Morbilliviruses possess a wide host spectrum, including various mammals, birds and reptiles, and are capable of causing systemic infections in the animals, with respiratory, intestinal, neurological, reproductive, and cutaneous clinical signs⁵. The initial case-control study indicated a correlation between FeMV infection and chronic tubulointerstitial nephritis (TIN), the most prevalent histopathological finding in feline chronic kidney disease². FeMV has been predominantly identified in the kidneys^3,10 and has also been detected in other organs, such as lymph nodes, lungs, liver, spleen, and brain^7,11-13. In most of the previous studies, FeMV has been detected in urine samples by reverse transcriptase polymerase chain reaction (RT-PCR) indicating a high prevalence in the urine^2,6,12,14,15.

In high-density cat populations, such as animal shelters, in addition to the increased risk of direct transmission of infectious agents, chronic stress enhances susceptibility to disease and facilitates both viral replication and shedding. Deaths have been reported in cats younger than four months, particularly from feline parvovirus (FPV); in middle-aged cats, from feline leukemia virus (FeLV) and urinary tract diseases; and in cats over eight years old, from tumours^16-18. Infections caused by FeLV, feline immunodeficiency virus (FIV), feline herpesvirus-1 (FHV-1), and feline calicivirus (FCV) are among the most frequently reported viral pathogens, especially in densely populated feline colonies¹⁹. However, data investigating their association with FeMV remain limited^3,12,20.

This study aimed to determine the prevalence of FeMV in urine samples collected from naturally infected cats, to perform molecular characterization and phylogenetic analysis of the virus, and to identify risk factors in FeMV-positive cats.

The study material consisted of cats housed in an animal shelter in Erzurum from 2022 to 2023. A total of 78 cats were initially included in the study both with or without clinical signs; however, the analyses were conducted on 28 cats, as urine samples were successfully obtained only from these individuals. Signalment data (gender, age, and breed) and clinical signs were recorded for each cat, and blood, urine, and conjunctival and oropharyngeal swab samples were subsequently collected for analysis. Urine samples were investigated for FeMV, and dipstick analysis was performed. In addition, conjunctival and oropharyngeal swab, and blood samples obtained from FeMV-positive cats were used to identify possible co-infections. Blood samples were also utilized for hematological and serum biochemical assessments. The cats were classified as kittens (under 1 year old), young adults (aged 1?6 years), mature adults (aged 7-10 years) or seniors (over 10 years old) ²¹.

Sample Collection, Blood Analysis and Urinalysis: Conjunctival and oropharyngeal swab samples were obtained from the cats using sterile swabs. Five milliliters of blood were collected from the cat?s vena cephalica antebrachii. Blood count analyses were conducted on samples collected in EDTA vacutainers (BD Vacutainer®, K2E 3.6 mg, BD Plymouth, UK) and analyzed using a hematology analyzer (Abacus Junior Vet5, Diatron, Hungary). The reference values for the hematological analysis were based on the set-up reference ranges of the hematology analyzer.

To avoid invasive procedures, urine samples were collected by manual bladder compression from cats in shelter conditions. However, insufficient urine could be obtained from some cats due to stress and behavioral factors, and it was impossible to safely collect samples from others. Consequently, urine samples could only be obtained from 28 animals. To evaluate the impact of FeMV on kidney function, samples were analysed immediately using a dipstick test (Combur 9 test strips, Roche Diagnostics, USA). An aliquot of each urine sample was stored at +4 °C for FeMV RNA detection. Normal urinalysis values were considered to be a pH range of 5-7, with negative results for protein, glucose, and ketones²².

Blood samples were collected in serum separator vacutainers (BD Vacutainer®, SST II Advance, BD-Plymouth, UK) and centrifuged at 3,000 rpm for 10 minutes to prepare them for biochemical analysis. The serum samples were stored at -80 °C until they were analyzed. Serum albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GGT), total protein, blood urea nitrogen (BUN), creatinine, iron, and total iron binding capacity were analyzed using an automated biochemistry analyzer (Beckman Coulter® AU5800, Inc., USA). Serum AST, ALT, GGT, total protein, BUN, iron, and total iron-binding capacity reference values were derived from Tvedten^23, Klaassen ²⁴ and Gest, et al. ²⁵. The upper limit for blood creatinine levels was considered at 1.7 mg/dL²⁶.

