Evolution of an Extended Pathogenicity Motif in VP2 of Infectious Pancreatic Necrosis Virus Isolates from Farmed Rainbow Trout in Turkey.

Infectious pancreatic necrosis virus (IPNV) causes economic losses with a highly variable mortality rate worldwide, especially in rainbow trout. The virus has a double-stranded bi-partite RNA genome designated segment A and B. New complete genome sequences of nine rainbow trout isolates from Turkey were determined and subjected to phylogenetic analysis, identifying all as genotype 5 (serotype Sp). A time-dependent change in the extended pathogenicity motif of VP2 from P217T221A247 (PTA) to PTE P217T221E247 over a period of 10 years was identified. A wider analysis of 99 IPNV sequences from Turkey and Iran revealed the emergence of the motif PTE from 2007 to 2017, inducing significant morbidity in fry by 2013. In fact, displacement of the PTA motif, by the PTE motif in IPNV isolates appeared to be connected to a production peak of rainbow trout in 2013. An additional CAI analysis provided more evidence, indicating that rainbow trout culture in Turkey has an influence on the evolution of IPNV.

The first serological detection of West Nile virus infection among residents living in northern Turkey.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is transmitted by a mosquito-borne virus whose natural reservoir is birds. Humans and horses are considered accidental hosts. Even if the vast majority of WNV infections in humans have asymptomatic or mild disease settings, serious neurological disorders with lethal outcomes can also be observed in around 1% of the cases. We aimed to serologically investigate the presence of WNV in humans living in Black sea of Turkey, and to obtain epidemiological data that will contribute to the implementation of public health policies to control and prevent potentially other life-threatening arboviral infections. METHODS: In the current study, a total of 416 human sera were collected from native patients of Samsun and its boroughs attending Samsun Training and Research Hospital; these sera were tested for WNV with pooling method, using anti-IgM and IgG ELISA commercial kits. All pools that were found positive for both IgM and IgG were individually retested for the detection of positive WNV sera. After that, all positive samples were tested using real-time PCR to detect the presence of WNV-RNA particles. RESULTS: Total seropositivity rates of WNV in terms of IgM and IgG were found as 0.96% and 0.72%, respectively. No presence of WNV-RNA could be detected in positive samples. INTERPRETATION & CONCLUSION: According to the data, further studies should be conducted to better understand the epidemiological dynamics of WNV in Turkey. It is recommended that other antigenically related flaviviruses which can give cross-reaction with WNV should also be investigated.

First isolation and molecular characterization of canine parvovirus-type 2b (CPV-2b) from red foxes (Vulpes vulpes) living in the wild habitat of Turkey.

BACKGROUND: The canine parvovirus, with its many variants, is responsible for a pivotal and common viral infection affecting millions of dogs and other carnivore species worldwide, particularly the wild ones, which are considered as the main reservoir hosts. To that end, this study investigated the presence of canine parvovirus (CPV) in red foxes (Vulpes vulpes) living in wild habitats of several regions of Turkey. METHODS: We randomly collected 630 archival fox stool specimens from rural areas of 22 provinces and used real-time PCR to detect CPV. RESULTS: Two of the 630 (0.3%) stool samples were positive for CPV-DNA, named Tr-Fox/128(Aydin) and Tr-Fox/159(Manisa). We attempted to isolate the virus in a MDCK cell line, and cytopathic effects were observed four days post-inoculation. Three regions corresponding to the CPV capsid protein VP2 gene from extracted DNA of positive samples were amplified by conventional PCR, and the products were visualised, purified, and Sanger sequenced. Three overlapping DNA raw sequence fragments, were read, assembled, and aligned to obtain approximately 1.5 kb-long regions that cover most of the VP2 gene, then deposited in GenBank. After comparing the isolates with parvovirus sequences data of domestic and wild carnivores by BLAST processing, our isolates' similarity rate with each other was 99.40%, with base differences in 9 nucleotide positions. They were classified as 2b variant closely related to isolates from dogs in Turkey, Egypt, Iraq, Italy, Thailand, and China. CONCLUSION: This study presents evidence of interspecies transmission of CPV, of which there are no reports on prevalence in wildlife carnivores of our country. Identification of CPV in red foxes threatens local and hunting dogs, which may contract the infection or disseminate it to other wild animal species or vice-versa.

