Generation and Characterization of a Humanized Lung Xenograft Mouse Model for Studying Henipavirus Pathogenesis.

The development of humanized mouse models has recently opened new avenues in the field of infectious diseases. These models allow research on many human viruses that were once difficult to study, because finding suitable animal models of infection can be challenging, cost prohibitive, and often do not entirely recapitulate all parameters of the disease. Here, we describe the procedure of human immune system reconstitution (humanization) of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice by the bone marrow, liver, and thymus (BLT) reconstitution method as well as the process of human lung engraftment. We then describe how to infect these human lung grafts with the paramyxovirus Nipah virus (NiV) that can cause lethal respiratory disease in humans, and for which there is only limited understanding of pathogenesis to acute lung injury.

The Susceptibility of BALB/c Mice to a Mouse-Adapted Ebola Virus Intravaginal Infection.

Ebola virus (EBOV) causes Ebola virus disease (EVD), which is characterized by hemorrhagic fever with high mortality rates in humans. EBOV sexual transmission has been a concern since the 2014-2016 outbreak in Africa, as persistent infection in the testis and transmission to women was demonstrated. The only study related to establishing an intravaginal small animal infection model was recently documented in IFNAR-/- mice using wild-type and mouse-adapted EBOV (maEBOV), and resulted in 80% mortality, supporting epidemiological data. However, this route of transmission is still poorly understood in women, and the resulting EVD from it is understudied. Here, we contribute to this field of research by providing data from immunocompetent BALB/c mice. We demonstrate that progesterone priming increased the likelihood of maEBOV vaginal infection and of exhibiting the symptoms of disease and seroconversion. However, our data suggest subclinical infection, regardless of the infective dose. We conclude that maEBOV can infect BALB/c mice through vaginal inoculation, but that this route of infection causes significantly less disease compared to intraperitoneal injection at a similar dose, which is consistent with previous studies using other peripheral routes of inoculation in that animal model. Our data are inconsistent with the disease severity described in female patients, therefore suggesting that BALB/c mice are unsuitable for modeling typical EVD following vaginal challenge with maEBOV. Further studies are required to determine the mechanisms by which EVD is attenuated in BALB/c mice, using maEBOV via the vaginal route, as in our experimental set-up.

Hydrogen Sulfide Ameliorates SARS-CoV-2-Associated Lung Endothelial Barrier Disruption.

Recent studies have confirmed that lung microvascular endothelial injury plays a critical role in the pathophysiology of COVID-19. Our group and others have demonstrated the beneficial effects of H2S in several pathological processes and provided a rationale for considering the therapeutic implications of H2S in COVID-19 therapy. Here, we evaluated the effect of the slow-releasing H2S donor, GYY4137, on the barrier function of a lung endothelial cell monolayer in vitro, after challenging the cells with plasma samples from COVID-19 patients or inactivated SARS-CoV-2 virus. We also assessed how the cytokine/chemokine profile of patients' plasma, endothelial barrier permeability, and disease severity correlated with each other. Alterations in barrier permeability after treatments with patient plasma, inactivated virus, and GYY4137 were monitored and assessed by electrical impedance measurements in real time. We present evidence that GYY4137 treatment reduced endothelial barrier permeability after plasma challenge and completely reversed the endothelial barrier disruption caused by inactivated SARS-CoV-2 virus. We also showed that disease severity correlated with the cytokine/chemokine profile of the plasma but not with barrier permeability changes in our assay. Overall, these data demonstrate that treatment with H2S-releasing compounds has the potential to ameliorate SARS-CoV-2-associated lung endothelial barrier disruption.

Characterization of an air-liquid interface primary human vaginal epithelium to study Ebola virus infection and testing of antivirals.

