Indian Journal of Medical Research

: 2014  |  Volume : 139  |  Issue : 5  |  Page : 782--785

Pathogenicity of avian influenza H11N1 virus isolated from wild aquatic bird Eurasian Spoonbill (Platalea leucorodia)

Santosh S Koratkar, Shailesh D Pawar, Vijay N Shelke, Sandeep D Kale, Akhilesh C Mishra 
 Avian Influenza Group, National Institute of Virology-Microbial Containment Complex (ICMR), 131/1, Sus Road, Pashan, Pune 411 021, India

Correspondence Address:
Shailesh D Pawar
Avian Influenza Group, National Institute of Virology-Microbial Containment Complex (ICMR), 131/1, Sus Road, Pashan, Pune 411 021

How to cite this article:
Koratkar SS, Pawar SD, Shelke VN, Kale SD, Mishra AC. Pathogenicity of avian influenza H11N1 virus isolated from wild aquatic bird Eurasian Spoonbill (Platalea leucorodia).Indian J Med Res 2014;139:782-785

How to cite this URL:
Koratkar SS, Pawar SD, Shelke VN, Kale SD, Mishra AC. Pathogenicity of avian influenza H11N1 virus isolated from wild aquatic bird Eurasian Spoonbill (Platalea leucorodia). Indian J Med Res [serial online] 2014 [cited 2020 Aug 11 ];139:782-785
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Several outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus from poultry have been reported from India during the period spanning from 2006 to 2012 [1],[2],[3] . The National Institute of Virology (NIV), Pune, India, conducted surveys in poultry, wild and migratory birds for detection and isolation of avian influenza (AI) viruses during 2006-2010. During these surveys influenza A virus was isolated from a wild aquatic bird, Eurasian Spoonbill (Platalea leucorodia), Rui-Chhatrapati village, Maharashtra, India, which was identified as AI H11N1 virus [4] . The isolation of AI H11N1 virus has been recently reported in Northern pintails (Anas acuta) in Japan [5] . There are reports of prevalence of AI H11N1 virus from Hong Kong, the United States of America and Japan from Black duck, chicken, duck, gull, mallard and ruddy turnstone [4] .

The evidence of past influenza A H11 infection in persons who were routinely, heavily exposed to wild ducks and geese through recreational activities (duck hunting) or through their employment (bird banding) has been shown in a cross-sectional seroprevalence study [6] . In addition, serum surveillance demonstrated that Lebanese backyard poultry growers, who were frequently exposed to chickens, may have been infected with H11 AI virus. These events have indicated that H11 influenza virus may possess the ability to cross the species barrier to infect humans [7] . Therefore, in the current scenario of emerging influenza viruses, it is necessary to understand virological characteristics, pathogenesis of AI viruses to study animal-human interface. The present study was undertaken to assess pathogenicity of H11N1 virus.

The virus isolates A/Aquatic Bird/India/NIV-0717095/2007-H11N1 was used in the study (GenBank Accession Nos. CY055172 to CY055179). The virus was inoculated in 10-day old embryonated chicken eggs (Venkateshwara Hatcheries Pvt Ltd, Pune). After incubation at 37 o C for 72 h, allantoic fluids were collected, aliquoted and stored at -80 o C for further study [8] .

Virus titration was performed by inoculating 10 -1 to 10 -10 virus dilutions in 10-day old embryonated chicken eggs. Each dilution was inoculated by the allantoic route in four embryonated chicken eggs. Allantoic fluids from the inoculated eggs were harvested and tested by the haemagglutination (HA) assay. Allantoic fluids showing ≥ 2 HA titre were considered positive for virus replication. Fifty per cent egg infectious dose (EID 50 ) titres were calculated using the Reed and Muench method [9] .

The pathogenicity of virus was assessed by intra-venous pathogenicity index (IVPI) assay in ten 5-week old chickens. Two chickens were kept as controls. The IVPI of AI H11N1 virus was determined as described in the Manual of Standards for Diagnostic Tests and Vaccines [7] . The Institutional Animal Ethical Committee approved the animal experiments. The chickens were housed in an animal isolator (Montair Process Technology, The Netherlands) under negative pressure. IVPI was calculated according to illness severity and the viability period following inoculation of the virus isolate.

