Chicken H5N1 Research Articles

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Seroprevalence of Avian Influenza virus subtype H5N1 in chickens of the two main poultry market in Surakarta area, Central Java

Avian influenza (AI) subtype H5N1 virus is very contagious among domestic poultry such as chicken (Gallus gallus) and potentially infecting humans through direct or indirect contact with the infected animal. In the Surakarta area, Central Java, there are two major poultry markets, Silir poultry market and Jumantono farm. Here, we determined the seroprevalence of the AI virus in chickens sold in both poultry markets. The method was conducted using an explorative experiment by collecting 140 samples of chicken blood serum. The presence of antibodies to the AI virus subtype H5N1 was measured through a standard serological test via hemagglutination-inhibition (HI) assay. The HI assay detected a large antibody difference against AI from samples of both locations; 4.28% from Silir poultry market and 48.57% from Jumantono farm. Because the chickens from the Silir poultry market were not vaccinated, the level of antibody detected indicatesd that some chickens were exposed to the virus through infection. Meanwhile, the high positivity rate in Jumantono farm might reflect the presence of neutralizing antibody, since the chickens from this farm received periodic vaccinations. Continuous quality surveillance in the environment of the poultry market is essential to reduce the impact of AI outbreaks against chickens and humans.

Serological survey for avian influenza virus infection of backyard poultry and poultry workers in Baghdad and Basrah provinces, Iraq

The aim of this study was to determine the prevalence of avian influenza virus (AIV) subtype H5N1 among backyard birds and poultry workers. The study included backyard birds (chickens and ducks) in rural areas of Baghdad and Basrah provinces, Iraq. Rural areas were divided into wet land and dry land. Total egg samples were 368, divided into 184 eggs (92 chicken eggs and 92 duck eggs) from each province. The total number of human sera was 180 (45 poultry worker and 45 non-poultry worker samples from each province). Competitive ELISA tests were used for detection of specific antibodies against influenza A virus and influenza subtype H5N1 in all samples. Seroprevalence against AIV type A in chickens was 62.5% (115/184) while ducks showed seroprevalence of 40.8% (75/184). The seropositivity among the backyard birds in wet land rural areas was higher than that in dry land rural areas. The samples positive for subtype H5N1 in chickens were 15.7% (18/115) while in ducks: 12% (9/75) with highest seropositivity among chickens in wet land rural areas (18.8%; 12/64). The seropositivity of avian influenza H5N1 was 6% (4/67) among AIV-positive poultry workers and 2.9% (1/34) among non-poultry workers.

Highly Pathogenic Avian Influenza H5N1 in Chickens in Upper Egypt

Highly Pathogenic Avian Influenza H5N1 in Chickens in Upper Egypt

Highly Pathogenic Avian Influenza H5N1 in Chickens in Upper Egypt

One-year surveillance of HPAI (H5N1) virus infection in different chicken flocks (141 flock) from different Governorates in Upper Egypt was carried out, during the period from January 2015 to January 2016. The detection rate of highly pathogenic avian influenza (HPAI) H5N1 virus was 21.3% (30/141) which was highest during the winter season compared with the other seasons. The investigated flocks suffered from respiratory and nervous signs and cyanosis of the comb and wattles was observed in some birds as well as subcutaneous hemorrhages in shanks. The thirty positive H5N1 samples were tested for the existence of other respiratory viruses. The results were as follows: single H5N1 virus infection was only detected in 5 flocks (16.6%). The mixed H5N1 with H9N2 in 2 flocks (6.6%); H5N1with NDV in 12 farms (40%); H5N1 with IBV in 7 farms (23.3%) while H5N1, H9N2 and NDV were detected together in 4 flocks (13.3 %). The sequence analysis of the HA gene from five selected H5N1 isolates revealed that these strains were clustered with Egyptian classical H5N1 viruses (sub-clade 2.2.1.2) of the Eurasian origin. It is concluded that, the five H5N1 subtype isolates of HPAI are clustered with classical H5N1 viruses to subclade 2.2.1.2 of Eurasian origin. Therefore, vaccination of the backyard chicken as well as chicken farms with suitable homologues vaccine to eliminate HPAI virus from Egypt is recommended.

Immune Responses Induced by Recombinant Bacillus Subtilis Expressing the Hemagglutinin Protein of H5N1 in chickens.

