A Temperature-Sensitive Recombinant of Avian Coronavirus Infectious Bronchitis Virus Provides Complete Protection against Homologous Challenge.

Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.

Adenoviruses of the most ancient primate lineages support the theory on virus-host co-evolution.

The scarcity or complete lack of information on the adenoviruses (AdVs) occurring in the most ancient non-human primates resulted in the initiation of a study for exploring their abundance and diversity in prosimians and New World monkeys (NWMs). In order to assess the variability of these AdVs and the possible signs of the hypothesised virus-host co-evolution, samples from almost every family of NWMs and prosimians were screened for the presence of AdVs. A PCRscreening of 171 faecal or organ samples from live or dead, captive or wild-living prosimians and NWMs was performed. The PCR products from the gene of the IVa2 protein were sequenced and used in phylogeny calculations. The presence of 10 and 15 new AdVs in seven and ten different species of prosimians and NWMs was revealed, respectively. Phylogenetic analysis indicated that the tentative novel AdVs cluster into two separate groups, which form the most basal branches among the primate AdVs, and therefore support the theory on the co-evolution of primate AdVs with their hosts. This is the first report that provides a comprehensive overview of the AdVs occurring in prosimians and NWMs, and the first insight into the evolutionary relationships among AdVs from all major primate groups.

A Highly Potent SARS-CoV-2 Blocking Lectin Protein.

The COVID-19 (coronavirus disease-19) pandemic affected more than 180 million people around the globe, causing more than five million deaths as of January 2022. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the new coronavirus, has been identified as the primary cause of the infection. The number of vaccinated people is increasing; however, prophylactic drugs are highly demanded to ensure secure social contact. A number of drug molecules have been repurposed to fight against SARS-CoV-2, and some of them have been proven to be effective in preventing hospitalization or ICU admissions. Here, we demonstrated griffithsin (GRFT), a lectin protein, to block the entry of SARS-CoV-2 and its variants, Delta and Omicron, into the Vero E6 cell lines and IFNAR-/- mouse models by attaching to the spike protein of SARS-CoV-2. Given the current mutation frequency of SARS-CoV-2, we believe that GRFT protein-based drugs will have a high impact in preventing the transmission of both the Wuhan strain as well as any other emerging variants, including Delta and Omicron variants, causing the high-speed spread of COVID-19.

[New-onset diabetes mellitus and liver transplantation, with special consideration of recurrent hepatitis C]. / De novo diabetes és májátültetés, különös tekintettel a hepatitis C-vírus kiújulására.

UNLABELLED: New-onset diabetes is a common complication after liver transplantation. AIM: We aimed to analyze the incidence and rate of known risk factors and the impact of new-onset diabetes mellitus on postoperative outcome. METHODS: We retrospectively evaluated the files of 310 patients who underwent liver transplantation between 1995 and 2009. Definition of new-onset diabetes included: repeated fasting serum glucose >6.8 mmol/l and/or sustained antidiabetic therapy that was present 3 months after transplantation. RESULTS: New-onset diabetes occurred in 63 patients (20%). Differences between the new-onset and the control group were the donor body mass index (24+/-3 vs. 22.4+/-3.6 kg/m 2 , p = 0.003), donor male gender (58% vs. 33%, p = 0.002), and recipient age (47.6+/-7.2 vs. 38.3+/-14.6 year, p<0.001), body mass index (26.7+/-3.8 vs. 23.3+/-5.6 kg/m 2 , p<0.001), male gender (60% vs. 44%, p = 0.031). The 66% of patients with new-onset diabetes were transplanted with cirrhosis caused by hepatitis C virus infection, while in the control group the rate was 23% (p<0.001). Cumulative patient survival rates at 1, 3, 5 and 8 year were 95%, 90.6%, 88% and 88% in the control group, and 87%, 79%, 79% and 64% in the de novo group, respectively (p = 0.011). Cumulative graft survival rates at 1, 3, 5 and 8 year in the control group were 92%, 87%, 86% and 79%, in the de novo diabetes group the rates were 87%, 79%, 79%, 65%, respectively (p = NS). In case of early recurrence (in 6 months), majority of patients developed new-onset diabetes (74% vs. control 26%, p = 0.03). More patients had more than 10 times higher increase of the postoperative virus titer correlate to the preoperative titer in the de novo diabetes group (53% vs. 20%, p = 0.028). Mean fibrosis score was higher in new-onset group one year after the beginning of antiviral therapy (2.05+/-1.53 vs. 1.00+/-1.08, p = 0.039). CONCLUSIONS: Risk factors for new-onset diabetes after transplantation are older age, obesity, male gender and cirrhosis due to hepatitis C infection. The early recurrence, viremia and more severe fibrosis after antiviral therapy have an impact on the occurrence of new-onset diabetes in hepatitis C positive patients.

