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Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene
Abstract
The H5 hemagglutinin (HA) gene of a highly pathogenic avian influenza virus (AIV) isolate (A/chicken/Italy/8/98) was cloned and sequenced, and inserted at the non-essential UL50 (dUTPase) gene locus of a virulent strain of infectious laryngotracheitis virus (ILTV). Northern and Western blot analyses of the obtained ILTV recombinants demonstrated stable expression of the HA gene under control of the human cytomegalovirus immediate-early gene promoter. In vitro replication of the HA-expressing ILTV mutants was not affected, and infection of chickens revealed a reduced but still considerable virulence, similar to that of a UL50 gene deletion mutant without foreign gene insertion. The immunized animals produced specific antibodies against ILTV and AIV HA, and were protected against challenge infections with either virulent ILTV, or two different highly pathogenic AIV strains (A/chicken/Italy/8/98, A/chicken/Scotland/59). After challenge, no ILTV could be reisolated from protected animals, and shedding of AIV was considerably reduced. Thus, although attenuation remains to be improved, genetically engineered ILTV live-virus vaccines might be used as vectors to protect chickens also against other pathogens.
Supplementary resource (1)
Nucleotide Sequence
February 2001
... Most of these genetic manipulations led to a significant and irreversible reduction of virulence in chickens, and, therefore, provided vaccines which are safer than classically attenuated and genetically uncharacterized ILTV strains [reviewed in 61]. In the genomes of ILTV recombinants lacking the non-essential dUTPase (UL50) or UL0 genes, the coding sequences of HA subtypes H5 and H7 have been expressed under control of the HCMV-IE promoter [79][80][81]. A single ocular immunization of chickens with 10 3 -10 5 plaque-forming units of the HA-expressing recombinants protected the animals against challenge with pathogenic ILTV, or lethal doses of HPAIV of the respective subtypes. ...
... Antibody titers and degree of protection were dependent on vaccine doses, and only the highest doses prevented any clinical signs of subsequent HPAIV infections [79]. Homology between the HA genes of vaccine and challenge virus is also relevant, since equal doses of the H5-expressing ILTV recombinant completely protected chickens against the homologous Italian H5N2 HPAIV, whereas transient disease was observed in animals challenged with an older Scottish H5N1 virus possessing 94% of identical HA amino acids [80]. Remarkably, only 50% of immunized chickens survived challenge infection with an Asian H5N1 HPAIV, although sequence homology between the HA proteins was also 94% [Veits et al., unpubl. ...
... This finding indicated that different virulence of challenge viruses might also influence the level of protection, and emphasizes the necessity to construct vector vaccines expressing the HA gene of currently relevant AIV isolates. Nevertheless, HA-expressing ILTV recombinants are promising candidates for emergency vaccines against fowl plague, since the protective effect of the described H5-expressing mutant against homologous H5N2 HPAIV was equivalent to that of inactivated AIV vaccines or HA-expressing NDV mutants [53,80]. This also applied to the substantial reduction of challenge virus shedding which demonstrated the low risk of influenza virus transmission by healthy vaccinated individuals. ...
Germany was among the first countries to be hit by highly pathogenic avian influenza (HPAI) or fowl plague virus soon after it first surfaced in Italy at the end of the 19th century. Subsequently, only very few sporadic outbreaks occurred despite the continuous presence of low pathogenic avian influenza viruses in wild birds in Germany. However, fowl plague episodes in poultry occurred in 1979 derived from a lowpathogenic strain originating from wild birds, in 2003 in the context of the Dutch epidemic and again in 2006 and 2007 by the introduction of HPAI virus (HPAIV) subtype H5N1 of Asian origin. The latter was associated with the westward spread of HPAIV H5N1 Asia out of China into Europe. In total, 343 (2006) and 326 (2007) wild birds, respectively, as well as one bird kept in a zoo, three cats and one stone marten as well as one commercial poultry holding were found infected with HPAIV H5N1 Asia. Molecular epidemiological analyses demonstrated that two distinct introductions occurred into Germany in spring 2006 resulting in a Northern and Southern German lineage. The last demonstration of HPAIV H5N1 in 2006 in Germany occurred in August. However, in June 2007, HPAIV H5N1 resurfaced in wild birds and also affected several poultry holdings leading to the destruction of more than 350,000 animals. Molecular epidemiological analyses demonstrated that in 2006 two genetically distinct viruses circulated in Germany, whereas in 2007 a third genotype was detected. This indicated a total of three separate introductions of HPAI H5N1 into Germany in 2006 and 2007. The arrival of HPAIV H5N1 in Germany together with restrictions in keeping poultry outdoors renewed requests for vaccination. However, currently available inactivated vaccines suffer from several shortcomings including the necessity for individual application and lack of an easy and sensitive differentiation between vaccinated and infected animals. Thus, the development of novel vaccines against HPAIV has become of prime importance. The most promising approaches include the use of viral vectors expressing the protective influenza virus hemagglutinin. They are primarily based on recombinant adeno-, pox-, paramyxo- or herpesviruses. In particular, the use of recombinant Newcastle disease virus as carrier for influenza hemagglutinin offers the advantage to be able to protect simultaneously against two of the most important chicken pathogens.
... Marek's disease can be effectively prevented by vaccination with attenuated MDV or herpesvirus of turkey (HVT) [12,13]. As with most herpesviruses, MDV exhibits several characteristics that make it a good vector for recombinant vaccines [14][15][16]. MDV has a large genome with several regions that are nonessential for viral replication, allowing the expression of foreign antigen genes from other pathogens [15,16]. Furthermore, MDV establishes a persistent infection in the lymphoid tissues and can induce long-term immune responses [11]. ...
