Avian influenza A H5N1 infections in cats

Although cats had been considered resistant to disease from influenza virus infection, domestic cats and large felids are now known to be naturally und experimentally susceptible to infection with highly pathogenic avian influenza virus H5N1 (HPAIV H5N1). The virus causes systemic infection, lung and liver being the mainly affected organs. Infected cats show fever, depression, dyspnoea, and neurological signs, but subclinical infections have also occurred. Mostly, cats have been infected by direct contact with affected birds, especially by eating raw poultry; transmission from cat to cat may also occur. Little is known about the role of cats in the epidemiology of the virus. So far, no reassortment between avian and mammalian influenza viruses has occurred in cats, but experts fear that cats might give the virus an opportunity to adapt to mammals. This publication gives a review on avian influenza in cats with a focus on practical aspects for veterinarians.     

"Constanze": a trinational project on avian influenza in wild birds at Lake Constance

When highly pathogenic avian influenza H5N1 (HPAI H5N1) arrived at Lake Constance in February 2006, little was known about its ecology and epidemiology in wild birds. In order to prevent virus transmission from wild birds to poultry, the adjacent countries initiated the tri-national, interdisciplinary research program ?Constanze? to investigate avian influenza infections in water birds at Lake Constance. In collaboration with government agencies scientists examined the prevalence of AI virus in the region of Lake Constance for a period of 33 months, compared the effectiveness of different surveillance methods and analysed the migration behaviour of water birds. Although virus introduction from regions as far as the Ural Mountains seemed possible based on the migration behaviour of certain species, no influenza A viruses of the highly pathogenic subtype H5N1 (HPAIV) was found. However, influenza A viruses of different low pathogenic subtypes were isolated in 2.2 % of the sampled birds (swabs). Of the different surveillance methods utilised in the program the sampling of so called sentinel birds was particularly efficient.     

Distribution of lesions and antigen of highly pathogenic avian influenza virus A/Swan/Germany/R65/06 (H5N1) in domestic cats after presumptive infection by wild birds

In early 2006, the highly pathogenic avian influenza virus (HPAIV) H5N1 of the Asian lineage caused the death of wild aquatic birds in Northern Germany. In the mainly affected areas, a trans-species transmission of HPAIV H5N1 to mammals occurred between birds and domestic cats and 1 Stone Marten (Martes foina), respectively. Here, we report lesions and distribution of influenza virus antigen in 3 cats infected naturally with HPAIV H5N1 A/swan/Germany/R65/06. The hemagglutinin partial nucleotide sequences of the viruses were genetically closely related to a H5N1 HPAIV obtained from a dead Whooper Swan (Cygnus cygnus) of the same area. At necropsy, within the patchy dark-red and consolidated lungs, there was granulomatous pneumonia caused by Aelurostrongylus sp. Histologically, the main findings associated with influenza in all cats were bronchointerstitial pneumonia and marked random hepatic necrosis. In addition, all animals displayed lymphoid necrosis in the spleen and Peyer's patches and necrosis of the adrenal cortex. Immunohistochemically, nucleoprotein of HPAIV was present intralesionally in the lungs, liver, adrenal glands, and lymphoid tissues. Oropharyngeal swabs were shown to be suited to detect HPAIV by quantitative real-time polymerase chain reaction (RT-PCR) in these cats, despite the paucity of influenza virus antigen in the upper respiratory tract by means of immunohistochemistry. The results show that outdoor cats in areas affected by HPAIV in wild birds are at risk for lethal infection. In conclusion, hepatic necrosis was, besides bronchointerstitial pneumonia, the primary lesion, suggesting that in naturally infected cats, damage to the liver plays an important role in the pathogenesis of H5N1 influenza.     

High doses of highly pathogenic avian influenza virus in chicken meat are required to infect ferrets

High pathogenicity avian influenza viruses (HPAIV) have caused fatal infections in mammals through consumption of infected bird carcasses or meat, but scarce information exists on the dose of virus required and the diversity of HPAIV subtypes involved. Ferrets were exposed to different HPAIV (H5 and H7 subtypes) through consumption of infected chicken meat. The dose of virus needed to infect ferrets through consumption was much higher than via respiratory exposure and varied with the virus strain. In addition, H5N1 HPAIV produced higher titers in the meat of infected chickens and more easily infected ferrets than the H7N3 or H7N7 HPAIV.     

A sero-survey of subtype H3 influenza A virus infection in dogs and cats in Japan

A sero-epidemiological survey of human and equine H3 influenza A virus infections in dogs and cats using the hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests was conducted. Serum samples were collected from 582 dogs and 237 cats in Japan during the periods 2002-2008 and 1997-2008, respectively. Although no HI antibodies against equine H3 virus were detected, 9 (3.8%) from cats and 12 (2.1%) from dogs were HI-positive against human H3 virus. Only one serum each from dogs and cats was NI-positive against N2 virus. These findings suggest that although equine H3 influenza virus infections have not been prevalent in companion animals, human H3N2 influenza A virus infections have occurred in dogs and cats in recent years in Japan.     

