Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

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.

Nipah Virus Bangladesh Infection Elicits Organ-Specific Innate and Inflammatory Responses in the Marmoset Model.

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

Differences in Genetic Diversity of Mammalian Tick-Borne Flaviviruses.

The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.

Evaluations of rationally designed rift valley fever vaccine candidate RVax-1 in mosquito and rodent models.

Rift Valley fever (RVF) is a mosquito-borne zoonosis endemic to Africa and the Arabian Peninsula, which causes large outbreaks among humans and ruminants. Single dose vaccinations using live-attenuated RVF virus (RVFV) support effective prevention of viral spread in endemic countries. Due to the segmented nature of RVFV genomic RNA, segments of vaccine strain-derived genomic RNA could be incorporated into wild-type RVFV within co-infected mosquitoes or animals. Rationally designed vaccine candidate RVax-1 displays protective epitopes fully identical to the previously characterized MP-12 vaccine. Additionally, all genome segments of RVax-1 contribute to the attenuation phenotype, which prevents the formation of pathogenic reassortant strains. This study demonstrated that RVax-1 cannot replicate efficiently in orally fed Aedes aegypti mosquitoes, while retaining strong immunogenicity and protective efficacy in an inbred mouse model, which were indistinguishable from the MP-12 vaccine. These findings support further development of RVax-1 as the next generation MP-12-based vaccine for prevention of Rift Valley fever in humans and animals.

Five years of outpatient parenteral antibiotic therapy with ceftriaxone in the paediatric emergency department: what clinical features are associated with need for admission?

BACKGROUND: More children presenting to Emergency Departments (EDs) with acute infections are now directly referred for outpatient parenteral antibiotic therapy (OPAT). Sparse data exist on what clinical features in these children are associated with OPAT failure. We hypothesised that children who were younger or presented with systemic features of infection would be more likely to need admission. METHODS: We conducted a service evaluation over a 5-year period (12 September 2015-12 September 2020) at a single UK tertiary centre paediatric ED formally known as the Royal Hospital for Sick Children Edinburgh. All children referred from the ED for OPAT with ceftriaxone were included. OPAT failure was defined as a decision by a senior clinician of need for admission. Univariate statistical testing and multivariate logistic regression modelling were performed. RESULTS: 754 children received OPAT in the ED over a 5-year period. 95 children (13%) required admission for inpatient management. Need for admission was independently associated with having a positive blood culture (adjusted OR (aOR) 8.9; 95% CI 1.49 to 47; p=0.01) or an ultrasound performed (aOR 6.8; 95% CI 3.74 to 12.7; p<0.001). We observed no significant association between age and systemic features (fever, white cell count or C reactive protein) with need for admission in our multivariate analysis. CONCLUSION: In children presenting with acute infections to our paediatric ED who were deemed suitable by senior clinicians to be managed using OPAT with ceftriaxone, younger age (above 3 months) and the presence of systemic features were not independently associated with need for admission. Overall, our service was safe and no child came to harm from early ambulation during this evaluation.

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.

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.

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

Tilorone-Dihydrochloride Protects against Rift Valley Fever Virus Infection and Disease in the Mouse Model.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East that can affect humans and ruminant livestock. Currently, there are no approved vaccines or therapeutics for the treatment of severe RVF disease in humans. Tilorone-dihydrochloride (Tilorone) is a broad-spectrum antiviral candidate that has previously shown efficacy against a wide range of DNA and RNA viruses, and which is clinically utilized for the treatment of respiratory infections in Russia and other Eastern European countries. Here, we evaluated the antiviral activity of Tilorone against Rift Valley fever virus (RVFV). In vitro, Tilorone inhibited both vaccine (MP-12) and virulent (ZH501) strains of RVFV at low micromolar concentrations. In the mouse model, treatment with Tilorone significantly improved survival outcomes in BALB/c mice challenged with a lethal dose of RVFV ZH501. Treatment with 30 mg/kg/day resulted in 80% survival when administered immediately after infection. In post-exposure prophylaxis, Tilorone resulted in 30% survival at one day after infection when administered at 45 mg/kg/day. These findings demonstrate that Tilorone has potent antiviral efficacy against RVFV infection in vitro and in vivo and supports further development of Tilorone as a potential antiviral therapeutic for treatment of RVFV infection.

