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.

A prevalence study of COVID-19 among healthcare workers in a pandemic hospital in the Samsun province of Turkey.

Among populations globally, many healthcare workers have been disproportionally impacted by the COVID-19 pandemic because of their above average exposure to people infected with SARS-CoV-2. Exposure to asymptomatic or pre-symptomatic individuals is particularly challenging, if those individuals continue to work, not knowing that they are potentially infectious. This study aimed to measure the level of asymptomatic infection in a cohort of workers in a healthcare setting in Turkey during the second major wave of infection in late 2020. Blood samples were collected and tested by electrochemiluminescence immunoassay for SARS-CoV-2 IgM and IgG antibodies. Nasal and throat swabs were performed in a subset of this cohort and RT-qPCR was used to search for the presence of SARS-CoV-2 RNA. The results showed that approximately 23% of the cohort were positive for anti-SARS-CoV-2 IgM antibodies and approximately 22% were positive for anti-SARS-CoV-2 IgG antibodies despite no reported history of COVID-19 symptoms. Just less than 30% of a subset of the group were positive for the presence of SARS-CoV-2 RNA indicating the likelihood of a current or recent infection, again despite a lack of typical COVID-19 associated symptoms. This study indicates a high rate of asymptomatic infection and highlights the need for regular testing of groups such as healthcare workers when community prevalence of disease is high and there is a desire to limit entry of virus into settings where vulnerable people may be present, because symptoms cannot be relied on as indicators of infection or infectiousness.

A retrospective molecular investigation of selected pigeon viruses between 2018-2021 in Turkey.

A recent first detection of pigeon aviadenovirus-1 and pigeon circovirus co-infection associated with Young Pigeon Disease Syndrome (YPDS) in a pigeon flock in Turkey, prompted a study focused on documenting the distribution of Pigeon aviadenovirus (PiAdV-1 and PiAdV-2), Pigeon circovirus (PiCV), Columbid alphaherpesvirus 1 (pigeon herpesvirus (PiHV)) and Fowl aviadenovirus (FAdV) in the country. These viruses were selected as they are associated with severe disease in pigeons across the world. A total of 192 cloacal swabs were collected from young (<1 year old) pigeons from 16 different private pigeon flocks across Turkey, between 2018 and 2021 as part of routine diagnostic sampling. PiCV genetic material was the most frequently detected 4/16 (25%), PiAdV-1 and CoHV-1 DNA were both found in one flock each, while neither PiAdV-2 and FAdV were detected in any of the studied pigeon flocks. PiCV and PiHV genetic material were both detected in the same pigeon flock's cloacal samples as a co-infection with the identification of PiHV being a first in Turkey.

First detection and molecular characterisation of a pigeon aviadenovirus A and pigeon circovirus co-infection associated with Young Pigeon Disease Syndrome (YPDS) in Turkish pigeons (Columba livia domestica).

Pigeon aviadenovirus A and Pigeon circovirus are both DNA viruses, infect and cause severe clinical diseases in pigeons. These viruses are associated with an immunosuppression syndrome similar to 'Young Pigeon Disease Syndrome' (YPDS). This study reports the identification of a natural co-infection, with severe clinical signs (crop vomiting, watery diarrhoea, anorexia and sudden death) of Pigeon aviadenovirus A and Pigeon circovirus in a breeding pigeon flock in Central Anatolia, Turkey. Both viruses were isolated from pigeons pooled internal organs using primary chicken embryo kidney cell cultures (CEKC) and specific pathogen-free (SPF) embryonated chicken eggs. Also, both viruses were identified by PCR amplification followed by Sanger sequencing whereas histopathological examination showed degenerated hepatocytes with basophilic intranuclear viral inclusions. As known, both viruses typically have similar transmission characteristics and common clinical manifestations; however, co-infection may exacerbate the disease with devastating outcomes. This is the first report of its kind in Turkey for those viruses and is essential for the protection against these kinds of infections in pigeons.

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.

Is SARS-CoV-2 Neutralized More Effectively by IgM and IgA than IgG Having the Same Fab Region?

Recently, recombinant monoclonal antibodies (mAbs) of three Ig isotypes (IgG, IgA, and IgM) sharing the same anti-spike protein Fab region were developed; we evaluated their neutralizing abilities using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein and ACE2-transfected Crandell-Rees feline kidney cells as the host cell line. Although each of the anti-SARS-CoV-2 mAbs was able to neutralize the spike-coated lentiviruses, IgM and IgA neutralized the viral particles at 225-fold and 125-fold lower concentrations, respectively, than that of IgG. Our finding that the neutralization ability of Igs with the same Fab domain was dramatically higher for IgM and IgA than IgG mAbs suggests a strategy for developing effective and affordable antibody therapies for COVID-19. The efficient neutralization conferred by IgM and IgA mAbs can be explained by their capacity to bind multiple virions. While several IgG mAbs have been approved as therapeutics by the FDA, there are currently no IgM or IgA mAbs available. We suggest that mAbs with multiple antigen-binding sites such as IgM and IgA could be developed as the new generation of therapy.

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.

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.

Development of a multiplex RT-PCR assay for the routine detection of seven RNA viruses in Apis mellifera.

