Arch Virol (2014) 159:2877–2882 DOI 10.1007/s00705-014-2119-y

ORIGINAL ARTICLE

Serological report of pandemic and seasonal human influenza virus infection in dogs in southern China Xin Yin • Fu-Rong Zhao • Dong-Hui Zhou Ping Wei • Hui-Yun Chang

•

Received: 1 March 2014 / Accepted: 11 May 2014 / Published online: 26 June 2014 Ó Springer-Verlag Wien 2014

Abstract From January to July 2012, we looked for evidence of subclinical A (H1N1) pdm09 and seasonal human influenza viruses infections in healthy dogs in China. Sera from a total of 1920 dogs were collected from Guangdong, Guangxi, Fujian and Jiangxi provinces. We also examined archived sera from 66 dogs and cats that were collected during 2008 from these provinces. Using hemagglutination inhibition (HI) and microneutralization (MN) assays, we found that only the dogs sampled in 2012 had elevated antibodies (C1:32) against A(H1N1)pdm09 virus and seasonal human influenza viruses: Of the 1920 dog sera, 20.5 % (n = 393) had elevated antibodies against influenza A(H1N1) pdm09 by the HI assay, 1.1 % (n = 22), and 4.7 % (n = 91) of the 1920 dogs sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus by the HI assay. Compared with dogs that were raised on farms, dogs that were raised as pets were more likely to have elevated antibodies against A(H1N1)pdm09 and seasonal human influenza viruses. Seropositivity was highest among pet dogs, which likely had more diverse and frequent exposures to humans than farm dogs. These findings will help us better understand which influenza A X. Yin and F.-R. Zhao contributed equally to this study. X. Yin
D.-H. Zhou
P. Wei (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin 150001, People’s Republic of China e-mail: [email protected] F.-R. Zhao
D.-H. Zhou
H.-Y. Chang (&) State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People’s Republic of China e-mail: [email protected]

viruses are present in dogs and will contribute to the prevention and control of influenza A virus. Moreover, further in-depth study is necessary for us to understand what roles dogs play in the ecology of influenza A.

Introduction Influenza A viruses belong to the family Orthomyxoviridae and infect both mammalian and avian species. Susceptibility to influenza A virus infection is dependent on the characteristics of the virus and the host [1, 2]. Influenza A viruses are subtyped on the basis of the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) glycoproteins expressed on the viral surface. Until now, 18 HA and 9 NA subtypes have been identified. Numerous subtypes of influenza A viruses, including influenza A(H1N1) pdm09, have been documented to cross the species barrier. This virus was first detected in humans in China in 2009 [3]. Since then, it has continued to circulate among humans along with a number of other seasonal influenza viruses in southern China [4–10]. Due to frequent cohabitation and interactions with humans and other animals, dogs are uniquely positioned to serve as reservoirs for influenza virus infection, both within a household and within a dog farm [11]. Different subtypes of influenza viruses are naturally transmitted to dogs from other species worldwide, including human (pandemic H1N1/09; H1N1 and H3N2), equine (H3N8) and avian (H3N2 and H5N1) viruses [7, 9, 12, 13]. As one of the most common companion animals in China, dogs are potentially at risk of acquiring human pathogens because of their close proximity and frequent contact with humans [14]. The close contact between humans and dogs poses a threat to public health because of the potential for zoonotic transmission of

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influenza viruses to humans. In China, avian-origin H3N2 and pandemic H1N1/09 influenza viruses have been isolated from dogs [9]. Currently, seasonal H3N2 influenza virus is predominately prevailing in humans in China. Therefore, we examined the seroprevalence of pandemic H1N1/09, human seasonal H1N1, and human seasonal H3N2 influenza viruses among dogs without clinical signs of influenza in the densely populated provinces of southern China from January 2012 to June 2012.

