Curating the Evidence About COVID-19 for Frontline Public Health and Clinical Care: The Novel Coronavirus Research Compendium.

The public health crisis created by the COVID-19 pandemic has spurred a deluge of scientific research aimed at informing the public health and medical response to the pandemic. However, early in the pandemic, those working in frontline public health and clinical care had insufficient time to parse the rapidly evolving evidence and use it for decision-making. Academics in public health and medicine were well-placed to translate the evidence for use by frontline clinicians and public health practitioners. The Novel Coronavirus Research Compendium (NCRC), a group of >60 faculty and trainees across the United States, formed in March 2020 with the goal to quickly triage and review the large volume of preprints and peer-reviewed publications on SARS-CoV-2 and COVID-19 and summarize the most important, novel evidence to inform pandemic response. From April 6 through December 31, 2020, NCRC teams screened 54 192 peer-reviewed articles and preprints, of which 527 were selected for review and uploaded to the NCRC website for public consumption. Most articles were peer-reviewed publications (n = 395, 75.0%), published in 102 journals; 25.1% (n = 132) of articles reviewed were preprints. The NCRC is a successful model of how academics translate scientific knowledge for practitioners and help build capacity for this work among students. This approach could be used for health problems beyond COVID-19, but the effort is resource intensive and may not be sustainable in the long term.

Curating and translating the evidence about SARS-CoV-2 and COVID-19 for frontline public health and clinical care: The Novel Coronavirus Research Compendium (NCRC).

The public health crisis created by the SARS-CoV-2 pandemic has spurred a deluge of scientific research aimed at informing public health and medical response to the COVID-19 pandemic. However, those working in frontline public health and clinical care had insufficient time to parse the rapidly evolving evidence and use it for decision making. Academics in public health and medicine were well-placed to translate the evidence for use by frontline clinicians and public health practitioners. The Novel Coronavirus Research Compendium (NCRC), a group of >50 faculty and trainees, began in March 2020 with the goal to quickly triage and review the large volume of preprints and peer-reviewed publications on SARS-CoV-2 and COVID-19, and to summarize the most important, novel evidence to inform pandemic response. From April 6, 2020 through January 1, 2021, 54,192 papers and preprints were screened by NCRC teams and 527 were selected for review and uploaded to the NCRC website for public consumption. The majority of papers reviewed were peer-reviewed publications (n=395, 75%), published in 102 journals; 25% (n=132) of papers reviewed were of preprints. The NCRC is a successful model of how academics can support practitioners by translating scientific knowledge into action and help to build capacity among students for this work. This approach could be used for health problems beyond COVID-19, but the effort is resource intensive and may not be sustainable over the long term.

Generation and propagation of recombinant mumps viruses exhibiting an additional U residue in the homopolymeric U tract of the F gene-end signal.

As a member of the family paramyxoviridae, subfamily paramyxovirinae, the genome of mumps virus (MuV) is postulated to be polyhexameric in length in order to be able to replicate efficiently. While all natural MuV strains sequenced so far obey to this "rule of six," we describe here the isolation of recombinant MuVs that appeared to contain an additional U residue in the homopolymeric tract of the F gene-end signal, resulting in a genome length of 6n + 1. Sequencing of several plaque-purified viruses from these preparations did not reveal the existence of length-correcting mutations, suggesting that they are violators of the rule of six. Employing high-throughput sequencing technology, we provide evidence that the insertion of an additional U residue is mainly the result of the rescue system used that relies on T7 RNA polymerase. Limited in vitro and in vivo testing of the viruses did not reveal any significant impact of the longer genome on virus replication or virulence, suggesting that the rule of six is not a strict requirement for MuV replication.

Recent mumps outbreaks in vaccinated populations: no evidence of immune escape.

Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation.

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.

Presence of lysine at aa 335 of the hemagglutinin-neuraminidase protein of mumps virus vaccine strain Urabe AM9 is not a requirement for neurovirulence.

The recent global resurgence of mumps has drawn attention to the continued need for robust mumps immunization programs. Unfortunately, some vaccines derived from inadequately attenuated vaccine strains of mumps virus have caused meningitis in vaccinees, leading to withdrawal of certain vaccine strains from the market, public resistance to vaccination, or in some cases, cessation of national mumps vaccination programs. The most widely implicated mumps vaccine in cases of postvaccination meningitis is derived from the Urabe AM9 strain, which remains in use in some countries. The Urabe AM9 vaccine virus has been shown to exhibit a considerable degree of nucleotide and amino acid heterogeneity. Some studies have specifically implicated variants containing a lysine residue at amino acid position 335 in the hemagglutinin-neuraminidase (HN) protein with neurotoxicity, whereas a glutamic acid residue at this position was associated with attenuation. To test this hypothesis we generated two modified Urabe AM9 cDNA clones coding either for a lysine or a glutamic acid at position 335 in the HN gene. The two viruses were rescued by reverse genetics and characterized in vitro and in vivo. Both viruses exhibited similar growth kinetics in neuronal and non-neuronal cell lines and were of similar neurotoxicity when tested in rats, suggesting that amino acid 335 is not a crucial determinant of Urabe AM9 growth or neurovirulence.