To develop an effective, safe, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI) H5N1, we have constructed a recombinant Bacillus subtilis strain (B.S.-HA) expressing the hemagglutinin (HA) protein. Then we evaluated the immune function in chicken bone marrow derived dendritic cells (BM-DCs), and the immune response after oral immunization. Our results show that the recombinant Bacillus subtilis B.S.-HA could be sampled by BM-DCs in vitro and increase the BM-DCs major histocompatibility complex (MHC) II phenotype. The weight, height of the small intestine villus, and lymphoid tissue area of the ileum increased significantly in B.S.-HA immunized chickens (P < 0.05 or P < 0.01). B.S.-HA induced the secretion of cytokines and the expression of Toll-like receptors in the trachea and small intestine (P < 0.05 or P < 0.01). In addition, B.S.-HA elevated the specific IgA titers in the trachea, IgG and HI antibody titers in serum (P < 0.05 or P < 0.01). Therefore, B.S.-HA provides a potential novel strategy and approach for developing an H5N1 vaccine.

Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice.

In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity. Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.

Amino acid substitutions in PB1 of avian influenza viruses influence pathogenicity and transmissibility in chickens.

Amino acid substitutions were introduced into avian influenza virus PB1 in order to characterize the interaction between polymerase activity and pathogenicity. Previously, we used recombinant viruses containing the hemagglutinin (HA) and neuraminidase (NA) genes from the highly pathogenic avian influenza virus (HPAIV) H5N1 strain and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken and wild-bird hosts (LP and WB, respectively) to demonstrate that the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in chickens is regulated by the PB1 gene (Y. Uchida et al., J. Virol. 86:2686-2695, 2012, doi:http://dx.doi.org/10.1128/JVI.06374-11). In the present study, we introduced a C38Y substitution into WB PB1 and demonstrated that this substitution increased both polymerase activity in DF-1 cells in vitro and the pathogenicity of the recombinant viruses in chickens. The V14A substitution in LP PB1 reduced polymerase activity but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increased polymerase activity correlates directly with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis. We identified 2 novel amino acid substitutions in the avian influenza virus PB1 gene that affect the characteristics of highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. An amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens and was associated with changes in polymerase activity in vitro. A substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of internal genes.

The pathobiology of two Indonesian H5N1 avian influenza viruses representing different clade 2.1 sublineages in chickens and ducks

To determine the pathobiology of Indonesian H5N1 highly pathogenic avian influenza, two viruses representing clades 2.1.1 and 2.1.3 were inoculated into broiler chickens and Pekin ducks via the eyes, nostrils and oropharynx. In chickens, both viruses produced fulminant disease; tissue tropism was broad but predominantly endothelial and viral loads in tissues were high. Except for one case of meningoencephalitis, the infection in ducks was sub-clinical, leading only to seroconversion. In these ducks, virus and viral antigen occurred in lower amounts, mainly in the respiratory tract (airsac and sinuses), prior to day 7 after inoculation. During clinical disease, chickens shed high virus titres orally and cloacally. Ducks intermittently shed low virus titres from the oral route for up to 8 days post-inoculation. We discuss the significance of the data for understanding the pathogenesis and pathobiology of Indonesian H5N1 in chickens and ducks.

Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy

Background Currently, Asian lineage highly pathogenic avian influenza (HPAI) H5N1 has become widespread across continents. These viruses are persistently circulating among poultry populations in endemic regions, causing huge economic losses, and raising concerns about an H5N1 pandemic. To control HPAI H5N1, effective vaccines for poultry are urgently needed. Objective In this study, we developed HPAI virus‐like particle (VLP) vaccine as a candidate poultry vaccine and evaluated its protective efficacy and possible application for differentiating infected from vaccinated animals (DIVA). Methods Specific pathogen‐free chickens received a single injection of HPAI H5N1 VLP vaccine generated using baculovirus expression vector system. Immunogenicity of VLP vaccines was determined using hemagglutination inhibition (HI), neuraminidase inhibition (NI), and ELISA test. Challenge study was performed to evaluate efficacy of VLP vaccines. Results and Conclusions A single immunization with HPAI H5N1 VLP vaccine induced high levels of HI and NI antibodies and protected chickens from a lethal challenge of wild‐type HPAI H5N1 virus. Viral excretion from the vaccinated and challenged group was strongly reduced compared with a mock‐vaccinated control group. Furthermore, we were able to differentiate VLP‐vaccinated chickens from vaccinated and then infected chickens with a commercial ELISA test kit, which offers a promising strategy for the application of DIVA concept.

