Molecular characterization of G and F protein genes of bovine respiratory syncytial virus detected from dead calves caused by severe respiratory syndrome: emergence of novel mutations and their importance.

Bovine respiratory syncytial virus (BRSV) is an important viral agent in bovine respiratory disease complex affecting young calves from asymptomatic to fatal. Although BRSV is widely prevalent in Türkiye as in other parts of the world, there are limited molecular studies on BRSV in Türkiye. Therefore, in order to better understand the characteristics of circulating BRSV in Türkiye, a study based on the molecular analysis of both F and G proteins was performed. For this purpose, the presence of BRSV was investigated in 20 calves that died as a result of severe respiratory syndrome in the western region of Türkiye in 2020. Nested PCR was performed for both gene regions, and the products were sequenced. Four samples detected as BRSV positive were identified as genotype III according to both gene regions in molecular analysis. However, they were separated into two distinct clusters due to significant differences in nucleotide (90.09-99.54%) and amino acid (85.42-99.31%) similarities between them. Besides, two positive samples in the same cluster were even more different from previously detected Turkish isolates (90.78-92.17% nt and 87.50-89.58% aa). More over, we detected nine novel aa mutations in the extracellular domain, an immunologically important region in the G protein of the virus, that have not been reported in other world isolates found in Genbank until now. These findings suggest that there may be many different viruses in circulation that have the ability to escape the immune system. We recommend that these findings be taken into account in planning both vaccine and epidemiological studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00846-7.

The first detection of betanodavirus reassortant genotype (RGNNV/SJNNV) isolated from gilthead sea bream (Sparus aurata) in the Turkish coastlines: The importance of screening and monitoring studies for identifying the source of the infection.

Viral nervous necrosis (VNN) is now endemic in the Mediterranean basin and the RGNNV genotype betanodavirus has caused frequent epidemics in European sea bass for a long time. Unexpected and increasing VNN epidemics have been reported in gilthead sea bream (GSB) farms in the last few years, from which the RGNNV/SJNNV genotype has been mostly isolated. The aim of this study was to perform a molecular characterization of the betanodavirus isolated from GSB (weighing 90-100 g) in a marine fish farm in the Aegean Sea and also, as an early warning exercise, to investigate the presence/absence of the virus in associated nearby farms (n:20) and in hatcheries (n:3). No virus was detected in any of the nearby farms or two hatcheries. However, in one hatchery, betanodavirus was detected in a 160-day-old GSB. The identified betanodavirus was genotyped as reassortant RGNNV/SJNNV and was phylogenetically related to the virus detected in the farm located in the Aegean sea. There have been multiple detections of the RGNNV genotype in Turkish coastal waters; however, the RGNNV/SJNNV genotype has been detected for the first time and it should be an early warning to focus attention on betanodaviruses in Turkish aquaculture.

Investigation of betanodavirus in sea bass (Dicentrarchus labrax) at all production stages in all hatcheries and on selected farms in Turkey.

Viral nervous necrosis (VNN) is one of the most important problems in sea bass culture. Although there have been many studies on detection and molecular characterization of betanodavirus, the causative agent of VNN, there has been little focus on understanding its prevalence to create epidemiological maps. The purpose of this study was to investigate the prevalence of betanodavirus in active sea bass hatcheries and on selected farms in Turkey by RT-qPCR. A total of 2460 samples, including fertilized eggs, prelarvae, postlarvae, fry, and fingerlings, were collected from 16 hatcheries to cover all production stages. A total of 600 sea bass were also collected from 20 farms. Betanodavirus was detected in one hatchery (1/16) in fingerling-sized sea bass, and the prevalence of betanodavirus at the hatchery level was calculated to be 6.25%. Betanodavirus was also detected on one farm (1/20) in fingerling-sized sea bass, and the prevalence of betanodavirus at the farm level was calculated to be 5%. Virus isolation initially could not be achieved in E-11 cells, but later, SSN-1 cells were used successfully. Partial genome sequence analysis of the RNA1 and RNA2 segments of the viruses revealed that they were of the red-spotted grouper nervous necrosis virus genotype, which is endemic in the Mediterranean basin. The absence of mortality related to VNN in the hatcheries and on the farms, the healthy appearance of the sea bass, the low viral load detected, and the results of retrospective epidemiological studies indicated that the infection was subclinical. Not detecting betanodavirus in other age groups where biosecurity was implemented indicates that there was no active infection. In light of these findings, it can be concluded that there was no betanodavirus circulating in hatcheries, and the virus might have been of seawater origin.

Detection of RGNNV genotype betanodavirus in the Black Sea and monitoring studies.

Viral nervous necrosis (VNN), caused by betanodavirus, is a significant viral infection that threatens marine aquaculture. Freshwater and marine fish farms in Turkey are subjected to annual pathogen screenings. In 2016, during the Nervous Necrosis Virus screening program conducted in the Black Sea, betanodavirus was unexpectedly detected using real-time reverse transcription-polymerase chain reaction in apparently healthy sea bass. Phylogenetic analysis of both the RNA1 and RNA2 segments of the virus determined that the betanodavirus detected was red-spotted grouper nervous necrosis virus genotype (RGNNV). Following the initial discovery of betanodavirus in the Black Sea, monitoring studies performed over a 3 yr period have not indicated any additional presence of the virus. The absence of clinical symptoms related to VNN disease in the area's marine fish farms and the surrounding detection zone, and the fact that the virus has not been detected anew in monitoring programmes conducted following the initial detection, indicate that there is no virus circulation in the detection zone.