Differential Growth Characteristics of Crimean-Congo Hemorrhagic Fever Virus in Kidney Cells of Human and Bovine Origin.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a lethal tick-borne zoonotic disease with severe clinical manifestation in humans but does not produce symptomatic disease in wild or domestic animals. The factors contributing to differential outcomes of infection between species are not yet understood. Since CCHFV is known to have tropism to kidney tissue and cattle play an important role as an amplifying host for CCHFV, in this study, we assessed in vitro cell susceptibility to CCHFV infection in immortalized and primary kidney and adrenal gland cell lines of human and bovine origin. Based on our indirect fluorescent focus assay (IFFA), we suggest a cell-to-cell CCHF viral spread process in bovine kidney cells but not in human cells. Over the course of seven days post-infection (dpi), infected bovine kidney cells are found in restricted islet-like areas. In contrast, three dpi infected human kidney or adrenal cells were noted in areas distant from one another yet progressed to up to 100% infection of the monolayer. Pronounced CCHFV replication, measured by quantitative real-time RT-PCR (qRT-PCR) of both intra- and extracellular viral RNA, was documented only in human kidney cells, supporting restrictive infection in cells of bovine origin. To further investigate the differences, lactate dehydrogenase activity and cytopathic effects were measured at different time points in all mentioned cells. In vitro assays indicated that CCHFV infection affects human and bovine kidney cells differently, where human cell lines seem to be markedly permissive. This is the initial reporting of CCHFV susceptibility and replication patterns in bovine cells and the first report to compare human and animal cell permissiveness in vitro. Further investigations will help to understand the impact of different cell types of various origins on the virus-host interaction.

Guidance on the special care of liver or kidney transplant recipients diagnosed with COVID-19 / Az új koronavírus (SARS-CoV-2) okozta fertozésben szenvedo vese- és májátültetett betegek ellátásának speciális szempontjai. (A COVID-19-pandémia orvosszakmai kérdései)

Due to the COVID-19 pandemic caused by infection with the novel, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), transplant medicine also had to face a new, hitherto unknown challenge. To be prepared for any possibility, we consider it important to summarize the current knowledge regarding COVID-19 of liver and kidney transplant patients. Very early reports from Spanish and French registry recorded fatality rates of 18.6% and 13%, respectively, in renal patients which suggests a moderately worse outcome compared to the general population. In patients with positive PCR test but not showing clinical signs, the reduction of immunosuppression is not advised. In the case of gastrointestinal or respiratory signs with fever, the discontinuation of mycophenolate or mTOR inhibitors is recommended with decrease of the trough levels of calcineurin inhibitors to the lowest effective limit. Stop (kidney transplanted patients) or decrease (liver transplanted patients) immunosuppression and maintain corticosteroids when pulmonal injury develops and consider anti-IL1 and anti-IL6 monoclonal antibody use when hyperinflammatory syndrome is evolving. No proven effective treatment for SARS-CoV-2 exists currently. The use of lopinavir/ritonavir should be avoided because of the severe drug interaction with calcineurin inhibitors. The efficacy and tolerability of hidroxychloroquin remains to be also questionable; enroll patients into clinical trial with remdesivir or favipiravir if available. COVID-19 is characterized by virus-induced endothelial dysfunction, procoagulant state and renin-angiotensin-aldosteron system imbalance. Early thromboprofilaxis combination with low-molecular-weight heparin and low-dose aspirin is strongly recommended with the maintenance of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin-II-receptor blocker (ARB) therapy when they were prescribed earlier. Orv Hetil. 2020; 161(32): 1310-1321.

