Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the central nervous system (CNS), were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, VEEV genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates.

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques.

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2-3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.

SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts.

Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an species to study mild/subclinical COVID-19 disease and with possible insights into live virus shedding. Future vaccine evaluation can be performed in AGM with correlates of efficacy being lung lesions by PET/CT, virus shedding, and tissue viral load.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients.

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Cooperativity between the 3' untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3' untranslated region (3' UTR) of the virus. Three of the sites are "canonical" with all 7 seed sequence residues complimentary to miR-142-3p while one is "non-canonical" and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.

Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants.

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.

The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent.

Interferon alpha/beta (IFN-α/ß) is a critical mediator of protection against most viruses, with host survival frequently impossible in its absence. Many studies have investigated the pathways involved in the induction of IFN-α/ß after virus infection and the resultant upregulation of antiviral IFN-stimulated genes (ISGs) through IFN-α/ß receptor complex signaling. However, other than examining the effects of genetic deletion of induction or effector pathway components, little is known regarding the functionality of these responses in intact hosts and whether host genetic or environmental factors might influence their potency. Here, we demonstrate that the IFN-α/ß response against multiple arthropod-vectored viruses, which replicate over a wide temperature range, is extremely sensitive to fluctuations in temperature, exhibiting reduced antiviral efficacy at subnormal cellular temperatures and increased efficacy at supranormal temperatures. The effect involves both IFN-α/ß and ISG upregulation pathways with a major aspect of altered potency reflecting highly temperature-dependent transcription of IFN response genes that leads to altered IFN-α/ß and ISG protein levels. Discordantly, signaling steps prior to transcription that were examined showed the opposite effect from gene transcription, with potentiation at low temperature and inhibition at high temperature. Finally, we demonstrate that by lowering the temperature of mice, chikungunya arbovirus replication and disease are exacerbated in an IFN-α/ß-dependent manner. This finding raises the potential for use of hyperthermia as a therapeutic modality for viral infections and in other contexts such as antitumor therapy. The increased IFN-α/ß efficacy at high temperatures may also reflect an innate immune-relevant aspect of the febrile response.IMPORTANCE The interferon alpha/beta (IFN-α/ß) response is a first-line innate defense against arthropod-borne viruses (arboviruses). Arboviruses, such as chikungunya virus (CHIKV), can infect cells and replicate across a wide temperature range due to their replication in both mammalian/avian and arthropod hosts. Accordingly, these viruses can cause human disease in tissues regularly exposed to temperatures below the normal mammalian core temperature, 37°C. We questioned whether temperature variation could affect the efficacy of IFN-α/ß responses against these viruses and help to explain some aspects of human disease manifestations. We observed that IFN-α/ß efficacy was dramatically lower at subnormal temperatures and modestly enhanced at febrile temperatures, with the effects involving altered IFN-α/ß response gene transcription but not IFN-α/ß pathway signaling. These results provide insight into the functioning of the IFN-α/ß response in vivo and suggest that temperature elevation may represent an immune-enhancing therapeutic modality for a wide variety of IFN-α/ß-sensitive infections and pathologies.

What the radiologist needs to know about urolithiasis: part 1--pathogenesis, types, assessment, and variant anatomy.

OBJECTIVE: This article reviews types of urinary calculi and their imaging appearances, presents direct and secondary imaging findings of urolithiasis, and provides an overview of treatment methods. Pertinent imaging findings that impact clinical management are highlighted. The implications of complex or variant genitourinary anatomy are reviewed. We outline a standard format for the reporting of urolithiasis to facilitate informed clinical management decisions. CONCLUSION: Unenhanced CT is the preferred examination for evaluation of urolithiasis because of its availability, ease of performance, and high sensitivity. An awareness of the important imaging findings to report allows appropriate and efficient therapy.

What the radiologist needs to know about urolithiasis: part 2--CT findings, reporting, and treatment.

OBJECTIVE: This article reviews types of urinary calculi and their imaging appearances, presents direct and secondary imaging findings of urolithiasis, and provides an overview of treatment methods. Pertinent imaging findings that affect clinical management are highlighted. The implications of complex or variant genitourinary anatomy are reviewed. We outline a standard format for the reporting of urolithiasis to facilitate informed clinical management decisions. CONCLUSION: Unenhanced CT is the preferred examination for evaluation of urolithiasis because of its availability, ease of performance, and high sensitivity. An awareness of the important imaging findings to report allows appropriate and efficient therapy.

Enhancement of anti-DIII antibodies by the C3d derivative P28 results in lower viral titers and augments protection in mice.