A one-year extensive molecular survey on SARS-CoV-2 in companion animals of Turkey shows a lack of evidence for viral circulation in pet dogs and cats.

Current evidence have now demonstrated that SARS-CoV-2 infects a wide array of mammalian animals; however, the full range of hosts and the viral circulation in companion animals remains to be clarified. In this context, as no such evidenced cases have been reported from Turkey, we aimed to screen for SARS-CoV-2 nucleic acid in housed dogs and cats clinically evaluated for respiratory symptoms and reared in different locations of Samsun province in the black sea region of Turkey from July 2020 to July 2021. Nasal swabs were collected from a total of 415 pets (65 cats and 350 dogs) aged between 1 and 9 years old. All the specimens were tested for SARS-CoV-2 RNA presence by real-time RT-PCR targeting two genomic regions of SARS-CoV-2, but none showed positive results. Our findings suggest that SARS-CoV-2 does not circulate in local pets and is not responsible for respiratory symptoms. However, further comprehensive molecular and serological surveys are required to have a better picture of the zoonotic, reverse zoonotic and pathogenic consequences of the ongoing COVID-19 pandemic in Turkey.

Development of a recombinase polymerase amplification assay for viral haemorrhagic septicemia virus.

Viral diseases of fish cause significant economic losses in the aquaculture industry. Viral haemorrhagic septicemia virus (VHSV) is one of the most important viral diseases that affects more than 80 fish species. Detection of the disease, especially in the field, is critical to managing disease prevention and control programmes. Recombinase polymerase amplification (RPA) is an isothermal method with a very short amplification period and a single incubation temperature ranging from 37 to 42°C, which is a good alternative to the polymerase chain reaction (PCR). This study aimed to develop an RPA assay as sensitive as a real-time RT-PCR to detect VHSV. For this purpose, primers and probes are designed for the same targeted region of gG of VHSV. The ssRNA standards were prepared to find the detection limits of the assay. Detection limits were found ten-fold differences between real-time RT-PCR and real-time RT-RPA. While the detection limit of the RT-PCR was found as 95.5 viral RNA molecules/reaction in 95% probit value, the detection limit of RT-RPA was found as 943.75 viral RNA molecules/reaction in 95% probit value using ssRNA standards. These results show that RPA is a suitable test for VHSV Ie detection.

Determination of VP2 sequence-based virulence motifs and phylogenetic analysis of domestic Turkish IPNV isolates.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish and is one of the most severe economic diseases in aquaculture. In Turkey, an increase in infectious pancreatic necrosis virus (IPNV) outbreaks in freshwater rainbow trout have been reported in recent years. This study aimed to analyze the VP2 gene from recent IPNV isolates from Turkey to determine whether there are epidemiological links between IPNV isolates from rainbow trout (Oncorhynchus mykiss; 62) and sea bass (Dicentrarchus labrax; 1), wild turbot (Scophthalmus maximus; 1) and the environment in order to investigate potential wild and farmed fish interactions. In this study, 62 Turkish IPNV isolates collected over 10 years (2005-2014) from rainbow trout, sea bass and turbot were genotypically characterized. The phylogenetic analysis indicated that Turkish IPNV isolates are closely related to strains from Denmark, Iran and Spain and that all Turkish IPNV isolates belong to genogroup V, serotype A2 (Sp strain). Furthermore, low genetic diversity was found among the Turkish isolates (identity, 95.5%-100% nucleotides and 97.8%-100% amino acids). The result of the analysis of the amino acid residues found at positions 217, 221 and 247 (proline, threonine and alanine, respectively) could be associated with virulence.

