Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.

Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi's sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein-Barr virus (EBV); however, the requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV + single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV + /EBV + PEL cell lines with an EBV-specific CRISPR/ Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein-Barr nuclear antigen 1 (EBNA-1) in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.O ne almost never finds two different viruses in the same cell. SignificancePrimary effusion lymphoma (PEL) is a B cell lymphoma; the prognosis is poor, with a median survival around 6 months. PEL is always associated with the presence of Kaposi's sarcomaassociated herpesvirus and in most cases is coinfected with Epstein-Barr virus (EBV); however, the role of EBV in the pathogenesis of the tumor is still not clear. This study demonstrates the intricate interaction between the two herpesviruses, which exacerbate tumorigenesis by mutually reinforcing the persistence of each latent genome and by altering cell proliferation. It establishes curing EBV and inhibiting Epstein-Barr nuclear antigen 1 as a potential treatment for PEL.

29 30 SARS-CoV-2 is constantly evolving. Prior studies have focused on high case-density locations, such 31 as the Northern and Western metropolitan areas in the U.S. This study demonstrates continued 32 SARS-CoV-2 evolution in a suburban Southern U.S. region by high-density amplicon sequencing of 33 symptomatic cases. 57% of strains carried the spike D614G variant. The presence of D614G was 34 associated with a higher genome copy number and its prevalence expanded with time. Four strains 35 carried a deletion in a predicted stem loop of the 3' untranslated region. The data are consistent with 36 community spread within the local population and the larger continental U.S. No strain had mutations 37 in the target sites used in common diagnostic assays. The data instill confidence in the sensitivity of 38 current tests and validate "testing by sequencing" as a new option to uncover cases, particularly those 39 not conforming to the standard clinical presentation of COVID-19. This study contributes to the 40 understanding of COVID-19 by providing an extensive set of genomes from a non-urban setting and 41 further informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate 42 in the U.S.

Primary effusion lymphoma (PEL) is an aggressive and incurable malignancy, which is usually characterized by lymphomatous effusions in body cavities without tumor masses. PEL has no established treatment and a poor prognosis, with a median survival time shorter than 6 months. PEL usually develops in the context of immunosuppression, such as HIV infection or post-organ transplantation. The optimal treatment for PEL has not been established, as PEL is generally resistant to traditional chemotherapy. The molecular drivers for PEL are still unknown; however, PEL displays a constitutively active mammalian target of rapamycin (mTOR) pathway, which is critical for metabolic and cell survival mechanisms. Therefore, the evaluation of novel agents targeting the mTOR pathway could be clinically relevant for the treatment of PEL.