Functional analysis of conserved motifs within herpes simplex virus regulatory protein ICP0

No abstract available

The ‘‘ICE’’ Study: Feasibility of Inexpensive Commercial Coolers on Mobile EMS Units

Introduction: Prehospital postresuscitation induced hypothermia (IH) has been shown to reduce neurological complications in comatose cardiac-arrest survivors. Retrofitting ambulances to include equipment appropriate to initiate hypothermia, such as refrigeration units for cooled saline, is expensive. The objective of this nonhuman subject research study was to determine if inexpensive, commercially available coolers could, in conjunction with five reusable ice packs, keep two 1 L bags of precooled 0.9% normal saline solution (NSS) at or below 48C for an average shift of eight to 12 hours in a real-world environment, on board in-service Emergency Medical Service (EMS) units, over varying weather conditions in all seasons. Methods: The coolers were chosen based on availability and affordability from two nationally available brands: The Igloo MaxxCold (Igloo Products Corp., Katy, Texas USA) and Coleman (The Coleman Company, Wichita, Kansas USA). Both are 8.5 liter (nine-quart) coolers that were chosen because they adequately held two 1 L bags of saline solution, along with the reusable ice packs designated in the study design, and were small enough for ease of placement on ambulances. Initial testing of the coolers was conducted in a controlled environment. Thereafter, each EMS unit was responsible to cool the saline to less than 48C prior to shift. Data were collected by emergency medical technicians, paramedics, and resident physicians working in seven different ambulance squads. Data analysis was performed using repeated measurements recorded over a 12-hour period from 19 individual coolers and were summarized by individual time points using descriptive statistics. Results: Initial testing determined that the coolers maintained temperatures of 48C for 12 hours in a controlled environment. On the ambulances, results based on the repeated measurements over time revealed that the saline solution samples as defined in the protocol, remained consistently below 48C for 12 hours. Utilizing the lower bound of the 2- sided 95% exact binomial confidence intervals, there was less than a five percent chance that saline samples could not be maintained below 48C for 12 hours, even during the summer months. Conclusions: Simple, commercially available coolers can maintain two 1 L bags of 0.9% NSS at 48C for 12 hours in ambulances in varying environmental conditions. This suggests that EMS agencies could inexpensively initiate prehospital IH in appropriate cases

End of 2022/23 season influenza vaccine effectiveness in primary care in Great Britain

Background The 2022/23 influenza season in the United Kingdom saw the return of influenza to prepandemic levels following two seasons with low influenza activity. The early season was dominated by A(H3N2), with cocirculation of A(H1N1), reaching a peak late December 2022, while influenza B circulated at low levels during the latter part of the season. From September to March 2022/23, influenza vaccines were offered, free of charge, to all aged 2–13 (and 14–15 in Scotland and Wales), adults up to 49 years of age with clinical risk conditions and adults aged 50 and above across the mainland United Kingdom. Methods End-of-season adjusted vaccine effectiveness (VE) estimates against sentinel primary-care attendance for influenza-like illness, where influenza infection was laboratory confirmed, were calculated using the test negative design, adjusting for potential confounders. Results In the mainland United Kingdom, end-of-season VE against all laboratory-confirmed influenza for all those > 65 years of age, most of whom received adjuvanted quadrivalent vaccines, was 30% (95% CI: −6% to 54%). VE for those aged 18–64, who largely received cell-based vaccines, was 47% (95% CI: 37%–56%). Overall VE for 2–17 year olds, predominantly receiving live attenuated vaccines, was 66% (95% CI: 53%–76%). Conclusion The paper provides evidence of moderate influenza VE in 2022/23

Dysregulated metabolism of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production.

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production

Multiple post-transcriptional strategies to regulate the herpes simplex virus type 1 vhs endoribonuclease

The HSV1 virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in shut-off of host protein synthesis. Hence its unrestrained activity is considered to be lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from co-transfected plasmids were also retained in the same nuclei where vhs mRNA was located, while polyA binding protein (PABP) was relocalised to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Co-expression of VP16 and VP22 rescued cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5’ region of vhs that blocked its translation and, when transferred to a heterologous GFP transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and auto-induced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex

Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins.

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins-VP22 and VP16 -are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus by a vhs-dependent mechanism, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production

Coding changes found in the genome sequences of the four Δ22 rescue viruses.

Genome sequences were aligned with the strain 17 reference sequence (JN555585.1). Coding changes from the parental Δ22 virus are highlighted in bold.</p

HSV1 spreads in the absence of VP22 without causing CPE.

(A) HFFF cells were infected with Wt s17 or Δ22 virus at a multiplicity of 0.01, supernatant (s/n) and cell-associated (c/a) virus harvested every day for 4 days and titrated onto Vero cells (mean±SEM, n = 3). (B) HFFF cells were infected with approximately 20 pfu of HSV1 GFP- Δ22 virus and brightfield and GFP images acquired 3 days later. Scale bar = 100 μm. (C) HFFF cells were infected with approximately 20 pfu of HSV1 GFP-22 or Δ22 viruses in the presence of 1% human serum and representative brightfield and GFP images acquired every day for 3 days. Scale bar = 100 μm. (D) Confluent HFFF cells were infected with approximately 20 pfu of Δ34 virus and representative brightfield and GFP images acquired at days 1 and 3. Scale bar = 100 μm.</p

The mutant vhs proteins in rescued Δ22 viruses maintain the ability to degrade cellular mRNA.

(A) HFFF cells infected with the indicated viruses at a multiplicity of 2 were harvested for total RNA at 16 h and analysed by qRT-PCR for a range of virus transcripts. Results are represented as Log2 fold change to Wt (ΔΔCT), and the mean ± standard error for n = 3 is shown. Transcripts are colour-coded as blue (immediate-early) red (early), green (late) and pink (true late). Part of this data has been presented in a previous publication [4]. (B) HFFF cells were infected with indicated viruses at a multiplicity of 2. At 6 hours, the cells were either harvested for total RNA, or actinomycin D was added (5 μg/ml) and the infection left for a further 4 hours before harvesting total RNA. qRT-PCR was carried out on all samples for the indicated virus transcripts. Note that in this graph, results are expressed as the log2 FC to the samples harvested at 6 hours (ΔΔCT). The mean and +/- standard error for n = 3 is shown. (C) The relative levels of the UL41 transcript were measured from the samples in (B). (D) HFFF cells infected with the indicated viruses at a multiplicity of 2 were harvested for total RNA at 16 h and analysed by qRT-PCR for the cellular MMP1 and MMP3 transcripts. Results are represented as Log2 fold change to uninfected (ΔΔCT), and the mean ± standard error for n = 3 is shown. (E) HFFF cells were infected with Wt, Δ22, Δvhs or Δ22* viruses at a multiplicity of 2, and total RNA was harvested at the indicated times (in hours). qRT-PCR was carried out on MMP1 and MMP3 cellular transcripts with relative levels expressed as log2 FC to uninfected (ΔΔCT) over time. The mean and ± standard error for n = 3 is shown. The data for the Δ22* virus in 6A and 6E was acquired from RNA samples isolated at the same time as those for the Wt, Δ22 and Δvhs viruses, but the latter three were also presented in a previous publication [4].</p

Relative compartmentalisation of PABPC1 in HFFF cells infected with Δ22 rescue viruses.

HFFF cells infected with the indicated viruses at MOI 2 were fixed at 10 h and 16 h, stained with an antibody for PABPC1 (green) and nuclei stained with DAPI (blue). Scale bar = 50 μm.</p