Bioluminescent images of viral replication in live animals (in vivo) reveal disease dynamics and effects of medical countermeasures over time. After selecting an appropriate orthopoxvirus animal model for the study, a recombinant virus with the firefly luciferase gene inserted in the genome is used to infect the animals. On the day of bioluminescent imaging, the substrate, D-luciferin, is prepared; animals are sedated and injected with the substrate and IVIS imager is utilized; various bioluminescent images are acquired; then animals recover and are able to continue in the study. Ex vivo imaging can also be completed after animals are euthanized at experimental endpoint. This approach allows real-time imaging of viral kinetics within an animal, and analysis of images can provide an additional quantitative measure throughout the study. Bioluminescent imaging not only provides scientific benefits but also benefits to animal welfare. For these reasons, bioluminescent imaging should be considered for any in vivo orthopoxvirus study.

The antiviral drug tecovirimat* has been used extensively to treat U.S. mpox cases since the start of a global outbreak in 2022. Mutations in the mpox viral protein target (F13 or VP37) that occur during treatment can result in resistance to tecovirimat† (1,2). CDC and public health partners have conducted genetic surveillance of monkeypox virus (MPXV) for F13 mutations through sequencing and monitoring of public databases. MPXV F13 mutations associated with resistance have been reported since 2022, typically among severely immunocompromised mpox patients who required prolonged courses of tecovirimat (3-5). A majority of patients with infections caused by MPXV with resistant mutations had a history of tecovirimat treatment; however, spread of tecovirimat-resistant MPXV was reported in California during late 2022 to early 2023 among persons with no previous tecovirimat treatment (3). This report describes a second, unrelated cluster of tecovirimat-resistant MPXV among 18 persons with no previous history of tecovirimat treatment in multiple states.

