Establishment of a Poliovirus Containment Program and Containment Certification Process for Poliovirus-Essential Facilities, United States 2017-2022.

Upon declaration of poliovirus (PV) type 2 eradication in 2015, the World Health Organization (WHO) published PV containment requirements in the Global Action Plan III (GAPIII) for mitigating the risk of a facility-associated release post eradication. In 2018, the 71st World Health Assembly resolution urged member states retaining PV to appoint a National Authority for Containment (NAC), reduce the number of PV facilities, and submit applications for containment certification. The United States (US) NAC was established in 2018 for containment oversight, and two paths to WHO GAPIII containment certification were developed. Facilities retaining PV were identified through national poliovirus containment surveys. The US NAC conducted 27 site visits at 18 facilities (20 laboratories: A/BSL-2 (65%), A/BSL-3 (20%), and storage-only (15%)) to verify the implementation of US NAC's preliminary containment measures. The NAC identified areas for improvement in seven categories: primary containment, decontamination, hand hygiene, security, emergency response, training, and immunization practices. Sixteen facility applications were endorsed to pursue poliovirus-essential facility (PEF) certification, whereas four facilities opted to withdraw during the containment certification process. The US made noteworthy progress in PV containment to enhance biosafety and biosecurity practices at US PV facilities to safeguard the polio eradication effort.

Transfer of Select Agents and Toxins: 2003-2013.

The Federal Select Agent Program, which is composed of the Centers for Disease Control and Prevention Division of Select Agents and Toxins and the Animal and Plant Health Inspection Service Agricultural Select Agent Services, regulates entities that possess, use, or transfer biological select agents and toxins in the United States and must preapprove all transfers within or into the US. The requirement to preapprove transfers allows the Federal Select Agent Program to monitor and track shipments to receive alerts of theft, loss, or release during shipment, thereby protecting public health and safety. As part of the program, the Division of Select Agents and Toxins regulates biological select agents and toxins that have been identified by the US government as posing a severe threat to public health and safety. The division analyzed 4,402 transfers that occurred between March 2003 and December 2013 to identify frequently transferred biological select agents and toxins and the types of entities involved in transfers. During the study period, 1 package was lost during shipment and it was determined not to pose a threat to public health. The Federal Bureau of Investigation investigated the loss and concluded that the package was most likely damaged by the commercial carrier and discarded. Further, there were no reports of theft or release associated with biological select agents and toxins shipments. This report represents the first in-depth review of biological select agent and toxin transfers that were approved by the Division of Select Agents and Toxins.

Kaposi sarcoma-associated herpesvirus g protein-coupled receptor enhances endothelial cell survival in part by upregulation of bcl-2.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) encoded G protein-coupled receptor (vGPCR) is a constitutively active lytic phase protein with significant homology to the human interleukin-8 receptor. vGPCR is necessary and sufficient to induce angiogenesis as well as the spindle cell proliferation characteristic of Kaposi sarcoma (KS) lesions. We previously demonstrated that Bcl-2, an antiapoptotic protein, is upregulated in KS lesions. The aim of this study was to determine if vGPCR enhances endothelial cell survival through upregulation of Bcl-2 expression and to elucidate the signaling pathways involved. METHODS: Primary human umbilical vein endothelial cells were transduced with a recombinant retrovirus expressing vGPCR and then subjected to serum starvation. Cell viability and apoptosis were analyzed by fluorescence-activated cell sorting. Bcl-2 expression was determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblotting. Specific pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) were employed to elucidate the signaling pathways involved. Bcl-2 expression was knocked down using small interfering RNA (siRNA). RESULTS: Endothelial cells expressing vGPCR showed increased survival after serum starvation and upregulation of Bcl-2 messenger RNA (mRNA) and protein. The vGPCR-induced increases in both Bcl-2 mRNA and protein levels were dependent on PI3K signaling but not on mTOR. Moreover, siRNA inhibition of Bcl-2 resulted in significant abrogation of the observed vGPCR-mediated cell survival advantage. CONCLUSIONS: Taken together, the results demonstrate that Bcl-2 is a mediator of vGPCR-induced endothelial cell survival and is a downstream effector of Akt in this process.