Serological study on the presence of some alpha-herpesviruses in goats of northern Anatolia, Turkey.

The aim of this study was to investigate the presence of caprine herpes virus-1 (CpHV-1) and bovine herpes virus-1 (BoHV-1) in 269 goat sera collected from small-scale family farms located in six provinces within the Black Sea region of northern Turkey. The overall seropositivity for alpha-herpesvirus in the native goats was found as 19.33% using BoHV-1 glycoprotein B (gB)-blocking enzyme-linked immunosorbent assay (ELISA). Additionally, the seroprevalence of BoHV-1 was determined in 5.20% of the goats using virus neutralization test. To distinguish between CpHV-1 and BoHV-1, the combinations of gB/gE-blocking ELISA tests were performed. Of tested samples, 15.24% were CpHV-1 seropositive; whereas, 4.09% were BoHV-1 seropositive. The results indicated that CpHV-1 is in circulation among local goats of northern Turkey. Considering the close relationship between BoHV-1 and CpHV-1, the transmission of BoHV-1 via goats may also be one of the predisposing factors involving in the spread of virus among the surrounding cattle.

Sentinel serosurveillance of backyard hens proved West Nile virus circulation in the western provinces of Turkey.

West Nile virus (WNV) is a mosquito-borne virus of a re-emergence importance with a wide range of vertebrate hosts. Granted, it causes asymptomatic infection, but fatal cases and neurologic disorders were also recorded, especially in humans, horses and some exposed birds. The virus is globally spread and birds are considered an amplifying and reservoir host of WNV, helping to spread the disease due to their close contact with main hosts. In this study, we aimed to detect the presence of antibodies against WNV in backyard hens that were reared in the western Anatolian part of Turkey. A total of 480 chicken sera were randomly collected from six provinces in the west of Turkey (Mugla, Izmir, Aydin, Afyonkarahisar, Kutahya and Manisa) with 80 samples from each province (40 in spring and 40 in fall seasons). They were tested by using a competitive ELISA method to identify the specific avian antibodies of IgG that produced against the WNV envelope proteins (pr-E). Twelve of 480 (2.5%) sera were found seropositive, three of these positive sera were detected from the Izmir province (3.75%) collected in the spring session and the other nine positive sera were detected from the Mugla province (11.25%) collected in the fall session. Both of these provinces are located seaside and have suitable climate conditions for vectors of infection. The results indicated that WNV infection is in circulation in these provinces, and that may put the other susceptible vertebrates under risk of infection.

Inactivated infectious pancreatic necrosis virus (IPNV) vaccine and E.coli-expressed recombinant IPNV-VP2 subunit vaccine afford protection against IPNV challenge in rainbow trout.

Infectious pancreatic necrosis (IPN) is a highly contagious disease causing high mortality in juvenile trouts. Since there is no effective way to treatment against IPNV, early diagnosis and prevention play an important role in combating the disease. The different types of IPNV vaccines (inactive, live, recombinant, DNA, etc) have been produced from local isolates and have been used in developed countries. In Turkey, there is no commercial licensed vaccines against IPNV. Due to this reason, IPNV vaccine is needed in Turkey. The production of recombinant VP2 subunit vaccine (IPNV-VP2) and inactivated whole particle virus vaccine (IPNV-WPV) were attempted from selected isolate belong to sp serotype. For this purpose; the virus was produced in RTG-2 cell line and RT-PCR amplification was performed by using primers with restriction enzymes. The whole VP2 gene was cloned into a plasmid vector and VP2 was expressed by using E. coli expression system. A trial was conducted to determine the immunity ability of IPNV-VP2 and IPNV-WPV in rainbow trout. According to the SN50 assay, the IPNV-WPV stimulates immune response faster than the IPNV-VP2 vaccine. Besides, the relative percent of Survive (RPS) was detected as 79% in fish vaccinated with IPNV-WPV and 70% in fish vaccinated with IPNV-VP2. Thus, we can say that the recombinant vaccine of IPNV-VP2 is almost protected against IPNV infection as well as the inactive vaccine.