BALB/c male mice (5-6 wk old) were used to study the pathogenicity. Mice were inoculated with 10 4.[4] EID 50 of H11N1 virus by intranasal (i.n.) route (50 μl/mouse). Each mice group contained eight mice. Control mice received uninfected allantoic fluid (50 μl/mouse). Prior to inoculation, mice were anaesthetized using CO 2 and were housed in individually ventilated cages (IVCs) [11] . These mice were daily observed for signs of disease, mortality and weights were monitored up to the post-infection day (PID) 14.

The mice organs namely liver, spleen, heart, lung, trachea, thymus, kidney, small and large intestine, pancreas and brain were collected on PID 3 and 6. Organs were triturated to prepare 10 per cent individual suspensions in virus transport medium, diluted 1:1 v/v and were inoculated in 10-day old embryonated chicken eggs. After incubation at 37 o C for 72 h, eggs were kept at 4 o C overnight and allantoic fluids were harvested. HA assay was performed to detect the presence of virus growth. The lung suspension was further titrated to determine infections virus titre (50% egg infectious dose - EID 50 ) using embryonated chicken eggs [9] .

HA assay was performed as described in the WHO manual on animal influenza diagnosis and surveillance [8] . Blood was collected from mice on PID 14. Serum was separated and treated with receptor destroying enzyme (Denka Seiken Co., Ltd, Japan) to remove non-specific inhibitors. All serum samples were tested by haemagglutination inhibition (HI) assay using 0.5 per cent turkey RBCs to detect serum antibody levels in mice [8] .

For histopathology examination, inoculated and uninoculated control mice were euthanized on PID 3 and 6 and organs were collected in 10 per cent neutral buffered formalin. After necropsy, these tissues were immediately fixed for a minimum period of 48 h. Tissues were processed for paraffin embedding, sectioned at 4 μm, stained with hematoxylin and eosin for examination by light microscopy. Selected sections from lung, liver and spleen were also stained with periodic acid-schiff stain. Tissues examined by light microscopy included liver, spleen, heart, lung, trachea, thymus, kidney, stomach, small and large intestine, pancreas and brain.

Per cent weight loss or gain in mice was calculated by comparing average weight of eight mice in each group before virus infection with average weight of the same mice group on each PID. Per cent weight loss, mean and standard error of mean were calculated using PASW version 18 software (USA). Geometric mean titres (GMT) of antibodies were calculated for mice groups.

AI H11N1 virus grew in 10-day old embryonated chicken eggs. No mortality was observed in chick embryos. The allantoic fluid from infected eggs showed ≥256 HA titre.

In the IVPI assay, AI H11N1 virus did not show any signs of sickness, respiratory illness or mortality in the inoculated chickens during 10 days observation period. The IVP index was 0.0/3.0. Thus, this was low pathogenic avian influenza (LPAI) virus in chickens.

No mortality was observed in infected mice. Mice showed weight loss up to PID 7 and remained underweight till PID 14. n0 one of the negative control mice exhibited weight loss. The virus was isolated from lungs and trachea of infected mice. All other organs were negative for virus isolation. The lungs of inoculated mice showed 2.19 and 1.25 EID 50 /100 μl infectious virus titre on PID 3 and PID 6, respectively. Infected mice were positive for presence of antibodies by HI assay on PID 14, showed antibody titre ranging from 10 to 40.

AI H11N1 virus showed pathological changes only in the airways and lungs; no specific abnormalities were identified in other organs. In the respiratory system, pathological lesions caused by H11N1 virus were less severe. The histopathological findings observed in the lung tissues of infected mice were focal non-coalescent small areas of consolidation, mild intra-alveolar, intrabronchiolar, intraseptal, and interstitial mononuclear cell (MNC) inflammatory infiltrate. Peribronchiolar and perivascular cuff of mild MNC infiltrate was also evident. Bronchiolar epithelial cells appeared taller with apical capping of secretions. The alveoli adjacent to the bronchioles showed mild congested blood vessels with accumulation of occasional lymphocyte and macrophage in the alveolar lumina. Alveolar septa appeared broadened and thickened due to mild MNC infiltration and dilated and congested blood vessels. Features were not up to the severity to satisfy the definition of diffused alveolar damage. Trachea showed mild MNC infiltrate in submucosa with intact mucosa and scant inflammatory exudates in the lumen. Lungs and trachea from uninoculated mice showed normal histology [Figure 1].{Figure 1}

There are no data available on AI H11N1 virus pathogenicity in chickens and mice. The present study showed that the virus infected chickens did not show any sickness and mortality, thus the virus was of low-pathogenicity. A mouse is often used as a model to study influenza viruses; however, mice differ in their susceptibilities to human influenza virus strains [12] . The H11N1 virus caused mild infection with weight loss and mice remained underweight as compared to uninfected mice. No mortality was seen. The mice organs other than lungs were negative for virus isolation, confirming that this virus did not cause systemic infection. Histologically, the virus under study showed predominant changes in the airways and lungs.

