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Transmission of avian H9N2 influenza viruses in a murine model
Veterinary Microbiology 142 (2010) 211–216
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Transmission of avian H9N2 influenza viruses in a murine model Rui Wu a,c,1, Zhiwei Sui b,1, Zewen Liu a, Wangwang Liang a, Keli Yang a, Zhongliang Xiong a, Diping Xu a,c,* a
Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China Shijingshan Center for Diseases Control and Prevention in Beijing City, Beijing 100043, China c Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Wuhan 430064, Hubei, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 19 September 2009 Accepted 23 September 2009
Avian H9N2 influenza viruses have circulated widely in domestic poultry around the world, resulting in occasional transmission of virus from infected poultry to humans. However, it is unknown whether H9N2 influenza virus has acquired the ability to be transmitted from human to human. Here, we report that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. To evaluate the transmissibility of mouse-adapted H9N2 influenza viruses, we carried out transmission studies in mice using both contact and respiratory droplet routes. Our results indicate that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. This suggests that once H9N2 influenza viruses adapt to new host, they should present potential public health risks, therefore, urgent attention should be paid to H9N2 influenza viruses. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Avian influenza virus H9N2 subtype Transmission Contact spread Droplet spread
1. Introduction Avian H9N2 influenza viruses have become panzootic in Eurasia over the last decade and have circulated widely around the world (Capua and Alexander, 2004; Chen et al., 2003). Human disease associated with H9N2 viruses was documented in Hong Kong in 1999, which suggested that H9N2 avian influenza virus can cross the species barrier to humans (Peiris et al., 1999). Prior to that event, human infection with H9N2 avian influenza virus had been reported in mainland China (Guo et al., 1999). Subsequently, a strain of H9N2 avian influenza virus has been isolated repeatedly from the human population in mainland China (Guo et al., 2000). In November 2003, an H9N2 avian influenza virus was isolated from a child with a flu-
* Corresponding author at: Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China. Tel.: +86 27 8738 0195; fax: +86 27 8738 0195. E-mail address: [email protected] (D. Xu). 1 The authors contribute the same work to this study. 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.068
like illness in Hong Kong (Butt et al., 2005). These documented human cases of avian influenza infection in Hong Kong and mainland China aroused global concern regarding the role of H9N2 avian influenza viruses in human disease. There is evidence that the interspecies transmission of H9N2 influenza virus from avian to mammalian hosts continues to occur. This allows us to raise the hypothesis that H9N2 influenza virus is a potential candidate for the next influenza pandemic in humans (Choi et al., 2004; Guan et al., 2000, 1999; Li et al., 2003). For a strain of influenza virus to cause a human pandemic, it must be (i) novel to the human immune system, (ii) virulent in the human host, and (iii) transmissible from person to person (Maines et al., 2006). Limited evidence suggests that H5N1 influenza viruses are probably transmitted from human to human (Olsen et al., 2005; Ungchusak et al., 2005), but that they do not transmit efficiently among humans (Lowen et al., 2006; Maines et al., 2006). It has been suggested that it is not impossible that H9N2 influenza virus shows a low level of human-to-human transmission (Butt et al., 2005; Guo
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et al., 1999; Peiris et al., 1999). However, it is not clear whether H9N2 influenza virus has indeed acquired a means of transmission among humans. The efficiency of transmission in humans is a key factor that determines the severity of influenza epidemics. An understanding of the mechanisms that underlie the transmission of H9N2 influenza virus would allow optimal control measures to be taken that may limit viral spread in pandemic years. Accumulated evidence suggests that influenza virus infection of humans can occur either through contact (direct or indirect) or respiratory droplet (droplet or droplet nuclei) transmission (Bridges et al., 2003; Tellier, 2006). However, the relative contribution of each of these modes of transmission is unknown. Using several animal models, previous studies have investigated the transmissibility of various human influenza viruses and H5N1 avian influenza virus (Bouvier et al., 2008; Herlocher et al., 2002; Herlocher et al., 2001; Lowen et al., 2006; Maines et al., 2006; Schulman, 1968; Schulman and Kilbourne, 1963a). The results indicated that, in general, human influenza viruses can transmit efficiently between hosts, while H5N1 avian influenza virus spread inefficiently (Lowen et al., 2006; Maines et al., 2006). In the face of the persistent threat of H9N2 avian influenza virus to humans, it is important to question whether the virus can transmit efficiently among humans. In this study, we determined