Immunological Analysis of a CCHFV mRNA Vaccine Candidate in Mouse Models.

Development of new vaccine platforms against viral diseases is considered urgent. In recent years, mRNA constructs have attracted great interest in this field due to unique advantages over conventional gene transfer platforms. In the present study, we developed a new naked conventional mRNA vaccine expressing the non-optimized small (S) segment of the Ank-2 strain of Crimean-Congo Hemorrhagic Fever virus (CCHFV). We then analyzed its single and booster dose immunogenicity and protection potential in the challenge assay in two mice models, including IFNα/ß/γR-/- and C57BL/6. The results obtained from the immunological assays, namely IL-4 and IFN-gamma ELISPOT, intracellular IFN-gamma staining, in-house sandwich ELISA, and survival data, demonstrated that our construct elicited the production of anti-nucleocapsid (N) specific immune responses in both mice models. A 100% protection rate was only obtained in the booster dose group of IFNα/ß/γR-/- mice, indicating that this platform needs further optimization in future studies. In conclusion, we assessed a novel approach in CCHFV vaccination by introducing a conventional mRNA platform which can be considered in future experiments as an efficient and safe way to battle this disease.

Co-Delivery Effect of CD24 on the Immunogenicity and Lethal Challenge Protection of a DNA Vector Expressing Nucleocapsid Protein of Crimean Congo Hemorrhagic Fever Virus.

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection, with an eminent risk of fatal human disease. The imminent public health threat posed by the disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing a nucleocapsid protein (N) of CCHFV (pV-N13), and investigated its potential to stimulate the cytokine and total/specific antibody responses in BALB/c and a challenge experiment in IFNAR-/- mice. Because of a lack of sufficient antibody stimulation towards the N protein, we have selected cluster of differentiation 24 (CD24) protein as a potential adjuvant, which has a proliferative effect on B and T cells. Overall, our N expressing construct, when administered solely or in combination with the pCD24 vector, elicited significant cellular and humoral responses in BALB/c, despite variations in the particular cytokines and total antibodies. However, the stimulated antibodies produced as a result of the N protein expression have shown no neutralizing ability in the virus neutralization assay. Furthermore, the challenge experiments revealed the protection potential of the N expressing construct in an IFNAR -/- mice model. The cytokine analysis in the IFNAR-/- mice showed an elevation in the IL-6 and TNF-alpha levels. In conclusion, we have shown that targeting the S segment of CCHFV can be considered for a practical way to develop a vaccine against this virus, because of its ability to induce an immune response, which leads to protection in the challenge assays in the interferon (IFN)-gamma defective mice models. Moreover, CD24 has a prominent immunologic effect when it co-delivers with a suitable foreign gene expressing vector.

Bovine Herpesvirus Type 4 (BoHV-4) Vector Delivering Nucleocapsid Protein of Crimean-Congo Hemorrhagic Fever Virus Induces Comparable Protective Immunity against Lethal Challenge in IFNα/ß/γR-/- Mice Models.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne infection with a significant mortality rate of up to 40% in endemic areas, with evidence of geographical expansion. Due to a lack of effective therapeutics and control measures, the development of a protective CCHFV vaccine remains a crucial public health task. This paper describes, for the first time, a Bovine herpesvirus type 4 (BoHV-4)-based viral vector (BoHV4-∆TK-CCHFV-N) and its immunogenicity in BALB/c and protection potential in IFNα/ß/γR-/- mice models in comparison with two routinely used vaccine platforms, namely, Adenovirus type 5 and a DNA vector (pCDNA3.1 myc/His A), expressing the same antigen. All vaccine constructs successfully elicited significantly elevated cytokine levels and specific antibody responses in immunized BALB/c and IFNα/ß/γR-/- mice. However, despite highly specific antibody responses in both animal models, the antibodies produced were unable to neutralize the virus in vitro. In the challenge experiment, only the BoHV4-∆TK-CCHFV-N and Ad5-N constructs produced 100% protection against lethal doses of the CCHFV Ank-2 strain in IFNα/ß/γR-/- mice. The delivery platforms could not be compared due to similar protection rates in IFNα/ß/γR-/- mice. However, during the challenge experiment in the T cell and passive antibody transfer assay, BoHV4-∆TK-CCHFV-N was dominant, with a protection rate of 75% compared to others. In conclusion, vector-based CCHFV N protein expression constitutes an effective approach for vaccine development and BoHV-4 emerged as a strong alternative to previously used viral vectors.