... Five fosmids (195,214,14,96, and 103) ( Figure 1B) containing sequences spanning the entire genome of MDV1 strain 814 ( Figure 1A) were constructed in our preliminary studies for the rescue of the parental virus. To facilitate the insertion of foreign genes into the MDV genome, a dual selection cassette encoding the KanR and ccdB markers and flanked with the attR1 and attR2 sequences was inserted into the US2 site in the MDV genome using the Counter Selection BAC Modification Kit (Gene Bridges, Berkeley, CA, USA) according to the manufacturer's instructions. ...
Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek’s disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.
... Earlier studies have shown that the infectious laryngotracheitis virus with large genomes also has the potential as a viral vector, which has been used to express the H5 or H7 protein of the influenza virus and obtain effective immune protection [157][158][159]. Furthermore, ILTV live vaccines are easily administered to large populations of animals through convenient methods such as eye drops, aerosol, and drinking water. ...
Inactivated and live attenuated vaccines are the mainstays of preventing viral poultry diseases. However, the development of recombinant DNA technology in recent years has enabled the generation of recombinant virus vector vaccines, which have the advantages of preventing multiple diseases simultaneously and simplifying the vaccination schedule. More importantly, some can induce a protective immune response in the presence of maternal antibodies and offer long-term immune protection. These advantages compensate for the shortcomings of traditional vaccines. This review describes the construction and characterization of primarily poultry vaccine vectors, including fowl poxvirus (FPV), fowl adenovirus (FAdV), Newcastle disease virus (NDV), Marek’s disease virus (MDV), and herpesvirus of turkey (HVT). In addition, the pathogens targeted and the immunoprotective effect of different poultry recombinant virus vector vaccines are also presented. Finally, this review discusses the challenges in developing vector vaccines and proposes strategies for improving immune efficacy.
... Viral vectored vaccines could be an alternate approach to control ARV infection. A number of attenuated poultry vaccine viruses, such as Newcastle disease virus (NDV) [14][15][16][17], fowl pox virus (FPV) [18][19][20], adenovirus [21], herpesvirus of turkeys (HVT) [22,23], infectious laryngotracheitis virus (ILTV) [24,25] and Marek's disease virus (MDV) [26][27][28] have been genetically modified in order to use them as vaccine vectors for developing combinations of live attenuated vaccines for controlling the diseases of poultry. Among these viruses, NDV is an attractive vaccine vector for both human and animal diseases. ...
Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds.
... In endemic area, vaccination of poultry flocks by inactivated or gene vaccines become the only solution in the long-term strategy. Vaccination is targeting to lower losses from mortality, reduce the viral load in the environment and the risk of human infection as well as eradication of positive cases (Lushow et al., 2001). ...
... In endemic area, vaccination of poultry flocks by inactivated or gene vaccines become the only solution in the long-term strategy. Vaccination is targeting to lower losses from mortality, reduce the viral load in the environment and the risk of human infection as well as eradication of positive cases (Lushow et al., 2001). ...
... 119,120 However, ILTV should be attenuated sufficiently, as pathogenicity caused by the vector was still observed in a single study. 119 ILTV vaccines expressing the NA gene were also generated but were poorly immunogenic; co-expression of HA genes was always required to obtain protective immunity. 121 Another alphaherpesvirus of poultry, turkey herpesvirus (HVT) has also been extensively studied as influenza vaccine vector in chickens. ...
Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population, complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.
... Although enforcement of biosecurity measures and an eradication strategy of an infected flock should be the basic line in any control against H5N1 virus infections (Capua and Marangon 2007); Vaccination as a "tailored synergy" has been implemented as a main tool to confront the disease in many of developing countries and to mitigate the impact of the unbearable pre-emptive culling of infected birds (van den Berg et al., 2008;Swayne, 2009 (Beard et al., 1991;Webster et al., 1991;Crawford et al., 1999;Swayne et al., 2000;Lüschow et al., 2001;Bublot et al., 2006;Gao et al., 2006;Veits et al., 2006;Toro et al., 2008;van den Berg et al., 2008;Soto, 2011). Prevention of the clinical signs, mortality, reduced shedding of the virus in the environment, increased the resistance of birds to an infection, decreased bird-to-bird transmission and limited decrease in the egg production are the main advantages of the AI vaccines (Capua and Marangon 2007;van den Berg et al., 2008;Swayne, 2009). ...
... Eine flächendeckende Impfung vermindert lediglich die Virusvermehrung, verhindert jedoch nicht die Infektion, sodass geimpfte Tiere, ohne ernsthaft zu erkranken, trotzdem das Virus weiter ausscheiden können.Garcia et al., 1998) und Pakistan (Naeem, 1998) vor. Darüber hinaus wurden Rekombinante-Impfstoffe sowohl unter experimentellen als auch Praxisbedingungen erprobt (Crawford et al., 1999; Boyle et al., 2000; Lüschow et al., 2001; Villareal und Cruz, 2002). In Zukunft muss eine sachliche emotionslose Diskussion über die Ausmerzungsstrategien von bestimmten Krankheiten, welche mit Vakzination in Kombination mit guten Diskriminierungs-Untersuchungsprogrammen unter Kontrolle gebracht werden könnten, auf globaler Ebene geführt werden.. 42, 248-256. ...