that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. We then conducted transmission studies in mice, using both contact and respiratory droplet routes, to evaluate the transmissibility of mouse-adapted H9N2 influenza viruses. Our results indicate that transmission of mouse-adapted H9N2 influenza virus occurs between mice. 2. Materials and methods 2.1. Viruses Mouse-adapted avian influenza variant A/Chicken/ Jiangsu/7/2002 (Ck/Js/11/2002, H9N2) is pathogenic for BALB/c mice and has been described previously (Qiu et al., 2006; Wu et al., 2009). The A/Chicken/Hubei/C1/2007 (H9N2) is not lethal to mice and has also been described previously (Wu et al., 2008). These H9N2 influenza viruses were plaque purified three times and propagated in MDCK cells in MEM containing 1.0 mg/ml trypsin. The infectivity titers of the viruses were determined by calculating the 50% egg infectious dose (EID50). 2.2. Adaptation of A/Chicken/Hubei/C1/2007(H9N2) to murine lungs The A/Chicken/Hubei/C1/2007 (H9N2) virus did not cause the deaths of BALB/c mice, but replication of the virus in the lungs of mice was detected after infection. To enhance its virulence, the A/Chicken/Hubei/C1/2007 (H9N2) virus was passaged in mice. The BALB/c mice were anesthetized and inoculated with 20 ml of the viral suspension (105 EID50) by intranasal drip. Three days after inoculation, the mice were sacrificed, and their trachea and lungs were removed and washed three times with a total of
2 ml of PBS containing 0.1% BSA. The bronchoalveolar lavage fluid was collected and used for infecting the next batch of mice. The lung-to-lung passage tests were repeated eight times, and the mouse-adapted A/Chicken/ Hubei/C1/2007 (H9N2) virus (Ck/HB/C1/07, H9N2) from passage 8 was harvested, purified, and proliferated to a large quantity in MDCK cells. The infectivity titers of the virus were determined by EID50 assay. 2.3. Model of infection Mice aged 6–8 weeks were anesthetized and then inoculated intranasally with 20 ml virus at 105 EID50. Lung tissue specimens were collected from mice on day 3 postinfection; the virus was titrated on 10-day-old embryonated eggs. The body weight and survival of mice were recorded daily for 21 days after infection. To evaluate the tissue distribution of the virus in mice, samples of heart, kidney, liver, spleen and brain were collected for virus titration 3 days post-infection. The viral titer in different tissues, expressed as the EID50, was calculated by the Reed–Mu¨ench method. 2.4. Transmission experiments The transmission experiments in SPF Kunming mice were performed as described previously (Lowen et al., 2006; Schulman and Kilbourne, 1963a). For the transmission experiments using respiratory droplets, the Kunming mice were housed in adjacent transmission cages; the transfer of respiratory droplets through the air was performed while direct contact and indirect contact were prohibited. A total of thirty Kunming mice were used for each transmission experiment. Fifteen Kunming mice were inoculated intranasally with 105 EID50 of virus and the other 15 Kunming mice were placed in a cage adjacent to the inoculated mice. For the contact transmission experiments, 15 Kunming mice were inoculated intranasally with 105 EID50 of virus, and another 15 Kunming mice were placed in the same cage 24 h post-infection. Clinical signs and changes in body weight were monitored daily in all mice for at least 21 days after infection. For the determination of virus titers, five inoculated or in-contact mice were killed on day 4 post-infection; the lung tissues were collected and homogenized. The tissue homogenates were titrated for virus infectivity in eggs. 2.5. HI assays The hemagglutinin inhibition (HI) assay was performed as described previously (Qiu et al., 2006) by using convalescent sera collected from mice 21 days after infection. Sera were serially diluted two-fold with PBS in a 96-well polystyrene microtiter plate; 25 ml was added to each well. A portion of 25 ml of virus suspension containing four hemagglutinin units (HAU) was added to each well. After incubation of the plate at 4 8C for 1 h, 50 ml of 0.5% (v/ v) chicken red blood cells was added to each well. After incubation at 4 8C for 1 h, the HI titers were determined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.
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3. Results 3.1. Characteristics of mouse-adapted viruses in Kunming mice It has been described previously that the Ck/Js/11/2002 virus is fatal for all infected female BALB/c mice. To investigate whether the Ck/Js/11/2002 virus can infect and cause death in other strains of mice, we inoculated female Kunming mice aged 6–8 weeks intranasally with Ck/JS/11/ 2002 virus at 105 EID50. All the mice infected with the Ck/ JS/11/2002 virus exhibited clinical signs of influenza, lost weight progressively, and died within 10 days of infection (Fig. 1A–C). To test whether the sex of the Kunming mice influenced the susceptibility to infection, we inoculated male Kunming mice at the same dose (105 EID50/mouse). All the infected male Kunming mice exhibited morbidity