Efficiency of lornoxicam in lung and trachea injury caused by peroxynitrite.

Peroxynitrite is involved in the pathogenesis of pulmonary diseases such as asthma, occupational pulmonary diseases and acute respiratory distress syndrome (ARDS) due to excessive production of nitric oxide or superoxide or both. Lornoxicam, a new oxicam derivative, is a potent anti-inflammatory agent. In this study, we evaluated the role of lornoxicam in a peroxynitrite-induced pulmonary and tracheal injury model by measuring myeloperoxidase (MPO) activity, malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) levels in lung tissue and bronco-alveolar lavage fluid. The study protocol was based on three experimental groups as treatment (T), control (C) and peroxynitrite (P). Each group was subdivided into three subgroups as 2nd, 24th and 48th hour groups. P and T groups were injected intratracheal peroxynitrite. The T group received intraperitoneal lornoxicam before and 24h after peroxynitrite installation. Tissue and serum MDA, MPO values and tissue 3-NT value of the treatment and control groups were found significantly lower than the peroxynitrite group at the 2nd, 24th and 48th hours (p<0.05). Similarly, values obtained from bronco-alveolar lavage fluid examination in the control and treatment groups were significantly less than those in the peroxynitrite group (p<0.01). Therefore, Lornoxicam has been found to be effective in attenuating peroxynitrite induced pulmonary and tracheal injury in rats.

Oleic acid-induced lung injury in rats and effects of caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant and is an active anti-inflammatory component of honeybee propolis. The authors evaluated the effects of CAPE on oxidative stress and lung damage in an oleic acid (OA)-induced lung-injury model. Rats were divided into 5 groups as sham, OA, CAPE, pre-OA-CAPE, and post-OA-CAPE. Acute lung injury was induced by intravenous administration of 100 mg/kg of OA. Pre-OA-CAPE group received CAPE (10 micromol/kg. intravenously) 15 minutes before OA infusion and post-OA-CAPE group received CAPE 2 hours after OA administration. Malondialdehyde (MDA) level of plasma, bronchoalveolar lavage fluid (BALF), and lung tissue; myeloperoxidase activity of BALF and lung tissue; Na(+)-K(+) ATPase activity of lung tissue; and total protein content of BALF were measured. Light microscopic analyses of lung specimens were performed. The increased MDA levels in lung homogenates (47.98+/-13.75 nmol/mL), BALF (31.12+/-3.07 nmol/mL), and plasma (61.84+/-15.34 nmol/mL) decreased significantly to 24.33+/-3.09 nmol/mL (P = 0.000), 23.19+/-4.97 nmol/mL (P = 0.002), and 27.36+/-5.37 nmol/mL (P = 0.000), respectively, following CAPE administration in pre-OA-CAPE group. Another important finding was the restoration of the enzymatic activity of Na(+)-K(+) ATPase from a value of 203.89+/-32.18 nmol Pi/mg Protein/h in OA group, to a value of 302.17+/-51.90 nmol Pi/mg Protein/h (P = 0.012) in pre-OA-CAPE group with CAPE treatment. CAPE has been shown to have a clear attenuating effect on oxidative damage in experimental animal studies. However, further investigations are necessary to suggest CAPE as a treatment agent in critically ill patients with lung injury.

Rho-kinase (ROCK-1 and ROCK-2) upregulation in oleic acid-induced lung injury and its restoration by Y-27632.

The possible contribution of Rho/Rho-kinase signalling in oleic acid (100 mg kg-1, i.v., for 4 h)-induced lung injury was investigated in rats. Furthermore, the possible protective effect of the administration of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 0.5-5 mg kg-1, i.v., 15 min before the administration of oleic acid), was also examined. Western blot analysis as well as histopathological examination revealed that Rho-kinase (ROCK-1 and ROCK-2) was upregulated in lungs obtained from oleic acid-administrated rats. In addition, the markers of oxidative and nitrosative stress, i.e., malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate, in serum and lung tissue were also increased in the injury group. Treatment of rats with 5 mg kg-1 Y-27632 reversed the oleic acid-induced lung damage, which was demonstrated by histopathological assessment and confirmed in Western blot experiments: ROCK-blots were more intense in the oleic acid group than in control and Y-27632 treatment reversed ROCK upregulation. In addition, malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate were also normalized after the administration of Y-27632 (0.5 mg kg-1 and 5 mg kg-1). These findings suggest that ROCK-1 and ROCK-2 are involved in oleic acid-induced lung damage in rats, and that inhibition of this enzyme by Y-27632 may have a protective effect against such damage. Consequently, Rho kinase inhibitors may be potential therapeutic agents in the treatment of acute respiratory distress syndrome (ARDS).