promoter as described previously35. In brief, viral RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN). cDNA was synthesized by using a Lunar-Script RT SuperMix Kit (New England BioLabs) and subjected to a multiplexed PCR in two pools using ARTIC-N1 primers v5 and Q5 Hot Start DNA polymerase (New England BioLabs). The DNA libraries for Illumina NGS were prepared from pooled amplicons by using a QIAseq FX DNA Library Kit (QIAGEN) and then analyzed by using the iSeq100 System in 150-bp paired-end mode using an iSeq 100 i1 Reagent v2 (300-cycle) kit (Illumina). The reads were assembled by CLC Genomics Workbench . The average coverage depths of the wild-type virus, L50F/E166V, and L50F/E166A/L167F were 8,078×, 10,742×, and 8,731×, respectively.The whole genome of SARS-CoV-2 was amplified by using a modified ARTIC network protocol in which some primers were replaced or added36. Briefly, VeroE6-TMPRSS2-T2A-ACE2 cells in 96-well plates were infected with SARS-CoV-2 at 100–400 focus forming unit/well. After a 1-h incubation at 37 °C, the inoculum was replaced with 1% Methyl Cellulose 400  in culture medium containing serial dilutions of the antiviral compounds. The cells were incubated for 18 h at 37 °C and then fixed with formalin. The cells were stained with a mouse monoclonal antibody against SARS-CoV-2 nucleoprotein, clone N45 , followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories Inc.). Foci were visualized by using TrueBlue Substrate (SeraCare Life Sciences). The focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The 50% inhibitory concentration (IC50) values and 95% confidence intervals were calculated by using GraphPad Prism 9.3.0 (GraphPad Software).Antiviral susceptibilities were determined by using a focus reduction assay as previously reportedVeroE6/TMPRSS2 cells were infected with the indicated virus at a multiplicity of infection (MOI) of 0.001. After a 1-h incubation at 37 °C, the inoculum was replaced with medium ± 20 μM nirmatrelvir. Cell culture supernatants were collected at 8, 16, 24, 32, 40, 48, 56, and 72 h post-infection. Virus titers were determined by the use of a plaque assay in VeroE6/TMPRSS2 cells.5 plaque forming unit (PFU) of the indicated virus. Body weights were measured daily before inoculation and 10 days after inoculation. Respiratory parameters [Penh (a nonspecific assessment of breathing patterns) and Rpef (a measure of airway obstruction)] were also measured by using a whole-body plethysmography system (PrimeBioscience) as previously described38.Five- to six-week-old male Syrian hamsters (Japan SLC) were used in this study. Since SARS-CoV-2 occasionally fails to replicate in female hamsters, we used only male hamsters in this study. Under isoflurane anesthesia, five hamsters per group were intranasally inoculated with 105 PFU of the indicated virus. At 3 and 6 days post-infection, the animals were euthanized and nasal turbinates and lungs were collected. The virus titers in these organs were determined by use of plaque assays on VeroE6/TMPRSS2 cells.For the virus titration, five hamsters per group were intranasally infected with 1040. Briefly, five hamsters per group were intranasally infected with 105 PFU of each indicated virus and placed in cages. One day after infection, naïve hamsters were placed in adjacent cages, 5-cm apart, separated by a double-layer partition with unidirectional air flow. Infected and exposed hamsters were euthanized at 4 days post-infection or 3 days after exposure. Nasal turbinates and lungs were collected for virus titration on VeroE6/TMPRSS2 cells.The transmission study was performed as previously described5 PFU) was inoculated into ten hamsters per group. One day after inoculation, five hamsters per group were treated orally with nirmatrelvir at 250 mg/kg twice daily as previously described42. The remaining five hamsters per group were left untreated. The animals were euthanized at 4 dpi and their nasal turbinates and lungs were collected for deep sequence analysis. The ratio of wild-type to Nsp5-L50F/E166V or Nsp5-L50F/E166A/L167F was calculated from the differences at positions 10202 and 10551 or 10202, 10551, and 10555, respectively. Samples with read depths of more than 1000 were analyzed.For the co-infection study, wild-type virus and Nsp5-L50F/E166V or Nsp5-L50F/E166A/L167F were mixed at a ratio of 3:7 based on their titers, and the virus mixture (total 2 × 10P values. We compared virus titers in hamsters with the control by using a one-way ANOVA followed by Tukey’s multiple comparisons. Body weight and virus growth kinetics were compared by using a two-way ANOVA followed by Tukey’s multiple comparisons. Transmission efficiency was compared by using a two-sided Fisher’s exact test. The frequency of wild-type virus in the nasal turbinate and lungs of the treated hamsters was compared with that of the untreated hamsters by using the Mann–Whitney test followed by the two-stage step-up procedure of the Benjamini, Krieger, and Yekutieli test. Differences between groups were considered significant for P values < 0.05.GraphPad Prism software version 9.3.0 was used to calculate All reagents described in this paper are available through Material Transfer Agreements.Further information on research design is available in the Reporting SummaryPeer Review FileSouce data file


