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Viral respiratory diseases (ILT, aMPV infections, IB): are they ever under control?11

Abstract 1. The use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases infectious laryngotracheitis (ILT), avian metapneumovirus (aMPV) infections and infectious bronchitis (IB). This paper appraises the current methods of vaccine control in the light of the nature of each virus and epidemiological factors associated with each disease.2. Infectious laryngotracheitis virus (ILTV) exists as a single type with a wide range of disease severity. It is a serious disease in certain regions of the world. Recent work has distinguished molecular differences between vaccine and field strains and vaccine virus can be a cause of disease. Vaccines have remained unaltered for many years but new ones are being developed to counter vaccine side effects and reversion and reactivation of latent virus.3. Avian metapneumoviruses, the cause of turkey rhinotracheitis and respiratory disease in chickens exists as 4 subtypes, A, B, C and D. A and B are widespread and vaccines work well provided that accurate doses are given. Newer vaccine developments are designed to eliminate reversion and possibly counter the appearance of newer field strains which may break through established vaccine coverage.4. IB presents the biggest problem of the three. Being an unstable RNA virus, part of the viral genome that codes for the S1 spike gene can undergo mutation and recombination so that important antigenic variants can appear irregularly which may evade existing vaccine protection. While conventional vaccines work well against homologous types, new strategies are needed to counter this instability. Molecular approaches involving tailoring viruses to suit field challenges are in progress. However, the simple use of two genetically different vaccines to protect against a wide range of heterologous types is now a widespread practice that is very effective.5. None of the three diseases described can claim to be satisfactorily controlled and it remains to be seen whether the newer generations of vaccines will be more efficacious and cost effective. The importance of constant surveillance is emphasised and the testing of novel vaccines cannot be achieved without the use of vaccine-challenge experiments in poultry.


INTRODUCTION
Viral respiratory diseases of poultry constitute an important cause of economic loss to the poultry industry worldwide in terms of impaired growth, reduced egg production and quality, mortality, slaughter downgrading and several ancillary factors including diagnoses, vaccines, and antimicrobials to treat inter-current bacterial infections etc. Those diseases with the potential to cause the most severe losses in terms of mortality are very virulent Newcastle disease and highly pathogenic avian influenza. Three other viral respiratory diseases constitute what might be called the ''second tier'' of importance in terms of disease severity, namely infectious laryngotracheitis (ILT), avian metapneumovirus (aMPV) infections (the cause of infections turkey rhinotracheitis, avian rhinotracheitis) and infectious bronchitis (IB). Of these, IB is the most economically important and in the UK alone, it has been reported to be responsible for losses amounting to some £23 million annually. For all three, disease severity can be exacerbated and prolonged by concurrent infections with bacteria including E. coli and Mycoplasma gallisepticum or if the birds are immunosuppressed by infectious bursal disease or chicken anaemia virus infection. However, this paper will concentrate on the uncomplicated infections alone.
For all three, live vaccines are available and widely used but control is variable. This paper attempts to assess how successful control by vaccination is, taking into account the nature of the viruses and the different epidemiological features which are influential in this respect.

The viruses
The three viruses are unrelated, belonging to different groups in the virus classification. Infectious laryngotracheitis virus (ILTV) is an alpha-herpes virus assigned to the Iltovirus genus ( Figure 1). ILT was one of the earliest diseases described for poultry (1925) and affects chickens and pheasants but rarely turkeys. Avian metapneumovirus (aMPV, Figure 2) belongs to the Paramyxoviridae family and is the cause of turkey rhinotracheitis, originally reported in South Africa in the late 1970s, but it affects chickens also and is the ''youngest'' of the three. IB, caused by a type 3 coronavirus (Figure 3), was first reported in 1931 but still flourishes. Despite years of attempting to control the diseases caused by these viruses by the use of vaccines, they are still present.

INFECTIOUS LARYNGOTRACHEITIS
ILT occurs in chickens with a wide spectrum of disease severity, ranging from subclinical to  peracute with significant mortalities (Guy & Garcia, 2008). Unlike IB and aMPV infections, ILT appears to solely affect the respiratory tract and does not spread rapidly. Disease, sometimes very severe, occurs in certain regions of the world where it usually remains endemic, while in other countries it is unknown or of little importance.

