Respiratory syncytial virus (RSV) was discovered in 1956 by the laboratory of Robert Chanock after its isolation from children with upper respiratory infections. Here, we review the events leading to its discovery including its prior isolation as chimpanzee coryza virus and its subsequent association with human disease.

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In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells. ABSTRACT hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment. IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.

We are public health scientists who have closely followed the emergence of 2019 novel coronavirus disease (COVID-19) and are deeply concerned about its impact on global health and wellbeing. We have watched as the scientists, public health professionals, and medical professionals of China, in particular, have worked diligently and effectively to rapidly identify the pathogen behind this outbreak, put in place significant measures to reduce its impact, and share their results transparently with the global health community. This effort has been remarkable. We sign this statement in solidarity with all scientists and health professionals in China who continue to save lives and protect global health during the challenge of the COVID-19 outbreak. We are all in this together, with our Chinese counterparts in the forefront, against this new viral threat. The rapid, open, and transparent sharing of data on this outbreak is now being threatened by rumours and misinformation around its origins. We stand together to strongly condemn conspiracy theories suggesting that COVID-19 does not have a natural origin. Scientists from multiple countries have published and analysed genomes of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),1Gorbalenya AE Baker SC Baric RS et al.Severe acute respiratory syndrome-related coronavirus: the species and its viruses—a statement of the Coronavirus Study Group.bioRxiv. 2020; (published online Feb 11. DOI: 2020.02.07.937862 (preprint).)Google Scholar and they overwhelmingly conclude that this coronavirus originated in wildlife,2Zhou P Yang X-L Wang X-G et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; (published online Feb 3.)DOI:10.1038/s41586-020-2012-7Crossref Scopus (14410) Google Scholar, 3Lu R Zhao X Li J et al.Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.Lancet. 2020; (published online Jan 30.)https://doi.org/10.1016/S0140-6736(20)30251-8Summary Full Text Full Text PDF Scopus (8212) Google Scholar, 4Zhu N Zhang D Wang W et al.A novel coronavirus from patients with pneumonia in China, 2019.NEJM. 2020; (published online Jan 24.)DOI:10.1056/NEJMoa2001017Crossref PubMed Scopus (18588) Google Scholar, 5Ren L Wang Y-M Wu Z-Q et al.Identification of a novel coronavirus causing severe pneumonia in humans: a descriptive study.Chin Med J. 2020; (published online Feb 11.)DOI:10.1097/CM9.0000000000000722Crossref Scopus (862) Google Scholar, 6Paraskevis D Kostaki EG Magiorkinis G Panayiotakopoulos G Tsiodras S Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event.Infect Genet Evol. 2020; (published online Jan 29.)DOI:10.1016/j.meegid.2020.104212Crossref PubMed Scopus (496) Google Scholar, 7Benvenuto D Giovanetti M Ciccozzi A Spoto S Angeletti S Ciccozzi M The 2019-new coronavirus epidemic: evidence for virus evolution.J Med Virol. 2020; (published online Jan 29.)DOI:10.1002/jmv.25688Crossref Scopus (452) Google Scholar, 8Wan Y Shang J Graham R Baric RS Li F Receptor recognition by novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS.J Virol. 