Materials and methods Specimen collection During 2012, we conducted this survey in domestic and farm-raised dogs in Guangdong, Guangxi, Fujian, and Jiangxi provinces in southern China. We wanted to study dogs without clinical signs of influenza that might have contact with large and diverse populations of humans and animals. We selected the cities based on their large and dense human, poultry, and pig populations, which we reasoned may be prone to cross-species influenza transmission. In these provinces, we studied dogs from a total of 64 pet hospitals and 10 dog farms (dogs raised for commercial purposes). In each province, we selected the single largest dog farm. The selected dogs from veterinary clinics were chosen based on the following criteria: the veterinary clinics were located in diverse geographical areas, they had treated more than 3,000 canine patients during the last year, the dogs were more than six months old, and the dogs had no history of canine influenza vaccination or clinical signs of influenza in the preceding three months. At each site, individual dogs were selected by a random-number procedure among the dogs without apparent clinical signs of influenza. Before sampling, we collected demographic information about the dogs. All the owners of the dogs gave permission for their animals to be sampled in this study. Sera were stored at -20 °C until they could be tested for antibodies against influenza A virus. In addition, we also studied 66 archived sera from 45 dogs and 21 cats collected during 2008 from two pet hospitals in Guangdong Province. Our sampling processes were assisted by local authorities and licensed veterinarians. The animal research in this study was reviewed and approved by Guangdong Province Animal Disease Control Center. Serological tests All samples were tested by hemagglutination inhibition (HI) and microneutralization (MN) assay according to recommended procedures. The influenza virus isolates used in this study were A/California/7/2009(H1N1pdm09) and

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A/Brisbane/59/2007(H1N1) for a human seasonal H1N1 influenza virus and A/Brisbane/10/2007(H3N2) for a human seasonal H3N2 influenza virus. Prior to running the HI assay, study sera were treated with receptor-destroying enzyme (RDE, prepared by CNIC, Beijing) and hemadsorbed to remove nonspecific inhibitors. RDE was added to each serum specimen to prevent nonspecific reactions, and the specimen was incubated at 37 °C overnight, followed by incubation at 56 °C for 30 min to inactivate RDE. Sera were next incubated and mixed with erythrocytes for 30 min at 4 °C. Then, the treated sera were centrifuged, and the sera were examined for hemagglutination inhibition according to the recommendations of the World Health Organization: Twenty-five microliters of serial twofold dilutions of the treated serum samples was mixed with four hemagglutinin units (HAU) of virus in V-shaped microtiter plates and incubated at room temperature for 30 min. Then, 50 ll of 1 % chicken red blood cells (RBCs) were added to each well, and the plate was incubated at room temperature for 30 min. The HI titer was calculated as the highest serum dilution that completely inhibited hemagglutination of 4 HAU of virus. Each of the serum samples was tested in duplicate, and the average of the duplicates was used. We also tested the sera for antibodies against two other viruses: A/chicken/Guangdong/V/2008(H9N2) (GenBank accession no. JQ639786), because it is similar to A/Chicken/ Beijing/1/94(H9N2), the most prevalent subtype detected among poultry in southern China, and A/canine/Guangdong/2/2011(H3N2), an H3N2 canine influenza virus (CIV) that has been circulating recently in dogs in China. WHO guidelines for vaccine evaluation suggest that HI antibody titers C40 indicate 50 % protection against influenza A virus [15, 16]. Therefore, we defined HI antibody titers C32 as the cutoff levels to estimate infection rates [17]. For the MN assay, we followed procedures recommended by the World Health Organization. We calculated the 50 % tissue culture infectious dose (TCID50) of the studied viruses according to the method of Reed and Muench [18]. Madin-Darby canine kidney (MDCK) cells were obtained from the CNIC, Beijing, China, and used for a maximum of 25 passages. Briefly, sera were treated with RDE, and twofold serial dilutions were performed in 96-well polystyrene immunoassay plates (Nunclon Delta surface, Nunc, Denmark). Then, an equal volume of virus diluent (DMEM containing 1 % bovine serum albumin [Gibco, Grand Island, NY, USA], TPCK trypsin [2 lg/mL; Worthington Biochemical Corp., Lakewood, NJ. USA] and antibiotics) containing influenza virus at 100 TCID50/50 ll was mixed with the diluted sera. Each plate included four control wells of virus plus virus diluent (VC) or virus diluent alone (CC). After incubation at 37 °C in a 5 % CO2