''Highly pathogenic'' avian influenza viruses cause fowl plague. The disease is a notifiable disease. In 2003 infections with highly pathogenic avian influenza were reported in the Netherlands, Belgium and Germany. Influenza viruses seem to be host specific. The risk of infection of humans with avian influenza A viruses is very low, however, in some cases people can attract infections. This observation should be seriously evaluated. The measures adopted to control and eradicate avian influenza are based on the strategy of stamping-out infected flocks and controlling the movement of poultry, poultry products and other contaminated materials. In general vaccinations against HPAI are only allowed as a supplement to the control measures. The decision to introduce the vaccine is accompanied with several restrictions. This paper explores the characteristics of avian influenza viruses, epidemiology, the disease and public health aspects, Finally, the current control strategy in the European communities is discussed.
... Des don-nées expérimentales ont également été obtenues pour les virus recombinants de la variole aviaire exprimant l'antigène H7 (Boyle et al., 2000). D'autres vecteurs ont été utilisés avec succès pour produire les antigènes H5 et H7, notamment en utilisant le virus de la laryngo-trachéite infectieuse (Luschow et al., 2001). Ce type de vaccin n'a pas reçu à ce jour d'autorisation de mise sur le marché par l'Union européenne. ...
Since mid-december 2003, an epizootic of highly pathogenic avian influenza (type A, sub-type H5N1) occurs in eastern and south-eastern Asia. This epizootic is historically unprecedented in its virulence, geographical spread, and economic consequences for the agricultural sector. Implications for human health were registered in Vietnam and in Thailand. This paper summarizes the current knowledge about the risk evaluation of the transmission of avian influenza virus to humans. The current asian epizootic has highlighted the key role of global health information systems and also the need for exhaustive notification of human and animal cases. It reinforces the concept of veterinary public health.
... A live attenuated fowlpox virus was originally developed which expressed the HA molecule from a highly virulent H5 strain of avian influenza (Taylor et al, 1988;Swayne, 2009 (Bublot et al, 2006;Swayne and Kapczynski, 2008). Other examples of live attenuated viruses used to deliver an influenza HA gene include vaccinia virus (Chambers et al, 1988), adenovirus (Gao et al, 2006;Tang et al, 2009), Rous sarcoma virus (Hunt et al, 1988), infectious laryngotracheitis virus (Luschow et al, 2001;Pavlova et al, 2009), Venzuelan equine encephalitis virus and Newcastle disease virus (Ge et al, 2007;Nayak et al, 2009). ...
The influenza virus, a member of the Orthomyxoviridae family, causes regular,
large-scale morbidity and mortality in birds and humans and significant human
suffering and economic loss. The primary aim of this study was to develop a novel
influenza vaccine. Vaccines are an essential tool for the control of influenza because
they increase resistance to infection, prevent illness and death and help to limit virus
transmission to other birds and mammals, including humans. By reducing the
environmental contamination of influenza virus in global poultry stocks, the risk of a
new pandemic virus being generated by the human-avian link is diminished.
Marek’s Disease is a common lymphoproliferative disease of poultry that is readily
controlled worldwide using the live attenuated vaccine, CVI988. The Marek’s
Disease Virus (MDV) CVI988 viral genome, available as a Bacterial Artificial
Chromosome (BAC), forms viable infectious viral particles when transfected into
Chicken Embryo Fibroblast (CEF) cells. Using BAC mutagenesis, two non-essential
genes in the MDV CVI988 BAC (UL41 and US10), were identified and replaced by
the low pathogenic influenza haemagglutinin 10 (H10) gene. These live recombinant
MDV-H10 vectors will allow simultaneous vaccination against both pathogens. In
addition, the non-essential genes were also replaced with GFP creating MDV-GFP
constructs. Both genes were expressed initially using a CMV promoter, although this
disrupted the MDV CVI988 BAC; a second promoter, PGK-1, proved more
successful. A third MDV gene (UL50) was deleted, but severe attenuation prevented
the incorporation of H10 into this open reading frame.
Future work to test the MDV-HA constructs in vivo will be carried out in
collaboration with the Istituto Zooprofilattico Sperimentale delle Venezie in Italy. In
addition, development of MDV constructs containing multiple HA genes (H10 and
H5) linked by the 2A polyprotein can be developed with the goal of establishing
heterosubtypic immunity.
... Des don-nées expérimentales ont également été obtenues pour les virus recombinants de la variole aviaire exprimant l'antigène H7 (Boyle et al., 2000). D'autres vecteurs ont été utilisés avec succès pour produire les antigènes H5 et H7, notamment en utilisant le virus de la laryngo-trachéite infectieuse (Luschow et al., 2001). Ce type de vaccin n'a pas reçu à ce jour d'autorisation de mise sur le marché par l'Union européenne. ...
Since mid-december 2003, an epizootic of highly pathogenic avian influenza (type A, sub-type H5N1) occurs in eastern and south-eastern Asia. This epizootic is historically unprecedented in its virulence, geographical spread, and economic consequences for the agricultural sector. Implications for human health were registered in Vietnam and in Thailand. This paper summarizes the current knowledge about the risk evaluation of the transmission of avian influenza virus to humans. The current asian epizootic has highlighted the key role of global health information systems and also the need for exhaustive notification of human and animal cases. It reinforces the concept of veterinary public health.
... The Gallid Herpesvirus (infectious laryngotracheitis virus (ILTV)) was used as a vector for the insertion of the HA-gene of an H5 or H7 virus at the non-essential UL50 gene locus [157,161]. Immunized chickens produced specific antibodies against ILTV and AIV HA, and were protected against challenge infections with either virulent ILTV, or two different highly pathogenic AIV strains, but developed minimal clinical signs. Therefore, Pavlova et al. designed an attenuated ILTV variant for the insertion of the H5 (or N1) gene, which proved to offer protection of chickens, when vaccinated with the H5-expressing vector or the combination of the H5 and N1 expressing ILTVs, without causing clinical signs [162]. ...