Proteomic analysis of the lungs of mice infected with different pathotypes of H5N1 avian influenza viruses

The virulence of influenza virus is determined by viral and host factors. Data on the genetic basis of the virulence of H5N1 influenza viruses have increased over the past decade; however, the contributions of host factors to the outcomes of H5N1 infection remain largely unknown. Here, we tested two chicken H5N1 viruses in mice and found that A/chicken/VN1214/2007 was nonlethal in mice and only replicated in the lung, whereas A/chicken/VN1180/2006 was highly lethal and replicated systemically in mice. To investigate the host response against these two different virus infections, we performed proteomic analysis by using 2D DIGE on the lung tissues of mice collected on days 1 and 3 postinoculation with different viruses or PBS as a control. Thirty-nine differentially expressed (DE) proteins related to "immune and stimulus response," "macromolecular biosynthesis and metabolism," and "cellular component and cytoskeleton" were identified in the virus-inoculated groups. Moreover, 13 DE proteins were identified between the two virus-inoculated groups, implying that these proteins may play important roles in the different outcomes of infection with these two viruses. Our data provide important information regarding the host response to mild and lethal H5N1 influenza virus infection.

Genome Replikin Count Predicts Increased Infectivity/Lethality of Viruses

The genomes of all groups of viruses whose sequences are listed on Pubmed, specimens since 1918, analyzed by a software from Bioradar UK Ltd., contain Replikins which range in concentration from a Replikin Count (number of Replikins per 100 amino acids) of less than 1 to 30 (see accompanying communications for higher Counts in tuberculosis, malaria, and cancer, associated with higher lethality). Counts of less than 4.0 were found in ‘resting’ virus states; Counts greater than 4.0, found to be associated with rapid replication, were found invariably to accompany or to predict virus outbreaks, by as much as two years, in viral hosts examined from salmon, to birds, to livestock, to humans. X-ray diffraction showed Replikins to be on the surface of the hemagglutinin gene of influenza and to spread as the Count increased from 3.2 to 10.1, prior to, then during, the 2009 H1N1 influenza pandemic. The degree of lethality of these outbreaks was found to be a function of the statistically significant increase in Replikin Count, particularly in the influenza polymerase gene p B1 or its equivalent in other viruses. Prediction up to two years in advance of the outbreak, and the geographic location of the outbreak, now done in 7/7 trials (see Bogoch, Nature Precedings), has permitted the solid phase synthesis of Replikin vaccines in 7 days, with time to permit manufacture, adequate testing for safety and efficacy, and distribution freeze-dried to all populations. These completely synthetic Replikins vaccines so far have been shown to be effective against Taura Syndrome virus in shrimp, and H5N1 in chickens. Thus for the first time this new technology provides the practical possibility to prevent pandemics rather than just to react to them.

Genome Replikin Count Predicts Increased Infectivity/Lethality of Viruses

AbstractThe genomes of all groups of viruses whose sequences are listed on Pubmed, specimens since 1918, analyzed by a software from Bioradar UK Ltd., contain Replikins which range in concentration from a Replikin Count (number of Replikins per 100 amino acids) of less than 1 to 30 (see accompanying communications for higher Counts in tuberculosis, malaria, and cancer, associated with higher lethality). Counts of less than 4.0 were found in ‘resting’ virus states; Counts greater than 4.0, found to be associated with rapid replication, were found invariably to accompany or to predict virus outbreaks, by as much as two years, in viral hosts examined from salmon, to birds, to livestock, to humans. X-ray diffraction showed Replikins to be on the surface of the hemagglutinin gene of influenza and to spread as the Count increased from 3.2 to 10.1, prior to, then during, the 2009 H1N1 influenza pandemic. The degree of lethality of these outbreaks was found to be a function of the statistically significant increase in Replikin Count, particularly in the influenza polymerase gene p B1 or its equivalent in other viruses. Prediction up to two years in advance of the outbreak, and the geographic location of the outbreak, now done in 7/7 trials (see Bogoch, Nature Precedings), has permitted the solid phase synthesis of Replikin vaccines in 7 days, with time to permit manufacture, adequate testing for safety and efficacy, and distribution freeze-dried to all populations. These completely synthetic Replikins vaccines so far have been shown to be effective against Taura Syndrome virus in shrimp, and H5N1 in chickens. Thus for the first time this new technology provides the practical possibility to prevent pandemics rather than just to react to them.

Bogoch Replikins Pandemic Prevention: Increase of Strain-Specific Influenza Genomic Replikin Counts, Having Predicted Outbreaks and their Location Seven Times Consecutively, Up to Two Years in Advance, Provides Time for Prevention of Pandemics

AbstractEarlier studies have shown that the increased concentration of a new class of virus genomic peptides, Replikins, precedes and predicts virus outbreaks. We now find that the area in the genome of the highest concentration of Replikins, and the country in which this peak exists in scout viruses, have permitted in the past five years seven consecutive accurate predictions of the geographic localization of coming outbreaks, including those now realized in Mexico for H1N1, and in Cambodia for H5N1. Real-time Replikin analysis of the evolution of the virus genome identified both mutations and structural reorganization of the hemagglutinin and p B1 genes over several years before each outbreak. This information, together with the specific Replikin sequences so obtained, permitted solid-phase synthesis of Replikin vaccines in seven days, which blocked H5N1 in chickens. The information also now provides up to two years of time to thoroughly test and distribute vaccines to high risk individuals in the countries identified; thus for the first time, a quantitative genomic Replikins method to both predict initial outbreaks and to prevent the development of a pandemic.

