Obesity fosters severe disease outcomes in a mouse model of coronavirus infection associated with transcriptomic abnormalities.

Obesity has been identified as an independent risk factor for severe outcomes in humans with coronavirus disease 2019 (COVID-19) and other infectious diseases. Here, we established a mouse model of COVID-19 using the murine betacoronavirus, mouse hepatitis virus 1 (MHV-1). C57BL/6 and C3H/HeJ mice exposed to MHV-1 developed mild and severe disease, respectively. Obese C57BL/6 mice developed clinical manifestations similar to those of lean controls. In contrast, all obese C3H/HeJ mice succumbed by 8 days postinfection, compared to a 50% mortality rate in lean controls. Notably, both lean and obese C3H/HeJ mice exposed to MHV-1 developed lung lesions consistent with severe human COVID-19, with marked evidence of diffuse alveolar damage (DAD). To identify early predictive biomarkers of worsened disease outcomes in obese C3H/HeJ mice, we sequenced RNA from whole blood 2 days postinfection and assessed changes in gene and pathway expression. Many pathways uniquely altered in obese C3H/HeJ mice postinfection aligned with those found in humans with severe COVID-19. Furthermore, we observed altered gene expression related to the unfolded protein response and lipid metabolism in infected obese mice compared to their lean counterparts, suggesting a role in the severity of disease outcomes. This study presents a novel model for studying COVID-19 and elucidating the mechanisms underlying severe disease outcomes in obese and other hosts.

Experimental SARS-CoV-2 Infection of Elk and Mule Deer.

To assess the susceptibility of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) to SARS-CoV-2, we performed experimental infections in both species. Elk did not shed infectious virus but mounted low-level serologic responses. Mule deer shed and transmitted virus and mounted pronounced serologic responses and thus could play a role in SARS-CoV-2 epidemiology.

Preexisting inter-serotype immunity drives antigenic evolution of dengue virus serotype 2.

Dengue virus (DENV) infects roughly 400 million people annually, causing febrile and hemorrhagic disease. While preexisting inter-serotype immunity (PISI) provides transient protection, it may drive severe disease over time. PISI's impact on virus evolution, however, is less understood. Retrospective epidemiological analyses suggest that PISI may drive DENV evolution. Using in vitro directed evolution, we explored how DENV2 evolves in the presence of DENV3/4 convalescent serum. Two post-passaging mutations (E-I6M and E-N203D) were then studied for fitness effects in mammalian and insect hosts and immune escape. E-I6M resisted neutralization, altered fitness in mammalian cell culture models, and had no effect in Aedes albopictus mosquitoes. E-N203D showed no change in neutralization sensitivity, reduced fitness in a DENV-naïve epithelial model, and no effects in the other models. These results align with surveillance data, where E-I6M emerged and disappeared, while E-203D and E-203 N cocirculate, thus suggesting that PISI can drive DENV evolution.

An in vitro workflow to create and modify infectious clones using replication cycle reaction.

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

Replication in the presence of dengue convalescent serum impacts Zika virus neutralization sensitivity and fitness.

Introduction: Flaviviruses like dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause febrile, hemorrhagic, and neurological diseases in humans, resulting in 400 million infections annually. Due to their co-circulation in many parts of the world, flaviviruses must replicate in the presence of pre-existing adaptive immune responses targeted at serologically closely related pathogens, which can provide protection or enhance disease. However, the impact of pre-existing cross-reactive immunity as a driver of flavivirus evolution, and subsequently the implications on the emergence of immune escape variants, is poorly understood. Therefore, we investigated how replication in the presence of convalescent dengue serum drives ZIKV evolution. Methods: We used an in vitro directed evolution system, passaging ZIKV in the presence of serum from humans previously infected with DENV (anti-DENV) or serum from DENV-naïve patients (control serum). Following five passages in the presence of serum, we performed next-generation sequencing to identify mutations that arose during passaging. We studied two non-synonymous mutations found in the anti-DENV passaged population (E-V355I and NS1-T139A) by generating individual ZIKV mutants and assessing fitness in mammalian cells and live mosquitoes, as well as their sensitivity to antibody neutralization. Results and discussion: Both viruses had increased fitness in Vero cells with and without the addition of anti-DENV serum and in human lung epithelial and monocyte cells. In Aedes aegypti mosquitoes-using blood meals with and without anti-DENV serum-the mutant viruses had significantly reduced fitness compared to wild-type ZIKV. These results align with the trade-off hypothesis of constrained mosquito-borne virus evolution. Notably, only the NS1-T139A mutation escaped neutralization, while E-V335I demonstrated enhanced neutralization sensitivity to neutralization by anti-DENV serum, indicating that neutralization escape is not necessary for viruses passaged under cross-reactive immune pressures. Future studies are needed to assess cross-reactive immune selection in humans and relevant animal models or with different flaviviruses.

Noble Metal Organometallic Complexes Display Antiviral Activity against SARS-CoV-2.

SARS-CoV-2 emerged in 2019 as a devastating viral pathogen with no available preventative or treatment to control what led to the current global pandemic. The continued spread of the virus and increasing death toll necessitate the development of effective antiviral treatments to combat this virus. To this end, we evaluated a new class of organometallic complexes as potential antivirals. Our findings demonstrate that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(1,3-dicyclohexylimidazol-2-ylidene)Cl2 (complex 2) and Cp*Rh(dipivaloylmethanato)Cl (complex 4), have direct virucidal activity against SARS-CoV-2. Subsequent in vitro testing suggests that complex 4 is the more stable and effective complex and demonstrates that both 2 and 4 have low toxicity in Vero E6 and Calu-3 cells. The results presented here highlight the potential application of organometallic complexes as antivirals and support further investigation into their activity.

Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones.

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.