2020; (published online Jan 29.)DOI:10.1128/JVI.00127-20Crossref Scopus (3161) Google Scholar, 9US Center for Disease Control and PreventionCoronavirus disease 2019 (COVID-19) situation summary.https://www.cdc.gov/coronavirus/2019-nCoV/summary.htmlDate: Feb 16, 2020Date accessed: February 8, 2020Google Scholar, 10Andersen KG Rambaut A Lipkin WI Holmes EC Garry RF The proximal origin of SARS-CoV-2.http://virological.org/t/the-proximal-origin-of-sars-cov-2/398Date: Feb 16, 2020Date accessed: February 17, 2020Google Scholar as have so many other emerging pathogens.11Bengis R Leighton F Fischer J Artois M Morner T Tate C The role of wildlife in emerging and re-emerging zoonoses.Rev Sci Tech. 2004; 23: 497-512PubMed Google Scholar, 12Woolhouse ME Gowtage-Sequeria S Host range and emerging and reemerging pathogens.Emerg Infect Dis. 2005; 11: 1842-1847Crossref PubMed Scopus (1029) Google Scholar This is further supported by a letter from the presidents of the US National Academies of Science, Engineering, and Medicine13NASEMThe National Academies of Science Engineering and Medicine of the USA. NAS, NAE, and NAM presidents' letter to the White House Office of Science and Technology Policy.https://www.nationalacademies.org/includes/NASEM%20Response%20to%20OSTP%20re%20Coronavirus_February%206,%202020.pdfDate: Feb 6, 2020Date accessed: February 7, 2020Google Scholar and by the scientific communities they represent. Conspiracy theories do nothing but create fear, rumours, and prejudice that jeopardise our global collaboration in the fight against this virus. We support the call from the Director-General of WHO to promote scientific evidence and unity over misinformation and conjecture.14WHODirector-General's remarks at the media briefing on 2019 novel coronavirus on 8 February 2020.https://www.who.int/dg/speeches/detail/director-general-s-remarks-at-the-media-briefing-on-2019-novel-coronavirus---8-february-2020Date: Feb 8, 2020Date accessed: February 18, 2020Google Scholar We want you, the science and health professionals of China, to know that we stand with you in your fight against this virus. We invite others to join us in supporting the scientists, public health professionals, and medical professionals of Wuhan and across China. Stand with our colleagues on the frontline! We speak in one voice. To add your support for this statement, sign our letter online. LM is editor of ProMED-mail. We declare no competing interests. Download .pdf (.12 MB) Help with pdf files Chinese translation of full text Addendum: competing interests and the origins of SARS-CoV-2In February, 2020, 27 public health experts co-authored a Correspondence in The Lancet ("Statement in support of the scientists, public health professionals, and medical professionals of China combatting COVID-19"),1 supporting health professionals and physicians in China during the early stages of the COVID-19 pandemic. In this letter, the authors declared no competing interests. Some readers have questioned the validity of this disclosure, particularly as it relates to one of the authors, Peter Daszak. Full-Text PDF Science, not speculation, is essential to determine how SARS-CoV-2 reached humansOn Feb 19, 2020, we, a group of physicians, veterinarians, epidemiologists, virologists, biologists, ecologists, and public health experts from around the world, joined together to express solidarity with our professional colleagues in China.1 Unsubstantiated allegations were being raised about the source of the COVID-19 outbreak and the integrity of our peers who were diligently working to learn more about the newly recognised virus, SARS-CoV-2, while struggling to care for the many patients admitted to hospital with severe illness in Wuhan and elsewhere in China. Full-Text PDF

Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals.IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpesviruses (oHSV) has a major impact on cancer therapy marred by rare failures of healthy organs. Furthermore, tumors vary with respect to susceptibility to oncolytic effects of oHSV. Here we report the construction and properties of 3 families of oncolytic herpes simplex viruses expressing no immunomodulatory genes (T1 series), murine IL-12 (T2 series) or murine or human IL-12 and anti PD-1 antibody (T3 series). We report that insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). The T3 oHSV expressed IL-12, PD-1 Ab were restricted to the tumor bed whereas the induced IFN-γ accumulated to high levels both in tumor bed and in blood. Furthermore, the T3 oHSV was superior to systemic administration of IL-12 and antibody to PD-1. This report also shows that the oncolytic activity of T3 oHSV in a relatively resistant tumor was enhanced by concurrent intratumoral administration of genetically engineered exosomes carrying miRNA targeting CTLA-4 checkpoint.

Abstract Our Phase I trials of experimental virotherapy for recurrent glioblastoma (GBM) have shown that inoculation with a conditionally replication-competent early generation oncolytic herpes simplex virus (oHSV), G207, is safe. However, while 17 of 37 subjects experienced objective clinical responses, the highly attenuated oHSV did not uniformly improve survival. We sought to identify predictors that would identify mechanisms contributing to survival and improve future trial design, by studying accrued samples. We analyzed pre-treatment biopsy and post-G207-treatment tumor samples (collected D2-5 post injection) banked from the patients enrolled in the phase IB G207 trial. The key findings from these patients suggest that productive G207 infection and G207-induced changes in gene expression were predictive of oHSV therapeutic success. RNAseq-based transcriptome analysis of these samples revealed that both the intrinsic IFN mediated antiviral response and adaptive immune functional response in patients correlated significantly with improved survival following G207 inoculation. Further, GBM tissue stained using multiplex fluorescent immunohistochemistry supported differences in the tumor microenvironments that were identified from RNAseq data analysis. Our data indicate that both viral gene expression and the resulting intrinsic anti-viral and recruited adaptive response were critical for survival after G207 inoculation and predict survival with this early generation oHSV in patients with recurrent malignant glioma.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.

The key mission of viruses is to spread within its ecological niche. To achieve their mission viruses must multiply efficiently, be available at appropriate body sites for transmission and yet not kill the host since extreme virulence that kills the host curtails the spread of viruses in a population as illustrated by SARS and Ebola viruses. HSV infects a very large fraction of human population and is therefore a successful pathogen. To achieve it mission the virus encodes functions of omission that fail to block host immune responses and functions of commission that specifically curtail its replication. The net effect is the evolution of a pathogen that controls its replication so as to maintain a high level of contacts between infected and uninfected individuals.

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: (i) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNβ. The genes that were not activated encoded HDAC4 and NF-κB1. (ii) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. (iii) IFNβ produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. (iv) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. (v) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.

Significance Nuclear domain 10 (ND10) bodies are both sensors and responders to many viruses that infect human cells. The key components of ND10 bodies are PML and SP100. Their importance in innate immune responses to infection is underscored by the observation that numerous viruses have evolved means to degrade, disperse, or at least inactivate PML and SP100. This report focuses on SP100, which, along with PML, is degraded by a viral E3 ligase in HSV-1–infected cells. The question posed here is the function of SP100 and PML in the course of HSV infection. The results indicate that the function of SP100 is different than, but complementary to, that of PML and ranks high in the cellular defense mechanisms against infection.

SignificancePromyelocytic leukemia protein (PML) is a component of nuclear domain 10 (ND10) bodies and an antiviral effector of IFN-β. A herpes simplex virus (HSV) protein, ICP0, interacts with PML, merges with ND10 bodies, degrades PML, and ultimately takes over the domain of ND10 bodies. Here we show that viral gene expression and growth are reduced inPML−/−cells infected at low ratios of virus to cells. In essence, the results indicate that HSV-1 evolved the means to take advantage of an inimical cellular defense mechanism to degrade it and at the same time use it to gain access to a nuclear domain essential for efficient replication.

We report on the properties and function of two herpes simplex virus-1 (HSV-1) microRNAs (miRNAs) designated "miR-H28" and "miR-H29." Both miRNAs accumulate late in productive infection at a time when, for the most part, viral DNA and proteins have been made. Ectopic expression of miRNA mimics in human cells before infection reduced the accumulation of viral mRNAs and proteins, reduced plaque sizes, and at vey low multiplicities of infection reduced viral yields. The specificity of the miRNA mimics was tested in two ways. First, ectopic expression of mimics carrying mutations in the seed sequence was ineffective. Second, in similar tests two viral miRNAs made early in productive infection also had no effect. Both miR-H28 and miR-H29 are exported from infected cells in exosomes. A noteworthy finding is that both miR-H28 and miR-H29 were absent from murine ganglia harboring latent virus but accumulated in ganglia in which the virus was induced to reactivate. The significance of these findings rests on the principle that the transmission of HSV from person to person is by physical contact between the infected tissues of the donor and those of uninfected recipient. Diminished size of primary or recurrent lesions could be predicted to enhance person-to-person transmission. Reduction in the amount of reactivating latent virus would reduce the risk of retrograde transport to the CNS but would not interfere with anterograde transport to a site at or near the site of initial infection.