Bogoch Replikins Pandemic Prevention: Increase of Strain-Specific Influenza Genomic Replikin Counts, Having Predicted Outbreaks and their Location Seven Times Consecutively, Up to Two Years in Advance, Provides Time for Prevention of Pandemics

Earlier studies have shown that the increased concentration of a new class of virus genomic peptides, Replikins, precedes and predicts virus outbreaks. We now find that the area in the genome of the highest concentration of Replikins, and the country in which this peak exists in scout viruses, have permitted in the past five years seven consecutive accurate predictions of the geographic localization of coming outbreaks, including those now realized in Mexico for H1N1, and in Cambodia for H5N1. Real-time Replikin analysis of the evolution of the virus genome identified both mutations and structural reorganization of the hemagglutinin and p B1 genes over several years before each outbreak. This information, together with the specific Replikin sequences so obtained, permitted solid-phase synthesis of Replikin vaccines in seven days, which blocked H5N1 in chickens. The information also now provides up to two years of time to thoroughly test and distribute vaccines to high risk individuals in the countries identified; thus for the first time, a quantitative genomic Replikins method to both predict initial outbreaks and to prevent the development of a pandemic.

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (>104 median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy.

A Latex Agglutination Test for the Rapid Detection of Avian Influenza Virus Subtype H5N1 and its Clinical Application

A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as HIN1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test,and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.

Insights from modeling the 3D structure of H5N1 influenza virus neuraminidase and its binding interactions with ligands

The highly pathogenic H5N1 influenza virus, which is rapidly mutating and becoming increasingly drug-resistant, was investigated by means of structure–activity relationship between NA (neuraminidase) and three inhibitors, i.e., DANA (2,3-didehydro-2-deoxy- N-acetylneuraminic acid), zanamivir, and oseltamivir. A homology model of the H5N1-NA from the highly pathogenic chicken H5N1 A viruses isolated during the 2003–2004 influenza outbreaks in Japan was built based on the crystal structure of N9-NA complexed with DANA (PDB code: 1F8B). It was found that the traditional constituent residues around the active site of NA family are highly conserved in the H5N1-NA. However, a partially lipophilic pocket composed by Ala248 and Thr249 in N9-NA becomes a hydrophilic pocket because the two residues in the H5N1-NA are replaced by hydrophilic residues Ser227 and Asn228, respectively. On the other hand, two hydrophilic residues Asn347 and Asn348 in the N9-NA are replaced by two lipophilic residues Ala323 and Tyr324 in the H5N1-NA, respectively, leading to the formation of a new lipophilic pocket. This kind of subtle variation not only destroys the original lipophilic environment but also changes the complement interaction between the H5N1-NA and DANA. Such a finding might provide insights into the secret why some of H5N1 strains bear high resistance for existing NA inhibitors, and stimulate new strategies for designing new drugs against these viruses.

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.

Infection Hazards of Xenotransplantation

Zoonoses are a major area of concern, with the growing pressure to carry out clinical xenotransplantation, the grafting of animal cells, tissues and organs into humans.1–3 While the benefit of the xenograft to the individual patient might outweigh the risk of infection, it is the fear of setting off a new epidemic in the human population that distinguishes the infection hazards of xenotransplantation from the well-known infection hazards of allografts and of blood transfusion. Of course, animals have transmitted viruses and other infectious pathogens to humans ever since we learnt to hunt or husband them, yet we continue to meet nasty surprises. The AIDS pandemic is caused by viruses which have only recently jumped host species from chimpanzees (HIV-1) and sooty mangabey monkeys (HIV-2).4 New variant Creutzfeld–Jakob disease is linked to bovine spongiform encephalopathy. The current Malaysian epidemic of Nipah virus probably has a reservoir in fruitbats, but is spread by pigs.5 The lack of onward, humanto-human transmission of chicken H5N1 influenza virus was a fortunate, unpredictable feature of a zoonosis which killed 6 of 18 infected individuals. If zoonoses happen so readily, why should we be concerned over an added risk of this occurring through xenotransplantation? In fact, such zoonoses are fairly rare events, but xenotransplantation could increase the risk of occurrence for three reasons:-