Influenza A viruses are important pathogens with a very broad host spectrum including domestic poultry and swine. For preventing clinical disease and controlling the spread, vaccination is one of the most efficient tools. Classical influenza vaccines for domestic poultry and swine are conventional inactivated preparations. However, a very broad range of novel vaccine types ranging from (i) nucleic acid-based vaccines, (ii) replicon particles, (iii) subunits and virus-like particles, (iv) vectored vaccines, or (v) live-attenuated vaccines has been described, and some of them are now also used in the field. The different novel approaches for vaccines against avian and swine influenza virus infections are reviewed, and additional features like universal vaccines, novel application approaches and the "differentiating infected from vaccinated animals" (DIVA)-strategy are summarized.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... This vaccine provided protection against challenge with a virulent ILTV strain and against lethal challenge with H7 AIV [59]. In addition, vaccination with H5 HA expressing recombinant ILTV vaccines that are constructed by inserting the H5 HA gene into the UL50 gene locus can confer effective protection against challenges with both homologous and heterologous H5 viruses [60][61][62] (Table 1). The efficacy of the H5 HA expressing ILTV vaccine is enhanced when the chickens are co-immunized with a recombinant ILTV expressing the NA gene of the N1 subtype [61] ( Table 1). ...
H5N1 avian influenza viruses (AIVs) have spread widely to more than 60 countries spanning three continents. To control the disease, vaccination of poultry is implemented in many of the affected countries, especially in those where H5N1 viruses have become enzootic in poultry and wild birds. Recently, considerable progress has been made toward the development of novel avian influenza (AI) vaccines, especially recombinant virus vector vaccines and DNA vaccines. Here, we will discuss the recent advances in vaccine development and use against H5N1 AIV in poultry. Understanding the properties of the available, novel vaccines will allow for the establishment of rational vaccination protocols, which in turn will help the effective control and prevention of H5N1 AI.
... Recombinant vaccines used ILTV as a viral vector to contain H5 or H7 genes of highly pathogenic AIVs. These recombinant ILTV may protect birds from ILT and pathogenic AIV [76,82] . Recently, a new ILTV vector had a HPAI H5 gene inserted into a deleted UL50 gene region. ...
Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.
... Des don-nées expérimentales ont également été obtenues pour les virus recombinants de la variole aviaire exprimant l'antigène H7 (Boyle et al., 2000). D'autres vecteurs ont été utilisés avec succès pour produire les antigènes H5 et H7, notamment en utilisant le virus de la laryngo-trachéite infectieuse (Luschow et al., 2001). Ce type de vaccin n'a pas reçu à ce jour d'autorisation de mise sur le marché par l'Union européenne. ...
... Remarkably, the resulting vaccine Vaxxitex R HVT + IBD is so far the only licensed and commercialised animal herpesvirus vector based product. Further developments of vectored bivalent vaccines against poultry diseases include expression of haemagglutinin genes encoding H5 and H7 of highly pathogenic avian influenza viruses by deletion mutants of infectious laryngotracheitis virus [107]. As mentioned above, PRV was one of the first animal herpesvirus suggested to be suitable as bivalent vaccine vector. ...
The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.
... Alternatives for the traditional vaccine preparation have been developed recently. Using molecular biology, recombinant fowlpox vaccines, recombinant infectious laryngotracheitis virus and recombinant Newcastle disease virus containing an H5 insert have been developed [57][58][59] . Vaccination with inactivated whole-virus vaccines or recombinant vaccines prevents clinical disease and mortality, decrease susceptibility of poultry to infection with avian infl uenza viruses and decrease shedding from the respiratory and intestinal tract. ...
Influenza A virus behoort tot de familie van Orthomyxoviridae. Infl uenza A virussen
zijn onregelmatig gevormde virussen van ongeveer 120 nm groot. Het genoom van
influenza A virussen is gesegmenteerd en bestaat uit negatief-strengs RNA. De
acht gensegmenten coderen voor 11 verschillende eiwitten. Infl uenza A virussen
worden onderverdeeld op basis van de oppervlakte eiwitten; hemagglutinine (HA,
een eiwit dat zorg draagt voor de binding van het virus aan en binnendringen
van de gastheercel) en neuraminidase (NA, een eiwit dat zorgt voor de efficiënte
verspreiding na vermenigvuldiging van het virus). Er waren tot voor kort 15
verschillende subtypen van HA geïdentificeerd en 9 verschillende subtypen van
NA. Dit leidt tot 135 potentiële subtype combinaties zoals H1N1, H3N2, H5N1 en
H7N7. Aviaire influenza virussen zijn verder in te delen op grond van biologische
eigenschappen: laag pathogene aviaire influenza virussen (LPAI), die geen tot milde
ziekteverschijnselen veroorzaken en hoog pathogene aviare influenza virussen
(HPAI), die massale sterfte onder pluimvee kunnen veroorzaken (ook bekend als
vogelpest).
Influenza A virus is voornamelijk bekend als veroorzaker van de drie pandemieën
(wereldwijde uitbraken) van de afgelopen eeuw: de H1N1 Spaanse griep in 1918,
de H2N2 Aziatische griep in 1957 en de H3N2 Hong Kong griep in 1968. Daarnaast
is het ook bekend van de jaarlijks terugkerende griepepidemieën (lokale uitbraken)
en van uitbraken van vogelpest. Hoewel het bekend is dat alle infl uenza A virussen
hun oorsprong hebben in wilde vogels, is er weinig bekend over het voorkomen van
aviaire influenza virussen in wilde vogels in Europa. Het onderzoek beschreven in dit
proefschrift richtte zich in eerste instantie op het in kaart brengen van de complexe
ecologie van aviaire influenza virussen in hun natuurlijke gastheer, de wilde vogels,
en vervolgens op de veranderingen die ten grondslag liggen aan het vermogen van
deze virussen om overgedragen te kunnen worden naar de mens.
