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Turkish Journal of Zoology Turk J Zool(2018) 42: 245-251© TÜBİTAKdoi:10.3906/zoo-1706-3

The prevalence and genetic diversity of feline immunodeficiency virusand feline leukemia virus among stray cats in Harbin, China

Mei-qiao PAN, Jiu-cheng WANG, Ya-jun WANG*Laboratory of Veterinary Medicine, College of Wildlife Resources, Northeast Forestry University, Harbin, P.R. China

* Correspondence: 18246194641@163.com

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are typical retroviruses that are transmitted horizontally among felines (Lutz et al., 2009). FIV was first described as an infectious disease of domestic cats in 1986 (Bendinelli et al., 1995; Bachmann et al., 1997). The infection in domestic cats is characterized by a long period without symptoms, followed by increased susceptibility to opportunistic infections leading to an AIDS-like disease. It has been well studied not only as a feline immunodeficiency pathogen but also as a pathogenic model for human immunodeficiency virus type 1 (HIV-1). FeLV was first described in 1964 and is one of the most common fatal pathogens affecting felines worldwide (Jarrett et al., 1964). FIV and FeLV are horizontally transmitted through saliva or other body fluids, such as in mutual grooming and sharing of food dishes and water bowls (Munro et al., 2014), while FIV is transmitted mostly through bite wounds and fighting of the animals, especially in male animals as they are more aggressive. Therefore, the infection incidence could be higher in males. Vertical transmission may also occur occasionally.

The FIV genome contains three main structural genes, termed gag, pol, and env, which encode the

internal structural proteins, viral enzymes, and envelope proteins, respectively (Talbott et al., 1989). The env gene plays an important role in subtype identification and the pathogenesis of the virus (Pancino et al., 1993; Bachmann et al., 1997). Phylogenetic analysis of FIV sequences is considered vital for understanding the transmission, geographical distribution, and evolution of the virus. The env protein has been deemed as a dominant antigen in immune responses to FIV. Therefore, the study of FIV diversity has focused on the env gene (Rigby et al., 1993; Sodora et al., 1994; Kraase et al., 2010; Beczkowski et al., 2014). FIV falls into five subtypes (A, B, C, D, and E) with a worldwide distribution according to variation of the env V3–V5 region (Greene et al., 1993; Langley et al., 1994; Sodora et al., 1994; Kakinuma et al., 1995; Pecoraro et al., 1996; Kraase et al., 2010; Samman et al., 2011). Since env, the major target gene for viral neutralization, displays immense diversity, it has been proposed that it may be necessary to create regional vaccines that are based on the sequences of the predominant circulating viruses in particular regions or countries (Pistello et al., 1997; Steinrigl et al., 2010). In this context, genetic diversity of the FIV genome, especially the env region, needs to precisely described from all geographic locations where

Abstract: Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are typical retroviruses that are transmitted horizontally among felines. FIV results in symptoms of immunodeficiency such as weight loss, chronic lesions, opportunistic infections, and neurological abnormalities, while FeLV is known to cause a variety of neoplastic and nonneoplastic diseases in cats, such as thymic lymphoma, multicentric lymphoma myelodysplastic syndromes, acute myeloid leukemia, aplastic anemia, and immunodeficiency. The prevalence and genetic diversity of FIV and FeLV in stray cats have not been investigated. In this study, polymerase chain reaction and enzyme-linked immunosorbent assay were used to test 198 blood samples from stray cats from Harbin, China, for FIV infection, FeLV seropositivity, and the prevalent subtypes of FIV. The gag capsid amino terminus and env V3–V5 regions of FIV samples were used to perform phylogenetic analysis. In total, 3% (6/198) of the stray cats from five districts of Harbin was PCR- were ELISA-positive for FIV. The FeLV positivity rate was 1% (2/198). Phylogenetic analysis of the gag and env sequences showed that cats were infected with FIV subtype A. This is the first study examining the prevalence rates of FIV and FeLV and the prevalent subtypes of FIV of stray cats in Harbin. These studies will enrich the epidemiological understanding of FIV and FeLV.

