Establishment of reverse genetics system for PPR virus to develop recombinant vaccines

Across the developing world peste des petits ruminants virus (PPRV) places a huge disease burden on small ruminant agriculture. PPR is mainly controlled by vaccinating animals with live attenuated vaccines. However, the current PPR vaccines and companion serological tests do not enable serological differentiation between naturally infected and vaccinated animals (DIVA), therefore a meaningful serological assessment of vaccine coverage and epidemiological surveillance is not possible. Therefore, the main objective of this PhD study was to establish a reverse genetics system for PPRV, so that a marker vaccine could be developed to enable the serological differentiation between vaccination and infection, alongside developing proof of concept for increasing the valency of the existing vaccines. Initially, as a prerequisite to full genome synthesis the full genome sequence for a PPRV vaccine strain was confirmed. An efficient reverse genetics system for the PPRV Nigeria75/1 vaccine strain was established in this study and 3 recombinant PPRVs were rescued including a faithful clone of the vaccine strain (rPPRV Nigeria75/1), a clone expressing GFP as a heterologous protein (rPPRV+GFP Nigeria75/1) and a negatively marked vaccine containing mutations to the haemagglutinin (H) gene (rPPRV-C77 Nigeria75/1). All 3 rescued viruses showed similar growth characteristics in vitro when compared to the parental vaccine strain and, following in vivo assessment the H mutant provided full protection in goats upon virulent virus challenge. Although the mutations made to H abrogated in vitro binding of C77, the mutations made were not sufficient to enable DIVA in vivo. Finally proof of concept was developed for the segmentation of PPRV and expression of heterologous proteins in an effort to generate a multivalent vaccine. A recombinant two-segmented version of PPRV was successfully rescued that expressed GFP from one segment and the bluetongue virus VP2 from the other. This virus was partially characterised in vitro and demonstrates the potential for this approach in the development of multivalent vaccines for small ruminants

Suppression of DC-SIGN and gH Reveals Complex, Subset-Specific Mechanisms for KSHV Entry in Primary B Lymphocytes

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple cancers in immunocompromised patients including two lymphoproliferative disorders associated with KSHV infection of B lymphocytes. Despite many years of research into the pathogenesis of KSHV associated diseases, basic questions related to KSHV molecular virology remain unresolved. One such unresolved question is the cellular receptors and viral glycoproteins needed for KSHV entry into primary B lymphocytes. In this study, we assess the contributions of KSHV glycoprotein H (gH) and the cellular receptor DC-SIGN to KSHV infection in tonsil-derived B lymphocytes. Our results show that (1) neither KSHV-gH nor DC-SIGN are essential for entry into any B cell subset, (2) DC-SIGN does play a role in KSHV entry into tonsil-derived B cells, but in all B cell subtypes alternative entry mechanisms exist, (3) KSHV-gH can participate in KSHV entry into centrocytes via a DC-SIGN independent entry mechanism, and (4) in the absence of KSHV-gH, DC-SIGN is required for KSHV entry into centrocytes. Our results provide a first glimpse into the complexity of KSHV entry in the lymphocyte compartment and highlight that multiple subset-dependent entry mechanisms are employed by KSHV which depend upon multiple cellular receptors and multiple KSHV glycoproteins

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro. Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro. Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells. IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

A Multivalent Kaposi Sarcoma-Associated Herpesvirus-Like Particle Vaccine Capable of Eliciting High Titers of Neutralizing Antibodies in Immunized Rabbits

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60–90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo

Kaposi Sarcoma-associated Herpesvirus Glycoprotein H is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal, and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types including epithelial, endothelial, and fibroblasts. MPORTANCE: All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

Intra-aortic balloon pump use does not affect the renal function in patients undergoing off pump coronary artery bypass surgery

Renal dysfunction is known to occur during cardiac surgery. A few factors such as perioperative hypotension, use of potential nephrotoxic therapeutic agents, radio opaque contrast media in the recent past, intra-aortic balloon pump (IABP) and cardiopulmonary bypass have been blamed as the contributing factors to the causation of postoperative renal dysfunction in cardiac surgical patients. At times, in patients with renal failure and low cardiac output status, one may face the dilemma if the use of IABP is safe. We undertook this prospective observational study to determine the degree of possible renal injury when IABP is used by measuring serial values of serum creatinine and Cystatin C. Elective patients scheduled for off-pump coronary artery bypass surgery requiring preoperative use of IABP were included in this study. Cystatin C and serum creatinine levels were checked at fixed intervals after institution of IABP. Twenty-two patients were eligible for enrolment to the study. There was no significant change in the values of serum creatinine; from the basal value of 1.10 &#177; 0.233 to 0.98 &#177; 0.363 mg /dL (<i>P</i> value &gt;0.05). Cystatin C levels significantly decreased from the basal level of 0.98 &#177; 0.29 to 0.89 &#177; 0.23 (<i>P</i> value &lt;0.05). Contrary to the belief, Cystatin C, the early indicator of renal dysfunction decreases suggesting absence of renal injury after the use of IABP. Absence of elevation of cystatin C levels in our study suggests the lack of potential of the IABP to cause renal dysfunction in patients who received elective IABP therapy preoperatively