Epidemiological features
ILTV exists as a single serotype, an advantage from the point of view of control by vaccines and this is also helpful in serological diagnosis. Different strains (e.g. wild and vaccine viruses) can be differentiated by molecular methods.
One aspect of ILT epidemiology relating to its being a herpes virus is that it becomes latent within the chicken (Guy & Garcia, 2008). Essentially this means that when affected birds have recovered from the acute phase of infection, the virus assumes a non-infectious state and resides in the trigeminal ganglia (Williams et al., 1993), though the flock appears to be in normal health. Live ILT vaccines can also become latent (Hughes et al., 1991). The consequences are that latent virus can become reactivated by factors such as stress or onset of lay (Hughes et al., 1989), so that such a flock can become an unrecognised source of infection (Williams et al., 1992). There are no known wild bird reservoirs of this virus and there is no evidence that it is egg transmitted (Bagust & Johnson, 1995).
Control is by the use of live vaccines in endemic areas and flock owners are ''afraid to stop'' vaccinating. Initial studies hinted that promoted by the nature of latency, ILT vaccines themselves were responsible for some outbreaks of disease in naive flocks. With improved molecular techniques, it has now been confirmed that this does happen, but other field strains of virus are unrelated to vaccine strains (Ojkic et al., 2006;Oldoni et al., 2008;Neff et al., 2008).

Recent findings
Recent molecular investigations have shown differences between strains of ILT viruses in different regions. It is worth pointing out that the molecular groupings have been given different labels according to the genetic comparisons (Table). In Europe, a study of strains collected over 35 years showed that 94% (98/104) of strains were related to vaccines (Neff et al., 2008). A similar study in the USA (Oldoni et al., 2009) found that of 9 ''genotypes'' reported, 63% were related to vaccine strains. In Australia, 5 ''genetic groups'' have been established but in contrast, most ILT was considered not to be due to vaccine types (Kirkpatrick et al., 2006). New strains have been reported to appear recently in Australia and ILT is considered an ''emerging disease'', but no one knows from where they have originated.

Control of ILT
Empirical attenuated ILT vaccines either of chick embryo origin (CEO) or tissue culture origin (TCO) have been used for many years with success (Guy & Garcia, 2008). Some time ago we discovered that the commercial vaccine used in the UK behaved in the same way as a field virus, that is, it had the same latency characteristics as wild type ILTV in the bird and persisted for life (Hughes et al., 1991). Thus, it was perceived that a significant proportion of disease could be due to vaccine virus becoming ''wild'' and recently referred to in the USA as ''vaccinal laryngotracheitis''. Molecular analysis, as shown above, has confirmed that this is the case. However, Korean workers found that one of four conventional ILT vaccines protects against virus latency (Han & Kim, 2003). Despite the presence of ILTV strains genetically different from vaccine strains,  Problems associated with conventional ILT vaccines have included vaccinal reactions, especially where other respiratory agents are present, reversion of vaccine viruses to virulence and latency with later reactivation. In addition, because ILT vaccines are not usually given until 10 weeks of age or later, birds are vulnerable up to this point. As a result, several new approaches have been explored to produce a new generation of safer vaccines. These include the production of a glycoprotein G-deficient vaccine (Devlin et al., 2008), G being associated with latency and a fowl pox vector carrying ILTV genes (Davison et al., 2006) or glycoprotein G together with the haemagglutinin of Newcastle Disease virus (Sun et al., 2008). It remains to be seen whether vaccines of this type will prove to be more efficacious than the empirically derived vaccines.
A new vaccine recently trialled in USA and South America consists of the Marek's disease turkey herpesvirus (THV) vaccine used as a vector containing ILTV genes gD and gI (I. Tarpey, personal communication). The vaccine is given by injection at one-day-old and since the THV vaccine persists in the chicken, it is claimed to give lifelong immunity against ILT. Because it contains only defined ILT genes, there are no problems with respiratory reactions or latency.
Finally, it is worth noting that Bagust and Johnson (1995) suggested that ILTV could be a suitable candidate for eradication, since it has a number of properties appropriate for this approach to control. These include its existence as a single serotype, lack of vertical transmission and no apparent wildlife reservoir. They proposed the use of a genetically defined marker vaccine such as mentioned above, together with a matched ELISA. The latter would distinguish vaccinal response from field infection and permit differentiation of birds which are naturally infected from those given vaccine.