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and mortality similar to that shown by the female Kunming mice (Fig. 1A–C), which suggested that the sex of the mice did not influence the susceptibility to infection. To test whether a different strain of virus produced the same results, we investigated the pathogenicity and replication of the mouse-adapted Ck/HB/C1/07 virus in Kunming mice. Infection with the parent Ck/HB/C1/07 virus was not lethal for BALB/c mice, but the virus could cause the deaths of BALB/c mice after lung-to-lung passage. Next we inoculated Kunming mice with the mouse-adapted Ck/HB/C1/07 at 105 EID50, and observed the morbidity and mortality. Both female and male Kunming mice showed severe clinical signs and died within 10 days of infection (Fig. 1A–C). 3.2. Replication of mouse-adapted viruses in Kunming mice To test the ability of mouse-adapted viruses to replicate in Kunming mice, Kunming mice aged 6–8 weeks were inoculated intranasally with Ck/JS/11/2002 or Ck/HB/C1/ 07 virus at 105 EID50. To determine the viral titers, we collected samples of heart, kidney, lung, liver, spleen and brain on day 3 post-infection. The Ck/JS/11/2002 and Ck/ HB/C1/07 viruses replicated successfully in both female and male Kunming mice; the lung titers ranged from 6.96 0.32 to 7.49 0.28 log10 EID50/ml (Fig. 1A). Furthermore, the Ck/JS/11/2002 was also able to replicate in the heart and kidney, and the Ck/HB/C1/07 virus could also replicate in the heart, kidney and liver (Table 1). However, the two mouse-adapted viruses could not replicate in spleen and brain. These results suggest that the two mouse-adapted viruses have the ability to replicate in other organs in addition to the lung in Kunming mice. 3.3. Morbidity and mortality caused by mouse-adapted viruses in C578BL/6 and NIH mice In addition to BALB/c and Kunming mice, we wished to investigate whether the two mouse-adapted viruses can replicate and cause death in other strains of mice. To estimate their ability to replicate and cause disease in C578BL/6 and NIH mice, we inoculated C578BL/6 and NIH mice intranasally with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. All mice infected with the two mouse-adapted viruses exhibited clinical signs of influenza, including decreased activity, huddling, hunched posture, and ruffled fur. In addition, all infected mice lost weight progressively, and all died within 10 days of infection (Fig. 2B and C). The two mouse-adapted viruses showed high levels of replication at day 3 post-infection; the lung titers ranged from 6.25 0.42 to 7.05 0.38 log10 EID50/ml (Fig. 2A). These results indicate that the two mouse-adapted viruses have gained high virulence and were lethal to mice of both sexes and both strains.
Fig. 1. Change in weight, virus replication, and mortality in Kunming mice inoculated with avian H9N2 influenza viruses. Kunming mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. On day 3 after infection, viruses in mouse lungs (A) were titrated by EID50 assay on 10-day-old chicken embryos. Body weight (B) and survival (C) of mice were recorded daily for 21 days after infection.
3.4. Transmissibility of mouse-adapted viruses between cocaged Kunming mice To assess whether the two mouse-adapted viruses could be transmitted from infected to uninfected mice under conditions of direct contact between the two
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Table 1 Viral distribution of avian H9N2 influenza virus in different tissues in Kunming micea. Virus titer (log10 EID50/g, mean SD)
Sex
Virus
Heart
Kidney
Liver
Spleen
Brain
Male
Ck/JS/11/2002 Ck/HB/C1/07
3.02 0.34 3.12 0.50
3.37 0.28 3.52 0.19
–b 1.25 0.45
– –
– –
Female
Ck/JS/11/2002 Ck/HB/C1/07
3.53 0.25 3.62 0.41
3.92 0.40 3.09 0.22
– 1.83 0.56
– –
– –
a Kunming mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. On day 3 after infection, five mice from each group were killed for virus titration. Results are expressed as means SD. b –, not detectable.
animals, 15 Kunming mice were infected intranasally with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. At day 1 post-infection, one uninfected animals was added to the cage of each infected animal. All mice inoculated with the two mouse-adapted viruses exhibited clinical signs of
influenza, lost weight progressively, and all died within 10 days of infection (date not shown). The viral titers in the lungs of the inoculated mice reached 7.05 0.32 or 7.37 0.45 log10 EID50/ml (date not shown), respectively, on day 4 post-infection. Both viruses were efficiently
Fig. 2. Change in weight, virus replication, and mortality in different strains of mouse inoculated with avian H9N2 influenza viruses. C578BL/6 and NIH mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/ C1/07 virus at 105 EID50. On day 3 after infection, viruses in mouse lungs (A) were titrated by EID50 assay on 10-day-old chicken embryos. Body weight (B) and survival (C) of mice were recorded daily for 21 days after infection.
Fig. 3. Transmissibility of direct contact and respiratory droplet of avian H9N2 influenza viruses in Kunming mice. The transmission experiments in mice were performed as described previously (Lowen et al., 2006; Schulman and Kilbourne, 1963a). On day 4 after infection, viruses in mouse lungs (A) were determined by EID50 assay on 10-day-old chicken embryos. Body weights (B) of mice were recorded daily for 21 days after infection. HI assays (C) were performed with homologous virus and RBCs derived from cock.
