Gene-edited and -engineered stem cell platform drives immunotherapy for brain metastatic melanomas.

Oncolytic virus therapy has shown activity against primary melanomas; however, its efficacy in brain metastases remains challenging, mainly because of the delivery and immunosuppressive nature of tumors in the brain. To address this challenge, we first established PTEN-deficient melanoma brain metastasis mouse models and characterized them to be more immunosuppressive compared with primary melanoma, mimicking the clinical settings. Next, we developed an allogeneic twin stem cell (TSC) system composed of two tumor-targeting stem cell (SC) populations. One SC was loaded with oncolytic herpes simplex virus (oHSV), and the other SC was CRISPR-Cas9 gene-edited to knock out nectin 1 (N1) receptor (N1KO) to acquire resistance to oHSV and release immunomodulators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). Using mouse models of brain metastatic BRAFV600E/PTEN-/- and BRAFV600E/wt/PTEN-/- mutant melanomas, we show that locoregional delivery of TSCs releasing oHSV and GM-CSF (TSC-G) activated dendritic cell- and T cell-mediated immune responses. In addition, our strategy exhibited greater therapeutic efficacy when compared with the existing oncolytic viral therapeutic approaches. Moreover, the TSCs composed of SC-oHSV and SCN1KO-releasing GM-CSF and single-chain variable fragment anti-PD-1 (TSC-G/P) had therapeutic efficacy in both syngeneic and patient-derived humanized mouse models of leptomeningeal metastasis. Our findings provide a promising allogeneic SC-based immunotherapeutic strategy against melanomas in the CNS and a road map toward clinical translation.

Prevalance of Schmallenberg orthobunyavirus (SBV) infection in sampled ruminants in Turkey's Eastern Mediterranean region between 2015 and 2017.

Schmallenberg orthobunyavirus (SBV), first discovered in 2011, belongs to the Orthobunyavirus genus of the Peribunyaviridae family. SBV, which predominantly infects ruminants, can cause severe fetal malformations when pregnant animals are infected during a critical phase of gestation. In this study, 1590 blood serum samples from cattle, sheep, and goats were obtained for serological investigation and 1604 specimens for virological investigation (including 1414 whole blood with EDTA, 165 vaginal swab samples from aborting animals, and tissue samples from 25 dead and/or aborted fetuses) in private and family-type ruminant establishments in Turkey's Eastern Mediterranean region. All the blood serum samples were tested for the presence of antibodies using ELISA, which showed SBV antibodies in 29.11% (95% CI: 26.89%-31.35%). The virological samples were tested using real-time RT-PCR for SBV nucleic acid presence, which showed 3.17% (95% CI:2.32%-4.04%) were positive. Finally, 10 different Culicoides species (a total of 29,156 Culicoides, including 16,005 females and 13,151 males) were tested to identify the vectors thought to carry infections in the region. However, no SBV nucleic acid was detected in the Culicoides pools.

Development of a BoHV-4 viral vector expressing tgD of BoHV-1 and evaluation of its immunogenicity in mouse model.

In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD∆TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgDΔTK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.

Akabane virus infection in Eastern Mediterranean Region in Turkey: Culicoides (Diptera: Ceratopogonidae) as a possible vector.

Akabane virus (AKAV), which causes Akabane disease, is an arthropod-borne virus (arbovirus) transmitted by Culicoides biting midges and mosquitoes. AKAV is an important pathogen that causes abortion and congenital anomalies in ruminants. In this study, we determined the prevalence of AKAV infection and identified possible viral vectors in Turkey's Eastern Mediterranean region. The presence and prevalence of AKAV infection were assessed using serological and virological methods. Serologically, the prevalence of AKAV antibodies in cattle, sheep and goats were 44.74% (400/894), 22.90% (60/262) and 14.52% (63/434), respectively, while the total prevalence was 32.89% (523/1590). AKAV-specific nucleic acid amplicons were obtained by real-time RT-PCR from 1.13% (9/799) and 1.74% (5/288) of the cattle and sheep tested, respectively. No goats were positive for AKAV RNA. Overall, AKAV-specific nucleic acid amplicons were detected in 0.87% (14/1604) of the sampled ruminants. In addition, specimens of the assumed vector, Culicoides, were caught using light traps and identified. Ten Culicoides species were detected in the area, of which Culicoides schultzei complex was the dominant species although 32 specimens could not be identified at the species level. These were defined as Culicoides spp. AKAV nucleic acid was detected in C. schultzei, Culicoides longipennis and Culicoides circumscriptus. Phylogenetic analysis indicated two different AKAV genogroups (genogroups Ib and genogroups II) while potential AKAV vectors in this region are C. schultzei complex, C. longipennis and C. circumscriptus.

A protocol for displaying viral envelope glycoproteins on the surface of vesicular stomatitis viruses.

We describe the production of single-cycle (sc) and replication-competent recombinant vesicular stomatitis viruses (rcVSVs) displaying heterologous envelope glycoproteins (Envs) on their surface. We prepare scVSVs by transiently expressing HIV-1 Envs or SARS-CoV-2 spike followed by infection of the cells with scVSV particles, which do not carry the vsv-g gene. To prepare rcVSVs, we replace the vsv-g with a specific env-encoding gene, transfect cells with multiple plasmids for production of the genomic RNA and viral proteins, and rescue replication-competent viruses.

Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

Novel Tick Phlebovirus Genotypes Lacking Evidence for Vertebrate Infections in Anatolia and Thrace, Turkey.

We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kirklareli, and Tekirdag (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Sanliurfa (Southeastern Anatolia) provinces from 2013-2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequences in three pools. A comprehensive analysis revealed three distinct, monophyletic virus genotypes, comprised of previously-described viruses from Anatolia and the Balkans, with unique fingerprints in conserved amino acid motifs in viral replicase. A novel tick phlebovirus group was discovered circulating in the Balkans and Turkey, with at least three genotypes or species. No evidence for replication in vertebrates or infections in clinical cases could be demonstrated.