Conclusions on ILT control
ILTV viruses have stayed remarkably stable over the years compared to say IBV, but they can now be distinguished by molecular methods. ILT continues to cause disease problems, especially in the Americas and Australia. Over the years, vaccines, derived from egg or tissue culture passage, have remained mostly unaltered but there have been problems with vaccine reaction, reversion of vaccine virus to virulence and latency.
New genotypes different from vaccines have emerged, particularly in Australia. In the USA, conventional vaccines have been shown to be effective against genetic variants (Rodríguez-Avila et al., 2004) but will future variants emerge which evade vaccinal protection? Another question is, since no known wildlife reservoirs are recognised, from where do the variants originate? To date, ILT may be considered to have been restrained rather than well controlled. Perhaps the new vaccines such as the turkey herpes vector vaccine will allow much better control.

AVIAN METAPNEUMOVIRUS INFECTIONS
Avian metapneumovirus is the cause of turkey rhinotracheitis (TRT), first seen in South Africa in the 1970s (Buys et al., 1989). It was soon shown to cause respiratory disease in chickens too (avian rhinotracheitis, ART) and sometimes swollen head syndrome (SHS) (Gough & Jones, 2008). aMPV soon spread to Europe and the Middle East and most parts of the world where commercial poultry are kept, apart from Australia and North America. Initially, two subtypes of virus were recognised, A and B, based on differences in the surface glycoprotein gene sequences (Juhasz & Easton, 1994). In 1996, a third subtype, C was identified in the USA in turkeys in Colorado and later in the important turkey producing state of Minnesota (Seal, 1998). In the USA, the disease is called ''avian pneumovirus infection of turkeys'' and subtype C does not appear to be important in chickens. Subsequently, a subtype D was described in France (Bäyon-Auboyer et al., 2000) and a variant C appeared in ducks in the same country (Toquin et al., 2008). Surprisingly, a subtype C has been reported in South Korea in pheasants in a market (Lee et al., 2007). aMPV primarily affects the respiratory tract, especially the upper tissues (Figure 4), and is able to replicate in the oviduct, the latter resulting in loss of egg production and quality (Gough & Jones, 2008).

Epidemiological features
Since the first reports of aMPV in South Africa and its subsequent appearance in most parts of the world, the mode of spread of these viruses has been a source of conjecture. How the virus reached Europe, the Middle East and further afield from South Africa is unknown. One suggestion has been transmission by long-distance migratory birds (Jones, 1996), although efforts to find evidence of infection in wild European species have not been fruitful. Perhaps even more puzzling is the fact that while subtype C has been present in turkeys in the USA, principally Minnesota, with its many lakes, while A and B are present in Central and South America, there is no apparent mixing of subtypes in the two regions, despite the migratory flight paths of wild waterfowl between the north and south. Evidence of aMPV infection in wild birds has been reported in the USA (Shin et al., 2002;Turpin et al., 2008), but this in itself does not mean that infected species are able to transmit infection to susceptible poultry.
The isolation of aMPV from very young turkeys (Gough et al., 1988) and the presence of abundant amounts of virus in the oviducts of infected laying turkeys (Jones et al., 1988) suggest that egg transmission may occur from time to time.