Increasing food prices have implications for basic subsistence, have a strong price visibility and symbolic value, and are characterized by high volatility and inelasticity of demand. Research thus assumes that food price is an important trigger for unrest. Yet, whether food is an especially potent driver for people's willingness to engage in collective action, or whether it concerns grievances about general inflation, is unknown. Does food have a greater effect on the willingness to participate in unrest? The paper investigates the relative importance of food in mobilization potential by using unique data from a survey experiment in Johannesburg, South Africa. The experiment collects information on how price increases in food, fuel, and electricity affects respondents' willingness to engage in unrest. The results show a higher willingness to engage in collective action when presented with increasing living expenses, regardless of whether it is food, fuel or electricity, compared to stable prices. We also consider the level of risk exposure to price hikes, and find that those who report going hungry in the last year have a higher willingness to engage in unrest than those who do not. Thus, food access influences the willingness to partake in unrest during price hikes, also for commodities seemingly unrelated to food. This suggests that for those who are most affected by a price hike it is less important what type of commodity it is. The question is whether it introduces further strain on an already hard-stretched budget. Los precios en aumento de los productos de alimentacion tienen implicaciones sobre la subsistencia basica. Ademas, estos precios en aumento tambien tienen una fuerte visibilidad en materia de precio y un valor simbolico, y se caracterizan por una alta volatilidad e inelasticidad de la demanda. Por lo tanto, la investigacion asume que el precio de los alimentos es un desencadenante importante de agitacion social. Sin embargo, no sabemos si el precio de los alimentos es una causa lo suficientemente relevante para que las personas decidan participar en acciones colectivas, o si estas acciones estan mas relacionadas con quejas sobre la inflacion general. Tienen los precios de los alimentos un mayor efecto sobre la disposicion de la poblacion a participar en disturbios sociales? Este articulo investiga este asunto utilizando datos unicos de un experimento de encuesta en Johannesburgo, Sudafrica. Este experimento recoge informacion sobre como los aumentos de precios de los alimentos, del combustible y de la electricidad influyen sobre la voluntad de los encuestados a participar en disturbios sociales. Los resultados muestran una mayor disposicion a participar en acciones colectivas cuando existe un aumento de los costes de vida, pero este efecto no es mas fuerte para el precio de los alimentos en comparacion con otros productos basicos. Tambien tenemos en cuenta el nivel de exposicion al riesgo de las subidas de precios, y descubrimos que quienes declaran haber pasado hambre en el ultimo ano tienen una mayor disposicion a participar en disturbios sociales que los que no lo hacen. Por lo tanto, el acceso a los alimentos tiene potencial en materia de movilizacion, no solo debido a los precios, sino tambien en terminos de como influye en la disposicion a participar en disturbios sociales durante los aumentos de precios. Esto tambien ocurre para productos basicos aparentemente no relacionados con los alimentos. Esto sugiere que para aquellas personas que se ven mas afectadas por un aumento de precios, el tipo de producto no resulta tan importante. La pregunta que nos hacemos es si esto crea mas presion sobre una situacion que ya es vulnerable y sobre un presupuesto muy ajustado. La hausse des prix de l'alimentation entraine des consequences pour les moyens de subsistance de base. Ces prix sont particulierement visibles et leur valeur -symbolique, mais ils se caracterisent egalement par une volatilite elevee et un manque d'elasticite de la demande. Aussi la recherche presuppose-t-elle que le prix de l'alimentation constitue un facteur declencheur important d'agitation urbaine. Pourtant, nous ne savons pas avec certitude si l'alimentation, a elle seule, pousse effectivement les gens a l'action collective, ou s'il s'agit davantage d'un mecontentement quant a l'inflation generale. L'alimentation incite-t-elle davantage a l'action ? Cet article s'interesse a cette question a l'aide de donnees uniques issues d'une experience de sondage a Johannesburg, en Afrique du Sud. L'experience recueille des informations sur l'effet de l'augmentation des prix de la nourriture, du carburant et de l'electricite sur le desir du participant de se joindre a l'agitation. D'apres les resultats, ce desir augmente quand le cout de la vie augmente, mais l'effet ne se renforce pas pour le prix de la nourriture, compare a d'autres marchandises. En prenant aussi en compte le niveau d'exposition au risque de hausse des prix, nous observons que les personnes signalant avoir souffert de la faim durant l'annee passee sont plus enclines a participer a l'agitation sociale que les autres. Ainsi, l'acces a la nourriture possede un potentiel de mobilisation vis-a-vis de son prix, mais aussi parce qu'il a une incidence sur la volonte de prendre part a l'agitation lors des hausses de prix, et meme pour des marchandises qui n'auraient aucun rapport avec la nourriture. Aussi, pour les personnes les plus affectees par les hausses de prix, le type de marchandise aurait une importance moindre. Une questions apparait alors : ces situations de vulnerabilite et d'insuffisance budgetaire ne s'en trouvent-elles pas aggravees ?

We assessed mpox virus prevalence in blood, pharyngeal, and rectal specimens among persons without characteristic rash presenting for JYNNEOS vaccine. Our data indicate that the utility of risk-based screening for mpox in persons without skin lesions or rash via pharyngeal swabs, rectal swabs, and/or blood is likely limited.