OPAT decreases length of stay/inpatient costs while benefiting patients. However, OPAT costs incurred in the ambulatory setting are poorly quantified. We evaluated unaccounted costs and potential savings from OPAT delivered via patient self-administration (S-OPAT), home care agencies/hemodialysis centers (HH-OPAT), and skilled nursing facilities (SNF-OPAT).The electronic health record (EHR) for all adult patients discharged on OPAT from Parkland Hospital (PH), a 900-bed safety-net hospital, during April - June 2021 and January - March 2022 was reviewed for the number and duration of antibiotics administered and post-discharge non-billable encounters (defined as an encounter on any day without a corresponding billable visit). Encounters on the day of a billable visit were excluded. Inpatient days avoided were defined as days post-discharge on which the patient received OPAT. An average daily hospital adjusted expense per inpatient day of $3,764 for Texas state hospitals and wholesale acquisition cost data from the Texas Department of State Health Services were used to estimate cost savings from OPAT. 340B pricing was used to calculate the cost of drugs provided to S-OPAT patients by PH. Antibiotics with different formulations were converted to equivalent daily doses and costs were averaged to estimate a daily antibiotic cost. The institutional IRB approved this study.Of 340 patients, 84 (25%) received SNF-OPAT, 115 (34%) HH-OPAT, and 141 (41%) S-OPAT. There were 255 non-billable encounters in the S-OPAT group, 242 in the HH-OPAT, and 220 in the SNF-OPAT group, with the highest rate per 100 person-days in SNF-OPAT recipients (Table 1), nearly twice that of S-OPAT. Antibiotic costs avoided by discharging patients on OPAT were $383,240 for HH-OPAT patients and $505,588 for SNF-OPAT patients (Table 2). For S-OPAT patients, costs incurred by PH based on 340B pricing were $33,785.All OPAT care models saved costs compared to hospitalization. Our study likely underestimates the unaccounted costs associated with OPAT; however, costs incurred may be significant and differ between care models. Personnel time and non-billable work should additionally be considered in determining true OPAT cost.All Authors: No reported disclosures


Due to error-prone replication, RNA viruses such as Zika virus (ZIKV), West Nile virus (WNV), influenza A virus (IAV), and simian and human immunodeficiency viruses  exist in nature as genetically and phenotypically complex mutant swarms –3. The aFig 1). While technical approaches to generating barcoded virus populations vary by virus and study, barcoded viruses generally contain a series of coding-neutral alterations to the genome [Fig 1). Current applications of barcoded virus technology use degenerate nucleotides that allow all possible mutations to occur at the site of insertion and, upon amplification and rescue of the recombinant virus, can theoretically generate several million unique barcodes without the burden of generating clones individually [A barcoded virus is engineered to contain a sequence motif  that can be used to distinguish one otherwise identical virus genome from another , stochastic forces, such as bottlenecks, have a significant impact on virus evolution within individual hosts . BarcodeThe predominant use of barcoded viruses is as mimics of highly diverse virus populations in studies of population dynamics during infection, transmission, and treatment. Early work successfully demonstrated the value of synthetic swarm viruses as analogues for virus populations by using marked clone populations to characterize the impact of stochastic forces, such as bottlenecks , on poliovirus populations during neuroinvasion in mice, and on WNV and Venezuelan equine encephalitis virus (VEEV) populations within relevant mosquito vectors ,11,12. TbcZIKV. Barcoded Zika virus (bcZIKV) was used to characterize ZIKV infection dynamics in pregnant and nonpregnant macaques [in vivo to examine virus populations throughout infection and highlighted the potential of barcoded viruses to probe the impacts of anatomical reservoirs, and bottlenecks on virus populations. bcZIKV also identified a cumulative reduction in bcZIKV population diversity associated with intrahost bottlenecks during infection in Aedes aegypti mosquitoes [Fig 2A). bcZIKV has also been instrumental in determining the impact of transmission modes between vertebrates and mosquitoes on ZIKV evolution and was used to demonstrate how direct vertebrate transmission chains could promote enhanced ZIKV virulence [macaques . Low commacaques . This wosquitoes  irulence . Furtherirulence .bcIAV. Barcoded influenza A virus (bcIAV) has been employed to study infection routes and their associated bottlenecks, evolution of the NS1 gene, virus replication in different regions of the respiratory tract, and the impact of compartmentalized replication on virus population dynamics [Fig 2B). Additionally, bcIAV infection of ferret lungs revealed that a series of bottlenecks within the lungs results in genetically distinct virus population “islands” that are heavily impacted by founder effects [dynamics ,10,19. S effects .bcCVB. Coxsackievirus B3 (CVB3) is an enterovirus that can penetrate the gastrointestinal tract and cause systemic infection [nfection . A highlnfection .bcSIV and bcHIV. Barcoded SIV and HIV  have been used in numerous studies to explore retrovirus population dynamics during transmission, infection, immune escape, treatment, and reactivation and rebound following latency [ latency ,16,22–25 latency ,22,23. FbcAAV. Adeno-associated viral (AAV) vectors are a clinically relevant mode of therapeutic gene transfer that have had success in reprogramming certain cell types in animal models [l models . Given tl models –29. Barcl models ,29.The barcoded virus approach to studying virus evolution is reliable, allows for investigation of population dynamics with unprecedented depth and ease, and has been successfully adapted to study these phenomena in a wide range of virus families and hosts. Barcoded viruses have proven to be valuable tools in assessing viral replication dynamics, progeny production, and polyinfection  at the single-cell level ,31. Fina