29 research outputs found
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Molecular genetic analysis of vaccinia virus genes which confer resistance to alpha-amanitin
Several laboratories have shown that the VV
replication cycle is dependent on the host cell nucleus.
The basic biological observation is that if the host cell
nucleus is physically removed by cytochalasin B-mediated
enucleation, functionally inactivated by UV-irradiation, or
selectively blocked with a-amanitin, the results are the
same: W is able to initiate infection and carry out
macromolecular syntheses, but no infectious progeny virions
are assembled.
To elucidate the molecular mechanism of this virus-host
cell interaction, one approach is to identify the
viral gene(s) responsible for interaction with the host
nucleus and subject them to a detailed molecular analyses. The genomic location of the gene(s) which provide an
α-amanitin resistant phenotype to viral mutants have been
mapped to Hindlll N/M region of the genome by the use of
marker rescue techniques [E.C. Villarreal and D.E. Hruby
(1986) Journal of Virology 57: 65-70] . In an attempt to
understand the genetic organization of this part of the
genome, the entire Hindlll N and part of the neighboring
Hindlll M DNA fragments have been sequenced. The sequencing
data revealed two complete leftward reading open reading
frames (ORFs, N2 and Ml). In order to determine which of
the two ORFs is responsible for confering resistance to
a-amanitin, marker rescue analyses on a VV dual mutant
(arts7) that has both temperature sensitive and resistant
to α-amanitin was carried out. This showed that N2 ORF is
the gene responsible for both phenotypes. To analyze
regulatory sequences responsible for expression of the N2
and Ml genes, putative cis-acting regulatory sequences
upstream from the N2 and Ml genes were isolated and abutted
it to a bacterial reporter gene [chloramphenicol acetyl
transferase (CAT)]. To assay for regulatory sequences,
these promoter:CAT plasmids were initially used in
transient expression protocols and subsequently these
plasmids were used to make recombinant viruses
Genetic Characterization of Nipah Virus, Bangladesh, 2004
Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks
An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice
Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Herein, we show that the ribonucleoside analog β-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (β-D-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple coronaviruses and oral bioavailability highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses
Broadening of Neutralization Activity to Directly Block a Dominant Antibody-Driven SARS-Coronavirus Evolution Pathway
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution
Multihospital Outbreak of a Middle East Respiratory Syndrome Coronavirus Deletion Variant, Jordan: A Molecular, Serologic, and Epidemiologic Investigation
Background
An outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Jordan in 2015 involved a variant virus that acquired distinctive deletions in the accessory open reading frames. We conducted a molecular and seroepidemiologic investigation to describe the deletion variant’s transmission patterns and epidemiology.
Methods
We reviewed epidemiologic and medical chart data and analyzed viral genome sequences from respiratory specimens of MERS-CoV cases. In early 2016, sera and standardized interviews were obtained from MERS-CoV cases and their contacts. Sera were evaluated by nucleocapsid and spike protein enzyme immunoassays and microneutralization.
Results
Among 16 cases, 11 (69%) had health care exposure and 5 (31%) were relatives of a known case; 13 (81%) were symptomatic, and 7 (44%) died. Genome sequencing of MERS-CoV from 13 cases revealed 3 transmissible deletions associated with clinical illness during the outbreak. Deletion variant sequences were epidemiologically clustered and linked to a common transmission chain. Interviews and sera were collected from 2 surviving cases, 23 household contacts, and 278 health care contacts; 1 (50%) case, 2 (9%) household contacts, and 3 (1%) health care contacts tested seropositive.
Conclusions
The MERS-CoV deletion variants retained human-to-human transmissibility and caused clinical illness in infected persons despite accumulated mutations. Serology suggested limited transmission beyond that detected during the initial outbreak investigation
Development of a Neutralization Assay for Nipah Virus Using Pseudotype Particles
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.This article is from Journal of Virological Methods 160 (2009): 1, doi:10.1016/j.jviromet.2009.02.025.</p
Persistence of Antibodies against Middle East Respiratory Syndrome Coronavirus
To determine how long antibodies against Middle East respiratory syndrome coronavirus persist, we measured long-term antibody responses among persons serologically positive or indeterminate after a 2012 outbreak in Jordan. Antibodies, including neutralizing antibodies, were detectable in 6 (86%) of 7 persons for at least 34 months after the outbreak