A serosurvey for bovine respirovirus 3 in Turkish domestic ruminants: The first comparison study of A and C genotypes.

Bovine parainfluenza virus-3 (BPIV-3), also known as bovine respirovirus 3, causes serious respiratory infection in ungulates, often involving other pathogens, such as viruses, bacteria and mycoplasmas. In this study, we evaluated antibody titers against virus genotypes A (BPIV-3a) and C (BPIV-3c). We conducted a serological survey and comparison analysis of archived serum samples from small and large ruminants reared in four Turkish provinces. A total of 1,307 samples, consisting of sheep (n = 444), cattle (n = 402), water buffalo (n = 261) and goat (n = 200) sera, were randomly selected from stock samples collected between 2015 and 2019 and screened by standard virus neutralisation assay. We found that 49.9% (653/1307) of all samples were positive for neutralising antibody titers. Goats had the highest titer, with total seropositivity of 63% (126/200), followed in descending order by cattle, sheep and water buffalo at 56.2% (226/402), 32.2% (143/444) and 26% (68/261) total seropositivity, respectively. BPIV-3c had the highest neutralising antibody rate at 34.3% (448/1307), whereas BPIV-3a had a 24.3% (317/1307) seropositivity rate. Neutralising antibody titers for positive samples ranged between 1/4 and 1/512 per the SN50 test. Seropositivity rates ranged from a low of 8.9% to a high of 18.3%. Our study was the first to compare antibody seroprevalence for two BPIV-3 genotypes in small and large domestic ruminants, which were shown to be more commonly exposed to BPIV-3c than BPIV-3a. This finding could have significant implications as current vaccines mainly use the BPIV-3a genotype. Further research can determine if current vaccines protect against different BPIV-3 virus genotypes.

Marek's disease virus in vaccinated poultry flocks in Turkey: its first isolation with molecular characterization.

Marek's disease (MD) is an important disease of avian species and a potential threat to the poultry industry worldwide. In this study, 16 dead commercial chickens from flocks with suspected MD were necropsied immediately after death. Pathological findings were compatible with MD, and gallid alphaherpesvirus 2 was identified in PCR of spleen samples. Virus isolation was performed in primary cell culture, and partial sequencing of the meq gene of the isolate revealed >99% nucleotide sequence identity to virulent and very virulent plus strains from a number of European countries, placing it in the same subclade of clade III as two virulent Italian strains and a very virulent plus Polish strain as well as virulent strains of geese and ducks. The data reported here indicate that a virulent strain of Marek's disease virus is circulating in Turkey and has not been stopped by the current national vaccination programme.

Circulation of Indigenous Bovine Respiratory Syncytial Virus Strains in Turkish Cattle: The First Isolation and Molecular Characterization.

Bovine respiratory disease (BRD) is a huge economic burden on the livestock industries of countries worldwide. Bovine respiratory syncytial virus (BRSV) is one of the most important pathogens that contributes to BRD. In this study, we report the identification and first isolation, with molecular characterization, of a new BRSV strain from lung specimens of three beef cows in Turkey that died from respiratory distress. After the screening of lung tissues for BRD-associated viruses using a multiscreen antigen-ELISA, a BRSV antigen was detected. This was then confirmed by real-time RT-PCR specific for BRSV. Following confirmation, virus isolation was conducted in MDBK cell cultures and clear CPE, including syncytia compatible with BRSV, were detected. RT-nested PCR, using F gene-specific primers, was performed on the cultured isolates, and the products were sequenced and deposited to Genbank with accession numbers MT179304, MT024766, and MT0244767. Phylogenetic analysis of these sequences indicated that the cattle were infected with BRSV from subgroup III and were closely related to previously identified American and Turkish strains, but contained some amino acid and nucleotide differences. This research paves the way for further studies on the molecular characteristics of natural BRSV isolates, including full genome analysis and disease pathogenesis, and also contributes to the development of robust national strategies against this virus.