Control of aMPV infections
The main approach to control of aMPV infections is by the use of live attenuated and killed vaccines, the former derived by attenuation in cell cultures or fertile eggs and tracheal organ cultures (Cook & Ellis, 1990;Williams et al., 1991). In chickens, live vaccines are incorporated into the vaccination programme at a different time from IB vaccines, since interference occurs (Cook et al., 2001), although aMPV vaccination simultaneously with Newcastle disease vaccines does not adversely affect the efficacy of either (Ganapathy et al., 2005). Killed vaccines are given by injection before the onset of lay but only after priming with live vaccines. Except for the USA, where subtype C is present, A and B are the prevalent subtypes elsewhere and vaccines against each of these two give good protection against the other (Cook et al., 1995). A and B type vaccines also give some protection against subtype C but not vice versa (Cook et al., 1999).
A recent survey of aMPVs in chickens and turkey flocks in Western Europe showed that subtype B predominates in the 6 countries studied, perhaps reflecting the wider use of subtype B vaccines (R.C. Jones and K.J. Worthington unpublished). This survey did not distinguish between vaccine and field viruses. Subtype A and B vaccines have been used successfully in most countries and in both species, where the disease is prevalent. In spite of this, several reports suggest that there is room for improved vaccines or at least closer attention to accurate vaccine delivery. For example in turkeys, some weeks after aMPV vaccination, respiratory disease sometimes occurs which has been diagnosed as TRT. This phenomenon been confirmed to be due to vaccine virus becoming virulent after recycling among birds which did not receive a dose of vaccine originally (Catelli et al., 2006).
A recent Israeli longitudinal study of aMPV infection in a small number of chicken and turkey flocks revealed that both vaccine and field strains of aMPV A and B subtypes could be recovered simultaneously (Banet-Noach et al., 2009). Recently in Brazil  and Italy (Cecchinato et al., 2009), field evidence has suggested that the existing vaccines are not fully effective against new strains of virus.
Though the empirical vaccines have been effective where given accurately, moves have been made to formulate novel vaccines which do not revert to virulence. These include vector vaccines, subunit vaccines and DNA vaccination -all only partially effective. One approach has involved the use of reverse genetics to make an infectious clone and looks promising (Naylor et al., 2004;Ling et al., 2008; C.J. Naylor and P. Brown, personal communication). In ovo vaccination has been shown to be a practical and effective form of delivery for protecting turkeys and chickens against aMPV infection (Worthington et al., 2003;Tarpey & Huggins, 2007).
Finally, in regions where flock density is low, it is apparent that aMPV infections can be eradicated with careful monitoring and strict attention to biosecurity. Examples are Sweden and Colorado, where subtype C was first reported in the USA. However, in the state of Minnesota, where C gained a strong foothold in the immense turkey industry, the disease has all but been eradicated (K. Nagaraja, personal communication). Serological monitoring has shown no evidence of aMPV for some time and the turkey industry has stopped vaccinating.

VIRAL RESPIRATORY DISEASES
No clinical cases and no isolations have been made for more than 4 years.

Conclusions on aMPV disease control
A small number of subtypes exist, A, B, C and D. D is rare, C appears to be important only in the USA, perhaps less and less so. A and B are the most widespread and important ones. Current vaccines are successful if administered accurately to each bird but problems can arise with reversion and there is evidence of possible virus evolution. New safer vaccines are being developed. However, the work of Banet-Noach et al. (2009) in Israel indicates that wild type virus can still be present in vaccinated flocks, illustrating that control is less than satisfactory with present vaccines.

INFECTIOUS BRONCHITIS
Infectious bronchitis is the most important of the viral respiratory diseases to be discussed here. It is considered to be the most important endemic viral disease of poultry in countries where highly pathogenic avian influenza or virulent Newcastle disease are not present (Cavanagh & Gelb, 2008). It is a disease of chickens and not turkeys. In addition to being a respiratory disease, affecting all ages and affecting growth, it is one of the most important causes of loss of egg production and quality (Cavanagh & Gelb, 2008). Some strains have a predilection for the kidneys and cause significant mortalities in young birds (Dhinakar Raj & Jones, 1997a). There is some evidence that IBV may sometimes be associated with enteritis and infertility in males (Villarreal et al., 2007a, b). Control of IB is by the use of live attenuated and killed vaccines. However, IB presents unique challenges regarding its control. Genomic diversity and the ability of the virus to rapidly change have created different genotypes of the virus that evade protection by conventional vaccines (Cavanagh & Gelb, 2008). Approaches to counteract this problem are discussed below.