Seroprevalence and risk factors of swine influenza in Spain

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI(95%): 89.1-98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI(95%): 60.2-64.3%) were seropositive. A total of 1622 animals (75.4%; CI(95%): 73.6-77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI(95%): 87.7-98.1%) had H1N1 seropositive animals; 63 (64.3%; CI(95%): 54.6-73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI(95%): 87.7-98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI(95%): 83.7-95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.     

Coexpression of avian influenza virus H5 and N1 by recombinant Newcastle disease virus and the impact on immune response in chickens

To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.     

A vaccine prepared from a non-pathogenic H5N1 influenza virus strain from the influenza virus library conferred protective immunity to chickens against the challenge with antigenically drifted highly pathogenic avian influenza virus

Inactivated influenza virus vaccine prepared from a non-pathogenic influenza virus strain A/duck/Hokkaido/Vac-1/2004 (H5N1) from the virus library conferred protective immunity to chickens against the challenge of antigenically drifted highly pathogenic avian influenza virus (HPAIV), A/whooper swan/Hokkaido/1/2008 (H5N1). The efficacy of the vaccine was comparable to that prepared from genetically modified HPAIV strain deltaRRRRK rg-A/ whooper swan/Mongolia/3/2005 (H5N1), which is more antigenically related to the challenge virus strain, in chickens.     

Avian influenza in birds and mammals

The disease syndromes caused by avian influenza viruses are highly variable depending on the host species infected, its susceptibility and response to infection and the virulence of the infecting viral strain. Although avian influenza viruses have a broad host range in general, it is rare for an individual strain or subtype to infect more than one species. The H5N1 highly pathogenic avian influenza virus (HPAIV) lineages of viruses that descended from A/goose/Guandong/96 (H5N1 HPAIV) are unusual in the diversity of species they have infected worldwide. Although the species affected by H5N1 HPAI in the field and those that have been experimentally studied are diverse, their associated disease syndromes are remarkably similar across species. In some species, multi-organ failure and death are rapid and no signs of the disease are observed. Most prominently in this category are chickens and other avian species of the order Galliformes. In other species, neurologic signs develop resulting in the death of the host. This is what has been reported in domestic cats (Carnivora), geese (Anseriformes), ratites (Struthioniformes), pigeons inoculated with high doses (Columbiformes) and ducks infected with H5N1 HPAIV isolated since 2002 (Anseriformes). In some other species, the disease is more prolonged and although multi-organ failure and death are the eventual outcomes, the signs of disease are more extensive. Predominantly, these species include humans (Primates) and the laboratory models of human disease, the ferret (Carnivora), mouse (Rodentia) and cynamologous macaques (Primates). Finally, some species are more resistant to infection with H5N1 HPAIV and show few or no signs of disease. These species include pigeons in some studies (Columbiformes), ducks inoculated with pre-2002 isolates (Anseriformes), and pigs (Artiodactyla).     

Transmission characteristics of low pathogenic avian influenza virus of H7N7 and H5N7 subtypes in layer chickens

Low pathogenic avian influenza virus (LPAIv) infections of H5 and H7 subtypes in poultry are notifiable to the OIE, hence surveillance programmes are implemented. The rate at which LPAIv strains spread within a flock determines the prevalence of infected birds and the time it takes to reach that prevalence and, consequently, optimal sample size and sampling frequency. The aim of this study was to investigate the transmission characteristics of an H7N7 and an H5N7 LPAIv in layer chickens. Two transmission experiments were performed, which consisted of 30 (first experiment) and 20 (second experiment) pairs of conventional layers, respectively. At the start of the experiments, one chicken per pair was inoculated with LPAIv and the other chicken was contact-exposed. Occurrence of infection was monitored by regularly collecting tracheal and cloacal swab samples, which were examined for the presence of virus RNA by RT-PCR. The results of the test were used to estimate the transmission rate parameter (β), the infectious period (T) and the basic reproduction ratio (R(0)). In addition, egg production and virus shedding patterns were quantified. For the H7N7 virus, the β, T and R(0) estimates were 0.10 (95% confidence interval (CI): 0.04-0.18) day(-1), 7.1 (95% CI: 6.5-7.8) days and 0.7 (95% CI: 0.0-1.7), respectively. With the H5N7 virus, only a few inoculated chickens (5 out of 20) became infected and no transmission was observed. This study shows that transmission characteristics of LPAIv strains may vary considerably, which has to be taken into account when designing surveillance programmes.     

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication,disease, and mortality in chickens

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10^6.9 EID₅₀) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10^5.3–5.5 EID₅₀) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users.     

Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection

Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0174-3) contains supplementary material, which is available to authorized users.     