Antibodies generated against West Nile virus (WNV) during infection are essential for controlling dissemination. Recent studies have demonstrated that epitopes in all three domains of the flavivirus envelope protein (E) are targets for neutralizing antibodies, with determinants in domain III (DIII) eliciting antibodies with strong inhibitory properties. In order to increase the magnitude and quality of the antibody response against the WNV E protein, DNA vaccines with derivatives of the WNV E gene (full length E, truncated E, or DIII region, some in the context of the pre-membrane [prM] gene) were conjugated to the molecular adjuvant P28. The P28 region of the complement protein C3d is the minimum CR2-binding domain necessary for the adjuvant activity of C3d. Delivery of DNA-based vaccines by gene gun and intramuscular routes stimulated production of IgG antibodies against the WNV DIII region of the E protein. With the exception of the vaccine expressing prM/E given intramuscularly, only mice that received DNA vaccines by gene gun produced protective neutralizing antibody titers (FRNT80 titer >1/40). Correspondingly, mice vaccinated by the gene gun route were protected to a greater level from lethal WNV challenge. In general, mice vaccinated with P28-adjuvated vaccines produced higher IgG titers than mice vaccinated with non-adjuvanted vaccines.

C3d adjuvant activity is reduced by altering residues involved in the electronegative binding of C3d to CR2.

The final degradation product of the complement protein C3, C3d, has been used as a molecular adjuvant to various antigens. Chimera proteins of the antigen and multiple copies of C3d were developed to test the adjuvant effect of this molecule. The main mechanism by which C3d enhances the immune response is interaction with CR2. In vitro studies showed that the avidity of C3d for CR2 is affected by residues located at the interacting surface (e.g. 170N) as well as by residues located in other areas. The role of the latter residues has been proposed to depend on the electrostatic nature of the C3d-CR2 interaction, where the charges of the whole molecules are responsible for their binding. C3d is primarily a negatively charged molecule, while CR2 is a positive one. Previous experiments demonstrated that elimination of a positive charge (K162A) in C3d enhanced its avidity for CR2, while elimination of negative charges or addition positives ones (D163A, N170R, respectively), impaired the avidity for CR2. Despite the extensive in vitro research, the role of these residues in the adjuvant effect of C3d is unclear. To study the role of residues at the interacting and non-interacting surface of C3d on the adjuvanticity, single as well as a double residue substitutions were engineered in the murine C3d (R162A, D163A, N170R and D163A-N170R) gene. Two copies of these mutant molecules were fused to HIV-1 Env(gp120) and the proteins were tested for their avidity to bind CR2 (sCR2). Later, these DNA constructs were tested in mice to determine their adjuvant capability. Mutation at residue 162 (R162A) neither enhanced nor impaired the avidity of Env(gp120)-C3d(2) for sCR2 in vitro. Mutations at residues D163A and N170R, on the other hand, reduced the binding affinity of Env(gp120)-C3d(2) for sCR2. Furthermore, these mutations synergized and abolished the interaction of C3d for CR2. The data correlated with the adjuvant capability of these molecules in the mouse model. In summary, residues that alter the electronegative status of C3d (D163A and N170R) impair the binding of chimera proteins to CR2, reducing the adjuvant activity of this molecule.

Local delivery of osteoprotegerin inhibits mechanically mediated bone modeling in orthodontic tooth movement.

INTRODUCTION: The RANKL-OPG axis is a key regulator of osteoclastogenesis and bone turnover activity. Its contribution to bone resorption under altered mechanical states, however, has not been fully elucidated. Here we examined the role of OPG in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. METHODS: The maxillary first molars of male Sprague-Dawley rats were moved mesially using a calibrated nickel-titanium spring attached to the maxillary incisor teeth. Two different doses (0.5 mg/kg, 5.0 mg/kg) of a recombinant fusion protein (OPG-Fc), were injected twice weekly mesial to the first molars. Tooth movement was measured using stone casts that were scanned and magnified. Changes in bone quantity were measured using micro-computed tomography and histomorphometric analysis was used to quantify osteoclasts and volumetric parameters. Finally, circulating levels of TRAP-5b (a bone resorption marker) was measured using enzyme-linked immunosorbent assay. RESULTS: The 5.0 mg/kg OPG-Fc dose showed a potent reduction in mesial molar movement and osteoclast numbers compared to controls (p<0.01). The molar movement was inhibited by 45.7%, 70.6%, and 78.7% compared to controls at days 7, 14, and 21 respectively, with the high dose of OPG. The 0.5 mg dose also significantly (p<0.05) inhibited molar movement at days 7 (43.8%) and 14 (31.8%). While incisor retraction was also decreased by OPG-Fc, the ratio of incisor to molar tooth movement was markedly better in the high-dose OPG group (5.2:1, p<0.001) compared to the control group (2.3:1) and the low-dose OPG group (2.0:1). CONCLUSIONS: Local delivery of OPG-Fc inhibits osteoclastogenesis and tooth movement at targeted dental sites.

Case report: subcapsular hepatic hematoma: retraction injury during laparoscopic adrenalectomy.

We present a case of subcapsular hepatic hematoma resulting from retraction of the liver during laparoscopic adrenalectomy. We discuss the management and prevention of this rare and important complication.