Kaposi's sarcoma associated herpesvirus G-protein coupled receptor activation of cyclooxygenase-2 in vascular endothelial cells.

BACKGROUND: Kaposi's sarcoma associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a highly vascularized neoplasm characterized by endothelial-derived spindle-shaped tumor cells. KSHV-infected microvascular endothelial cells demonstrate increased cyclooxygenase-2 (COX-2) expression and KS lesions have high levels of prostaglandin E2 (PGE2), a short-lived eicosanoid dependent on cyclooxygenase activity that has been linked to pathogenesis of other neoplasias. To determine whether increased COX-2 expression and PGE2 production is mediated by the angiogenic and tumorigenic KSHV-encoded G-protein coupled receptor (vGPCR), we developed a recombinant retrovirus to express vGPCR in Human Umbilical Vascular Endothelial Cells (HUVEC). RESULTS: In the present study, we show that vGPCR-expressing HUVEC exhibit a spindle-like morphology that is characteristic of KS endothelial cells and demonstrate selective induction of PGE2 and COX-2. By treating vGPCR-expressing HUVEC with selective and non-selective COX inhibitors, we show that vGPCR-induced PGE2 production is dependent on the expression of COX-2 but not COX-1. CONCLUSION: Taken together, these results demonstrate that vGPCR induces expression of COX-2 and PGE2 that may mediate the paracrine effects of this key viral protein in KS pathogenesis.

Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner.

We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s) from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s) from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

Activation of proMMP-2 and Src by HHV8 vGPCR in human pulmonary arterial endothelial cells.

Idiopathic pulmonary arterial hypertension (iPAH) is associated with human herpesvirus 8 (HHV8) infection and demonstrates pathological angiogenesis similar to that observed with another HHV8-linked disease, namely Kaposi Sarcoma (KS). Importantly, the HHV8 encoded viral G-protein-coupled receptor (vGPCR) induces KS lesions in a murine model. Investigating the impact of vGPCR expression on the angiogenic activity of human pulmonary arterial endothelial cells (HPAEC) can yield insight into the pathobiology of HHV8-associated vascular disorders, particularly PAH. Cultured HPAECs were transduced with retroviral vectors carrying either control or vGPCR coding regions. vGPCR expression selectively activated matrix metalloproteinase (MMP)-2, a pivotal matrix modulating enzyme during angiogenesis. A membrane type 1 MMP (MT1-MMP) neutralizing antibody and the tissue inhibitor of metalloproteinases-2 (TIMP-2) independently blocked vGPCR-induced MMP-2 activation. vGPCR expression concordantly promoted MMP-2 activation by increasing MT1-MMP expression while decreasing TIMP-2 expression. vGPCR activated Src kinase as demonstrated by phosphorylation of Src and its substrate focal adhesion kinase (FAK). vGPCR promoted angiogenesis of HPAECs as demonstrated by a substantial increase in tubulogenesis in vitro. The Src inhibitors PP2 and SU6656 significantly diminished vGPCR-induced MMP-2 activation and tubulogenesis. Our findings indicate that vGPCR induces MMP-2 activation in HPAECs through regulation of MT1-MMP and TIMP-2 expression. vGPCR activates Src and inhibition of such activation abrogates proMMP-2 activation and in vitro angiogenesis induced by vGCPR. The current study implicates vGPCR as an etiological agent in iPAH and identifies Src and MMP-2 as potential therapeutic targets in HHV8 associated KS and iPAH.

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Gamma-herpesvirus neoplasia: a growing role for COX-2.

Both human gamma-herpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) induce neoplasia. Burkitt's and Hodgkin's lymphomas harbor EBV sequences, while KSHV has been associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric castleman's disease (MCD). Each of these gamma-herpesvirus-associated malignancies displays typical characteristics of neoplasia, such as angiogenesis and cell survival. One enzyme commonly overexpressed in breast, prostate, and colon cancers is cyclooxygenase-2 (COX-2). Recently, COX-2 overexpression has been reported in herpesvirus infections in vitro. This review will outline potential mechanisms by which COX-2 may participate in herpesvirus-induced neoplasia.