Ebola virus (EBOV) is the causative agent of the often-fatal Ebola virus disease (EVD) characterized by hemorrhagic fever in humans and non-human primates. Sexual transmission from male survivors has been at the origin of multiple outbreak flare-ups between 2015 and 2021. However, this route is still poorly understood and the resulting EVD from it is also understudied. To support epidemiological studies documenting sexual transmission to women, and as a transition from previously using monolayer vaginal epithelial cells (VK2/E6E7), we first determined the biological relevance of two similar air-liquid interface models of the human vaginal epithelium (VEC and VLC Epivaginal™) and then characterized their susceptibility to EBOV and virus-induced inflammation. Finally, we evaluated toxicity of Polyphenylene Carboxymethylene (PPCM) microbicide in VLC and reassessed its antiviral effect. As expected, the VEC, but also VLC model showed stratified layers including a lamina propria under an epithelial structure similar to the full thickness of the human vaginal epithelium. However, we could not detect the immune cells featured in the most relevant model (VLC) of the vaginal epithelium using the dendritic cell CD1a and CD11c markers. Consistent with our previous work using the VK2/E6E7 cell line, infectious virus was detected from the apical side of both primary human cell systems, but only when using a high infective dose, with titers remaining at a constant level of 103-4 pfu/ml over 7 days suggesting lasting infectious virus shedding. In addition, infection caused disruption of the epithelium of both models and virus antigen was found from the apical superficial layers down to the lamina propria suggesting full virus penetration and overall confirming the susceptibility of the human vaginal tissue for EBOV. Just like previously seen in VK2/E6E7 cells, VLC infection also caused significant increase in inflammatory markers including IL-6, IL-8, and IP-10 suggesting vaginitis which is again consistent with tissue lesions seen in non-human primates. Finally, both virus infection and virus-induced inflammatory response in VLC could be prevented by a single 5-min PPCM microbicide treatment prior infection.

Polyphenylene carboxymethylene (PPCM) microbicide repurposed as antiviral against SARS-CoV-2. Proof of concept in primary human undifferentiated epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected over 200 million people throughout the world as of August 2021. There are currently no approved treatments providing high chance of recovery from a severe case of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, and the beneficial effect of Remdesivir and passive immunization therapies may only be seen when administered early on disease onset. The emergence of variants is also raising concerns regarding the efficacy of antibody therapies, antivirals, and vaccines. Therefore, there is still a need to develop new antivirals. Here, we investigated the suitability of primary human epithelial cells from the trachea/bronchia (NHBE) and small airway (SAEC) as lung models of SARS-CoV-2 infection to determine, whether the microbicide polyphenylene carboxymethylene (PPCM) has antiviral activity against SARS-CoV-2. Both NHBE and SAEC expressed proteins required for virus entry in lung epithelial cells. However, these cells were only low to moderately permissive to SARS-CoV-2 as titers increased at best by 2.5 log10 during an 8-day kinetic. Levels of replication in SAEC, unlike in NHBE, were consistent with data from other studies using human normal tissues or air-liquid interface cultures, suggesting that SAEC may be more relevant to use than NHBE for drug screening. PPCM EC50 against SARS-CoV-2 was between 32 and 132 µg/ml with a selectivity index between 12 and 41, depending on the cell type and the infective dose used. PPCM doses were consistent with those previously showing effect against other human viruses. Finally, PPCM antiviral effect observed in SAEC was in line with reduction of inflammatory markers observed overly expressed in severe COVID-19 patients. Altogether, our data support the fact that PPCM should be further evaluated in vivo for toxicity and antiviral activity against SARS-CoV-2.

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV).

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014-2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Polyphenylene carboxymethylene (PPCM) in vitro antiviral efficacy against Ebola virus in the context of a sexually transmitted infection.