Hoofdstuk 1 geeft een algemene inleiding en een overzicht van de huidige kennis
over het voorkomen van infl uenza A virussen in wilde vogels. Hierbij wordt ingegaan
op de rol die verschillende vogelfamilies spelen in de ecologie van infl uenza A
virussen zoals de Anseriformes (eenden, ganzen en zwanen) en Charadriformes
(meeuwen, alken en steltlopers).
... La Sota NDV vaccine expressing AI H5 (rLH5-5) was licensed and commercialized in China in 2005 [38,39]. Another avian herpesvirus, infectious laryngotracheitis virus, was also used for developing recombinant vaccines expressing HA genes encoding H5 [40] and H7 [41] of HPAIV, and the immunized animals produced specific antibodies against ILTV and AIV HA and were protected against challenge infections with either virulent ILTV, or two different highly pathogenic AIV strains. Recombinant duck enteritis virus vectored live vaccine provided fast and complete protection against lethal H5N1 avian influenza virus challenge in ducks [42]. ...
Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek's disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1.
A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose.
The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.
... So, long-term control of the H5N1 panzootic in these areas may well depend on controlling the infection in duck flocks along with other strategies. Recent field and experimental vaccines for HPAI in poultry include inactivated, conventional whole virus vac- cines [3, 10, 11]; inactivated vaccines developed using reverse genetics121314; in vitro baculovirus-expressed H5 antigen as subunit vaccines [10, 15]; in vivo vector-expressed vaccines including recombinant fowl poxvirus [16, 17], Newcastle Disease virus [18] or infectious laryngotracheitis virus vectors [19]; and DNA vaccines [20]. These vaccines have all conferred clinical protection and eliminated or reduced viral shedding following HPAI virus challenge. ...
The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.
... Another advantage of live vaccines is that they do not need to be adjuvant-formulated, thereby limiting production costs. Because the use of live influenza vaccines has obvious safety restrictions, alternative vaccines based on different heterologous viral vectors have been developed, including pox virus [1], adenovirus [2], infectious laryngotracheitis virus [3], baculovirus [4] and Newcastle disease virus (NDV) [5]–[11]. ...
Background:
Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity.
Methodology/principal findings:
In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited.
Conclusions/significance:
Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.
... To develop an improved vaccine against AI, recombinant DNA technology has been used to generate live virus vaccines. Fowlpox virus (FPV) [4] [5] [6] [7] [8] [9], adenovirus [10], infectious laryngotracheitis virus [11] [12], Newcastle disease virus [13] [14], and duck enteritis virus [15] have been used as vectors to develop recombinant vaccines. It has been reported that a FPV-vectored vaccine Abbreviations: AI, avian influenza; AIV, avian influenza virus; CEF, chicken embryo fibroblast; ChIL-6, chicken IL-6; EID50, 50% chicken embryo infection dose; E-rosette, erythrocyte rosette; FBS, fetal bovine serum; FPV, fowlpox virus; HA, haemagglutinin; HI, hemagglutination inhibition; HPAI, highly pathogenic avian influenza; IFA, indirect immunofluorescence assay; IL, interleukin; MOI, multiple of infection; PFU, Plaque forming units; rFPV, recombinant fowlpox virus; RT-PCR, reverse transcriptase-polymerase chain reaction; SPF, specific-pathogen-free. * Corresponding author at: 48 East Wenhui Road, Yangzhou, Jiangsu 225009, PR ...
Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.
... All influenza vaccines, which have been used since the 1940s until today as permitted vaccines for the treatment of humans and animals, consist of one or more virus strains De et al. 1988;Brown et al. 1992;Schultz-Cherry et al. 2000;Luschow et al. 2001;Gao et al. 2006;Chen and Bu 2009 which have been conventionally produced in the allantoic cavity of embryonated hens' eggs. Influenza viruses grown by this method are then used for producing live attenuated virus, killed whole virus, or subunit vaccines. ...
Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.
The different technology platforms used to make poultry vaccines are reviewed. Vaccines based on classical technologies are either live attenuated or inactivated vaccines. Genetic engineering is applied to design by deletion, mutation, insertion, or chimerization, genetically modified target microorganisms that are used either as live or inactivated vaccines. Other vaccine platforms are based on one or a few genes of the target pathogen agent coding for proteins that can induce a protective immune response (“protective genes”). These genes can be expressed in vitro to produce subunit vaccines. Alternatively, vectors carrying these genes in their genome or nucleic acid–based vaccines will induce protection by in vivo expression of these genes in the vaccinated host. Properties of these different types of vaccines, including advantages and limitations, are reviewed, focusing mainly on vaccines targeting viral diseases and on technologies that succeeded in market authorization.