Virological Analyses: The leukocyte layer (buffy coat) of the blood samples was separated by centrifugation and washed twice with phosphate-buffered saline (PBS). The conjunctival and oropharyngeal swab and urine samples were also centrifuged, and the precipitated layer at the bottom was collected and transferred to 2 ml sterile tubes, and all samples were kept at -80 °C until use. Viral RNA was extracted from leucocyte and urine samples using a viral nucleic acid extraction kit (GF-1 Viral Nucleic Acid Kit, Vivantis, Malaysia) according to the manufacturer's instructions. A first-strand cDNA synthesis kit (First strand cDNA synthesis kit, Thermo Scientific, USA) was used to obtain complementary DNA (cDNA), and the obtained cDNAs were used as templates in the nested RT-PCR analysis (Taq DNA Polymerase Kit, Thermo Scientific, USA). For the detection of FeMV, we used L gene-specific primers (Table 1) and thermal conditions reported by Furuya et al. ²⁷. Additionally, PCR analysis was performed for FHV-1, FeLV, FIV, and FCV, which are important viral infections in cats. The primers and conditions used in the study were the same as those in the articles on which the study was based (Table 1)^28,29.

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Table 1: Specific PCR primers and product sizes in the study

The PCR amplicons were analyzed by electrophoresis on a 1% agarose gel, and those ~401 bp in size were sequenced for FeMV. Sequence analysis was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, CA, USA). The obtained nucleotide sequences were aligned with the reference FeMV sequences in Gen-Bank using the BioEdit v.7.2.5 software³⁰. Phylogenetic analyses were carried out using the maximum likelihood method with 1,000 bootstrap replicates based on the Tamura 3-parameter model using MEGA X software to analyze feline morbilliviruses and some other paramyxoviruses³¹. Local FeMV sequences were compared with GenBank reference strains from Germany, Japan, Italy, Brazil, and Türkiye. For phylogenetic reconstruction, canine distemper virus (CDV; KJ466106) and measles virus (MV; OR453544), members of the family Paramyxoviridae, were designated as outgroup taxa.

Statistical Analysis: Analyses were conducted using SPSS version 20.0 (IBM, Chicago, IL, USA). The prevalence rate of FeMV and the rate of co-infections were presented as percentages. The distribution characteristics of the continuous variables were assessed using the Shapiro?Wilk test. Data that did not follow a normal distribution were expressed as the median and interquartile range (IQR). The Mann?Whitney U test was used to determine whether there were statistically significant differences in the median values of categorical variables that showed an abnormal distribution. Biochemical parameters and urine dipstick analysis for FeMV-positive cats could not be analyzed comparatively due to insufficient valid observations in the FeMV-negative group. Therefore, only descriptive statistics (median and interquartile range) for the FeMV-positive group are presented. The chi-square test (Fisher?s Exact Test) was used to evaluate the relationship between FeMV positivity and categorical variables such as age group and gender.

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Table 2: Signalment and viral co-infections in FeMV-positive cats

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Figure 1: A phylogenetic tree depicting the relationship between FeMV strains encoding the partial L gene. The analysis was performed using the Maximum Likelihood method, with bootstrap values calculated from 1000 replicates. The Tamura 3-parameter model was applied, and the analysis was conducted using MEGA X. Canine distemper virus and measles virus sequences obtained from GenBank were used as outgroups. The diamond (◆) symbols in the phylogeny represent the study samples.

Signalment and Co-infections of the FeMV-Positive Cats: The cats included in the study were young adults between one and five years of age, comprising 16 males and 12 females. Of these, six young adult mixed breed cats?three males and three females?were positive for FeMV. FeMV-positive cats were consecutively numbered (Cat 1? Cat 6). All cats that tested positive for FeMV were co-infected with other viruses, including FHV-1, FeLV and/or FIV (Table 2). No statistically significant relationship was found between FeMV positivity and the variables of age (p=1.00) and gender (p=0.062) (Table 3).

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Table 3: Evaluation of the relationship between FeMV positivity and age and gender

The clinical manifestations observed in FeMV-positive cats varied and included conjunctival hyperemia, ocular discharge, conjunctivitis, purulent nasal discharge, unilateral lymph adenomegaly, stomatitis, dehydration, lethargy, coughing, and sneezing. Among the six FeMV-positive cats, five exhibited one or more clinical signs, whereas one cat remained completely asymptomatic. All cases testing positive for FeLV or FIV were clinically asymptomatic, or presented with non-specific findings. No pathognomonic clinical signs specific to these infections were identified.

