A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

BACKGROUND:Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells. METHODOLOGY/PRINCIPAL FINDINGS:Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow. CONCLUSIONS/SIGNIFICANCE:The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections

Simultaneous evaluation of physical and social environmental correlates of physical activity in adults: A systematic review

Background: Ecological models of physical activity posit that social and physical environmental features exert independent and interactive influences on physical activity, but previous research has focussed on independent influences. This systematic review aimed to synthesise the literature investigating how features of neighbourhood physical and social environments are associated with physical activity when both levels of influence are simultaneously considered, and to assess progress in the exploration of interactive effects of social and physical environmental correlates on physical activity. Methods: A systematic literature search was conducted in February 2016. Articles were included if they used an adult (≥15 years) sample, simultaneously considered at least one physical and one social environmental characteristic in a single statistical model, used self-reported or objectively-measured physical activity as a primary outcome, reported findings from quantitative, observational analyses and were published in a peer-reviewed journal. Combined measures including social and physical environment items were excluded as they didn’t permit investigation of independent and interactive social and physical effects. Forty-six studies were identified. Results: An inconsistent evidence base for independent environmental correlates of physical activity was revealed, with some support for specific physical and social environment correlates. Most studies found significant associations between physical activity and both physical and social environmental variables. There was preliminary evidence that physical and social environmental variables had interactive effects on activity, although only 4 studies examined interactive effects. Conclusions: Inconsistent evidence of independent associations between environmental variables and physical activity could be partly due to unmeasured effect modification (e.g. interactive effects) creating unaccounted variance in relationships between the environment and activity. Results supported multiple levels of environmental influence on physical activity. It is recommended that further research uses simultaneous or interaction analyses to gain insight into complex relationships between neighbourhood social and physical environments and physical activity, as there is currently limited research in this area

Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons

Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons

Neurons containing latency-associated transcripts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice with herpes simplex virus type 1 mutant in1814

The herpes simplex virus type 1 (HSV-1) mutant in1814 lacks the ability to trans-activate immediate early gene transcription and enter lytic replication but it can establish and reactivate from latency. We therefore investigated the number of neurons that expressed latency-associated transcripts (LATs) in animals latently infected with in1814, the rescued revertant (1814R), or wild-type (wt) HSV-1. The percentage of LAT + neurons increased with increasing doses of each of the viruses. After inoculation of equal amounts of infectious virus many more LAT + neurons were observed in animals infected with in1814 than with 1814R or wt HSV-1. Whereas the LAT + neurons in animals infected with 1814R or wt HSV-1 were largely confined to lumbar dorsal root ganglia (DRG) L4/L5/L6 (those which innervate the lower leg), in animals infected with in1814 they were also present in DRG not directly involved with such innervation (thoracic 12 and 13, L1, L2 and L3). We concluded that the large number of LAT + neurons observed with in1814 was related to the high particle numbers in the inoculum and that spread of virus was related to limited replication as well as to the low neurovirulence of in1814. This spread was not unique to in1814 but when it occurred with more virulent viruses such as 1814R or wt HSV-1, it resulted in the death of the host

Reactivation in vivo and in vitro of herpes simplex virus from mouse dorsal root ganglia which contain different levels of latency-associated transcripts

In the dorsal root ganglia (DRG) of mice latently infected with the herpes simplex virus type 1 mutant in1814, there are more neurons that contain latencyassociated transcripts (LATs) than in DRG of mice infected with a dose of equal infectivity of either a revertant or a wild-type virus. We investigated whether higher levels ofLAT + neurons resulted in more extensive reactivation either in vivo following neurectomy of the sciatic nerve or in vitro after explantation into culture. Neurectomy appeared to induce expression of immediate early 1 mRNA (IElmRNA) in neurons of mice latently infected with each of three viruses. However IElmRNA was detected in no more than 0"25 % of the neurons of DRG from animals 2 to 4 days after neurectomy, irrespective of the percentage of LAT ÷ neurons present. Of the 22 neurons shown to express IElmRNA, none expressed LATs also. However the lack of expression of viral antigen and the absence of a reduced potential for reactivation on explantation suggested that neurectomy had not induced full reactivation involving lytic replication leading to the death of the latently infected neurons. When DRG were explanted into culture, the distribution of the frequency of reactivation was similar to the distribution of DRG that contained LAT + neurons. The presence of a high proportion of LAT + neurons was not directly associated with earlier detection of reactivation but such experiments cannot be regarded as quantitative. We therefore concluded that neurectomy did not result in a reduced reactivation potential as described by others and that the frequency of expression of IElmRNA following neurectomy did not correlate with the number of LAT + nuerons present