A metagenomic survey identifies Tamdy orthonairovirus as well as divergent phlebo-, rhabdo-, chu- and flavi-like viruses in Anatolia, Turkey.

We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional investigation of viruses in 10 tick pools, collected from Aegean, Mediterranean and central Anatolian locations in Turkey. Sequences from all genome segments of Tamdy orthonairovirus (family Nairoviridae) were characterized in ticks collected from a Meriones tristrami. We further obtained near-complete L and partial S segments of several tick-associated phleboviruses (family Phenuiviridae), including Tacheng tick virus 2 and a novel virus, tentatively named as the tick phlebovirus Anatolia. Partial NS5-coding region of recently-described flavi-like virus (Tacheng tick virus 8) was further detected. Moreover, near-complete and polymerase-coding regions of arthropod-associated rhabdoviruses as well as sequences closely-related to the members of the newly-proposed virus family, the Chuviridae, were characterized. Despite origins of the viral sequences could not be fully elucidated, the findings suggest the circulation of diverse arthropod and tick-associated viruses in Anatolia. Occurrence and outcome of vertebrate exposure and probable health impact of these viruses require further investigation. We also report the initial detection of Tamdy orthonairovirus, an established human pathogen, which should be included in the diagnostic workup of infections with unknown etiology.

Generic amplification and next generation sequencing reveal Crimean-Congo hemorrhagic fever virus AP92-like strain and distinct tick phleboviruses in Anatolia, Turkey.

BACKGROUND: Ticks are involved with the transmission of several viruses with significant health impact. As incidences of tick-borne viral infections are rising, several novel and divergent tick- associated viruses have recently been documented to exist and circulate worldwide. This study was performed as a cross-sectional screening for all major tick-borne viruses in several regions in Turkey. Next generation sequencing (NGS) was employed for virus genome characterization. Ticks were collected at 43 locations in 14 provinces across the Aegean, Thrace, Mediterranean, Black Sea, central, southern and eastern regions of Anatolia during 2014-2016. Following morphological identification, ticks were pooled and analysed via generic nucleic acid amplification of the viruses belonging to the genera Flavivirus, Nairovirus and Phlebovirus of the families Flaviviridae and Bunyaviridae, followed by sequencing and NGS in selected specimens. RESULTS: A total of 814 specimens, comprising 13 tick species, were collected and evaluated in 187 pools. Nairovirus and phlebovirus assays were positive in 6 (3.2%) and 48 (25.6%) pools. All nairovirus sequences were closely-related to the Crimean-Congo hemorrhagic fever virus (CCHFV) strain AP92 and formed a phylogenetically distinct cluster among related strains. Major portions of the CCHFV genomic segments were obtained via NGS. Phlebovirus sequencing revealed several tick-associated virus clades, including previously-characterized Antigone, Lesvos, KarMa and Bole tick viruses, as well as a novel clade. A wider host range for tick-associated virus strains has been observed. NGS provided near-complete sequences of the L genomic segments of Antigone and KarMa clades, as well as Antigone partial S segment. Co- infections of CCHFV and KarMa or novel phlebovirus clades were detected in 2.1% of the specimens. CONCLUSIONS: Widespread circulation of various tick-associated phlebovirus clades were documented for the first time in Anatolia. Genomes of CCHFV AP92 strains were identified in previously unexplored locations. NGS provided the most detailed genomic characterization of the Antigone and KarMa viruses to date. The epidemiological and health-related consequences must be elucidated.