PLoS One. 2015 Jun 2;10(6):e0128893. doi: 10.1371/journal.pone.0128893.

Host tissue and glycan binding specificities of avian viral attachment proteins using novel avian tissue microarrays.

Wickramasinghe IN1, de Vries RP2, Eggert AM1, Wandee N1, de Haan CA2, Gröne A1, Verheije MH1.
  • 1Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
  • 2Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.



The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens.

PMID: 26035584



Viral infections in birds can have enormous consequences. In poultry, viral diseases comprise the welfare of animals, and cause concomitant economic losses. In wild birds, the same viruses often cause asymptomatic infections, but these birds might form a reservoir, posing the risk of spreading the virus to other birds and mammals, including humans2. To develop novel intervention strategies, including vaccines, it is of importance to understand the tropism of avian viruses. However, the lack of infection model systems hampers the elucidation of critical virus-host interactions that determine the outcome of an infection.


HV Figure 1

Figure 1. Development of avian tissue microarrays (TMA). (A) Avian species present in TMA; (B) Collecting tissues from birds; (C) Grouping of tissues into paraffin blocks and tissues slides of TMA.


To fill this gap, we developed novel avian tissue microarrays (TMAs) from a collection of tissues of eleven different bird species (Fig. 1A and 1B). These tissues were combined in paraffin blocks in a multi-species (having one organ system of all birds, f.e. respiratory tract) or a single-species (with all tissues of one bird) array (Fig. 1C). Incubation of tissue slides with recombinant produced attachment proteins of avian viruses (Fig. 2A and 2B), including the hemagglutinin (HA) of avian influenza virus and the spike (S1) protein of avian coronavirus (CoVs), and subsequent detection of binding with an antibody against an in-frame tag can reveal binding profiles (see reference for details of the protocol3; example of the results shown in Fig. 2C).

Previously, we reported that the binding profile of the S1 protein of the prototype chicken CoV (infectious bronchitis virus, IBV) matches with those cells that are infected in chickens4. Furthermore, the binding affinity of S1 proteins from various IBV strains correlated with the reported pathogenicity of these viruses4. Finally, others and we have shown that glycan binding specificities can be revealed using recombinant viral attachment proteins4-6.

In the work featured in the publication addressed here7, we applied our viral protein binding strategy now using our recently developed avian tissue microarrays. In particular, we aimed at gaining knowledge on the host tropism of the prototype avian CoV IBV, which is thought to be chicken-specific. In addition, we aimed a defining the tissue tropism of other avian CoVs from which host and tissue tropisms are not known. Ultimately, we elucidated critical similarities and differences between interaction of various viral attachment proteins and host species.

The first important finding of our study was that the spike protein of the IBV CoV strain M41 attached to respiratory tissues of many birds other than chickens, including Canada goose, graylag goose, guineafowl, mallard duck, partridge, pheasant, quail, teal and turkey (examples shown in Fig. 3A). However, the S1 appeared to have a different preference for sialic acid subtype between species. While sialic acids type I lactosamine completely blocked the binding of S1 proteins to the trachea of Anseriformes (Canada goose, graylag goose, mallard duck, teal), partridge, and pigeon, it did not block binding to the trachea of guineafowl, pheasant, quail, and turkey. These data indicate that other birds do express a host attachment factor for the chicken CoV, but that these are likely different for at least some bird species. The actual susceptibility of other species for IBV remains, however, to be further investigated, as virus binding is a first and important, but not the only interaction with the host determining the outcome of the infection.


HV Figure 2

Figure 2. Viral attachment binding assay. (A) Expression construct to produce recombinant viral attachment proteins; (B) Schematic representation of the binding assay; (C) Example of viral protein binding to chicken trachea.


Secondly, we elucidated the tissue tropism of various avian CoVs using our single-species TMAs. The attachment proteins of the chicken, pigeon and partridge CoVs all showed primarily tropism to the respiratory system of their respective host. While chicken and partridge CoV S1 bound the trachea and lung of the chicken and partridge, respectively, the binding of pigeon CoV S1 was limited to pigeon lung (Fig. 3B). This might be related to the differential expression of receptors in the upper respiratory tract of these species1. While all S1 proteins required sialic acid for tissue attachment, binding of pigeon CoV S1 and partridge CoV S1 could not be blocked by sialic acids type I lactosamine, in contrast to that of chicken CoV S1. This indicates that these viruses have different sialic acid prerequisites that might determine their host specificity.

Binding of chicken, pigeon and partridge CoV S1 proteins correlated with the observation that these viruses are respiratory pathogens. In this respect, it is worth to mention that binding of spikes of avian CoVs causing enteric disease is primarily to the intestine of the respective birds8. For IBV M41, we observed in addition to the trachea binding to various other chicken tissues, including lung, kidney and intestine (Fig. 3C). It is known that M41 can replicate in these tissues, albeit without causing obvious disease. Taken together, binding profiles of S1 can provide novel insights in the preference of these viruses for organ systems.

Finally, our avian TMAs were applied to study the binding characteristics of other avian pathogens, like avian influenza virus H5N1. The H5 hemagglutinin of IAV H5N1 had high affinity for many different respiratory tract tissues in our multi-species TMA. As H5N1 is known to infect all hosts present in our multi- species TMA2, the tissue binding affinity of HA reflects the susceptibility of these avian species to the virus.

In conclusion, profiling tissue attachment characteristics using our novel developed avian TMA can expand our knowledge on the host and organ tropism of avian viruses. Such TMAs are new in the field of infection biology, and can increase insights in virus-host interactions that are critical in determining the outcome of the infection. This knowledge will also be of importance to estimate the risks of transmission of these viruses between birds. It needs to be stressed, however, that more data is required to ultimately conclude on the susceptibility of bird species for the various coronaviruses. Despite this, the novel avian TMA and the tissue-based method to profile attachment of viruses is an excellent method to assess the first step in the virus infection cycle for viruses for which limited model systems are available.


HV Figure 3

Figure 3. Profiling the binding of coronavirus spike proteins using TMAs. (A) Multi-species TMA showing binding of IBV M41 S1 to trachea of chicken, partridge, and turkey; (B) Single-species TMA showing partridge and pigeon CoV S1 binding to trachea and lung of partridge and pigeon, resp.; (C) Single-species TMA showing IBV M41 S1 binding to chicken lung, kidney and intestine.



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