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transmitted to each of the in-contact mice by day 4 postinfection, as demonstrated by detection of virus in the lungs (Fig. 3A) and seroconversion to HI antibody 21 days postinfection (Fig. 3 C) in all in-contact animals. In addition, all incontact animals exhibited signs of illness, and lost weight progressively, with 7.47% or 8.54% weight lost, respectively, on days 2–7 post-infection (Fig. 3B). Thus, the in-contact mice acquired influenza virus infection from their cage-mates, which demonstrates that both mouse-adapted H9N2 viruses were efficiently transmitted from inoculated to naive mice. 3.5. Transmission of mouse-adapted viruses by means of droplet spread We next tested the ability of the two mouse-adapted viruses to undergo respiratory droplet transmission in mice. Fifteen Kunming mice were inoculated intranasally with 105 EID50 of Ck/JS/11/2002 or Ck/HB/C1/07 virus, and, the next day, 15 uninfected Kunming mice were placed in adjacent transmission cages. The infection status of each animal was determined by observation of clinical signs, changes in weight, lung washings on day 4 post-infection, and seroconversion to HI antibody 21 days post-infection. The results indicated that the sentinel animals in each cage acquired influenza virus infection. All sentinel mice exhibited signs of illness, accompanied by progressive weight loss (Fig. 3B). Viral titers were detected in the lungs of the sentinel mice 4 days post-infection (Fig. 3A), and the HI antibody titers were detected in all sentinel mice at 21 days post-infection (Fig. 3C). These results indicate that the two mouse-adapted viruses can transmit efficiently between mice by the respiratory droplet route. 4. Discussion In 1999 and 2003, documented human infections with H9N2 influenza viruses were detected in Hong Kong and mainland China (Butt et al., 2005; Guo et al., 1999, 2000; Peiris et al., 1999). The occurrence of infection with H9N2 viruses raises the fear that H9N2 influenza virus may have the ability to undergo efficient and sustained transmission among humans. Previous studies indicated that the seroprevalence of H9 antibodies in southern China was at least 2% among the human population, suggesting that a low level of human-to-human transmission of H9N2 virus may occur (Butt et al., 2005; Guo et al., 1999; Peiris et al., 1999). In this study, we demonstrated that mouse-adapted H9N2 influenza viruses are lethal to different strains of mice, and can transmit efficiently in the murine model. Based on previous data and our own findings, it is possible that H9N2 influenza viruses may transmit among the human population (Butt et al., 2005). The avian H9N2 influenza viruses are characterized as low pathogenic avian influenza viruses; they generally infect waterfowl and cause no overt clinical signs in these birds (Capua and Alexander, 2004). In recent years, many reports have indicated that H9N2 viruses are virulent for chickens and other birds, and cause high morbidity and mortality in poultry (Aamir et al., 2007; Capua and Alexander, 2004; Choi et al., 2004; Guo et al., 2000). Further, H9N2 influenza viruses can replicate systemically
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in mice, and cause serious clinical signs in pigs (Choi et al., 2004; Guo et al., 2000; Xu et al., 2004). In humans, H9N2 influenza viruses usually cause moderate symptoms of influenza (Butt et al., 2005; Peiris et al., 1999). In this study, two mouse-adapted H9N2 viruses were shown to replicate efficiently in various tissues of Kunming mice, and to be lethal for Kunming, C578BL/6 and NIH mice. These findings suggest that the virulence of H9N2 influenza viruses has increased dramatically during the last two decades. Along with enhanced virulence of H9N2 influenza viruses, there is increased concern that they can infect humans easily and spread among the human population. Previous experimental studies have demonstrated that transmission of human influenza viruses occurs either through contact or respiratory droplet transmission (Bridges et al., 2003; Schulman and Kilbourne, 1962; Tellier, 2006). There is evidence that transmission of human influenza varies with different viruses, different strains of virus, different host susceptibility and age, environmental factors and so on (Lowen et al., 2007, 2008; Schulman, 1967, 1968; Schulman and Kilbourne, 1963a, 1963b). Our experiments confirm that, under certain conditions, mice infected experimentally with H9N2 avian influenza virus transmit the infection to susceptible cage-mates. In addition, mice can acquire influenza virus infection by transmission via respiratory droplets. In contrast, several studies have indicated that highly pathogenic H5N1 viruses cannot be transmitted in animal models (mice, guinea pig, and ferret) (Lowen et al., 2006; Maines et al., 2006), although H5N1 viruses exhibit efficient replication in those animals. Because H9N2 influenza viruses are of low pathogenicity for poultry, the extent of infection in both poultry and humans will probably remain underestimated. Furthermore, previous studies have indicated that some H9N2 viruses have continued to evolve and to reassort with other influenza viruses, and have acquired an affinity for the human receptor binding profile (Choi et al., 2004; Matrosovich et al., 2001). Therefore, H9N2 influenza viruses have the ability to be introduced into humans more efficiently than current H5N1 strains, and have the potential to be transmitted from human to human. The present study has exhibited the ability of mouse-adapted H9N2 influenza virus to be transmitted among mice. In conclusion, it is important to pay urgent attention to H9N2 influenza virus and to carry out more studies on the mechanisms underlying transmission of influenza viruses. 5. Conclusions We conclude that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. Acknowledgements We thank Ze Chen (from Wuhan Institute of Virology, Wuhan, Hubei, China) for the A/Chicken/Jiangsu/7/ 2002(H9N2) virus. This study was supported by Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding (2007ZD08).