Plataformas tecnológicas de vacunas avícolas
En este artículo se revisan las diferentes plataformas tecnológicas utilizadas para elaborar vacunas avícolas. Las vacunas basadas en tecnologías clásicas son vacunas vivas atenuadas o inactivadas. La ingeniería genética se aplica al diseño mediante eliminación, mutación, inserción o quimerización de microorganismos diana genéticamente modificados que se utilizan como vacunas vivas o inactivadas. Otras plataformas de vacunas se basan en uno o varios genes del agente patógeno objetivo que codifican proteínas que pueden inducir una respuesta inmunitaria protectora (“genes protectores”). Estos genes pueden expresarse in vitro para producir vacunas de subunidades. Alternativamente, los vectores que llevan estos genes en su genoma o las vacunas basadas en ácidos nucleicos inducirán protección mediante la expresión in vivo de estos genes en el huésped vacunado. Se revisan las propiedades de estos diferentes tipos de vacunas, incluidas sus ventajas y limitaciones, centrándose principalmente en las vacunas dirigidas a enfermedades virales y en las tecnologías que lograron la autorización de comercialización.
To develop an alternative vaccine against Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV), we firstly constructed a gC-deleted ILTV mutant based on an avirulent ILTV, and then obtained a recombinant ILTV expressing the fusion protein (F) of the genotype VII NDV (designated ILTV-ΔgC-F) based on the gC-deleted ILTV mutant. Expression of the NDV F protein in ILTV-ΔgC-F-infected LMH cells was examined by indirect immunofluorescence assay (IFA) and western blotting (WB). The recombinant ILTV-ΔgC-F retained the growth kinetics of parental ILTV with increased replication rate. The inserted F gene was stably maintained in the genome of ILTV-ΔgC-F and the F protein was be stably expressed. Based on the clinical signs and viral shedding detection of experimentally infected chickens, the recombinant ILTV-ΔgC-F is proven to be avirulent. Vaccination of specific pathogen-free (SPF) with recombinant ILTV-ΔgC-F induced ILTV-specific enzyme-linked immunosorbent assay (ELISA) antibody and provided complete clinical protection against virulent ILTV although viral shedding in respiratory tract and viral replication in the target tissues were detected at the early stage of infection in only s very small number of birds. In addition, a single vaccination with ILTV-ΔgC-F could provide complete clinical protection against virulent genotype VII NDV challenge although the level of NDV-specific ELISA antibody were low. Notably, the numbers of birds which were positive for virus detection and the replication level of the challenged NDV in the selected target tissues were significantly reduced in ILTV-ΔgC-F-vaccinated chickens, comparing to those of control birds. Our results indicate that ILTV-ΔgC-F could be considered as a bivalent vaccine candidate against both ILTV and NDV.
L'influenza aviaire hautement pathogène (IAHP), aussi connue sous le nom de peste aviaire, est une maladie virale touchant les oiseaux sauvages et domestiques.Il s'agit habituellement d'une affection difficilement transmissible à l'homme ; pourtant la souche A(H5N1) dont il est question dans ce travail, a contaminé dix-huit personnes et provoqué la mort de six personnes à Hong Kong.Dès lors, l'IAHP à A(H5N1) est devenue un sujet de préoccupation. Depuis, le virus s'est non seulement répandu au reste de l'Asie mais également en Europe et en Afrique.Les autorités sanitaires mondiales ont réagi en élaborant des stratégies visant à contenir l'épizootie et à éviter un passage du virus à l'homme.
Infectious laryngotracheitis (ILT) is a respiratory tract infection of chickens caused by Gallid herpesvirus type 1 (GaHV-1). This virus may cause severe production losses due to mortality and/or decreased egg production. The diagnosis of GaHV-1 requires laboratory assistance because a sole diagnosis based on clinical presentation can be misleading. Other respiratory pathogens of poultry can cause similar clinical signs and lesions. Cooperative control of ILT outbreaks by collaboration between government and industry is most desirable. In controlling ILT outbreaks, the most effective approach is a coordinated effort to obtain a rapid diagnosis, institute a vaccination program, and prevent further virus spread. Prior to implementation of any vaccination program it is important to review the country's regulations for the use of vaccines against GaHV-1. The importance of site quarantine and hygiene in preventing the movement of potentially contaminated personnel, feed, equipment, and birds is central to successful prevention and control of ILT.
Avian influenza (AI) vaccines have been developed and used to protect poultry and other birds in various countries of the world. Protection is principally mediated by an immune response to the subtype-specific hemagglutinin protein. AI vaccines prevent clinical signs of disease, death, drops in egg production, and bird-to-bird contact transmission, as well as decreasing the number of birds infected with AI virus (AIV), and reducing high pathogenicity (HP) AIV contamination of eggs and the quantity of AIV shed from the respiratory and alimentary tracts. AI vaccines are produced using various technologies, including inactivated whole AI vaccines, in-vitro expressed HA protein, in-vivo expressed HA protein in vectored systems, and HA-based DNA vaccines to induce protective immunity. AI vaccines must be periodically re-evaluated to determine whether they are still effective against circulating field virus strains, and should be replaced with updated strains if they are no longer protective under field conditions.
Total of 335 fecal and nasal swabs were collected from Broilers, Broiler Breeders, Layers and duck flocks from farms and backyard in different Provinces in the period from 2011 to the year 2013. All these flocks had history for previous vaccination against avian influenza and Newcastle disease viruses. All samples were inoculated in 9-day old specific pathogenic free (SPF) embryonated chicken eggs through allantoic sac. Rapid haemagglutination (HA) test was done for detection of haemagglutaning viruses. Results of Haemagglutination revealed that, 12 (3.6%) out of 335 collected samples were positive by using HA test. The results revealed that 4 (4.4%) out of 90 broiler samples from Fayoum Governorate were positive by HA test. One sample (6.6%) out of 15 samples; 2 samples (1.3%) out of 157 samples and 2 (3.4%) out of 58 were positive by HA test in broiler, breeder and layer flocks respectively from Sharkia Governorate. In Menofia Governorate, one sample (7.7%) out of 13 from backyard and 2 samples (100%) out of 2 collected from ducks were positive by HA test. HA positive samples either Newcastle disease and/or avian influenza virus. Results of virus detection by RT-PCR using F gene primers proved that all isolated viruses were ND negative. Also, the results of RT-PCR test for H5 and H9 genes proved that AIV H5N1 virus was detected in 8 isolates (66.6%) out of 12 HA positive samples, where 5 samples (41.6%) from Sharkia and 3 samples (25%) from Menofia. Also, AIV H9N2 was detected in 9 isolates (75%) out of 12 HA positive samples, where 5 (41.6%) samples from Sharkia and 4 samples (20%) from Fayoum.