Emergence of PPR and its threat to Europe

PPR is an important infectious viral disease of domestic and wild small ruminants, that threatens the food security and sustainable livelihood of farmers across Africa, the Middle East and Asia. Europe is free of the disease except in Thrace (European part of Turkey) and Israel where outbreaks occur. Following the successful eradication of RPV, PPR has been targeted by the OIE and FAO as the next viral pathogen to be eradicated by 2030. However, the recent outbreaks in Northen Africa and Thrace (European part of Turkey) represent a significant threat to mainland Europe, as a source of disease spread. We have discussed here the emergence of PPR worldwide since its discovery with particular reference to the recent outbreaks in Northen Africa and Thrace, and the potential for spread of the disease into Europe. (C) 2016 The Authors. Published by Elsevier B.V

Anesthetic implications of subxiphoid coronary artery bypass surgery

Background: Minimal invasive surgeries are carried out to benefit the patient with less pain, blood loss, mechanical ventilation and hospital stay; a smaller scar is not the aim. Minimal invasive cardiac surgeries are carried out via small sternotomy, small thoracotomy and via robotic arms. Subxiphoid route is a novel method and avoids sternotomy. Aim: This case series is an attempt to understand the anesthetic modifications required. Secondly, whether it is feasible to carry out subxiphoid coronary artery bypass surgery. Methods: Elective patients scheduled to undergo subxiphoid coronary artery bypass surgery were chosen. The surgeries were conducted under general anesthesia with left lung isolation via either endobronchial tube or bronchial blocker. Results: We conducted ten (seven males and 3 females) coronary artery bypass graft surgeries via subxiphoid technique. The mean EuroSCORE was 1.7 and the mean ejection fraction was 53.6. Eight patients underwent surgery via endobronchial tube, while, in the remaining two lung isolation was obtained using bronchial blocker. Mean blood loss intraoperatively was 300 ± 42 ml and postoperatively 2000 ± 95 ml. The pain score on the postoperative day ′0′ was 4.3 ± 0.6 and 2.3 ± 0.7 on the day of discharge. Length of stay in the hospital was 4.8 ± 0.9 days. There were no complications, blood transfusions, conversion to cardiopulmonary bypass. The modifications in the anesthetic and surgical techniques are, use of left lung isolation using either endobronchial tube or bronchial blocker, increased duration for conduit harvesting, grafting, requirement of transesophageal echocardiography monitoring in addition to hemodynamic monitoring. Other minor requirements are transcutaneous pacing and defibrillator pads, a wedge under the chest to ′lift′ up the chest, sparing right femoral artery and vein (to serve as vascular access) for an unlikely event of conversion to cardiopulmonary bypass. Any anesthesiologist wishing to start this technique must be aware of these modifications. Conclusions: Subxiphoid route is safe to carry out coronary artery bypass graft surgery using the minimal invasive cardiac surgery. It is reproducible and has undeniable benefits. We plan to conduct such surgeries in awake patients under thoracic epidural anesthesia thus making it even less invasive and amenable for fast tracking

Myocardial protection during off pump coronary artery bypass surgery: A comparison of inhalational anesthesia with sevoflurane or desflurane and total intravenous anesthesia

Aims and Objectives: The objective of the study was to evaluate the myocardial protective effect of volatile agents-sevoflurane and desflurane versus total intravenous anesthesia (TIVA) with propofol in offpump coronary artery bypass surgery (OPCAB) by measuring cardiac troponin-T (cTnT) as a marker of myocardial cell death. Materials and Methods: The study was conducted on 139 patients scheduled to undergo elective OPCAB surgery. The patients were randomly allocated to receive anesthesia with sevoflurane, desflurane or TIVA with propofol. The cTnT levels were measured preoperatively, at arrival in postoperative intensive care unit, at 8, 24, 48 and 96 hours thereafter. Results: The changes in cTnT levels at all time intervals were comparable in the three groups. Conclusion: The study did not reveal any difference in myocardial protection after OPCAB with either sevoflurane or desflurane or TIVA using propofol as assessed by measuring serial cTnT values