Previous studies have shown that the herpes simplex virus 1 gene UL26 encodes a 635-amino acid protease that cleaves approximately 20 amino acids from the carboxyl terminus of itself and of a 329-amino acid product of the UL26.5 gene. The results of studies with a variety of protease inhibitors showed that the UL26 protease was inhibited by serine protease inhibitors but not by inhibitors of cysteine protease, aspartic acid protease, or metalloprotease. Mutations resulting in amino acid substitutions, deletions, or insertion of stop codons in the gene or of 20-amino acid stretches into the protease have delineated the dispensable domains I and IV at the amino and carboxyl domains of the gene product. The essential carboxyl- proximal domain (III) can be separated from the essential amino-proximal domain (II) by at least 20 amino acids. The amino-proximal domain is the most conserved region among varicella-zoster virus and human cytomegalovirus homologues Of UL26. Of the conserved aspartic acid, histidine, or serine amino acids in this domain, only histidine-61 and -148 could not be replaced without impairment of the proteolytic activity.

ABSTRACT Herpes simplex viruses (HSV) package and bring into cells an RNase designated virion host shutoff (VHS) RNase. In infected cells, the VHS RNase targets primarily stress response mRNAs characterized by the presence of AU-rich elements in their 3′ untranslated regions (UTRs). In uninfected cells, these RNAs are sequestered in exosomes or P bodies by host proteins that bind to the AU-rich elements. In infected cells, the AU-rich RNAs are deadenylated and cleaved close to the AU-rich elements, leading to long-term persistence of nontranslatable RNAs consisting of the 5′ portions of the cleavage products. The host proteins that bind to the AU-rich elements are either resident in cells (e.g., TIA-1) or induced (e.g., tristetraprolin). Earlier, this laboratory reported that tristetraprolin binds VHS RNase. To test the hypothesis that tristetraprolin directs VHS RNase to the AU-rich elements, we mapped the domains of VHS and tristetraprolin required for their interactions. We report that VHS binds to the domain of tristetraprolin that enables its interaction with RNA. A single amino acid substitution in that domain abolished the interaction with RNA but did not block the binding to VHS RNase. In transfected cells, the mutant but not the wild-type tristetraprolin precluded the degradation of the AU-rich RNAs by VHS RNase. We conclude that TTP mediates the cleavage of the 3′ UTRs of stress response mRNAs by recruiting the VHS RNase to the AU-rich elements. IMPORTANCE The primary host response to HSV infection is the synthesis of stress response mRNAs characterized by the presence of AU-rich elements in their 3′ UTRs. These mRNAs are the targets of the virion host shutoff (VHS) RNase. The VHS RNase binds both to mRNA cap structure and to tristetraprolin, an inducible host protein that sequesters AU-rich mRNAs in exosomes or P bodies. Here we show that tristetraprolin recruits VHS RNase to the AU-rich elements and enables the degradation of the stress response mRNAs.

I was shaped by World War II, years of near starvation as a war refugee, postwar chaos, life in several countries, and relative affluence in later life. The truth is that as I was growing up I wanted to be a writer. My aspirations came to an end when, in order to speed up my graduation from college, I took courses in microbiology. It was my second love at first sight-that of my wife preceded it. I view science as an opportunity to discover the designs in the mosaics of life. What initiates my search of discovery is an observation that makes no sense unless there exists a novel design. Once the design is revealed there is little interest in filling all the gaps. I was fortunate to understand that what lasts are not the scientific reports but rather the generations of scientists whose education I may have influenced.

On entry into the body herpes simplex viruses (HSV) replicate in a series of steps that involves derepression of viral DNA activated by VP16, a virion protein, and sequential transcription of viral genes in a cascade fashion. HSV also enters into neurons in which viral DNA maintained as heterochromatin and with few exceptions viral gene expression is silenced. A third face of the interaction of HSV with its host cells takes place at the moment when the silenced viral genome in neurons is abruptly derepressed. The available data do no reveal evidence that HSV encodes different regulatory programs for each facet of its interaction with its host cells. Rather the data point to significant gaps in our knowledge of the mechanisms by which each facet is initiated and the roles of the infected cells at each facet of the interaction of viral gene products with the host cell.