Urine and Blood Analysis Findings in FeMV-Positive Cats: Urine dipstick analysis was only evaluated in the urine of five cats. Analyses showed proteinuria in five cats (5/5) that were FeMV positive and blood was detected in the urine of four cats (4/5). Other parameters were within the normal reference range. No statistically significant differences were found between the FeMV-positive and FeMV-negative groups in any of the hematological parameters examined (p>0.05) (Table 4). In terms of biochemical parameters, one cat had low albumin levels, three had low total protein levels, two had high GGT, one had high BUN, and four had low iron-binding capacity, while the albumin/globulin ratio (A/G ratio), AST, ALT, BUN, creatinine, iron levels were within normal limits in FeMV-positive cats (Table 5).

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Table 4: Comparison of hematological parameters in FeMV-positive and FeMV-negative cats

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Table 5: Biochemical findings in FeMV-positive cats

The recently discovered FeMV-1D is limited only in Italy, according to a more thorough analysis of Genotype 1³⁴. FeMV-1B was discovered to be more extensively distributed, whereas FeMV-1A was determined to consist of strains from Asia (Malaysia, Thailand, Japan, and Hong Kong). All FeMV strains obtained in this study were grouped in subgroup 1C together with strains obtained from Brazil, Italy, Germany, Türkiye and Japan. FeMV-1C has been recorded in many nations across the world, according to the phylogenetic tree, indicating a wide geographic distribution. Genotype 2, on the other hand, is still uncommon. With 99.6-100% nucleotide and 100% amino acid identity, the FeMV strains found in this investigation showed a remarkable level of genetic conservation. When compared with the FeMV strain previously reported in Türkiye, these strains showed 98.5% nucleotide and 100% amino acid similarity. Furthermore, within FeMV subgroup 1C, they shared 93.5-99.6% nucleotide and 98.9-100% amino acid similarity with other reference strains, indicating close phylogenetic relatedness despite minor genomic variations. The sequences obtained from this study were found to be very similar to those previously reported from Türkiye⁹.

In the present study, the molecular prevalence of FeMV was found to be 21.4%. Although studies analyzing FeMV RNA in cat urine reported relatively low prevalence rates in Japan [6.1%^27, 15.1% (35), and 13.7% ³⁶], Brazil (11.4%)^37, the United Kingdom (12.5%)^26, and Italy (7.3%)^34, a considerably higher prevalence rate has been reported in Malaysia (50.8%)³². These differences in prevalence rates might be associated with various factors, such as the lifestyle of the cats included in the studies (household, stray, shelter, or access to the street), their habitat (urban, suburban, or rural), and the testing methodologies employed. To date, only one study has been conducted in Türkiye (Istanbul), reporting a prevalence of 3.13% in urine⁹. In that study, samples were collected from cats presented to veterinary clinics, the majority of which had access to the street. In contrast, the present study revealed that 21.4% prevalence rate in shelter cats.

All FeMV-positive cats were found to be concurrently infected with additional viral agents such as FHV-1, FeLV, and FIV. There is still a lack of comprehensive knowledge regarding the relationship between FeMV infection and other feline pathogens. One study reported that cats positive for FIV antibodies and concurrently shedding FeMV RNA in their urine had higher viral loads than FIV antibody negative cats. Other studies have reported a concurrent association between FeMV-positive cats and both FIV and FeLV infections^9,12. FIV and FeLV co-infection in cats has been documented in multiple studies^38-40. In this study, consistent with previous reports, simultaneous detection of FeMV, FIV, and FeLV was observed in two cats, while co-detection of FeMV and FeLV was observed in another two cats. Additionally, two FeMV-positive cats were found to be concurrently infected with FHV-1, whereas FCV was not detected in any of the FeMV-positive cats. The presence of co-infections in all FeMV-positive cats may prevent the interpretation of clinical findings. Donato, et al. ¹² reported that cats positive for both FeMV and FIV were more affected by lymph node enlargement compared to cats positive for FeMV alone. Zahro, et al. ⁴¹ detected FeLV in the kidneys of four FeMV-positive cats and noted that FeLV, through systemic infection, can impact renal function, potentially confounding the interpretation of FeMV-specific pathology. Further studies are required to identify other factors (viral, bacterial, parasitic, etc.) that may cause co-infections with FeMV infection, and to evaluate the findings.