A Study on the Identification of Five Arboviruses from Hematophagous Mosquitoes and Midges Captured in Some Parts of Northern Turkey.

BACKGROUND: Whether zoonotic or not, arboviral infections are continuing to be a major threat to human health as well as the livestock industry all around the world. This project presented the results of the identification study on five arboviruses, including West Nile virus (WNV), Bovine ephemeral fever virus, Akabane virus, Bluetongue virus, and Epizootic hemorrhagic disease virus, in mosquitos and midges from eight provinces of the Black Sea Region. METHODS: During 2011 and 2012, 3193 mosquitoes were captured around natural streams, rivers, lakes, and ponds using dry-baited miniature light-traps. Identification studies were concluded by employing molecular methods. RESULTS: According to the morphological identification, blood-sucking mosquitoes and biting-midges belonged to Aedes (44.69%), Anopheles (28.34%), Culex (22.14%) and Culicoides (4.83%) species. Overall, 146 pools were made up of captured mosquitos and midges. None of the five viruses were directly identified by mosquitoes. CONCLUSION: Mosquitoes and midges have got a crucial role in the transmission of arboviruses. The risk of occurrence for the investigated arboviruses will continue depending upon many factors including the presence of these viruses in Turkey and its neighboring countries, uncontrolled livestock movements, global warming and climate changes.

Characterisation of the First Bovine Parainfluenza Virus 3 Isolate Detected in Cattle in Turkey.

A respiratory disease outbreak on a cattle farm in northern Turkey produced respiratory tract symptoms and severe pneumonia symptoms in 20 calves. Eight calves died, and a lung specimen from one carcass was analysed for bacteria and for viruses of the Bovine respiratory diseases complex. Bacteriological analysis was negative, but antigen detection ELISA and RT-PCR results indicated the presence of Bovine parainfluenza virus (BPIV). Virus isolation succeeded on Madin-Darby Bovine Kidney cells, and subsequent whole genome sequencing and phylogenetic analysis identified BPIV-3c. This is the first report of BPIV-3c isolation from cattle in Turkey, indicating the need for more virological and epidemiological studies.

Seroepidemiological study of west nile virus and rift valley Fever virus in some of Mammalian species (herbivores) in northern Turkey.

BACKGROUND: West Nile virus (WNV) and Rift Valley fever virus (RVFV) are mosquito-borne viral diseases. The objective of this study was to investigate the RVFV and WNV infections as serologically in different mammalian species (cattle, horse, goat, sheep and water buffalo) in the northern Turkey. METHODS: Blood samples randomly collected from 70 each cattle, horse, sheep, goat and water buffalo were analyzed for the presence of antibodies to RVFV and WNV using an competitive enzyme-linked immunosorbent assay (C-ELISA) in northern Turkey. RESULTS: None of the animals were positive for antibodies to RVFV. In contrast, WNV antibodies were found in two of 350 samples (0.57%). CONCLUSION: This may suggest that the RVFV disease is not present in northern Turkey.This is the first serological study on RVFV in Turkey.

Antibodies to Schmallenberg virus in domestic livestock in Turkey.

This, partly retrospective study, was designed to determine the seroprevalence of Schmallenberg virus (SBV), a new Orthobunyavirus first reported in Germany in late 2011, in domestic ruminants from the Middle Black Sea, West, and Southeast regions of Turkey. An indirect enzyme-linked immunosorbent assay was used to screen serum samples collected from slaughterhouse animals between 2006 and 2013. The overall seroprevalence was 335/1,362 (24.5 %) with 325/816 (39.8 %), 5/307 (1.6 %), 3/109 (2.8 %), and 2/130 (1.5 %) recorded in cattle, sheep, goats, and Anatolian water buffalo, respectively. This is the first study to demonstrate the presence of antibodies to SBV in Turkish ruminants; it indicates that cattle are more susceptible to infection than sheep, goats, or buffalo and that exposure of domestic ruminants to SBV in Turkey may have occurred up to 5 years prior to the first recorded outbreak of the disease in 2011.