Background During the 2022 multinational outbreak of monkeypox virus (MPXV) clade IIb, the antiviral drug tecovirimat (TPOXX®) was deployed in the US on a large scale for the first time ever. The MPXV F13L gene homolog encodes the target of tecovirimat, and single amino acid changes in the F13 protein are known to cause resistance to tecovirimat in orthopoxviruses (OPXV).Methods Whole genome metagenomic sequencing and amplicon-based sequencing targeting the F13L gene was used to identify nine mutations previously reported to cause resistance in other OPXV along with ten novel mutations that have been identified from the 2022 mpox outbreak. A cytopathic effect assay, previously established at CDC as part of WHO smallpox research, was adapted to MPXV for tecovirimat phenotype testing of virus isolated from mpox patients.Results As of March 2023, in total, 70 isolates from 40 patients were tested, and 50 of these isolates from 26 patients were found to have a resistant phenotype. Most resistant isolates were associated with severely immunocompromised mpox patients on multiple courses of TPOXX treatment; while isolates with F13 mutations identified by routine surveillance of patients not treated with TPOXX have remained sensitive.Conclusions These data indicate that tecovirimat resistance is developing in immunocompromised patients treated with TPOXX and that for isolates that we have analyzed, the frequency of resistant viruses remain relatively low (< 1%) compared to the total number of patients treated with TPOXX. These findings inform our understanding of when tecovirimat resistance is likely to occur and highlight the need for additional OPXV therapeutics.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study did not receive any funding### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The activities in this report were reviewed by the Human Subjects Advisor in the National Center for Emerging and Zoonotic Disease at the Centers for Disease Control and Prevention and determined that it does not meet the regulatory definition of research under provision 45 CFR 46.102(l); the activities fall under public health surveillance and do not require IRB review.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Tecovirimat (also known as TPOXX or ST-246) is a drug available for the treatment of mpox through the Centers for Disease Control and Prevention’s Expanded Access Investigational New Drug “compassionate use” protocol (https://www.cdc.gov/poxvirus/ monkeypox/clinicians/Tecovirimat.html). In Los Angeles County, a fatal case of mpox with tecovirimat resistance was previously reported (1). Epidemiologic surveillance in Los Angeles County has since identified additional cases of severe mpox that did not improve after multiple rounds of tecovirimat treatment, including one involving a person who succumbed to infection (Table 1). Consistent with reports describing severe manifestations of mpox within the current global outbreak (1, 2), the identified cases involved host immunodeficiency due to advanced HIV infection. Tecovirimat targets the conserved orthopoxvirus VP37 envelope protein required for extracellular virus particle generation, and previous studies from monkeypox virus (MPXV) and other orthopoxviruses identified more than 20 mutations in VP37 associated with tecovirimat resistance (https://www.fda.gov/emergency-preparedness-and-response/mcm-issues/ fda-mpox-response#therapeutics) (1, 3). To determine whether these identified cases involved tecovirimat resistance, MPXV specimens were subjected to whole-genome sequencing and examined for mutations in the homolog of the vaccinia virus Copenhagen F13L gene that encodes VP37 protein (1, 4). All specimens encoded lysine at amino acid position 353, which is prevalent within the current outbreak and does not affect tecovirimat sensitivity (5). Specimens from six cases harbored a cumulative total of eight VP37 mutations associated with resistance in prior studies: H238Q, P243S, N267D, A288P, A290V, D294V, A295E, and I372N (Table 2). Five previously undescribed mutations of unknown significance were also identified (T220A/I, T245I, A265D, and T289A), which may represent novel tecovirimat-interacting residues. Allele frequency analysis demonstrated resistance mutation heterogeneity within single lesion specimens, and different mutations were identified among distinct lesion specimens from the same person, suggesting mutations were acquired during treatment. Consistent with a de novo process, comparison of specimens collected before and after treatment in a single person (Patient F) showed resistance-associated mutations in VP37 only after treatment exposure (Table 2). Phenotypic testing of MPXV cultured from a subset of specimens (1, 5) displayed a wide range of tecovirimat resistance levels in vitro compared to wild-type isolates (Table 2), arguing these mutations confer resistance within currently circulating strains. For heterogenous cultures with multiple VP37 mutations, it is unclear howmuch individual mutations contribute to resistance and whether resistance is due to single or multiple mutations, especially in cases with low allele frequencies. It is also possible that uncharacterized mutations outside of VP37 enhance or diminish tecovirimat resistance. Copyright © 2023 American Society for Microbiology. All Rights Reserved. Address correspondence to Nicole M. Green, nicgreen@ph.lacounty.gov. The authors declare no conflict of interest. Published 20 June 2023

An Mpox-Related Death in the United States A death due to mpox in an immunocompromised patient in the United States is described. Evaluation revealed disseminated viral infection.