Ebola virus disease (EVD) is caused by Ebola virus (EBOV) and characterized in humans by hemorrhagic fever with high fatality rates. Human-to-human EBOV transmission occurs by physical contact with infected body fluids, or indirectly by contaminated surfaces. Sexual transmission is a route of infection only recently documented despite isolating EBOV virus or genome in the semen since 1976. Data on dissemination of EBOV from survivors remain limited and EBOV pathogenesis in humans following sexual transmission is unknown. The in vitro antiviral efficacy of polyphenylene carboxymethylene (PPCM) against EBOV was investigated considering the limited countermeasures available to block infection through sexual intercourse. PPCM is a vaginal topical contraceptive microbicide shown to prevent sexual transmission of HIV, herpes virus, and bacterial infections in several different models. Here we demonstrate its antiviral activity against EBOV. No viral replication was detected in the presence of PPCM in cell culture, including vaginal epithelial (VK2/E6E7) cells. Specifically, PPCM reduced viral attachment to cells by interfering with EBOV glycoprotein, and possibly through binding the cell surface glycosaminoglycan heparan sulfate important in the infection process. EBOV-infected VK2/E6E7 cells were found to secrete type III interferon (IFN), suggesting activation of distinct PRRs or downstream signaling factors from those required for type I and II IFN. The addition of PPCM following cell infection prevented notably the increase of these inflammation markers. Therefore, PPCM could potentially be used as a topical microbicide to reduce transmission by EBOV-positive survivors during sexual intercourse.

Filovirus Virulence in Interferon α/ß and γ Double Knockout Mice, and Treatment with Favipiravir.

The 2014 Ebolavirus outbreak in West Africa highlighted the need for vaccines and therapeutics to prevent and treat filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would facilitate the screening of anti-filovirus agents. To that end, we characterized knockout mice lacking α/ß and γ interferon receptors (IFNAGR KO) as a model for wild-type filovirus infection. Intraperitoneal challenge of IFNAGR KO mice with several known human pathogenic species from the genus Ebolavirus and Marburgvirus, except Bundibugyo ebolavirus and Taï Forest ebolavirus, caused variable mortality rate. Further characterization of the prototype Ebola virus Kikwit isolate infection in this KO mouse model showed 100% lethality down to a dilution equivalent to 1.0 × 10-1 pfu with all deaths occurring between 7 and 9 days post-challenge. Viral RNA was detectable in serum after challenge with 1.0 × 10² pfu as early as one day after infection. Changes in hematology and serum chemistry became pronounced as the disease progressed and mirrored the histological changes in the spleen and liver that were also consistent with those described for patients with Ebola virus disease. In a proof-of-principle study, treatment of Ebola virus infected IFNAGR KO mice with favipiravir resulted in 83% protection. Taken together, the data suggest that IFNAGR KO mice may be a useful model for early screening of anti-filovirus medical countermeasures.

Experimental Infection of Syrian Hamsters With Aerosolized Nipah Virus.

Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.

Host gene expression profiles in ferrets infected with genetically distinct henipavirus strains.

Henipavirus infection causes severe respiratory and neurological disease in humans that can be fatal. To characterize the pathogenic mechanisms of henipavirus infection in vivo, we performed experimental infections in ferrets followed by genome-wide gene expression analysis of lung and brain tissues. The Hendra, Nipah-Bangladesh, and Nipah-Malaysia strains caused severe respiratory and neurological disease with animals succumbing around 7 days post infection. Despite the presence of abundant viral shedding, animal-to-animal transmission did not occur. The host gene expression profiles of the lung tissue showed early activation of interferon responses and subsequent expression of inflammation-related genes that coincided with the clinical deterioration. Additionally, the lung tissue showed unchanged levels of lymphocyte markers and progressive downregulation of cell cycle genes and extracellular matrix components. Infection in the brain resulted in a limited breadth of the host responses, which is in accordance with the immunoprivileged status of this organ. Finally, we propose a model of the pathogenic mechanisms of henipavirus infection that integrates multiple components of the host responses.

Contribution of Human Lung Parenchyma and Leukocyte Influx to Oxidative Stress and Immune System-Mediated Pathology following Nipah Virus Infection.

Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD-SCID/γ mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the bone marrow, liver, and thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections. Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system-reconstituted mice in order to identify the overall effect of immune cells on NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in lung grafts. In addition, the presence of human leukocytes increased the expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI and inform efforts to identify therapeutic targets.IMPORTANCE Nipah virus (NiV) is an emerging paramyxovirus that can cause a lethal respiratory and neurological disease in humans. Only limited data are available on NiV pathogenesis in the human lung, and the relative contribution of the innate immune response and NiV to acute lung injury (ALI) is still unknown. Using human lung grafts in a human immune system-reconstituted mouse model, we showed that the NiV Bangladesh strain induced cytopathic lesions in lung grafts similar to those described in patients irrespective of the donor origin or the presence of leukocytes. However, the human immune system interfered with virus spread, responded to infection by leukocyte infiltration in the small airways and alveolar area, induced oxidative stress, and triggered the production of cytokines and chemokines that regulate inflammatory influx by leukocytes in response to infection. Understanding how leukocytes interact with NiV and cause ALI in human lung xenografts is crucial for identifying therapeutic targets.

Broad-Range Antiviral Activity of Hydrogen Sulfide Against Highly Pathogenic RNA Viruses.

Hydrogen sulfide is an important endogenous mediator that has been the focus of intense investigation in the past few years, leading to the discovery of its role in vasoactive, cytoprotective and anti-inflammatory responses. Recently, we made a critical observation that H2S also has a protective role in paramyxovirus infection by modulating inflammatory responses and viral replication. In this study we tested the antiviral and anti-inflammatory activity of the H2S slow-releasing donor GYY4137 on enveloped RNA viruses from Ortho-, Filo-, Flavi- and Bunyavirus families, for which there is no FDA-approved vaccine or therapeutic available, with the exception of influenza. We found that GYY4137 significantly reduced replication of all tested viruses. In a model of influenza infection, GYY4137 treatment was associated with decreased expression of viral proteins and mRNA, suggesting inhibition of an early step of replication. The antiviral activity coincided with the decrease of viral-induced pro-inflammatory mediators and viral-induced nuclear translocation of transcription factors from Nuclear Factor (NF)-kB and Interferon Regulatory Factor families. In conclusion, increasing cellular H2S is associated with significant antiviral activity against a broad range of emerging enveloped RNA viruses, and should be further explored as potential therapeutic approach in relevant preclinical models of viral infections.

Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

Oxidative stress in Nipah virus-infected human small airway epithelial cells.

Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.

Development and Characterization of Broadly Cross-reactive Monoclonal Antibodies Against All Known Ebolavirus Species.

As of 25 March 2015, the largest recorded outbreak of Ebola virus infection is ongoing, with almost 25 000 cases and >10 000 deaths. There are 5 genetically and antigenically distinct species within the genus Ebolavirus. Limited cross-reactivity and protection is observed between these 5 Ebolavirus species, which complicates vaccine development. However, on the basis of sequence homology between the 5 Ebolavirus species, we hypothesize that conserved epitopes are present on the viral glycoprotein (GP), which can be targeted by antibodies. In the current study, a panel of mouse monoclonal antibodies was isolated and characterized using an enzyme-linked immunosorbent assay (ELISA) to determine cross-reactivity, avidity, and competition for epitope binding; Western blot analysis was also performed. Four monoclonal antibodies were identified by ELISA as cross-reacting with the GPs of all 5 Ebolavirus species. The identification of cross-reactive antibodies that bind the GPs of all known Ebolavirus species will give us important insight into the presence of conserved epitopes on the viral GP. These data will be crucial for the development of novel therapeutics and diagnostic assays.

Pathogenesis of Hendra and Nipah virus infection in humans.

Hendra virus (HeV) and Nipah virus (NiV) are emerging zoonotic viruses that cause severe and often lethal respiratory illness and encephalitis in humans. Henipaviruses can infect a wide range of species and human-to-human transmission has been observed for NiV. While the exact route of transmission in humans is not known, experimental infection in different animal species suggests that infection can be efficiently initiated after respiratory challenge. The limited data on histopathological changes in fatal human cases of HeV and NiV suggest that endothelial cells are an important target during the terminal stage of infection; however, it is unknown where these viruses initially establish infection and how the virus disseminates from the respiratory tract to the central nervous system and other organs. Here we review the current concepts in henipavirus pathogenesis in humans.

Henipavirus pathogenesis in human respiratory epithelial cells.

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.

Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.