Colony losses in apiaries are frequently one of the most important problems in beekeeping. Colony loss is multifactorial with many reported disorders, Colony Collapse Disorder (CCD), is an increasingly recognised phenomenon which is thought to be caused by many pathogens, including viruses. The aim of this study was to develop a multiplex RT-PCR (mRT-PCR) test to obtain faster results in routine diagnostic laboratories for seven crucial bee viruses. Specific primers for seven RNA viruses, including Israeli acute bee paralysis virus (IAPV), deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), black queen cell virus (BQCV), kashmir bee virus (KBV) and chronic paralysis virus (CBPV), were used for testing procedure. The mRT-PCR assay can amplify seven plasmid DNA fragments from the pooled viral genomes and it was shown to be sensitive because virus copy numbers were detected to be 104 copies/µl when log10 serial dilutions were performed for the optimized mRT- PCR method. It is concluded that, mRT-PCR test can be used in routine analysis because this assay can perform specific, sensitive and reliable results also achieves economic gains and time due to detecting seven viral agents simultaneously.

Specific Substitutions in Region V2 of gp120 env confer SHIV Neutralisation Resistance.

A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 env). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of env of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region.

First report of fowl aviadenovirus serotypes FAdV-8b and FAdV-11 associated with inclusion body hepatitis in commercial broiler and broiler-breeder flocks in Turkey.

Inclusion body hepatitis (IBH), hydropericardium syndrome (HS), and gizzard erosion (GE) are all economically important diseases in the poultry industry worldwide and are all caused by fowl aviadenovirus (FAdV). It is important to identify the serotype of the virus to differentiate these diseases. In the present study, a total of six recent FAdV serotypes were isolated and identified in broiler and broiler-breeder flocks in Izmir, Manisa, and Aydin provinces of the Aegean region of Turkey between January and March 2019. The viruses were isolated from livers and pooled organs of chickens using primary chicken embryo kidney cell cultures (CEKC). Virus isolates were identified by PCR amplification of the loop 1 (L1) variable region of the hexon gene followed by Sanger sequencing. Sequence analysis revealed the presence of both FAdV-D (serotype 11) and FAdV-E (serotype 8b). The viruses that were isolated were associated with IBH, which is typically characterized by gross lesions such as enlarged and pale yellow liver with multiple petechial hemorrhages. Histopathological examination also showed necrotizing hepatitis with intranuclear inclusion bodies in hepatocytes. This study is the first report of the isolation and identification of FAdV serotypes associated with IBH in commercial broilers and broiler-breeder flocks in Turkey. The results of sequence analysis showed that FAdV-8b and FAdV-11 were the circulating serotypes that caused recent field outbreaks of IBH in the Aegean region between January and March, 2019.

Correction to: First report of fowl aviadenovirus serotypes FAdV-8b and FAdV-11 associated with inclusion body hepatitis in commercial broiler and broiler-breeder flocks in Turkey.

The co-author names were found missed in the original publication of this article and the complete lists of authors were updated here. The original article has been corrected.

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.

Does porphyrin suppres the apoptotic and necrotic effects of bovine herpes virus type-1(BoHV-1) and herpes simplex virus type-1(HSV-1)?

In this study, antiviral effect of porphyrin was investigated. Cooper strain of Bovine Herpes Virus type 1(BoHV-1) and Kos strain of Herpes Simplex Virus type-1 (HSV-1) were used to determine the potential of porphyrins to inhibit infection in vitro (with morphological and cytopathological criteria). Apoptotic and necrotic changes were determined by using DAPI and propidium staining. The non-cytotoxic dose of porphyrin (NCD-p) was initially calculated as 312.50µg/mL on MDBK and Vero cells. The apoptotic cell (APC) count was found 10% with BoHV-1 while it was 5.3% with BoHV-1 treated with porphyrin on MDBK cells between 6th to 24th hours post infection (hpi). Necrotic cell (NEC) count was 51% with BoHV-1 and 37.8% BoHV-1 treated with porphyrin on MDBK cells at 24th hpi. On the other hand, the APC count was found 23% with HSV-1, while 22% with the HSV-1 treated with porphyrin on Vero cells between 6th to 24th hpi. NEC count was 49% with HSV-1 and 34% HSV-1 treated with porphyrin on MDBK cells at 24th hpi. The results show that BoHV-1 was inhibited by porphyrin resulting in decreased apoptotic and necrotic changes in MDBK cells. On the contrary, porphyrine was not effective in the inhibition of HSV-1 in terms of apoptosis but it caused necrotic changes in Vero cells.

Maedi-visna virus infection in Karayaka and Amasya Herik breed sheep from provinces in northern Turkey.

Maedi-visna is an important virus infection of sheep having prolonged incubation period (slow disease) and reflecting two distinct forms clinically and pathologically. In this study, the presence of MVV was investigated serologically in 58 Amasya Herik sheep breed and 525 Karayaka sheep breed. Seropositivity rates in Amasya Herik sheep breed and Karayaka sheep breed were detected as 69.0% and 18.5%, respectively. MVV antibodies were found in 137 of 583 serum samples (23.5%). Positivity rates for the provinces varied and were as follows: Samsun 19.4%, Sinop 15.4%, Ordu 25.8%, Trabzon 26.7%, Rize 36.7%, Amasya 69.0% and Tokat 35.0%, however no antibody response was detected in all of the sheep in Giresun province.