Human influenza virus infection in dogs in southern China

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humidified atmosphere for an hour, 100 ll of MDCK cells at 1.5 9 105/ml was added to each well. After incubation for 18-22 hours at 37 °C and 5 % CO2, the monolayers of MDCK were washed with phosphate-buffered saline (PBS) and fixed in cold 80 % acetone for 10 min. Using influenza-A-virus-specific anti-nucleoprotein monoclonal antibodies (Millipore, Temecula, CA, USA), viral nucleoprotein (NP) was detected by enzyme-linkedimmunosorbent assay (ELISA) [19]. The wells were scored positive for virus growth if the absorbance reading was greater than three standard deviations above the mean absorbance of wells containing only MDCK cells. In the MN assay, each of the sera was tested in duplicate, and the average absorbance value for each pair of wells was used. We tested sera using the MN assay with the A/Guangdong/ 1057/2010(H1N1), A/Brisbane/59/2007(H1N1), and A/Brisbane/10/2007(H3N2) viruses. An MN titer of C1:32 was considered evidence of previous infection with an A (H1N1) pdm09 virus. Statistical analysis We performed descriptive statistics to explore potential risk factors for serological HI assay outcomes. A bivariate v2 test of independence or chi-square test was used to examine the association between demographic characteristics and serological outcome for the HI and MN tests. Covariates that had a p-value less than 0.25 in the bivariate analysis were entered into a multivariable logistic regression model. Keeping age in the model, we performed backward elimination of the covariates, keeping covariates

in the model that had a p-value less than 0.05. Final covariates were tested for goodness of fit.

Results From January to July 2012, we sampled 1920 dogs: 960 in Guangdong province and 320 in each of the other three provinces. Of these dogs, 1100 (57.3 %) were male. Most (65.6 %) were raised as pets. There was no statistically significant difference in infection rates with respect to gender. Overall, 20.5 % (n = 393) of the 1920 dog sera had elevated antibodies against influenza A(H1N1) pdm09 virus as determined by the HI assay, and 1.1 % (n = 21) and 4.7 % (n = 91) of the 1920 dogs sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus as determined by the HI assay. Comparing the four provinces and both serological methods, we consistently identified a higher prevalence of dogs with an elevated level of antibody against A (H1N1) pdm09 virus in all four provinces (Tables 1, 2). A total of 201 (88.2 %) of the 228 MNpositive samples were also HI positive. When considered as a binary outcome (elevated or not), the two tests had moderate to high agreement [Kappa 0.51; 96 % CI 0.32 – 0.43]. Seroprevalence estimated by the HI assay was significantly higher than the estimates from the MN assay (20.5 % vs. 11.9 %, p \ 0.01). Interestingly, only 35 dogs had an elevated antibody titer for both influenza A (H1N1) pdm09 and human seasonal influenza virus (data not shown).

Table 1 Results of serological assays carried out on serum specimens collected from dogs in Guangdong, Guangxi, Fujian, and Jiangxi from January 2012 to June 2012 Location

Guangdong Guangxi Fujian Jiangxi

Raising pattern

Number sampled

Number (%) with serological evidence of seasonal H1N1 infection

Number (%) with serological evidence of seasonal H3N2 infection

Number (%) with serological evidence of pdm09 H1N1 infection

HI assay C1:32

MN assay C1:32

HI assay C1:32

MN assay C1:32

HI assay C1:32

MN assay C1:32

Pet dog

660

9 (1.4)

7 (1.1)

45 (6.8)

34 (5.2)

185 (28.0)

72 (7.5)

Farm dog

300

1 (0.3)

0 (0.0)

4 (1.3)

2 (0.7)

52 (17.3)

32 (10.7) 37 (18.5)

Pet dog

200

4 (2.0)

2 (1.0)

14 (7.0)

9 (4.5)

47 (23.5)

Farm dog

120

0 (0.0)

0 (0.0)

2 (1.7)

0 (0.0)

8 (6.7)

7 (5.8)

Pet dog

200

3 (1.5)

1 (0.5)

11 (5.5)

7 (3.5)

43 (21.5)

35 (17.5)

Farm dog

120

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

9 (7.5)

7 (5.8)

Pet dog

200

4 (2.0)

1 (0.5)

12 (6.0)

9 (4.5)

39 (19.5)

30 (15.0)

Farm dog

120

0 (0.0)

0 (0.0)

3 (2.5)

1 (0.8)

10 (8.3)

8 (6.7)

1920

21 (1.1)

11 (0.6)

91 (4.7)

62 (3.2)

393 (20.5)

228 (11.9)