Tests for binding properties of various types of influenza viruses to sialic acid, sialoligosaccharides or gangliosides, have indicated that the binding pattern to different gangliosides by H11N9 viruses resembled most human viruses rather than avian isolates [13] . In receptor specificity assays, the H11N1 virus under study showed ability to bind both avian and mammalian sialic acid (SAα 2, 3-Gal and SA α2, 6-Gal) receptors [14] . The seroprevalence of H11 virus among Waterfowl hunters, wildlife professionals and chicken growers highlight probable potential of H11 viruses to cross species barrier [7] . Therefore, the present study underlines the need of continuous AI surveillance in wild and migratory birds and studies on animal-human interface.


Authors thank Shriyut RM Kolhapure, Avinash Patil for assistance in laboratory work, AM Walimbe for statistical analysis and acknowledge the Indian Council of Medical Research, New Delhi, for intramural funds.


1Chakrabarti AK, Pawar SD, Cherian SS, Koratkar SS, Jadhav SM, Pal B, et al. Characterization of the influenza A H5N1 viruses of the 2008-09 outbreaks in India reveals a third introduction and possible endemicity. PLoS One 2009; 4 : e7846.
2Pattnaik B, Pateriya AK, Khandia R, Tosh C, Nagarajan S, Gounalan S, et al. Phylogenetic analysis revealed genetic similarity of the H5N1 avian influenza viruses isolated from HPAI outbreaks in chickens in Maharashtra, India with those isolated from swan in Italy and Iran in 2006. Curr Sci 2006; 91 : 77-81.
3Ray K, Potdar VA, Cherian SS, Pawar SD, Jadhav SM, Waregaonkar SR, et al. Characterization of the complete genome of influenza A (H5N1) virus isolated during the 2006 outbreak in poultry in India. Virus Genes 2008; 36 : 345-53.
4Pawar S, Chakrabarti A, Cherian S, Pande S, Nanaware M, Raut S, et al. An avian influenza A (H11N1) virus from a wild aquatic bird revealing a unique Eurasian-American genetic reassortment. Virus Genes 2010; 41 : 14-22.
5Jahangir A, Watanabe Y, Chinen O, Yamazaki S, Sakai K, Okamura M, et al. Surveillance of avian influenza viruses in Northern pintails (Anas acuta) in Tohoku District, Japan. Avian Dis 2008; 52 : 49-53.
6Gill JS, Webby R, Gilchrist MJ, Gray GC. Avian influenza among waterfowl hunters and wildlife professionals. Emerg Infect Dis 2006; 12 : 1284-6.
7Kayali G, Barbour E, Dbaibo G, Tabet C, Saade M, Shaib HA, et al. Evidence of infection with H4 and H11 avian influenza viruses among Lebanese chicken growers. PLoS One 2011; 6 e26818.
8WHO. Manual on animal influenza diagnosis and surveillance, WHO/CDS/CSR/NCS/2002.5. Geneva, Switzerland.
9Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg 1938; 27 : 493-7.
10World Organization for Animal Health, Highly pathogenic avian influenza (fowl plague). In: Manual of standards for diagnostic tests and vaccines. Paris, France : Office International des Epizooties (OIE); 2001. p. 212-20.
11Lux X, Tumpey TM, Morken T, Zaki SR, Cox NJ, Katz JM. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans. J Virol 1999; 73 : 5903-11.
12Ibricevic A, Pekosz A, Walter MJ, Newby C, Battaile JT, Brown EG, et al. Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J Virol 2006; 80 : 7469-80.
13Li J, Cardona CJ, Xing Z, Woolcock PR. Genetic and phenotypic characterization of a low-pathogenicity avian influenza H11N9 virus. Arch Virol 2008; 153 : 1899-908.
14Pawar SD, Parkhi SS, Koratkar SS, Mishra AC. Receptor specificity and erythrocyte binding preferences of avian influenza viruses isolated from India. Virol J 2012; 9 : 251-7.