Pathogenesis and Improved Diagnosis and Control of Avian Influenza Infections
In recent years there has been a global increase in the number of outbreaks of highly pathogenic avian influenza (HPAI). Two devastating outbreaks have occurred in Europe - in Italy and the Netherlands - both resulting in major economic losses. In Asia an ongoing epizootic is causing major concern due to the number of human infections associated with a particular strain of H5N1 AI virus, sparking fears and considerable planning for a potential influenza pandemic. Millions of birds have been culled to control these outbreaks.
Much attention has focused on determining the best control strategies for dealing with outbreaks and new introductions of HPAI viruses in poultry. Successful control strategies depend upon a number of factors including the use of rapid and sensitive laboratory tests appropriately applied; knowledge about the infecting virus, e.g. the host species affected; rates and mechanisms of transmission between animals of the same or different species; and determinants for pathogenicity and the use of vaccination.
We have addressed some of these issues in the AVIFLU project: Rapid and sensitive laboratory tests for avian influenza (AI) for use in surveillance and controls programmes were developed and validated. A transmission model was used to study the efficacy of vaccination in poultry and ducks, AI marker vaccines were developed and evaluated, and reverse genetics was used to study the effect of molecular pathogenicity markers in vitro and in vivo.[+] Read More
Control of an infectious disease that can spread as rapidly as HPAI requires a rapid and harmonised response. It is critical that veterinarians and animal keepers recognise the clinical signs of the the disease and report suspected cases quickly. Suspected outbreaks must be backed up by rapid, sensitive and highly specific laboratory tests before control measures can be implemented.
Early warning systems such as surveillance in wild bird reservoirs and in poultry flocks also play an important part in the control of AI. This requires the testing of high volumes of samples in a timely manner and may require a different testing strategy to that of index cases. It is also crucial that we understand the mechanisms and markers for the emergence of HPAI and the impact vaccination can have on the spread of disease.
The AVIFLU project has now been completed and some elements of the work are now being explored further in the follow-on project, FLUAID. Highlights of our findings are summarised below:
Two ring trials were conducted that involved six EU national AI laboratories and a number of other invited laboratories, with the aim of establishing an agreed approach to AI diagnostic RT/PCR in the EU. The trials were designed to evaluate RT/PCR and real time RT/PCR protocols for the detection of H5 and H7 AI viruses that had been developed by each participating laboratory. The best performing protocols for sensitivity and specificity were then validated and circulated to all EU AI national laboratories. They are now included in the EU diagnostic manual which supports the new AI directive.
A generic AI antigen capture ELISA had been developed using as a key reagent AI Nuclear Protein (NP) specific monoclonal antibodies (Mabs) developed within the AVIFLU project. These NP Mabs have been evaluated for their potential as reagents in a penside antigen detection test, in the form of a Lateral Flow Device. Prototype devices were produced, tested and compared with other devices available commercially. The prototypes performed well in comparison with other devices for cloacal and oropharyngeal swabs collected from chickens and Pekin ducks.
An infection model was developed enabling the quantification of different intervention strategies on the transmission of avian influenza viruses. A stochastic susceptible, latently infected, infectious, recovered (SEIR) epidemic model was developed and used to analyse data generated with the infection model. The transmission experiments offer a way to measure the spread of virus under experimental conditions and also to quantify the effect of vaccination on the transmission characteristics of the viruses. The model was used to investigate the transmission characteristics of LPAI and HPAI viruses of the H7 and H5 subtypes in vaccinated and non vaccinated chickens, Pekin ducks, teal and golden pheaSants.
The major findings are:
A reverse genetics system was established based on viruses from the 1999-2000 Italian H7N1 LPAI and HPAI outbreaks. This was used to investigate the biological relevance of various mutations observed between these viruses. The findings were that additional glycosylation of the haemagglutinin near the receptor binding site promotes the spread of infection by HPAI virus, apparently due to increased virus release from cells and not due to changes in receptor specificity. Also, a shortened neuraminidase stalk is a prerequisite for adaptation to chickens. Viruses with a reconstituted full NA stalk had a loss of infectivity and grew to lower titres.
Mutations found in the NS1 proteins of high and low pathogenic viruses seem to be a critical determinant for host adaptation.
All the diagnostic tests and reagents (including monoclonal antibodies) developed in this project are now recommended for use in the national avian influenza reference laboratories of the EU. The protocols are available in the EU diagnostic manual. This effort has harmonised the approach taken by EU national AI laboratories and enabled the best molecular tests currently available to be used throughout the EU. The experience gained in running the ring-trials is now used by the AI Community Reference Laboratory to conduct the EU national AI laboratory annual proficiency panel testing for AI molecular tests. The strategy adopted for these trials will also ensure that improved tests will be identified, thus keeping our testing regimes up to date with advances in molecular diagnostics.
Monoclonal antibodies produced in this project have been evaluated for their potential as reagents in a penside antigen detection test, in the form of a Lateral Flow Device. The test is now being developed further and validated within the current FLUAID project. It is likely that this test will be marketed commercially in the future.
Differences in the transmission of highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) strains may play a role in the mechanism of selection by which HPAI viruses arise in the field. The mathematical infection model will contribute to an understanding of these processes. It is a tool that enables the effect of different intervention strategies to be measured, e.g. vaccination.
Our results indicate that vaccination of Pekin ducks with an inactivated, conventional product is successful in preventing clinical signs and mortality and in suppressing shedding of viable virus. This suggests that the perpetuation of the infectious cycle may be interrupted by optimum vaccination.
These findings have significant implications on practical aspects of AI control. It appears that following field challenge a vaccinated flock will not shed enough virus to infect either vaccinated or unvaccinated flock-mates. As the amount of virus shed in the environment is not sufficient to infect unvaccinated ducks, it could be speculated that such a practice would presumably reduce the risk of infection spilling over into the wild bird population, and possibly to mammalian hosts.
The vaccination protocol under study was also developed in order to evaluate a system that could be compatible with husbandry practices. Vaccination at one and 30 days old appears to be compatible with Pekin duck husbandry methods in Asia. The results indicate that such a programme could be used in the field to prevent primary introduction and secondary spread in nave Pekin ducks. A duration of immunity above threshold levels for more than five months was recorded, which appears to be longer than the economic life of a meat duck, approximately four months.
Vaccination may also prevent the establishment of viraemia and therefore prevent viral colonisation of internal organs, positively influencing the food security of duck products.
The reverse genetics system has already given some surprising insights into the determinants for host range and pathogenicity. Some of the markers identified may be relevant for the development of new diagnostic tests and for the surveillance of avian influenza.