Veterinary medicine has recently achieved important success by introducing viral marker vaccines and marker diagnostics able to differentiate between vaccinated and infected animals (DIVA). Using these new marker strategies, one of the most devastating infectious diseases in swine, Aujeszky's disease caused by the pseudorabies virus, has been eliminated from Germany. Similar approaches for eradication or control of other important infectious diseases such as bovine herpesvirus type 1 infection, classical swine fever, foot-and-mouth disease or fowl plague now appear feasible. The DIVA strategies for the different diseases are presented and possibilities as well as limitations of the concepts discussed.
Infectious bursal disease (IBD) is one of the economically important avian diseases for the poultry industry. In addition to traditional inactivated and attenuated vaccines, recent research has developed other types of vaccines to make IBD vaccines more adaptable for preventing different strain types and avoid the interference of maternal antibodies. Avian viral vector vaccines are emphasized in the present review. The background of IBD, virus and viral vectors is introduced. Different avian viral vectors for IBD, including their benefits and drawbacks, and the potential use of IBD virus as the viral vector are addressed.
This chapter reviews the direct and indirect control measures for influenza infections in animals: poultry, swine, dogs, and horses. Regulations and guidelines linked to the international movements of animals for trade or other purposes are also addressed, as well as statutory requirements whenever applicable.
In this study thebroiler chicks having maternal antibodies at 1 day 1.82± 2.60(H5N2) and 1.52± 0.64 (H5N1). The administration of H5N2 either half dose (0.25 ml) or full dose (0.5 ml) at the 1 or 7 day of life did st th not resulting in increased antibodies till the 14 day of life if compared with the maternal antibodies at 1 day th 1.82± 2.60(H5N2) and 1.52± 0.64 (H5N1). The revaccinated groups showed antibody response lower than the single vaccinated ones. ELISA test in AI-H5N2 vaccinated broiler chickens proved that no positive S/P ratio could be detected in all vaccinated birds at all doses and weeks post vaccination. Body weight of broiler chickens vaccinated with AI-H5N2 vaccine showed body weight higher than the non-vaccinated, but there was no marked effect of vaccine dose or age on the deference in body weight between vaccinated groups. Commercial broiler chickens vaccinated with AI-H5N1 full dose (0.5 ml) at 1 or 7 days of age showed higher HI titres, than those given 0.25 ml. Birds vaccinated with two half doses showed lower titres (2.95±1.95, 3.84± 1.83 and 4.31± 3.54) than those received full doses (2.83 ± 1.75, 4.11± 1.40 and 4.47± 2.26) at 21, 28 and 35 days of age; respectively. The protective titre was detected at 35 days of age (25 days after the 2 half dose) nd and birds given two full doses (0.5 ml) showed that the protective titre was detected from 18 days after the 2 dose. ELISA S/P ratios were interpreted as negative in groups received half dose, while 2/20 were positive nd at 21 days where full dose were given at one day and 1/20 in vaccination at 7 days. Single full doses showed positive s/p reties in (3/20 and 2/20) at 28 and 35 days of age and in birds given two full doses showed positive results (3/18, 5/17 and 7/19) at 21, 28 and 35 days of age (11, 18 and 25 days post 2 dose); respectively.
On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic Avian Influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters. These five promoters included three MDV endogenous promoters (the promoter for the gB gene and a bi-directional promoter in both directions for pp38 (ppp38) and 1.8kb RNA transcripts (p1.8kb)), and two exogenous promoters (CMV and SV40). Among these five promoters, the CMV promoter demonstrated the highest activity, followed by p1.8kb and SV40, which had a similar transcriptional activity level. Two of the MDV endogenous promoters showed much lower transcriptional activities, particularly the promoter ppp38, which had the lowest activity. The results of the in vivo experiment proved that none of the three recombinant viruses of rGX-CMV-HA, rGX-SV40-HA and rGX-p1.8kb-HA provided protection in SPF chickens. Chickens vaccinated with rGX-pPP38-HA induced 50% and rGX-gB-HA induced 25% protection against the challenge with H9N2, respectively.
In this book, the authors present current research in the study of the physiology, diseases and farming practices of chickens. Topics discussed include recent trends in the diagnosis and vaccines for poultry diseases; nutrient regulation of intestinal development and function in chickens; the role of selenium in chicken physiology; harvest and post-harvest practices for chickens; organ blood flow in the hen fetal and early postnatal periods; central regulation of physiological and metabolic responses in neonatal chicks; bacteriophages in the control of colibacillosis; and chicken egg size and embryonic development. (Imprint: Nova) http://goo.gl/zd8RDW.