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Transmission of avian H9N2 influenza viruses in a murine model Rui Wu a,c,1, Zhiwei Sui b,1, Zewen Liu a, Wangwang Liang a, Keli Yang a, Zhongliang Xiong a, Diping Xu a,c,* a
Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China Shijingshan Center for Diseases Control and Prevention in Beijing City, Beijing 100043, China c Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Wuhan 430064, Hubei, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 19 September 2009 Accepted 23 September 2009
Avian H9N2 influenza viruses have circulated widely in domestic poultry around the world, resulting in occasional transmission of virus from infected poultry to humans. However, it is unknown whether H9N2 influenza virus has acquired the ability to be transmitted from human to human. Here, we report that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. To evaluate the transmissibility of mouse-adapted H9N2 influenza viruses, we carried out transmission studies in mice using both contact and respiratory droplet routes. Our results indicate that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. This suggests that once H9N2 influenza viruses adapt to new host, they should present potential public health risks, therefore, urgent attention should be paid to H9N2 influenza viruses. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Avian influenza virus H9N2 subtype Transmission Contact spread Droplet spread
1. Introduction Avian H9N2 influenza viruses have become panzootic in Eurasia over the last decade and have circulated widely around the world (Capua and Alexander, 2004; Chen et al., 2003). Human disease associated with H9N2 viruses was documented in Hong Kong in 1999, which suggested that H9N2 avian influenza virus can cross the species barrier to humans (Peiris et al., 1999). Prior to that event, human infection with H9N2 avian influenza virus had been reported in mainland China (Guo et al., 1999). Subsequently, a strain of H9N2 avian influenza virus has been isolated repeatedly from the human population in mainland China (Guo et al., 2000). In November 2003, an H9N2 avian influenza virus was isolated from a child with a flu-
* Corresponding author at: Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China. Tel.: +86 27 8738 0195; fax: +86 27 8738 0195. E-mail address: [email protected] (D. Xu). 1 The authors contribute the same work to this study. 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.068
like illness in Hong Kong (Butt et al., 2005). These documented human cases of avian influenza infection in Hong Kong and mainland China aroused global concern regarding the role of H9N2 avian influenza viruses in human disease. There is evidence that the interspecies transmission of H9N2 influenza virus from avian to mammalian hosts continues to occur. This allows us to raise the hypothesis that H9N2 influenza virus is a potential candidate for the next influenza pandemic in humans (Choi et al., 2004; Guan et al., 2000, 1999; Li et al., 2003). For a strain of influenza virus to cause a human pandemic, it must be (i) novel to the human immune system, (ii) virulent in the human host, and (iii) transmissible from person to person (Maines et al., 2006). Limited evidence suggests that H5N1 influenza viruses are probably transmitted from human to human (Olsen et al., 2005; Ungchusak et al., 2005), but that they do not transmit efficiently among humans (Lowen et al., 2006; Maines et al., 2006). It has been suggested that it is not impossible that H9N2 influenza virus shows a low level of human-to-human transmission (Butt et al., 2005; Guo
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et al., 1999; Peiris et al., 1999). However, it is not clear whether H9N2 influenza virus has indeed acquired a means of transmission among humans. The efficiency of transmission in humans is a key factor that determines the severity of influenza epidemics. An understanding of the mechanisms that underlie the transmission of H9N2 influenza virus would allow optimal control measures to be taken that may limit viral spread in pandemic years. Accumulated evidence suggests that influenza virus infection of humans can occur either through contact (direct or indirect) or respiratory droplet (droplet or droplet nuclei) transmission (Bridges et al., 2003; Tellier, 2006). However, the relative contribution of each of these modes of transmission is unknown. Using several animal models, previous studies have investigated the transmissibility of various human influenza viruses and H5N1 avian influenza virus (Bouvier et al., 2008; Herlocher et al., 2002; Herlocher et al., 2001; Lowen et al., 2006; Maines et al., 2006; Schulman, 1968; Schulman and Kilbourne, 1963a). The results indicated that, in general, human influenza viruses can transmit efficiently between hosts, while H5N1 avian influenza virus spread inefficiently (Lowen et al., 2006; Maines et al., 2006). In the face of the persistent threat of H9N2 avian influenza virus to humans, it is important to question whether the virus can transmit efficiently among humans. In this study, we determined that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. We then conducted transmission studies in mice, using