During the 2022 multinational outbreak of monkeypox virus (MPXV) infection, the antiviral drug tecovirimat (TPOXX; SIGA Technologies, Inc., https://www.siga.com) was deployed in the United States on a large scale for the first time. The MPXV F13L gene homologue encodes the target of tecovirimat, and single amino acid changes in F13 are known to cause resistance to tecovirimat. Genomic sequencing identified 11 mutations previously reported to cause resistance, along with 13 novel mutations. Resistant phenotype was determined using a viral cytopathic effect assay. We tested 124 isolates from 68 patients; 96 isolates from 46 patients were found to have a resistant phenotype. Most resistant isolates were associated with severely immunocompromised mpox patients on multiple courses of tecovirimat treatment, whereas most isolates identified by routine surveillance of patients not treated with tecovirimat remained sensitive. The frequency of resistant viruses remains relatively low (<1%) compared with the total number of patients treated with tecovirimat.

Human rabies remains a globally significant public health problem. Replacement of polyclonal anti-rabies immunoglobulin (RIG), a passive component of rabies post-exposure prophylaxis (PEP), with a monoclonal antibody (MAb), would eliminate the cost and availability constraints associated with RIG. Our team has developed and licensed a human monoclonal antibody RAB1 (Rabishield © ), as the replacement for RIG where canine rabies is enzootic. However, for the highly diverse rabies viruses of North America, a cocktail containing two or more MAbs targeting different antigenic sites of the rabies glycoprotein should be included to ensure neutralization of all variants of the virus. In this study, two MAb cocktails, R172 (RAB1-RAB2) and R173 (RAB1-CR57), were identified and evaluated against a broad range of rabies variants from North America. R173 was found to be the most potent cocktail, as it neutralized all the tested North American RABV isolates and demonstrated broad coverage of isolates from both terrestrial and bat species. R173 could be a promising candidate as an alternative or replacement for RIG PEP in North America.

Monkeypox is a viral zoonotic disease endemic in Central and West Africa. In May 2022, dozens of non-endemic countries reported hundreds of monkeypox cases, most with no epidemiological link to Africa. We identified two lineages of monkeypox virus (MPXV) among two 2021 and seven 2022 US monkeypox cases: the major 2022 outbreak variant called B.1 and a minor contemporaneously sampled variant called A.2. Analyses of mutations among these two variants revealed an extreme preference for GA-to-AA mutations indicative of human APOBEC3 cytosine deaminase activity among Clade IIb MPXV (previously West African, Nigeria) sampled since 2017. Such mutations were not enriched within other MPXV clades. These findings suggest that APOBEC3 editing may be a recurrent and a dominant driver of MPXV evolution within the current outbreak.

The current worldwide monkepox outbreak has reaffirmed the continued threat monkeypox virus (MPXV) poses to public health. JYNNEOS, a Modified Vaccinia Ankara (MVA)-based live, non-replicating vaccine, was recently approved for monkeypox prevention for adults at high risk of MPXV infection in the United States. Although the safety and immunogenicity of JYNNEOS have been examined previously, the clinical cohorts studied largely derive from regions where MPXV does not typically circulate. In this study, we assess the quality and longevity of serological responses to two doses of JYNNEOS vaccine in a large cohort of healthcare workers from the Democratic Republic of Congo (DRC). We show that JYNNEOS elicits a strong orthopoxvirus (OPXV)-specific antibody response in participants that peaks around day 42, or 2 weeks after the second vaccine dose. Participants with no prior history of smallpox vaccination or exposure have lower baseline antibody levels, but experience a similar fold-rise in antibody titers by day 42 as those with a prior history of vaccination. Both previously naïve and vaccinated participants generate vaccinia virus and MPXV-neutralizing antibody in response to JYNNEOS vaccination. Finally, even though total OPXV-specific IgG titers and neutralizing antibody titers declined from their peak and returned close to baseline levels by the 2-year mark, most participants remain IgG seropositive at the 2-year timepoint. Taken together, our data demonstrates that JYNNEOS vaccination triggers potent OPXV neutralizing antibody responses in a cohort of healthcare workers in DRC, a monkeypox-endemic region. MPXV vaccination with JYNNEOS may help ameliorate the disease and economic burden associated with monkeypox and combat potential outbreaks in areas with active virus circulation.