Data on potential risk factors for FeMV exposure remain limited; however, male gender, young age, and multicat households have been reported to be associated with increased FeMV positivity rates^3,8,20,32,42. In contrast to previous studies reporting a higher prevalence of FeMV infection in male cats, FeMV RNA was detected in urine samples from three male and three female cats in the present study (p=0.062). All FeMV positive cats were young adults (p=1.00). Transmission is reported to occur readily among cats in multicat environments, where the virus can spread chronically, resulting in higher prevalence in these populations³². The high prevalence observed in this study is thought to be due to the sampling of cats living in shelter conditions, consistent with previous studies.

FeMV-positive cats showed positive reactions for protein (5/5) and blood (4/5) in their urine dipstick analyses. Chronic kidney disease is a long-term condition, particularly prevalent in older cats, characterised by the irreversible loss of kidney function, which usually develops without an identifiable cause⁴³. Therefore, it is difficult to distinguish the true cause of proteinuria clearly⁴⁴. For this reason, diagnosing chronic kidney disease requires more advanced techniques. However, when detected by dipstick in individuals at risk of chronic kidney disease, proteinuria is a clinically significant screening finding that may indicate the possibility of underlying renal disease. Dipstick results are therefore considered indicative of cases requiring further evaluation rather than a definitive diagnosis. In addition, some hematological and biochemical parameters were found to be above, within, or below the reference ranges. The results of this study are consistent with those reported by Muratore, et al. ^34, which suggested a urine pH of 5-7 and the presence of proteinuria, as well as findings by Darold, et al. ^20, which demonstrated that FeMV RNA shedding primarily occurs in the urine of cats without clinical, biochemical or ultrasonographical signs. Yilmaz, et al. ⁹ reported that urine, hematological, and biochemical parameters in morbillivirus positive cats did not differ from those in negative cats. However, Donato, et al. ¹² demonstrated a relationship between anemia, neutrophilia, basophilia, eosinopenia, monocytosis, thrombocytosis, and FeMV infection. In contrast, Ito et al. (7) reported significant leukopenia (7 out of 32) and thrombocytopenia (6 out of 32) in FeMV-infected cats. These differences in hematological and biochemical findings in FeMV-positive cats may be due to co-infections alongside FeMV. The relationship between the presence of a viral disease and clinical / biochemical / hematological findings needs further investigation, which may support to determine challenging treatment options for viral diseases in small animal practice^45,46.

Viruses are constantly evolving. Viral genomes can alter in ways that improve their capacity to infect new hosts through processes like genetic mutation, recombination, and reassortment. Further modifications could improve the virus's ability to replicate more effectively, escape host immune responses, or spread between hosts⁴⁷. Therefore, more thorough research on the evolutionary dynamics and host adaptability of the FeMV, a relatively recently discovered RNA virus, is necessary. This study is the second detection of FeMV in the Türkiye and the first in the eastern region, as FeMV has only been detected in the western region of Türkiye (Istanbul) thus far⁹. The finding of the virus indicates the existence and possible circulation of FeMV in the area, despite the fact that its detection was restricted to a single geographic location. To understand the potential therapeutic significance of FeMV, it is necessary to examine its genetic characteristics and epidemiological distribution. This study provides an important foundation for future research.

One of the main limitations of the study was that urine samples could only be obtained from a relatively small number of cats. Only twenty-eight of the seventy-eight cats included in the study provided urine samples. Additionally, statistical analysis could not be performed on the biochemical and urine analysis results due to the small quantity of blood and urine obtained from the cats. This reflects the difficulty of collecting urine samples under ideal conditions, particularly from uncooperative or asymptomatic cats.

In conclusion, the findings of this study reveal the presence and molecular characterization of FeMV in urine samples from cats and provide information about risk factors. The results of the virologic analysis, performed using RT-PCR, provide information on the distribution of four different phenotypes and demonstrate that FeMV subtype 1C is the most prevalent in the region. Furthermore, this study confirmed the presence of FeMV RNA in 6 (6/28) of the cat urine samples, indicating that the virus was actively circulating within the local cat population. No association was found between FeMV positivity and age or gender. It is also noteworthy that all cats infected with FeMV tested positive for one or more of the known immunosuppressive viruses FHV-1, FeLV, and/or FIV. These findings indicate that such viral agents may improve the spread of FeMV by predisposing cats to infection. The data obtained in this study provide valuable information about FeMV, a relatively new virus, and may contribute to directing future research in this field. FeMV infection should be investigated in larger populations through more comprehensive analyses to further elucidate the role of concurrent infections in its transmission and pathogenesis.

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