both contact and respiratory droplet routes, to evaluate the transmissibility of mouse-adapted H9N2 influenza viruses. Our results indicate that transmission of mouse-adapted H9N2 influenza virus occurs between mice. 2. Materials and methods 2.1. Viruses Mouse-adapted avian influenza variant A/Chicken/ Jiangsu/7/2002 (Ck/Js/11/2002, H9N2) is pathogenic for BALB/c mice and has been described previously (Qiu et al., 2006; Wu et al., 2009). The A/Chicken/Hubei/C1/2007 (H9N2) is not lethal to mice and has also been described previously (Wu et al., 2008). These H9N2 influenza viruses were plaque purified three times and propagated in MDCK cells in MEM containing 1.0 mg/ml trypsin. The infectivity titers of the viruses were determined by calculating the 50% egg infectious dose (EID50). 2.2. Adaptation of A/Chicken/Hubei/C1/2007(H9N2) to murine lungs The A/Chicken/Hubei/C1/2007 (H9N2) virus did not cause the deaths of BALB/c mice, but replication of the virus in the lungs of mice was detected after infection. To enhance its virulence, the A/Chicken/Hubei/C1/2007 (H9N2) virus was passaged in mice. The BALB/c mice were anesthetized and inoculated with 20 ml of the viral suspension (105 EID50) by intranasal drip. Three days after inoculation, the mice were sacrificed, and their trachea and lungs were removed and washed three times with a total of
2 ml of PBS containing 0.1% BSA. The bronchoalveolar lavage fluid was collected and used for infecting the next batch of mice. The lung-to-lung passage tests were repeated eight times, and the mouse-adapted A/Chicken/ Hubei/C1/2007 (H9N2) virus (Ck/HB/C1/07, H9N2) from passage 8 was harvested, purified, and proliferated to a large quantity in MDCK cells. The infectivity titers of the virus were determined by EID50 assay. 2.3. Model of infection Mice aged 6–8 weeks were anesthetized and then inoculated intranasally with 20 ml virus at 105 EID50. Lung tissue specimens were collected from mice on day 3 postinfection; the virus was titrated on 10-day-old embryonated eggs. The body weight and survival of mice were recorded daily for 21 days after infection. To evaluate the tissue distribution of the virus in mice, samples of heart, kidney, liver, spleen and brain were collected for virus titration 3 days post-infection. The viral titer in different tissues, expressed as the EID50, was calculated by the Reed–Mu¨ench method. 2.4. Transmission experiments The transmission experiments in SPF Kunming mice were performed as described previously (Lowen et al., 2006; Schulman and Kilbourne, 1963a). For the transmission experiments using respiratory droplets, the Kunming mice were housed in adjacent transmission cages; the transfer of respiratory droplets through the air was performed while direct contact and indirect contact were prohibited. A total of thirty Kunming mice were used for each transmission experiment. Fifteen Kunming mice were inoculated intranasally with 105 EID50 of virus and the other 15 Kunming mice were placed in a cage adjacent to the inoculated mice. For the contact transmission experiments, 15 Kunming mice were inoculated intranasally with 105 EID50 of virus, and another 15 Kunming mice were placed in the same cage 24 h post-infection. Clinical signs and changes in body weight were monitored daily in all mice for at least 21 days after infection. For the determination of virus titers, five inoculated or in-contact mice were killed on day 4 post-infection; the lung tissues were collected and homogenized. The tissue homogenates were titrated for virus infectivity in eggs. 2.5. HI assays The hemagglutinin inhibition (HI) assay was performed as described previously (Qiu et al., 2006) by using convalescent sera collected from mice 21 days after infection. Sera were serially diluted two-fold with PBS in a 96-well polystyrene microtiter plate; 25 ml was added to each well. A portion of 25 ml of virus suspension containing four hemagglutinin units (HAU) was added to each well. After incubation of the plate at 4 8C for 1 h, 50 ml of 0.5% (v/ v) chicken red blood cells was added to each well. After incubation at 4 8C for 1 h, the HI titers were determined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.
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3. Results 3.1. Characteristics of mouse-adapted viruses in Kunming mice It has been described previously that the Ck/Js/11/2002 virus is fatal for all infected female BALB/c mice. To investigate whether the Ck/Js/11/2002 virus can infect and cause death in other strains of mice, we inoculated female Kunming mice aged 6–8 weeks intranasally with Ck/JS/11/ 2002 virus at 105 EID50. All the mice infected with the Ck/ JS/11/2002 virus exhibited clinical signs of influenza, lost weight progressively, and died within 10 days of infection (Fig. 1A–C). To test whether the sex of the Kunming mice influenced the susceptibility to infection, we inoculated male Kunming mice at the same dose (105 EID50/mouse). All the infected male Kunming mice exhibited morbidity