Key words: Feline immunodeficiency virus, feline leukemia virus, env, gag, polymerase chain reaction, diagnosis

Received: 27.06.2017 Accepted/Published Online: 05.12.2017 Final Version: 21.03.2018

Short Communication

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the virus is identified. Harbin is a provincial capital city in northeast China with about 9.95 million residents. Domestic cat is the most popular pet after dogs, with an estimated number over 500,000. Stray cats are often seen around the city, living on trash, natural prey, and food provided by cat lovers. Stray cats in the city are expected to serve as a reservoir for FIV and FeLV. However, the prevalence and genetic diversity of FIV and FeLV have not been investigated. We carried out an investigation on the prevalence of FIV and FeLV and analyzed the genetic diversity of the env region of FIV genome.

All experimental protocols were approved by the Animal Ethics Committee of the Harbin Veterinary and Animal Ethics Committee of Northeast Forestry University and were performed accordingly. The blood samples for evaluation were collected randomly from 198 stray cats between June 2015 and June 2016 in Harbin, China. The sampled animals consisted of 116 males and 82 females. Age was successfully estimated by the adopter for 111 cats, leaving the ages of 87 cats unestimated. Cats were categorized into three age groups: kittens (<12 months, 34 samples), adults (1 to 10 years old, 71 samples), and old (>10 years old, 6 samples).

FIV and FeLV infections were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) to test the FeLV antigen and FIV specific antibody (SNAP Combo, IDEXX Laboratories, Inc., Westbrook, ME, USA) and polymerase chain reaction (PCR). Following the protocols of commercial DNA extraction kits (Biotech, Beijing, China), the proviral DNA was extracted from 200 µL of each whole-blood sample, eluted with 60 µL of ddH2O and stored at –20 °C. The primers for FIV (Pecoraro et al., 1996) and FeLV (Kim et al., 2014; Wilkes et al., 2015) were referred to in previous studies and were synthesized commercially.

The 328-bp gag fragment located in the CA amino terminus and the 551-bp V3–V5 loop of env were amplified by nested PCR. PCR amplifications of the gene regions were performed under the following conditions: 5 min at 98 °C; 35 cycles consisting of 10 s at 98 °C, 30 s of annealing at 59 °C (FeLV) or 53.8 °C (FIV env) or 58 °C (FIV, gag), and 1 min at 72 °C; and final extension for 10 min at 72 °C. PCR products were recovered using a commercial purification kit (Biotech) and sequenced using the Sanger method. The sequences generated were manually processed using editing software (Tamura et al., 2013). All the reference sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The data for env and gag were submitted to the GenBank and serial numbers were obtained (gag: KX258453–KX258458; env: KX055477–KX055482; available at https://www.ncbi.nlm.nih.gov/genbank). Primer binding sites were excluded from analysis. The final lengths were 328 bp for gag CA

and 551 bp for the V3–V5 loop of env. Phylogenetic trees were constructed by neighbor-joining method (Figures 1 and 2) using the best-models Tamura 3-parameter with a gamma distribution and evolutionarily invariable for the gag gene and the env gene, to analyze the subtypes of FIV. The statistical strength of the neighbor-joining method was assessed by bootstrap analysis (1000 replicates).

Results in Table 1 show that FeLV had a low ELISA positivity rate of 1% (2/198) in Harbin, while FIV was found in four males (HEB6, HEB11, SHC24, and SHC44) and two females (HEB13 and SH2363), yielding a positivity rate of 3% (6/198), as shown in Table 2. Positivity rates of FIV varied among districts in the city (Table 1): Xiangfang District (n = 47, 2%), Nangang District (n = 36, 3%), Daoli District (n = 60, 5%), Pingfang District (n = 38, 3%), and Daowai District (n = 17, 0%). FeLV was detected from one sample in Daoli District and one sample in Pingfang District, respectively. None of the cats were detected to carry both viruses at the time of testing. This rate is significantly lower than 6.6% in stray cats as reported in Italy (Munro et al., 2014) and 18% in the United States (Wang et al., 2010). Male cats were predisposed to FIV infection as transmission of FIV is mainly through bite wounds, such as in fighting for territory.