The virus
The cause of IB is a type 3 coronavirus. The virus, infectious bronchitis virus (IBV) is an enveloped RNA virus with a surface fringe of club-shaped spikes. The spike is in two parts, S1 and S2 and the gene coding for the S1 spike has a hypervariable region which is liable to mutate or undergo reassortment with other IBVs, thus generating novel variant IBVs. The S1 spike is important in viral attachment, is a major component in development of immunity and has traditionally been important for cross-neutralisation, the original basis for IBV classification. The different types of IBV generally give little or no protection against heterologous types, although there are some exceptions. Important variants appear from time to time in a very unpredictable manner and are a constant challenge to vaccine strategies. Hence there is a constant need for surveillance of prevalent types.
In a recent survey of IBV in some 5000 commercial chicken flocks in UK, France, Spain, Holland, Germany and Belgium between 2002(Worthington et al., 2008, 59% were positive for IBV. The most prevalent genotypes were Massachusetts and the major variant 793B (4/91 or CR88) ( Figure 5). On the basis of nucleotide sequence shared identity, approximately 50% of each were considered to be vaccine virus (H120, MA5, 4/91, IB88, etc). The next most common genotypes were two new important field types: Italy 02 and a virus originally detected in China called QX (Worthington et al., 2008). The origins of Italy 02 are unknown (Dolz et al., 2006) but QX originated in China in 1996 (Wang et al., 1998), though how it arrived in Europe is unknown. Italy 02 has behaved primarily as a respiratory pathogen, but QX has had more serious consequences, causing nephritis in young flocks and false layers in egg laying flocks. This survey is an example of the diversity of IBV genotypes in a region and the appearance of new ones. Considerable diversity has been well documented for USA, Australia and China and in recent years, reports of novel genotypes have been published from Brazil , Argentina (Rimondi et al., 2009), Thailand (Pohuang et al., 2009), Malaysia (Zulperi et al., 2009) and Africa (Ducatez et al., 2009).

Epidemiological features
IB viruses affect chickens but not turkeys like aMPV although turkeys and pheasants have their own different coronaviruses. IBV is not generally considered to be egg transmitted and while there is abundant virus in the oviduct of the hen shortly after infection (Jones & Jordan, 1972), very few chicks would be likely to hatch with virus.
Undoubtedly the biggest problem of control relates to the diversity of types and the appearance of new ones from time to time. Although in Europe, the number of important variants to appear in the last 50 years has been relatively small -Dutch types in the 1970s, 793B in the 1990s and two in this decade, Italy 02 and QXtheir emergence is unpredictable. Perhaps increasing poultry populations and greater use of vaccines will promote the more rapid generation of new types.
Some well-established genotypes are found worldwide (e.g. Massachusetts) and in part due to the widespread use of vaccines. However, we are largely ignorant as to how IBVs move around the world. For example, QX originated in China but is now established in Europe. It travelled across Asia in a similar time span to that taken by avian influenza H5N1 but, as far as we know, without the mediation of wild birds. There is increasing evidence that wild birds can be infected with coronaviruses (Jonassen et al., 2005;Woo et al., 2008) but with few exceptions (Liu et al., 2005;Sun et al., 2007;Hughes et al., 2009) they seem to be unrelated to IBV. As yet, there is no hard evidence that IBV is transmitted over long distances by wild species. Long-term persistence of IBV has been demonstrated after early infection but its significance is unknown (Jones & Ambali, 1987;Dhinakar Raj & Jones, 1997b).

Control of IB -how to deal with variants?
In regions where licensed vaccines are from the same genotype as the field challenge, then IB vaccines work well. However, where variants appear and persist and against which existing vaccines do not protect, then this presents a big problem. A number of possibilities exist for addressing the problem of a new important variant for which a new vaccine is perceived to be necessary.
(1) Test the existing repertoire of vaccines.
Sometimes protection can be offered by an unrelated vaccine. For example, D274 vaccine protects against 793B variant ( Figure 6). (2) Develop an empirical vaccine by attenuation in eggs or perhaps cell culture. This is the traditional ''fire brigade'' method but is a long tedious process. (3) Use molecular techniques to engineer a vaccine for the new challenge virus that will not revert to virulence. Recent examples of this are modified DNA vaccination (Tian et al., 2008;Tan et al., 2009) and a multi-epitope-based peptide vaccine . One of the most promising of the new generation of vaccine appears to be the ''spike swapping'' technology, whereby using reverse genetics, an infectious clone is produced into which specific S1 spike genes can be incorporated, appropriate to the new variant (Casais et al., 2001). Variations on this theme have also included incorporation of nucleocapsid genes or specific cytokines to induce a broader immunity (Cavanagh et al., 2007). (4) Use two heterologous IBV vaccines (Cook et al., 1999). This method has been shown to be very successful, even though the mechanisms have not been elucidated to date. It is usual to first administer a Massachusetts-type vaccine followed by a variant (793B-type in Europe) offers wide protection against types which are different again (Figure 7). Such a programme  Jones, 1996). . The use of two heterologous vaccines (Massachusetts H120) and 4/91 gives good protection against a strain of IBV unrelated to either (here A1121). Protection against this virus by either vaccine alone is poor. Protection was assessed as in Figure 6 ( from Cook et al., 1999).

