Murine norovirus (MNV) viral protein genome-linked (VPg) manipulates the cell cycle to induce a G0/G1 arrest and gain a beneficial replication environment. All viruses of the norovirus genus encode a VPg protein; however, it is unknown if the G0/G1 arrest induced by MNV VPg is conserved in other members of the genus. RNA transcripts encoding a representative viral VPg from five norovirus genogroups were transfected into RAW-Blue murine macrophages, and the percentage of cells in each phase of the cell cycle was determined. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins tested, and in the wider Caliciviridae family the arrest was also conserved in rabbit hemorrhagic disease virus (RHDV) VPg and human sapovirus (HuSV) VPg. Truncation of MNV VPg shows that the first 62 amino acids are sufficient for a cell cycle arrest, and alignment of VPg sequences revealed a conserved motif in the N-terminal region of VPg. Analysis of VPg constructs with single N-terminal region point mutations, or exchange of N-terminal regions between VPg proteins, confirmed the importance of the N-terminal region for cell cycle arrest. These results provide evidence that G0/G1 cell cycle arrest is a conserved function of norovirus VPg proteins that involves the N-terminal region of these proteins.

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We aimed to establish the utility of the Fast-track diagnostics Viral meningitis multiplex PCR kit for the diagnosis of central nervous system infection in infants. The multiplex assay had reduced sensitivity for the detection of enterovirus, the predominant pathogen in young infants, when compared to our in-house singleplex PCR. In our infant population, multiple singleplex PCR assays perform better than a multiplex assay for the detection of CSF viruses. J. Med. Virol. 89:559-561, 2017. © 2016 Wiley Periodicals, Inc.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

We examined the cultural, social, and economic aspects of livestock operations of ranchers who have Federal grazing permits (called permittees) on the Santa Fe and Carson National Forests of northern New Mexico. This study was an expansion of the 2003 pilot study and was designed to provide much-needed information concerning the culture and economic practices of the northern New Mexico region for USDA employees, policy makers, social science researchers, and the general public. The research focused on both the economic and noneconomic contributions of livestock ownership to local families and communities, and we explored ways in which ranching maintains traditional values and connects families to ancestral lands and heritage. Sense of place, attachment to land, and the value of preserving open space were common themes throughout the interviews. The importance of land and animals as means of maintaining culture and way of life figured repeatedly in permittee responses, as did the subjects of responsibility and respect for land, animals, family, and community. This report will assist agency land managers in the effective administration of forest lands by promoting greater cultural understanding of the local ranching community. It will also serve as an educational tool for the public, as many visitors and residents of New Mexico are unfamiliar with the primarily Hispanic culture and traditions of the region. Due to the history of land ownership in the region, many ranching operations rely on public lands for livestock grazing. Recognizing the importance of these small livestock operations to area communities and families is crucial to comprehending and resolving disputes over public land and resource use.

Murine norovirus (MNV) is a positive‐sense, plus‐stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal‐oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT‐qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1‐2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1‐2 in insect cells Support Protocol 3: Purification of the norovirus NS1‐2 protein Support Protocol 4: Expression of norovirus NS1‐2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus‐induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents

We report for the first time the antiviral activities of two iminovirs (antiviral imino-C-nucleosides) 1 and 2, structurally related to galidesivir (Immucillin A, BCX4430). An iminovir containing the 4-aminopyrrolo[2,1-f ][1,2,4-triazine] nucleobase found in remdesivir exhibited submicromolar inhibition of multiple strains of influenza A and B viruses, as well as members of the Bunyavirales order. We also report the first syntheses of ProTide prodrugs of iminovir monophosphates, which unexpectedly displayed poorer viral inhibition than their parent nucleosides in vitro. An efficient synthesis of the 4-aminopyrrolo[2,1-f ][1,2,4triazine]-containing iminovir 2 was developed to enable preliminary in vivo studies, wherein it displayed significant toxicity in BALB/ c mice and limited protection against influenza. Further modification of this anti-influenza iminovir will therefore be required to improve its therapeutic value.

Viral infections are one of the leading causes of acute morbidity in humans and much endeavour has been made by the synthetic community for the development of drugs to treat...