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis.

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT 50 ) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT 50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. IMPORTANCE: As COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. ARTICLE SUMMARY: A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro .

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.

A single-cycle replicable Rift Valley fever phlebovirus vaccine carrying a mutated NSs confers full protection from lethal challenge in mice.

Rift Valley fever phlebovirus (RVFV) is a pathogen of Rift Valley fever, which is a mosquito-borne zoonotic disease for domestic livestock and humans in African countries. Currently, no approved vaccine is available for use in non-endemic areas. The MP-12 strain is so far the best live attenuated RVFV vaccine candidate because of its good protective efficacy in animal models. However, there are safety concerns for use of MP-12 in humans. We previously developed a single-cycle replicable MP-12 (scMP-12) which lacks NSs gene and undergoes only a single round of viral replication because of its impaired ability to induce membrane-membrane fusion. In the present study, we generated an scMP-12 mutant (scMP-12-mutNSs) carrying a mutant NSs, which degrades double-stranded RNA-dependent protein kinase R but does not inhibit host transcription. Immunization of mice with a single dose (105 PFU) of scMP-12-mutNSs elicited RVFV neutralizing antibodies and high titers of anti-N IgG production and fully protected the mice from lethal wild-type RVFV challenge. Immunogenicity and protective efficacy of scMP-12-mutNSs were better than scMP-12, demonstrating that scMP-12-mutNSs is a more efficacious vaccine candidate than scMP-12. Furthermore, our data suggested that RVFV vaccine efficacy can be improved by using this specific NSs mutant.

Natural History and Pathogenesis of Wild-Type Marburg Virus Infection in STAT2 Knockout Hamsters.

Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.

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.

Favipiravir (T-705) protects against Nipah virus infection in the hamster model.

Nipah and Hendra viruses are recently emerged bat-borne paramyxoviruses (genus Henipavirus) causing severe encephalitis and respiratory disease in humans with fatality rates ranging from 40-75%. Despite the severe pathogenicity of these viruses and their pandemic potential, no therapeutics or vaccines are currently approved for use in humans. Favipiravir (T-705) is a purine analogue antiviral approved for use in Japan against emerging influenza strains; and several phase 2 and 3 clinical trials are ongoing in the United States and Europe. Favipiravir has demonstrated efficacy against a broad spectrum of RNA viruses, including members of the Paramyxoviridae, Filoviridae, Arenaviridae families, and the Bunyavirales order. We now demonstrate that favipiravir has potent antiviral activity against henipaviruses. In vitro, favipiravir inhibited Nipah and Hendra virus replication and transcription at micromolar concentrations. In the Syrian hamster model, either twice daily oral or once daily subcutaneous administration of favipiravir for 14 days fully protected animals challenged with a lethal dose of Nipah virus. This first successful treatment of henipavirus infection in vivo with a small molecule drug suggests that favipiravir should be further evaluated as an antiviral treatment option for henipavirus infections.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model.

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2-3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.

Attenuation and protective efficacy of Rift Valley fever phlebovirus rMP12-GM50 strain.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the differentiation from wild-type RVFV strains. In this study, we aimed to strengthen the attenuation profile of the MP-12 vaccine strain via the introduction of 584 silent mutations. To minimize the impact on protective efficacy, codon usage and codon pair bias were not de-optimized. The resulting rMP12-GM50 strain showed 100% protective efficacy with a single intramuscular dose, raising a 1:853 mean titer of plaque reduction neutralization test. Moreover, outbred mice infected with one of three pathogenic reassortant ZH501 strains, which encoded rMP12-GM50 L-, M-, or S-segments, showed 90%, 50%, or 30% survival, respectively. These results indicate that attenuation of the rMP12-GM50 strain is significantly attenuated via the L-, M-, and S-segments. Recombinant RVFV vaccine strains encoding similar silent mutations will be also useful for the surveillance of reassortant strains derived from vaccine strains in endemic countries.

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.

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.