A serological survey of selected pathogens in wild boar (Sus scrofa) in northern Turkey.

During the hunting season in March 2012, a total of 93 blood samples were collected from wild boars (Sus scrofa) shot in the area of northern Turkey (Samsun and Gumushane provinces). These blood samples were examined by enzyme immunoassay (ELISA) for the presence of antibodies to classical swine fever virus (CSFV), Aujeszky's disease virus (ADV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), swine influenza virus (SIV), porcine parvovirus (PPV), swine vesicular disease virus (SVDV), hepatitis E virus (HEV), African swine fever virus (ASFV), porcine rotavirus (PRV), transmissible gastroenteritis virus (TGEV) and bovine viral diarrhoea virus (BVDV). Out of 93 serum samples examined, 65 (69.9 %) were positive for PRV, 22 (23.7 %) were positive for ADV, 5 (5.4 %) were positive for BVDV, 4 (4.3 %) were positive for PPV and 2 (2.2 %) were positive for PRRSV. All sera were negative for ASFV, SVDV, HEV, SIV, PRCV, TGEV and CSFV. The results, recorded for the first time in Turkey, supported the hypothesis that wild boar act as a potential reservoir of selected viruses and thus have a role in the epidemiology of these diseases.

Molecular evolution of lineage 2 West Nile virus.

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Serosurvey and molecular detection of Crimean-Congo hemorrhagic fever virus (CCHFV) in northern Turkey.

In this study, the hard ticks, whole blood and serum samples collected from small ruminants (sheep and goat) in middle Black Sea region of Turkey where Crimean-Congo hemorrhagic fever (CCHF) human cases were observed in the past years were surveyed for the presence of RNA and specific IgG antibodies from CCFH virus (CCHFV). CCHFV RNA was found in 30 of 255 tick pools (11.76%) and nine of 105 (8.57%) leucocyte samples. No CCHFV genomic RNA was detected from animals in Yildizeli and Vezirkopru. However, CCHFV RNA was found from animals in Gerze and Resadiye. Seventy-eight of 105 goat and sheep blood serum samples tested were antibody-positive for CCHFV by enzyme-linked immunosorbent assay (ELISA) (goat: 42/63; sheep: 36/42). Viral RNA was detected from tick samples in all of four provinces. Positivity rates for the provinces varied and were as follows: Gerze 13.04%, Resadiye 35.41%, Vezirkopru 1.61% and Yildizeli 6.06%. CCHFV genomic RNA was detected in four of seven tick species tested. These results suggest that these hard ticks may act as a reservoir for CCHFV in northern Turkey.

The first investigation of west nile virus in horses using real time rt-PCR in middle black sea region in Turkey.

BACKGROUND: West Nile Virus (WNV) is a mosquito-borne disease that can cause fatal infection in mammals including humans, dogs, horses, birds and reptiles. Although West Nile Virus is an asymptomatic infection, especially it can cause neurologic disorders in humans and horses. The aim of this study was to the investigate virological presence of WNV in horses in the Black Sea Region of Turkey using real time RT-PCR (rRT-PCR). METHODS: Totally, 120 horse sera were collected equally from 4 provinces in Middle Black Sea Region of Turkey and investigated for WNV presence by Taqman based rRT-PCR. RESULTS: WNV nucleic acid was not detected in any horse serum sample. CONCLUSION: Although obtained result indicated no evidence of WNV-RNA in horses, Black Sea Region of Turkey is one of the suitable places for the WNV infection. For this reason, our research will continue for the determination of the viruses in vectors and susceptible animals such as horses, dogs, etc.