Evidence of pandemic H1N1 influenza exposure in dogs and cats,Thailand: A serological survey

Influenza A virus causes respiratory disease in both humans and animals. In this study, a survey of influenza A antibodies in domestic dogs and cats was conducted in 47 animal shelters in 19 provinces of Thailand from September 2011 to September 2014. One thousand and eleven serum samples were collected from 932 dogs and 79 cats. Serum samples were tested for influenza A antibodies using a multi‐species competitive NP‐ELISA and haemagglutination inhibition (HI) assay. The NP‐ELISA results showed that 0.97% (9/932) of dogs were positive, but all cat samples were negative. The HI test against pandemic H1N1, human H3N2 and canine H3N2 showed that 0.64% (6/932) and 1.20% (1/79) of dogs and cats were positive, respectively. It is noted that all six serum samples (5 dogs and 1 cat) had antibodies against pandemic H1N1. In summary, a serological survey revealed the evidence of pandemic H1N1 influenza exposure in both dogs and cats in the shelters in Thailand.     

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets,China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.     

Development of a pen-site test kit for the rapid diagnosis of H7 highly pathogenic avian influenza

As well as H5 highly pathogenic avian influenza viruses (HPAIV), H7 HPAIV strains have caused serious damages in poultry industries worldwide. Cases of bird-to-human transmission of H7 HPAIV have also been reported [11]. On the outbreak of avian influenza, rapid diagnosis is critical not only for the control of HPAI but also for human health. In the present study, a rapid diagnosis kit based on immunochromatography for the detection of H7 hemagglutinin (HA) antigen of influenza A virus was developed using 2 monoclonal antibodies that recognize different epitopes on the H7 HAs. The kit detected each of the tested 15 H7 influenza virus strains and did not react with influenza A viruses of the other subtypes than H7 or other avian viral and bacterial pathogens. The kit detected H7 HA antigen in the swabs and tissue homogenates of the chickens experimentally infected with HPAIV strain A/chicken/Netherlands/2586/03 (H7N7). The results indicate that the present kit is specific and sensitive enough for the diagnosis of HPAI caused by H7 viruses, thus, recommended for the field application as a pen-site test kit.     

(Highly pathogenic) avian influenza as a zoonotic agent

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.     

Serological Survey in Dogs and Cats for Influenza A(H1N1)pdm09 in Germany

A serological survey for the detection of antibodies to influenza A(H1N1)pdm09 was carried out in a population of dogs and cats in Germany. A total of 1150 sera collected in 2010 and 2011 were screened using an ELISA targeting anti‐nucleoprotein NP antibodies. Those initially screened positive samples were subsequently tested for antibodies to N1 neuraminidase followed by a virus neutralization test using A/Bayern/74/2009 strain. A prevalence of A(H1N1)pdm09‐specific antibodies of 0.13% and 1.93% was estimated among dogs and cats, respectively. Evidence of exposure to other influenza A virus subtypes was also observed.     

禽流感病毒H5N2亚型西藏株的分离鉴定与特性分析

从西藏地区病死鸡体内分离鉴定到1株H5N2亚型禽流感病毒,基因组序列测定与分析显示,此H5N2毒株可能是由H5NI和H9N22种亚型的病毒重配形成,其PB2、HA、NS片段与高致病性H5NI亚型高度同源,PBI、PA、NP、NA、M均与低致病性H9N2亚型亲缘关系相近。尽管HA蛋白切割位点有多个碱性氨基酸序列,但静脉接种指数只有0．59,表现为低致病力。     

Virological surveillance and phylogenetic analysis of the PB2 genes of influenza viruses isolated from wild water birds flying from their nesting lakes in Siberia to Hokkaido, Japan in autumn

Recent introduction of H5N1 highly pathogenic avian influenza virus (HPAIV) in wild birds from poultry in Eurasia signaled the possibility that this virus may perpetuate in nature. Surveillance of avian influenza especially in migratory birds, therefore, has been conducted to provide information on the viruses brought by them to Hokkaido, Japan, from their nesting lakes in Siberia in autumn. During 2008-2009, 62 influenza viruses of 21 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes were isolated. Up to September 2010, no HPAIV has been found, indicating that H5N1 HPAIV has not perpetuated at least dominantly in the lakes where ducks nest in summer in Siberia. The PB2 genes of 54 influenza viruses out of 283 influenza viruses isolated in Hokkaido in 2000-2009 were phylogenetically analysed. None of the genes showed close relation to those of H5N1 HPAIVs that were detected in wild birds found dead in Eurasia on the way back to their northern territory in spring.     

Serological evidence of pig-to-human influenza virus transmission on Thai swine farms

We investigated influenza interspecies transmission in two commercial swine farms in Thailand. Sera from swine-exposed workers (n=78), age-matched non-swine-exposed healthy people (n=60) and swine populations in both farms (n=85) were studied. Hemagglutination-inhibition (HI) assay was performed on Thai swine H1 viruses (swH1N1 and swH1N2) isolated from both farms. Thai human H1N1 (huH1N1) and pandemic H1N1 2009 (pH1N1) were also used as test antigens. The hemagglutinin (HA) 1 genes of swH1N1 and swH1N2 viruses were sequenced and shown to be genetically distinct from the Thai huH1N1 and pH1N1 viruses. Evidence of pig-to-human influenza virus transmission was found in farm workers with increased odds of elevated antibody titers to both swH1N1 (OR 42.63, 95% CI, 14.65-124) and swH1N2 (OR 58, 95% CI, 13.12-256.3) viruses. No evidence of human-to-pig influenza virus transmission was detected in this study.