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OBJECTIVES: To analyse nosocomial transmission in the early stages of the coronavirus 2019 (COVID-19) pandemic at a large multisite healthcare institution. Nosocomial incidence is linked with infection control interventions. METHODS: Viral genome sequence and epidemiological data were analysed for 574 consecutive patients, including 86 nosocomial cases, with a positive PCR test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first 19 days of the pandemic. RESULTS: Forty-four putative transmission clusters were found through epidemiological analysis; these included 234 cases and all 86 nosocomial cases. SARS-CoV-2 genome sequences were obtained from 168/234 (72%) of these cases in epidemiological clusters, including 77/86 nosocomial cases (90%). Only 75/168 (45%) of epidemiologically linked, sequenced cases were not refuted by applying genomic data, creating 14 final clusters accounting for 59/77 sequenced nosocomial cases (77%). Viral haplotypes from these clusters were enriched 1-14x (median 4x) compared to the community. Three factors implicated unidentified cases in transmission: (a) community-onset or indeterminate cases were absent in 7/14 clusters (50%), (b) four clusters (29%) had additional evidence of cryptic transmission, and (c) in three clusters (21%) diagnosis of the earliest case was delayed, which may have facilitated transmission. Nosocomial cases decreased to low levels (0-2 per day) despite continuing high numbers of admissions of community-onset SARS-CoV-2 cases (40-50 per day) and before the impact of introducing universal face masks and banning hospital visitors. CONCLUSION: Genomics was necessary to accurately resolve transmission clusters. Our data support unidentified cases-such as healthcare workers or asymptomatic patients-as important vectors of transmission. Evidence is needed to ascertain whether routine screening increases case ascertainment and limits nosocomial transmission.

Matthew B Phillips,1 Johnasha D Stuart,1 Roxana M Rodríguez Stewart,2 Jameson TL Berry,2 Bernardo A Mainou,2 Karl W Boehme1 1Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Atlanta, GA, USA; 2Department of Pediatrics, Emory University, Atlanta, GA, USA Abstract: Mammalian orthoreovirus (reovirus) is under development as a cancer virotherapy. Clinical trials demonstrate that reovirus-based therapies are safe and tolerated in patients with a wide variety of cancers. Although reovirus monotherapy has proven largely ineffective, reovirus sensitizes cancer cells to existing chemotherapeutic agents and radiation. Clinical trials are underway to test the efficacy of reovirus in combination with chemotherapeutic and radiation regimens and to evaluate the effectiveness of reovirus in conjunction with immunotherapies. Central to the use of reovirus to treat cancer is its capacity to directly kill cancer cells and alter the cellular environment to augment other therapies. Apoptotic cell death is a prominent mechanism of reovirus cancer cell killing. However, reoviruses can also kill cancer cells through nonapoptotic mechanisms. Here, we describe mechanisms of reovirus cancer cell killing, highlight how reovirus is used in combination with existing cancer treatments, and discuss what is known as to how reovirus modulates cancer immunotherapy. Keywords: virotherapy, cancer, immunotherapy, cell death, interferon

CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation., eLife digest Like many viruses, the genetic information of the human immunodeficiency virus (or HIV for short) is formed of molecules of RNA, which are sequences of building blocks called nucleotides. Once the virus is inside human cells, a protein called ZAP can identify viral RNAs by binding to a precise motif, a combination of two nucleotides called CpG. This allows the cell to destroy the viral RNA, thus preventing the virus from multiplying. However, HIV and other viruses that infect mammals are often able to ‘hide’ from ZAP because their genetic codes have many fewer CpG nucleotides than what would be expected by chance. ZAP by itself does not appear to be able to cut up RNA, so it is thought that it recruits other, as yet unidentified, proteins to destroy the genome of viruses. Here, Ficarelli et al. used genetic techniques to identify a new human protein called KHNYN that interacts with ZAP. First, a new version of the RNA genome of HIV was engineered, which contained higher numbers of CpGs: this CpG-enriched virus could be inhibited by ZAP in human cells. The experiments showed that increasing the amount of KHNYN protein led to lower levels of HIV genomes enriched in CpG. However, increasing the levels of KHNYN protein in mutant cells without ZAP had no effect on how well CpG-enriched HIV multiplied. CpG-enriched HIV and another related virus with many CpG nucleotides were able to multiply more successfully in mutant cells lacking the KHNYN protein than in normal cells. Further experiments also suggested that mutating a region of KHNYN which is likely to cut RNA prevented it from inhibiting HIV enriched with CpGs. Artificially manipulating the CpG nucleotide content of viral sequences could help create viruses useful for human health. For instance, weakened viruses could be designed for use in vaccines. Some human tumors have decreased levels of ZAP, and it could therefore be possible to build viruses that healthy cells can destroy, but which could multiply in and kill cancer cells. However, before these approaches can be developed, exactly how ZAP and KHNYN degrade strands of viral RNA needs to be characterized.

The immunoglobulin-like transcript (ILT) 7 is a surface molecule selectively expressed by human plasmacytoid dendritic cells (pDCs). ILT7 cross-linking suppresses pDC activation and type I interferon (IFN-I) secretion following Toll-like receptors (TLR)7/9 engagement. The bone marrow stromal cell antigen 2 (BST2, aka HM1.24, tetherin or CD317) is expressed by different cell types upon exposure to IFN-I and is a natural ligand for ILT7. Here we show that ILT7 expression decreased spontaneously in pDCs upon in vitro culture, which correlates with pDC differentiation measured as increased side scatter properties and CCR7 expression. TLR7/9 Ligands , as well as HIV, induced BST2 upregulation on all tested cell types except T cells, which required TCR stimulation to respond to TLR9L-induced IFN-I. IFN-γ, IL-4, IL-10 and TNF-α had only marginal effects on BST2 expression in blood leukocytes compared to TLR9L. Pre-incubation with ILT7-crosslinking Ab inhibited IFN-I production in PBMCs treated with TLR7/9L or HIV, whereas BST2 blockade did not affect IFN-I responses even when BST2 upregulation was further boosted with TCR agonists or immunoregulatory cytokines. Our data indicate that BST2-mediated ILT7 cross-linking may act as a homeostatic regulatory mechanism on immature circulating pDC, rather than a negative feedback for activated mature pDCs which have downregulated ILT7.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.