Total

Human seasonal H1N1, A/Brisbane/59/2007(H1N1); a human seasonal H3N2, A/Brisbane/10/2007(H3N2); pdm09 virus H1N1, A/California/7/ 2009 (H1N1) HI chicken RBC hemagglutination inhibition assay, MN microneutralization assay

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Sera were judged to have elevated antibodies against influenza virus if they had either an HI titer C1:32 or a MN titer C1:32; 95 % CI, 95 % confidence interval

1.8 [1.3-2.5] 1.4 [1.0-2.0] 1.6 [0.9-3.0] 4 (1.3) Jiangxi

1.1 [0.3-3.8]

1.2 [0.4-4.0]

15 (4.7)

1.6 [0.9-3.0]

49 (15.3)

1.6 [1.1-2.2] 1.7 [1.2-2.4] 1.2 [0.9-1.7] 1.4 [1.0-2.0] 1.5 [0.9-2.7] 2.3 [1.2-4.4] 4 (1.3) 3 (0.9) Guangxi Fujian

1.1 [0.3-3.8] 1.6 [0.4-6.0]

1.2 [0.4-4.0] 1.6 [0.4-6.0]

16 (5.0) 11 (3.4)

1.7 [1.0-2.9] 2.4 [1.3-4.5]

55 (17.2) 52 (16.3)

1.2 [1.0-1.6]

5.0 [3.9-6.5]

Reference Reference

1.2 [1.0-1.6] 120 (14.6)

237 (24.7)

1.2 [0.8-1.7]

Reference

1.2 [0.8-1.7]

Reference

55 (6.7)

49 (5.1) Reference

1.1 [0.7-1.9] 1.1 [0.7-1.9]

Reference 10 (1.0)

25 (3.0) Female

Guangdong Province

Reference Reference 37 (3.4) Sex

Male

Reference

Reference

87 (7.9)

Reference

191 (17.4)

5.2 [3.7-7.8]

Reference

9.5 [4.7-19.0]

79 (12.0)

Reference

9.5 [4.7-19.0] 9 (1.4) 20 [2.7-150.7]

Reference

1 (0.1)

Reference 20 (1.6)

Farm dog

Raising pattern

Pet dog

20 [2.7-150.7]

82 (6.5)

Reference

314 (24.9)

Unadjusted OR (95 % CI) No. positive (%) Adjusted OR (95 % CI) Unadjusted OR (95 % CI) Variable

No. positive (%)

Unadjusted OR (95 % CI)

Adjusted OR (95 % CI)

No. positive (%)

pdm09 H1N1 infection Human seasonal H3N2 infection Human seasonal H1N1 infection

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Reference

Table 2 Unadjusted and adjusted (logistic regression) associations of risk factors with elevated antibodies against human seasonal H1N1 virus, human seasonal H3N2 virus, and A(H1N1)pdm09 virus in dogs in southern China in 2012

Our final model estimated risk factors for the serological outcome of elevated antibody against influenza A (H1N1) pdm09 and human seasonal influenza viruses (Table 2). Dogs that were raised as pets were more likely to have elevated antibodies against influenza A (H1N1) pdm09 virus than dogs that were raised on farms. Dogs that were raised in Guangdong were more likely to develop antibodies against influenza A(H1N1)pdm09 virus than dogs that were from Guangxi (OR = 1.6; 95 % 1.1–2.2), Fujian (OR = 1.7; 95 % 1.2–2.4), or Jiangxi (OR = 1.8; 95 % 1.3–2.5) (Table 2). All 1920 serum samples were also studied with HI assays against canine H3N2 and avian H9N2 viruses. None had evidence of previous infection with H9N2. Dogs that were raised as pets were also more likely to have elevated antibodies against human seasonal influenza virus infection than dogs that were raised on farms (Table 2). A total of 323 (16.8 %) sera were positive by HI assay against H3N2 CIV (Table 3). Fifty-five out of 323 sera from the dogs had an elevated antibody titer for both influenza A (H1N1)pdm09 and H3N2 CIV. None of the 66 dog and cat sera from 2008 had elevated antibodies against influenza A(H1N1)pdm09 virus by the HI assay.