Identification of genetic mutations associated with attenuation and changes in tropism of Urabe mumps virus.

Although several effective mumps virus vaccines have been developed, almost nothing is known about the genetic changes responsible for loss of virulence. One vaccine, Urabe AM9, was withdrawn from the market because of insufficient attenuation. The vaccine was found to contain a mixture of viruses that could be distinguished based on the sequence of the hemagglutinin-neuraminidase gene (HN). Viruses containing lysine at HN amino acid position 335 were isolated from cases of post-vaccination parotitis or meningitis whereas viruses containing glutamic acid at this position were not associated with post-vaccination disease. Using a rat based model of mumps neurovirulence, we demonstrate that this latter virus is significantly attenuated compared to a virus isolated from a patient with post-vaccination meningitis. Complete sequence analysis of the genomes of the two viruses identified sixteen genetic differences, some or all of which must be responsible for differences in virulence. These same genetic differences also account for changes in tropism in cell culture.

Herpes simplex virus type 1 C-terminal variants of the origin binding protein (OBP), OBPC-1 and OBPC-2, cooperatively regulate viral DNA levels in vitro, and OBPC-2 affects mortality in mice.

Two in-frame, C-terminal isoforms of the herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), OBPC-1 and OBPC-2, and a unique C-terminal transcript, UL8.5, are specified by HSV-1 DNA. As the first isoform identified, OBPC-1 was initially assumed to be the product of the UL8.5 transcript. Recent evidence has demonstrated, however, that OBPC-1 is a cathepsin B-mediated cleavage product of OBP, suggesting that OBPC-2 is the product of the UL8.5 transcript. Because both OBPC-1 and -2 contain the majority of the OBP DNA binding domain, we hypothesized that both may be involved in regulating origin-dependent, OBP-mediated viral DNA replication. In this paper, we demonstrate that OBPC-2 is, indeed, the product of the UL8.5 transcript. The translational start site of OBPC-2 was mapped, and a virus (M571A) that does not express this protein efficiently was constructed. Using M571A, we have shown that OBPC-2 is able to bind origin DNA, even though it lacks seven N-terminal amino acid residues of the previously mapped OBP DNA binding domain, resulting in a revision of the limits of the OBP DNA binding domain. Consistent with their proposed roles in regulating viral DNA replication, OBPC-1 and -2 act together to down-regulate viral DNA replication in vitro. During functional studies in vivo, OBPC-2 was identified as a factor that increases mortality in the mouse ocular model of HSV-1 infection.

Cathepsin B mediates cleavage of herpes simplex virus type 1 origin binding protein (OBP) to yield OBPC-1, and cleavage is dependent upon viral DNA replication.

Although the seven viral proteins required for herpes simplex virus type 1 (HSV-1) DNA replication have been identified, the mechanism by which viral DNA synthesis is regulated is unclear. HSV-1 DNA replication is thought to occur in two stages: origin-dependent DNA replication (stage I) mediated by the origin binding protein (OBP), followed by origin- and OBP-independent DNA replication (stage II). The mechanism that facilitates the switch from stage I to stage II is unknown; however, it must involve the loss of OBP function or OBP itself from the replication initiation complex. Previous studies from this laboratory identified a transcript (UL8.5) and protein (OBPC) that are in frame with and comprise the C terminus of the gene specifying OBP. Because of its DNA binding ability, OBPC has been hypothesized to mediate the switch from stage I to stage II. Here, we identify a second protein (OBPC-2) that is also in frame with the C terminus of OBP but comprises a smaller portion of the protein. We demonstrate that the protein originally identified (OBPC-1) is a cathepsin B-mediated cleavage product of OBP, while OBPC-2 may be the product of the UL8.5 transcript. We further demonstrate that the cleavage of OBP to yield OBPC-1 is dependent upon viral DNA replication. These results suggest that cleavage may be a mechanism by which OBP levels and/or activity are regulated during infection.

Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase.

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.

The Arcanobacterium pyogenes collagen-binding protein, CbpA, promotes adhesion to host cells.

Arcanobacterium pyogenes is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys. A. pyogenes adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of A. pyogenes only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa A. pyogenes cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of A. pyogenes to adhere to HeLa and 3T6 cells, as a cbpA knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing cbpA on a multicopy plasmid. Furthermore, HIS-CbpA blocked A. pyogenes adhesion to HeLa or 3T6 cells in a dose-dependent manner. cbpA was only present in 48% of the A. pyogenes strains tested (n = 75), and introduction of plasmid-encoded cbpA into a naturally cbpA-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9- and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of A. pyogenes, plays a role in the adhesion of this organism to host cells.