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and mortality similar to that shown by the female Kunming mice (Fig. 1A–C), which suggested that the sex of the mice did not influence the susceptibility to infection. To test whether a different strain of virus produced the same results, we investigated the pathogenicity and replication of the mouse-adapted Ck/HB/C1/07 virus in Kunming mice. Infection with the parent Ck/HB/C1/07 virus was not lethal for BALB/c mice, but the virus could cause the deaths of BALB/c mice after lung-to-lung passage. Next we inoculated Kunming mice with the mouse-adapted Ck/HB/C1/07 at 105 EID50, and observed the morbidity and mortality. Both female and male Kunming mice showed severe clinical signs and died within 10 days of infection (Fig. 1A–C). 3.2. Replication of mouse-adapted viruses in Kunming mice To test the ability of mouse-adapted viruses to replicate in Kunming mice, Kunming mice aged 6–8 weeks were inoculated intranasally with Ck/JS/11/2002 or Ck/HB/C1/ 07 virus at 105 EID50. To determine the viral titers, we collected samples of heart, kidney, lung, liver, spleen and brain on day 3 post-infection. The Ck/JS/11/2002 and Ck/ HB/C1/07 viruses replicated successfully in both female and male Kunming mice; the lung titers ranged from 6.96 0.32 to 7.49 0.28 log10 EID50/ml (Fig. 1A). Furthermore, the Ck/JS/11/2002 was also able to replicate in the heart and kidney, and the Ck/HB/C1/07 virus could also replicate in the heart, kidney and liver (Table 1). However, the two mouse-adapted viruses could not replicate in spleen and brain. These results suggest that the two mouse-adapted viruses have the ability to replicate in other organs in addition to the lung in Kunming mice. 3.3. Morbidity and mortality caused by mouse-adapted viruses in C578BL/6 and NIH mice In addition to BALB/c and Kunming mice, we wished to investigate whether the two mouse-adapted viruses can replicate and cause death in other strains of mice. To estimate their ability to replicate and cause disease in C578BL/6 and NIH mice, we inoculated C578BL/6 and NIH mice intranasally with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. All mice infected with the two mouse-adapted viruses exhibited clinical signs of influenza, including decreased activity, huddling, hunched posture, and ruffled fur. In addition, all infected mice lost weight progressively, and all died within 10 days of infection (Fig. 2B and C). The two mouse-adapted viruses showed high levels of replication at day 3 post-infection; the lung titers ranged from 6.25 0.42 to 7.05 0.38 log10 EID50/ml (Fig. 2A). These results indicate that the two mouse-adapted viruses have gained high virulence and were lethal to mice of both sexes and both strains.
Fig. 1. Change in weight, virus replication, and mortality in Kunming mice inoculated with avian H9N2 influenza viruses. Kunming mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. On day 3 after infection, viruses in mouse lungs (A) were titrated by EID50 assay on 10-day-old chicken embryos. Body weight (B) and survival (C) of mice were recorded daily for 21 days after infection.
3.4. Transmissibility of mouse-adapted viruses between cocaged Kunming mice To assess whether the two mouse-adapted viruses could be transmitted from infected to uninfected mice under conditions of direct contact between the two
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Table 1 Viral distribution of avian H9N2 influenza virus in different tissues in Kunming micea. Virus titer (log10 EID50/g, mean SD)
Sex
Virus
Heart
Kidney
Liver
Spleen
Brain
Male
Ck/JS/11/2002 Ck/HB/C1/07
3.02 0.34 3.12 0.50
3.37 0.28 3.52 0.19
–b 1.25 0.45
– –
– –
Female
Ck/JS/11/2002 Ck/HB/C1/07
3.53 0.25 3.62 0.41
3.92 0.40 3.09 0.22
– 1.83 0.56
– –
– –
a Kunming mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. On day 3 after infection, five mice from each group were killed for virus titration. Results are expressed as means SD. b –, not detectable.
animals, 15 Kunming mice were infected intranasally with Ck/JS/11/2002 or Ck/HB/C1/07 virus at 105 EID50. At day 1 post-infection, one uninfected animals was added to the cage of each infected animal. All mice inoculated with the two mouse-adapted viruses exhibited clinical signs of
influenza, lost weight progressively, and all died within 10 days of infection (date not shown). The viral titers in the lungs of the inoculated mice reached 7.05 0.32 or 7.37 0.45 log10 EID50/ml (date not shown), respectively, on day 4 post-infection. Both viruses were efficiently
Fig. 2. Change in weight, virus replication, and mortality in different strains of mouse inoculated with avian H9N2 influenza viruses. C578BL/6 and NIH mice were intranasally inoculated with Ck/JS/11/2002 or Ck/HB/ C1/07 virus at 105 EID50. On day 3 after infection, viruses in mouse lungs (A) were titrated by EID50 assay on 10-day-old chicken embryos. Body weight (B) and survival (C) of mice were recorded daily for 21 days after infection.
Fig. 3. Transmissibility of direct contact and respiratory droplet of avian H9N2 influenza viruses in Kunming mice. The transmission experiments in mice were performed as described previously (Lowen et al., 2006; Schulman and Kilbourne, 1963a). On day 4 after infection, viruses in mouse lungs (A) were determined by EID50 assay on 10-day-old chicken embryos. Body weights (B) of mice were recorded daily for 21 days after infection. HI assays (C) were performed with homologous virus and RBCs derived from cock.
