The architecture of the simian varicella virus transcriptome

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed direct long-read RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Histopathological Analysis of Adrenal Glands after Simian Varicella Virus Infection

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with βIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with “long-hauler” symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Intratracheal inoculation of human varicella zoster virus (VZV; MAV strain) vaccine successfully induced VZV IgG antibodies in rhesus monkeys

Background The objective of this study was to investigate whether the use of live attenuated varicella zoster virus (VZV) MAV vaccination can efficiently induce VZV antibody production in naive rhesus monkeys as an approach to prevent simian varicella virus (SVV) reactivation in animals immunosuppressed for transplantation studies. Results Clinically available human VZV vaccine was used to induce the production of anti-VZV antibodies in rhesus monkeys. A vial of the vaccine was subcutaneously injected at 0 week, and the second and third vaccination was performed at 5 and 6 weeks by intratracheal inoculation. The titer of anti-VZV IgG was assessed at 0, 2, 4, 6, and 7 weeks. At 2 weeks, 3/16 were seropositive for VZV IgG. At 6 weeks, 9/16 were shown to be seropositive. At 7 weeks, 16/16 were found to be seropositive. Conclusions The VZV vaccine via intratrachael inoculation was shown to induce VZV IgG humoral immunity in rhesus monkeys and may be important immunosuppressed macaques for transplantation studies. Although the humoral immunity produced is an important finding, further studies will be necessary to confirm possible protection and it could protect probably against SVV infection in rhesus monkey.

A new isolated local varicella virus: isolation, identification, comparative growth characteristics and immunological evaluation on an animal model

A panel of 4 different cell lines was optimized for isolation, identification, and authentication of a VZV virus from a swab sample of an 8-year-old boy suspected to varicella zoster infection. The system enabled highly efficient and rapid isolation of viruses in 33°C by serial sub culturing to more than 25 passages. The technique relies on isolation of viral genes by increasing the number of particles that are statistically represented in cell culture and verified by CCID50, FAM-RT-PCR, and IE62 antibody in IF test. The viral genes (ORF38, ORF54) confirmed the new isolate as VZV and revealed the amino acid sequence of viral-encoded proteins after 27 passages, identical with positive control virus, in RFLP-PCR test. Utilization of successive serial passages at temperatures lower than the normal body temperature would reduce the virus virulence and directly cause virus attenuation. As a result, the attenuated virus is adapted to growth in vitro and presented higher replication at 33°C. Our goal was to determine if the targeted virus with a large double-stranded DNA genome, varicella virus, is isolated and can be attenuated by cold adapting in vitro, Using vOka as attenuated VZV golden standard, in two quantitative tests, including CCID50 and FAM-RT-PCR. Finally, when compared with the local isolated virus, these results were strongly confirmed. We recorded plaque forming assay to show phenotypic changing which encodes the attenuation regarding size of plaque. Although in plaque forming assay, the size of plaque seemed smaller at first glance, the statistical distribution of the plaque size did not show any change between the virus in the first and last passages. In cell culture, the local VZV isolated viruses formed clear plaques and grew to higher titers compared with lower passages as parental virus.Due to lack of access to human fetal lung cells (MRC-5) and an alternative to vaccine production in the future, a new authenticated local foreskin cell substrate (RFSC) was used for virus cultivation. In comparison to, MRC-5-optimized and cloned-viruses replicated in vitro with kinetics that were similar to those of the RFSC. Laboratory animals that were infected with the optimized virus fluid as vaccine showed a good neutralization antibody against local VZV isolated as compared to vOka as control positive virus. These results demonstrate that the virus isolated from swab sample was authenticated as VZV virus, and this cold adapted attenuated virus may be an applicable candidate for future plan.Author summaryWe used different cell substrates for isolation, identification, and attenuation of a new local VZV virus from vesicle swab of suspected patient to varicella zoster diseases. The technique involves serial sub culturing of virus in cell culture. Our goal was to determine if the targeted virus with a large double-stranded DNA genome, varicella virus, is isolated and can be attenuated by cold adapting in vitro, using vOka as attenuated VZV golden standard, The virus was cloned and purified by serial dilution cloning and plaque assay. Different techniques were used regarding verification as differentiation from other members in family, potency, and sterility for isolated virus. The virus can grow well in new local foreskin cell substrate and produce good titre as compared with MRC-5. The isolated VZV potentially induced neutralization antibody in animal model. The results of our study imply that local VZV might be an applicable isolate for future plan in research and developing a varicella vaccine.

Varicella Virus Vaccine Live: A 22-Year Review of Postmarketing Safety Data

Background Varicella, a contagious infectious disease caused by varicella zoster virus (VZV), can result in hospitalization and, occasionally, death. Varicella virus vaccine live (VVVL [VARIVAX]) was introduced in the United States in 1995. Methods This comprehensive review of the VVVL safety profile is based on 22 years of postmarketing adverse event (AE) data received through spontaneous and noninterventional study reports submitted by health care providers and on a review of the published literature (cumulatively from March 17, 1995, through March 16, 2017, during which period >212 million doses were distributed globally). Results The VVVL safety profile was consistent with previous publications, with common AEs including varicella, rash, and pyrexia. AE reports have decreased over time, from ~500 per million doses in 1995 to ~40 per million doses in 2016; serious AEs comprise 0.8 reports per million doses. Secondary transmission was rare (8 confirmed cases); polymerase chain reaction analysis indicated that 38 of the 66 reported potential secondary transmission cases of varicella were attributable to wild-type VZV. The prevalence of major birth defects in the Pregnancy Registry was similar to that in the general US population. In total, 86 cases of death were reported after vaccination with VVVL; immunocompromised individuals appeared to be most at risk for a fatal varicella- or herpes zoster–related outcome. Conclusions This comprehensive 22-year review confirms the overall safety profile for VVVL, with no new safety concerns identified. Since VVVL’s introduction in 1995, notable declines in varicella cases and in varicella-related deaths have occurred compared with the prevaccination period.