There was very good agreement between the results of ELISA and PCR for FIV specimens collected from Harbin. There were six samples with initial positive test results for both ELISA and PCR, while the other samples were negative and had negative virus isolation. There were two samples with negative PCR and positive ELISA results for FeLV from Daoli District and Pingfang District (Table 1). Considering the small sample size, the sensitivity and specificity of such assays were deemed satisfactory in this study. However, with an increase in sample size, the possibility of false positives or false negatives could increase, and false positive and false negative results have been reported in other studies. There are multiple potential reasons for inconsistent ELISA and PCR test results. For PCR testing, the viral load fluctuates during FIV infection, and in different populations, the circulating viral load could be too low to amplify during PCR (Beebe et al., 1994; Crawford and Levy, 2007; Nichols et al., 2017). Commercial PCR tests also differ with respect to primers and reaction protocols, and their sensitivity and specificity vary (Bienzle et al., 2004; Crawford et al., 2005; Nichols et al., 2017). For ELISA testing, cats can have positive ELISA test results when not infected with FIV as a result of maternal antibodies (young kittens) or vaccine-induced antibodies, and such cats would have negative PCR test results (Nichols et al., 2017). At present, there is no vaccine history in Harbin, China. The six positive samples had negative virus isolation results, possibly due to deterioration following long-term preservation of the

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Figure 1. Phylogenetic analysis of the FIV gag gene: neighbor-joining tree. Accession numbers: Subtype A: L06132 (FIVWo); M36968 (PPR); D37820 (Sendai-1); M25381 (Petaluma). Subtype B: AY139110 (TX200); AY139111 (TXTG); D37819 (Yokohama); D37824 (Aomori2); D37821 (Sendai2); D37816 (Aomori1). Subtype C: AB027298 (TI1); AB027301 (TI4). Subtype D: D37822 (Fukuoka); D37818 (Shizuoka). Subtype E: AB027304 (LP24); AB027303 (LP20); AB027302 (LP3); KX258453 (HEB11); KX258454 (SH2363); KX258455 (SHC4-4); KX258456 (SHC2-4); KX258457 (HEB13); KX258458 (HEB6).

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Figure 2. Phylogenetic analysis of the FIV env V3–V5 region: neighbor-joining tree. Accession numbers: Subtype A: D67052  (JN-BR1); AB010402 (SAP01); AB010401 (PTH-BM3); KX055477 (HEB13); KX055478 (HEB6); KX055479 (SHC24); KX055480 (SHC44); KX055481 (SH-2363); KX055482 (HEB11). Subtype B: AB010397 (AIC02); D67064 (TY1). Subtype C: AB016025 (TI1); AB016026 (TI2); AB016027 (TI3); AB016028 (TI4); AB083509 (VND-8); ABO83503 (VND-2); AB010396 (AIC01). Subtype D: AB010398 (KUM01); AB010399 (KUM02); AB083502 (VND-1). Subtype E: D84496 (Lp3); D84498 (Lp20); D84500 (Lp24).

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samples at –80 °C. A study with a larger number of stray cats with good consistency between the ELISA and PCR tests would be required to evaluate the accuracy of this PCR test for diagnosing FIV infection.

Genetic analysis of the FIV gag CA region, comprising 329 bp, and the env V3–V5 region, comprising 551 bp, showed that all the samples (Sub AC) displayed 3.7% genetic variability compared to gag CA region references and 12.2% genetic variability compared to env V3–V5 region references. Our six positive samples showed that the gag CA amino region was highly conserved, while the env gene was highly variable. Genetic analysis of the isolates from Harbin exhibited low genetic variability (approximately 3.7% in CA and 7.4% in V3–V4) in comparison to other subtypes, such as subtype B (16.1% and 19.7% variability for CA and V3–V4, respectively) (Table 3). All six env genes showed a high degree of homology with reference genes of five subtypes (Table 3), phylogenetic analysis clearly demonstrating that all six FIV isolates in this study were confirmed to belong to subtype A (Figures 1 and 2). The envelope protein is one of the major theoretical obstacles in generating an

effective vaccine against HIV infection. At present, there is only one registered FIV vaccine, which is composed of two FIV subtypes, A and D. The vaccine is reported to confer protection against subtypes A, B, and D (Pu et al., 2005; Hayward and Rodrigo, 2010), and it will be essential to know precisely the diversity of FIV strains present in Harbin and to tailor the immunogens accordingly.

The detection rate of 3% (6/198) in Harbin showed that stray cats often have a high prevalence of FIV. At present, there are a large number of stray cats surrounding the zoos, and they have opportunities to access the other felines in the zoos. Compared with the wild field, this phenomenon not only increases the probability of interspecific cross-infection but also raises the risk of same-species propagation. In particular, there have been reports of large felines infected with FIV in recent years, such as tiger (Panthera tigris), leopard cat (Prionailurus bengalensis), cougar (Puma concolor), and Asian golden cat (Catopuma temminckii), and these are important exhibition animals. In zoos where a large number of them were imported from Africa, South America, North America, and Europe without FIV detection, it can be inferred that the

Table 1. Samples and positivity rates in each district in Harbin.