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transmitted to each of the in-contact mice by day 4 postinfection, as demonstrated by detection of virus in the lungs (Fig. 3A) and seroconversion to HI antibody 21 days postinfection (Fig. 3 C) in all in-contact animals. In addition, all incontact animals exhibited signs of illness, and lost weight progressively, with 7.47% or 8.54% weight lost, respectively, on days 2–7 post-infection (Fig. 3B). Thus, the in-contact mice acquired influenza virus infection from their cage-mates, which demonstrates that both mouse-adapted H9N2 viruses were efficiently transmitted from inoculated to naive mice. 3.5. Transmission of mouse-adapted viruses by means of droplet spread We next tested the ability of the two mouse-adapted viruses to undergo respiratory droplet transmission in mice. Fifteen Kunming mice were inoculated intranasally with 105 EID50 of Ck/JS/11/2002 or Ck/HB/C1/07 virus, and, the next day, 15 uninfected Kunming mice were placed in adjacent transmission cages. The infection status of each animal was determined by observation of clinical signs, changes in weight, lung washings on day 4 post-infection, and seroconversion to HI antibody 21 days post-infection. The results indicated that the sentinel animals in each cage acquired influenza virus infection. All sentinel mice exhibited signs of illness, accompanied by progressive weight loss (Fig. 3B). Viral titers were detected in the lungs of the sentinel mice 4 days post-infection (Fig. 3A), and the HI antibody titers were detected in all sentinel mice at 21 days post-infection (Fig. 3C). These results indicate that the two mouse-adapted viruses can transmit efficiently between mice by the respiratory droplet route. 4. Discussion In 1999 and 2003, documented human infections with H9N2 influenza viruses were detected in Hong Kong and mainland China (Butt et al., 2005; Guo et al., 1999, 2000; Peiris et al., 1999). The occurrence of infection with H9N2 viruses raises the fear that H9N2 influenza virus may have the ability to undergo efficient and sustained transmission among humans. Previous studies indicated that the seroprevalence of H9 antibodies in southern China was at least 2% among the human population, suggesting that a low level of human-to-human transmission of H9N2 virus may occur (Butt et al., 2005; Guo et al., 1999; Peiris et al., 1999). In this study, we demonstrated that mouse-adapted H9N2 influenza viruses are lethal to different strains of mice, and can transmit efficiently in the murine model. Based on previous data and our own findings, it is possible that H9N2 influenza viruses may transmit among the human population (Butt et al., 2005). The avian H9N2 influenza viruses are characterized as low pathogenic avian influenza viruses; they generally infect waterfowl and cause no overt clinical signs in these birds (Capua and Alexander, 2004). In recent years, many reports have indicated that H9N2 viruses are virulent for chickens and other birds, and cause high morbidity and mortality in poultry (Aamir et al., 2007; Capua and Alexander, 2004; Choi et al., 2004; Guo et al., 2000). Further, H9N2 influenza viruses can replicate systemically
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in mice, and cause serious clinical signs in pigs (Choi et al., 2004; Guo et al., 2000; Xu et al., 2004). In humans, H9N2 influenza viruses usually cause moderate symptoms of influenza (Butt et al., 2005; Peiris et al., 1999). In this study, two mouse-adapted H9N2 viruses were shown to replicate efficiently in various tissues of Kunming mice, and to be lethal for Kunming, C578BL/6 and NIH mice. These findings suggest that the virulence of H9N2 influenza viruses has increased dramatically during the last two decades. Along with enhanced virulence of H9N2 influenza viruses, there is increased concern that they can infect humans easily and spread among the human population. Previous experimental studies have demonstrated that transmission of human influenza viruses occurs either through contact or respiratory droplet transmission (Bridges et al., 2003; Schulman and Kilbourne, 1962; Tellier, 2006). There is evidence that transmission of human influenza varies with different viruses, different strains of virus, different host susceptibility and age, environmental factors and so on (Lowen et al., 2007, 2008; Schulman, 1967, 1968; Schulman and Kilbourne, 1963a, 1963b). Our experiments confirm that, under certain conditions, mice infected experimentally with H9N2 avian influenza virus transmit the infection to susceptible cage-mates. In addition, mice can acquire influenza virus infection by transmission via respiratory droplets. In contrast, several studies have indicated that highly pathogenic H5N1 viruses cannot be transmitted in animal models (mice, guinea pig, and ferret) (Lowen et al., 2006; Maines et al., 2006), although H5N1 viruses exhibit efficient replication in those animals. Because H9N2 influenza viruses are of low pathogenicity for poultry, the extent of infection in both poultry and humans will probably remain underestimated. Furthermore, previous studies have indicated that some H9N2 viruses have continued to evolve and to reassort with other influenza viruses, and have acquired an affinity for the human receptor binding profile (Choi et al., 2004; Matrosovich et al., 2001). Therefore, H9N2 influenza viruses have the ability to be introduced into humans more efficiently than current H5N1 strains, and have the potential to be transmitted from human to human. The present study has exhibited the ability of mouse-adapted H9N2 influenza virus to be transmitted among mice. In conclusion, it is important to pay urgent attention to H9N2 influenza virus and to carry out more studies on the mechanisms underlying transmission of influenza viruses. 5. Conclusions We conclude that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. Acknowledgements We thank Ze Chen (from Wuhan Institute of Virology, Wuhan, Hubei, China) for the A/Chicken/Jiangsu/7/ 2002(H9N2) virus. This study was supported by Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding (2007ZD08).
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