Monoclonal antibodies are an efficacious therapy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, rapid viral mutagenesis led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of patients with convalescent COVID-19, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta and Omicron BA.1 and BA.2. Here, we describe an antibody cocktail, IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at nonoverlapping surfaces on the SARS-CoV-2 Spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD altered the conformation of the Spike Trimer, promoting the release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our preclinical data demonstrated that the three-antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies that demonstrated robust antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants. Here, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190 directly block spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its unique epitope on the outer surface of RBD, alters the conformation of the spike trimer, promoting release of spike monomers. These antibodies decreased SARS-CoV-2 infection in the lungs of Syrian golden hamsters, and efficacy in vivo efficacy was associated with broad antiviral neutralizing activity against multiple SARS-CoV-2 variants and robust antiviral effector function response, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrate that the three antibody cocktail IMM-BCP-01 shows promising potential for preventing or treating SARS-CoV-2 infection in susceptible individuals. One sentence summary IMM-BCP-01 cocktail triggers Spike Trimer dissociation, neutralizes all tested variants in vitro , activates a robust effector response and dose-dependently inhibits virus in vivo .

Community colleges play a vital role in preparing the highly skilled technical workforce needed to support the biotechnology industry. Community colleges offer students hands-on practical experience, certificates, and technical degrees. Students include high-school graduates, individuals changing careers, college graduates, and even PhD holders. As these colleges support the many facets of the biotechnology industry, their laboratories are equipped to teach modern techniques, including DNA sequencing, mass spectrometry, microscopy, chromatography, immunoassays, and bioinformatics. Many programs are also developing education skill standards and curriculum to support the latest biotechnology manufacturing that includes CRISPR-based gene therapies, CAR-T, immuno-therapeutics, and patient derived tissues. Some programs have established contract service organizations and business incubators to catalyze regional economic development and provide internships for students entering the workforce. These college-run organizations share many similarities with ABRF core facilities. Over the last 20+ years, community college biotechnology programs have come together to share experiences and learning through the Bio-Link network. Bio-Link was funded by the NSF-ATE (National Science Foundation Advanced Technological Education) program until the fall of 2018. In the fall of 2019, InnovATEBIO, a new national center for biotechnology education, was initiated through a five-year NSF-ATE award. InnovATEBIO will build on the Bio-Link foundation to further advance connections between high schools, community colleges, and the biotechnology industry to increase the number of highly trained biotechnology technicians in the United States. InnovATEBIO will support activities designed to increase authentic research and work-based experiences and seeks to develop collaborations with ABRF members supporting course development and partner on projects that could be funded by NSF or others.

Rabies is nearly 100% lethal in the absence of treatment, killing an estimated 59,000 people annually. Vaccines and biologics are highly efficacious when administered properly. Sixteen rabies-related viruses (lyssaviruses) are similarly lethal, but some are divergent enough to evade protection from current vaccines and biologics, which are based only on the classical rabies virus (RABV). Here we present the development and characterization of LyssaVax, a vaccine featuring a structurally designed, functional chimeric glycoprotein (G) containing immunologically important domains from both RABV G and the highly divergent Mokola virus (MOKV) G. LyssaVax elicits high titers of antibodies specific to both RABV and MOKV Gs in mice. Immune sera also neutralize a range of wild-type lyssaviruses across the major phylogroups. LyssaVax-immunized mice are protected against challenge with recombinant RABV and MOKV. Altogether, LyssaVax demonstrates the utility of structural modeling in vaccine design and constitutes a broadened lyssavirus vaccine candidate.

Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2-2 nM and 20-600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model (n = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.