VIRAL RESPIRATORY DISEASES
has been shown to be efficacious against for example Italy 02 and QX (Jones et al., 2005, Figure 8); (Terregino et al., 2008;De Wit & van de Sande, 2009). (5) In ovo vaccination. This is under development (Tarpey et al., 2006) and is dependent on the strain of IBV not killing embryos.

Conclusions on IB control
IB is highly infectious and maintaining virus-free flocks seems impracticable. The main choice for future strategies in IBV control seems to be as follows: (i) production of a specific tailored vaccine, produced by molecular technology and designed to protect against the variant challenge virus. This has the advantage that once the virus ''carrier'' is established, mechanisms for the insertion of the appropriate S1 spike gene (perhaps with accessory genes for cytokines etc., are now available. However, the question arises as to how many tailored vaccines the bird can take, if the numbers of variants in the challenge become large. (ii) The use of two different vaccines. This is simple to employ but is not guaranteed to succeed in every case and the mechanisms have not been fully established. However, unless a ''pan-IBV'' vaccine was to be produced (which is unlikely), this is a significant and very successful ''stop-gap'' approach. IBV shows viral evolution in action and is likely to remain one step ahead.

CONCLUDING REMARKS
Three viral respiratory diseases have been considered and they present different problems of control by vaccines. ILTV affects the chicken not the turkey, exists as a single serotype, but strains show differences in virulence. Genetic differences can be demonstrated but it is not clear how important they are in vaccine formulation. Vaccines have remained relatively unchanged but improved ones are needed for avoidance of vaccine reactions and problems of long immunity. In some countries, new strains are emerging but their origin is unknown. Four subtypes of aMPV exist. A and B are virtually worldwide, affecting chickens and turkeys. While existing vaccines are effective when given accurately, field evidence of reversion and perhaps virus evolution highlights the need for improved safer vaccines.
IB presents the biggest challenge, with erratic and unpredictable appearance by variants which may evade protection of existing vaccines. Likely solutions to vaccine control may be tackled by specialised type-specific engineered vaccines or by the use of double heterologous vaccination. But what is the answer in the longterm? IBV variants will continue to arise: perhaps this disease will never be completely controlled.
Because of the difficulties outlined above, it cannot be claimed that any of the three diseases is satisfactorily controlled by vaccination. The ideal scenario where producing a vaccine against a new disease means the end of the problem does not apply in reality, even though experimental trials sometimes produce perfect results, with sterile vaccination as the outcome. The field situation is quite different.
For all three viruses, despite the widespread use of live vaccines, there is evidence, mediated through refined molecular techniques, of wild virus in circulation. Poor vaccine delivery, overwhelming field challenge, intercurrent infections, viral evolution, new variants from outside the immediate regions all conspire to militate against vaccinal efficacy. Existing vaccines may be described as restraining rather than fully controlling these diseases. It remains to be seen whether the new generation of vaccines will be more satisfactory than the existing empirical ones and cost effective. I think we can expect a move to in ovo delivery because of its convenience, even though it has not yet been taken up widely in Europe.
Some final thoughts. Essential to good control is constant surveillance using the optimal techniques available for detecting viruses currently infecting poultry populations, with the added benefit of alerting diagnosticians to possible new variants of current viruses or indeed new disease agents. It must also be remembered that vaccine efficacy can only be determined by the use of vaccine-challenge experiments in birds.