District TotalFeLV(PCR/ELISA) FIV(PCR/ELISA) FIV and FeLV

Negative Positive Rate Negative Positive Rate Positive Negative

Daoli 60 59 (-/-) 1 (-/+) 2% 57 (-/-) 3 (+/+) 5% 0 60

Xiangfang 47 47 (-/-) 0 0 46 (-/-) 1 (+/+) 2% 0 47

Nangang 36 36 (-/-) 0 0 35 (-/-) 1 (+/+) 3% 0 36

Pingfang 38 37 (-/-) 1 (-/+) 3% 37 (-/-) 1 (+/+) 3% 0 38

Daowai 17 17 (-/-) 0 0 17 (-/-) 0 0 0 17

Total 198 198 (-/-) 2 1% 192 (-/-) 6 (+/+) 3% 0 198

FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; PCR, polymerase chain reaction.

Table 2. Characterization of FIV-positive samples obtained in districts in Harbin.

Sample Sex Age group Location Source Clinical symptom

HEB6 Male Adult Xiangfang Stray cat Stomatitis

HEB11 Male Old Pingfang Stray cat None

HEB13 Female Adult Nangang Stray cat Stomatitis

SH2363 Female Unknown Daoli Stray cat Unknown

SHC24 Male Adult Daoli Stray cat Unknown

SHC44 Male Adult Daoli Stray cat Unknown

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introduction of these feline individuals brings certain risks of FIV infection. In addition, there are 12 species of original felines in the Chinese mainland. When they are introduced into a zoo, these native FIV strains have the opportunity to interact with exotic strains, resulting in new variations, which have a wider impact on domestic felines. FIV in most naturally infected cats does not cause severe clinical symptoms and FIV-infected cats may live many years without any health problems. As a result, there are few reports of FIV infection of domestic cats, as they fail to get recognized. The clinical implications of FIV infection in feline species are still unclear, and thus further studies are needed to reach accurate conclusions. However, felines feeding in zoos and wildlife rescue centers have

many difficulties, such as difficultly in propagation, short lifespan, and high mortality rate. Therefore, it is necessary to investigate the FIV infection rates and epidemiological trends and to study genetic variation patterns in China to provide some epidemiological basis for better control and prevention of this disease and to protect these species.

AcknowledgmentsThis study was supported by Northeast Forestry University, China (grant number 41316425). We are deeply grateful to Dr Yumiao Tian at the Harbin Veterinary Research Institute, CAAS (Harbin, China), who provided laboratory facilities to isolate viruses. Our thanks also go to Dr Yajun Wang for assistance with blood sample collection.

Table 3. Pairwise distances matrix between groups of subtypes for env V3-V5 and gag CA regions.

Subtypes% Distance

SubA** SubAC** SubB** SubC** SubD** SubE**

Sub A* 3.70 16.50 15.20 16.00 15.90

Sub AC* 7.40 16.10 15.00 16.40 15.40

Sub B* 19.60 19.70 16.20 14.70 10.40

Sub C* 20.50 21.50 14.50 13.40 16.40

Sub D* 18.70 18.70 16.50 17.90 12.50

Sub E* 20.00 18.80 16.30 20.30 16.60

Pairwise distance matrix between groups of subtypes for the CA regions (upper-right diagonal half) and env V3–V5 (lower-left diagonal half) of FIV-positive cats from China and representatives of FIV subtypes with sequences in GenBank.*Env: Sub A, subtype A (JN-BR1, SAP01, PTH-BM3); Sub B, subtype B (AIC02, TY1); Sub C, subtype C (TI1, TI2, TI3, TI4, VND-8); Sub D, subtype D (KUM01, KUM02, VND-1); Sub E, subtype E (LP24, LP20, LP3); Sub AC (HEB11, SH2363, SHC4-4, SHC2-4, HEB13, HEB6).**Gag: Sub A, subtype A (FIV Wo, PPR, Sendai-1, Petaluma); Sub B, subtype B (TX200, TXTG, Yokohama, Sendai2, Aomori2, Aomori1); Sub C, subtype C (TI1, TI4); Sub D, subtype D (Fukuoka, Shizuoka); Sub E, subtype E (LP24, LP20, LP3); Sub AC (HEB11, SH2363, SHC4-4, SHC2-4, HEB13, HEB6).

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