The effects of a grain-based subacute ruminal acidosis (SARA) challenge (GBSC) and an alfalfa-pellet SARA challenge (APSC) on fermentation and endotoxins in the rumen and in the cecum, as well as on endotoxins in peripheral blood, were determined. Six nonlactating Holstein cows with cannulas in the rumen and cecum were used in the study. A 3×3 Latin square arrangement of treatments with 4-wk experimental periods was adopted. During the first 3 wk of each experimental period, all cows received a diet containing 70% forages [dry matter (DM) basis]. In wk 4 of each period, cows received 1 of the following 3 diets: the 70% forage diet fed during wk 1 to 3 (control), a diet in which 34% of the dietary DM was replaced with grain pellets made of 50% ground wheat and 50% ground barely (GBSC), or a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen pH was monitored continuously using indwelling pH probes, and rumen fluid, blood, cecal digesta, and fecal grab samples were collected immediately before feed delivery at 0900 h and at 6 h after feed delivery on d 3 and 5 of wk 4. The time for which rumen pH was below 5.6 was 56.4, 225.2, and 298.8 min/d for the control, APSC, and GBSC treatments, respectively. Compared with the control, SARA challenges resulted in similar reductions in cecal digesta pH, which were 7.07, 6.86, and 6.79 for the control, APSC, and GBSC treatments, respectively. Compared with the control, only GBSC increased starch content in cecal digesta, which averaged 2.8, 2.6, and 7.4% of DM for the control, APSC, and GBSC, respectively. Free lipopolysaccharide endotoxin (LPS) concentration in rumen fluid increased from 10,405 endotoxin units (EU)/mL in the control treatment to 30,715 and 168,391 EU/mL in APSC and GBSC, respectively. Additionally, GBSC increased the LPS concentration from 16,508 to 118,522 EU/g in wet cecal digesta, and from 12,832 to 93,154 EU/g in wet feces. The APSC treatment did not affect LPS concentrations in cecal digesta and feces. All concentrations of LPS in blood plasma were below the detection limit of >0.05 EU/mL of the technique used. Despite the absence of LPS in blood, only GBSC increased the concentration of LPS-binding protein in blood plasma, which averaged, 8.9, 9.5, and 12.1mg/L for the control, APSC, and GBSC treatments, respectively. This suggests that GBSC caused translocation of LPS from the digestive tract but that LPS was detoxified before entering the peripheral blood circulation. The higher LPS concentration in cecal digesta in the GBSC compared with the APSC suggests a higher risk of LPS translocation in the large intestine in GBSC than in APSC.

SUMMARY Previous efforts to identify cross-neutralizing antibodies to the receptor binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS, cross-neutralizes Ebola (EBOV), Sudan (SUDV), and to a lesser extent Bundibugyo viruses, and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™, is remarkably effective against EBOV (Zaire), but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.

COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection is a disease affecting several organ systems. A model that captures all clinical symptoms of COVID-19 as well as long-haulers disease is needed. We investigated the host responses associated with infection in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines (IL-6, IL-1beta, and TNF) and type II interferons whereas type I interferons were inhibited. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of myocarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2 (PASC).

The 2014–15 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp™, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. Here we show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV, variant Makona, the virus responsible for the ongoing 2014–15 outbreak, while a similar formulation of ZMapp™ protected 2 of 3 NHPs. The chimeric MIL77E mAb cocktail is produced in engineered CHO cells and is based on mAbs c13C6 and c2G4 from ZMapp™. The use of only 2 antibodies in MIL77E opens the door to a pan-ebolavirus cocktail.

Bundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25 to 36%. Despite having been known to the scientific and medical communities for almost 1 decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the order Mononegavirales. To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8 and 9 days, and a petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts and dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological, and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to what was seen with BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates and will accelerate efforts to understand filovirus pathogenesis and transmission as well as the development of specific vaccines and antivirals. IMPORTANCEThe 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genus Ebolavirus and has caused outbreaks in the past but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines and therapies and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection as well as to Ebola virus infection and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to that seen in humans and nonhuman primates. We believe that this will improve the ability to study BDBV and provide a platform to test vaccines and therapeutics.

Ebola outbreaks occur on a frequent basis, with the 2014-2015 outbreak in West Africa being the largest one ever recorded. This outbreak has resulted in over 11,000 deaths in four African countries and has received international attention and intervention. Although there are currently no approved therapies or vaccines, many promising candidates are undergoing clinical trials, and several have had success in promoting recovery from Ebola. However, these prophylactics and therapeutics have been designed and tested only against the same species of Ebola virus as the one causing the current outbreak. Future outbreaks involving other species would require reformulation and possibly redevelopment. Therefore, a broad-spectrum alternative is highly desirable. We have found that a flavonoid derivative called quercetin 3-␤-O-D-glucoside (Q3G) has the ability to protect mice from Ebola even when given as little as 30 min prior to infection. Furthermore, we have demonstrated that this compound targets the early steps of viral entry. Most promisingly, antiviral activity against two distinct species of Ebola virus was seen. This study serves as a proof of principle that Q3G has potential as a prophylactic against Ebola virus infection.

Infections with Sudan virus (SUDV), a member of the genus Ebolavirus, result in a severe hemorrhagic fever with a fatal outcome in over 50% of human cases. The paucity of prophylactics and therapeutics against SUDV is attributed to the lack of a small-animal model to screen promising compounds. By repeatedly passaging SUDV within the livers and spleens of guinea pigs in vivo, a guinea pig-adapted SUDV variant (SUDV-GA) uniformly lethal to these animals, with a 50% lethal dose (LD 50 ) of 5.3 ؋ 10 ؊2 50% tissue culture infective doses (TCID 50 ), was developed. Animals infected with SUDV-GA developed high viremia and died between 9 and 14 days postinfection. Several hallmarks of SUDV infection, including lymphadenopathy, increased liver enzyme activities, and coagulation abnormalities, were observed. Virological analyses and gross pathology, histopathology, and immunohistochemistry findings indicate that SUDV-GA replicates in the livers and spleens of infected animals similarly to SUDV infections in nonhuman primates. These developments will accelerate the development of specific medical countermeasures in preparation for a future disease outbreak due to SUDV. IMPORTANCE A disease outbreak due to Ebola virus (EBOV), suspected to have emerged duringDecember 2013 in Guinea, with over 11,000 dead and 28,000 infected, is finally winding down. Experimental EBOV vaccines and treatments were administered to patients under compassionate circumstances with promising results, and availability of an approved countermeasure appears to be close. However, the same range of experimental candidates against a potential disease outbreak caused by other members of the genus Ebolavirus, such as Sudan virus (SUDV), is not readily available. One bottleneck contributing to this situation is the lack of a small-animal model to screen promising drugs in an efficient and economical manner. To address this, we have generated a SUDV variant (SUDV-GA) that is uniformly lethal to guinea pigs. Animals infected with SUDV-GA develop disease similar to that of SUDV-infected humans and monkeys. We believe that this model will significantly accelerate the development of lifesaving measures against SUDV infections.