Reference

X. Yin et al.

Adjusted OR (95 % CI)

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Discussion To date, no human H1N1 or H3N2 influenza viruses have been isolated from cats or dogs. Since domestic dogs are in close contact with humans, they are subsequently exposed to human influenza viruses. Currently, seasonal H1N1 and H3N2 influenza viruses are predominately prevailing in humans in southern China. Our results showed a significantly higher seroprevalence than that reported by Sun et al. [20]. Their results showed that the seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5 %, 1.5 %, and 1.2 %, respectively. Maybe this is because our serum samples were from southern China, an area with a high incidence of influenza, and out results are more complete for surveying various subtypes of influenza A virus. We hypothesize the sustained transmission of human influenza viruses in the human population in our study area, as well as close and prolonged exposure of dogs to clinically ill individuals, could have led to infection of the dogs. Here, we conducted serosurveillance of the 2009 pandemic H1N1 and seasonal human influenza viruses over six months, and 20.5 % (n = 393) of the 1920 dog sera had elevated antibodies against influenza A (H1N1) pdm09 virus by the HI assay, whereas 1.1 % (n = 22) and 4.7 % (n = 91) of the 1920 dog sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus. All four provinces of southern China participating in the seroepidemiology study

Human influenza virus infection in dogs in southern China

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Table 3 Prevalence of elevated antibody titers in dogs in southern China in 2012 against an avian influenza H9N2 virus, a canine influenza H3N2 virus, A(H1N1)pdm09 virus, human seasonal H1N1 HI assay virus

Number

virus, and human seasonal H3N2 virus, as determined by hemagglutination inhibition (HI) assay

Antibody titer \1:8

1:16

1:32

1:64

C1:128

Number of specimens with titers C1:32 (%)

Number of specimens with titers C1:128 (%)

Avian H9N2

1920

1620

67

0

0

0

0 (0)

0 (0)

Canine H3N2

1920

787

637

173

63

87

323 (16.8)

87 (4.5)

pdmH1N1

1920

1073

464

210

141

32

393 (20.5)

32 (1.7)

Human seasonal H1N1

1920

1731

127

45

13

4

62 (3.2)

4 (0.2)

Human seasonal H3N2

1920

1574

204

82

42

18

142 (7.4)

18 (0.9)

Human seasonal H1N1, A/Brisbane/59/2007(H1N1); Human seasonal H3N2, A/Brisbane/10/2007(H3N2); pdm09 virus H1N1, A/California/7/ 2009 (H1N1); H9N2, A/chicken/Guangdong/V/2008(H9N2); H3N2, A/canine/Guangdong/2/2011(H3N2)

have now been confirmed to be seropositive for human influenza viruses in dogs, indicating that the human influenza viruses have already been prevailing among the different dog populations in southern China, not only in Guangdong Province. The seropositivity was highest among pet dogs, which likely had more diverse and frequent exposures to humans than did farm dogs. This study demonstrated a higher prevalence of influenza A (H1N1)pdm09 infections among pet dogs in China compared to the studies conducted earlier in different parts of the world [5, 6, 21, 22]. Previous studies influenza A (H1N1) pdm09 infections in dogs of have not found a high seroprevalence. We hypothesize that relatively frequent A (H1N1)pdm09 transmission may have occurred between humans and dogs during the peak period of virus infection in the human population [9]. This hypothesis is supported by our observation that pet dogs were more likely to have evidence of previous infection with A (H1N1) pdm09 that were farm dogs. Although transmission experiments have shown that human A (H1N1) pdm09 can infect dogs, transmission is thought to be inefficient between dogs [9]. Our findings were subject to several limitations. The possibility of cross-reactivity between H3 subtypes of other origin (including equine H3N8 subtype not tested in this study) in the serological assay cannot be excluded. This suggests that continued surveillance for influenza viruses among dogs should be performed more routinely in southern China. In summary, these study data suggest that dogs in these four province of southern China all had a relatively high prevalence of evidence of previous A(H1N1)pdm09 infection. We also investigated the seroprevalence of human seasonal H1N1 and human seasonal H3N2 in dogs in southern China. Since dogs may be exposed to different influenza virus subtypes, including avian- and human-origin influenza viruses [23, 24], coinfection of dogs with different influenza viruses might provide an opportunity for reassortment, leading to the emergence of novel reassortant strains with novel pandemic zoonotic potential. Their

potential role in the epidemiology of influenza virus should be investigated further. Acknowledgments This work was supported by National Transgenic Project of China (2011ZX08011-004) and Basic Conditions for Science and Technology Projects of Lanzhou City (2012-2-71).

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