The serologically diverse influenza A viruses, although transmissible among various susceptible species, mostly infect avian species. Avian influenza viruses (AIV) are also notorious for adapting to mammalian species, including humans. Although eradication of commercial birds infected with AIV is the preferred method of control, the ever presence of potential avian migratory, reservoir species makes worldwide spread inevitable and vaccine development a high priority for poultry. Live, attenuated vaccine strategies are of concern because of the potential of AIV to mutate, through point mutations and/or reassortment of their segmented genome. Both live and the safer, killed vaccines are of concern because of competition of natural and vaccine antigens in critical diagnostic assays. Subunit vaccines, which allow for protein distinction for diagnostic purposes, may consist of purified AIV protein or genes that encode individual viral proteins. Most vaccines have targeted the virus hemagglutinin protein, which is responsible for induction of the most effective neutralizing antibodies. Gene vaccines that include plasmid DNA and viral vector delivery of AIV genes allow for endogenous in vivo amplification of protein within cells. While fowlpox virus vectors have been licensed and proven to be efficacious even in field situations, other viral vectors that target the respiratory tract are in experimental development with promising practical application for poultry. Maximum exploitation of vectored vaccines may incorporate enhancing immune molecules.
Highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Far East Asia in 1996 and spread in three continents in a period of 10 or less years. Before this event, avian influenza infections caused by highly pathogenic viruses had occurred in many different countries, causing minor or major outbreaks, and had always been eradicated. The unique features of these H5N1 viruses combined to the geographic characteristics of the area of emergence, including animal husbandry practices, has caused this subtype to become endemic in several Asian countries, as well as in Egypt. Our aim is to review the direct and indirect control strategies with the rationale for use, advantages and shortcomings - particularly resulting from practicalities linked to field application and economic constraints. Certainly, in low income countries which have applied vaccination, this has resulted in a failure to eradicate the infection. Although the number of infected countries has dropped from over 40 (2006) to under 10 (2012), the extensive circulation of H5N1 in areas with high poultry density still represents a risk for public and animal health.
Vaccination of poultry for avian influenza virus (AIV) is a complex topic as there are numerous technical, logistic and regulatory aspects which must be considered. Historically, control of high pathogenicity (HP) AIV infection in poultry has been accomplished by eradication and stamping out when outbreaks occur locally. Since the H5N1 HPAIV from Asia has spread and become enzootic, vaccination has been used on a long-term basis by some countries to control the virus, other countries have used it temporarily to aid eradication efforts, while others have not used it at all. Currently, H5N1 HPAIV is considered enzootic in China, Egypt, Viet Nam, India, Bangladesh and Indonesia. All but Bangladesh and India have instituted vaccination programs for poultry. Importantly, the specifics of these programs differ to accommodate different situations, resources, and industry structure in each country. The current vaccines most commonly used are inactivated whole virus vaccines, but vectored vaccine use is increasing. Numerous technical improvements to these platforms and novel vaccine platforms for H5N1 vaccines have been reported, but most are not ready to be implemented in the field.
Influenza A viruses that infect poultry can be divided into two groups. Very virulent viruses cause highly pathogenic avian influenza (HPAI), with flock mortality as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease (LPAI), unless exacerbated. Until recently, HPAI viruses were rarely isolated from wild birds, but for LPAI viruses extremely high isolation rates have been recorded in surveillance studies. Influenza viruses may infect all types of domestic or captive birds in all areas of the world. The frequency with which primary infections occur in any type of bird usually depends on the degree of contact there is with feral birds. Secondary spread is typically associated with human involvement, either by birds or bird product movement or by transferring infective faeces from infected to susceptible birds, but potentially wild birds can be involved. In recent years the frequency of HPAI outbreaks appears to have increased and there have been particularly costly outbreaks of HPAI in densely populated poultry areas in Italy, The Netherlands and Canada. In each outbreak millions of birds were slaughtered to bring the outbreaks under control. Since the 1990s, AI infections due to two subtypes have been widespread in poultry across a large area of the World. LPAI H9N2 appears to have spread across the whole of Asia in that time and has become endemic in poultry in many of the affected countries. However, these outbreaks have been overshadowed by the H5N1 HPAI virus, initially isolated in China that has now spread in poultry and/or wild birds throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonosis threat. To date control methods seem to have been unsuccessful on the larger scale and HPAI H5N1 outbreaks continue to be reported.
The risk and the size of an outbreak of avian influenza virus (AIV) could be restricted by vaccination of poultry. A vaccine used for rapid intervention during an AIV outbreak should be safe, highly effective after a single administration and suitable for mass application. In the case of AIV, aerosol vaccination using live virus is not desirable because of its zoonotic potential and because of the risk for virus reassortment. The rational design of novel mucosal-inactivated vaccines against AIV requires a comprehensive knowledge of the structure and function of the lung-associated immune system in birds in order to target vaccines appropriately and to design efficient mucosal adjuvants. This review addresses our current understanding of the induction of respiratory immune responses in the chicken. Furthermore, possible mucosal vaccination strategies for AIV are highlighted.
Please cite this paper as: Nang et al. (2013) Live attenuated H5N1 vaccine with H9N2 internal genes protects chickens from infections by both Highly Pathogenic H5N1 and H9N2 Influenza Viruses. Influenza and Other Respiratory Viruses 7(2) 120–131.
Background The highly pathogenic H5N1 and H9N2 influenza viruses are endemic in many countries around the world and have caused considerable economic loss to the poultry industry.
Objectives We aimed to study whether a live attenuated H5N1 vaccine comprising internal genes from a cold-adapted H9N2 influenza virus could protect chickens from infection by both H5N1 and H9N2 viruses.
Methods We developed a cold-adapted H9N2 vaccine virus expressing hemagglutinin and neuraminidase derived from the highly pathogenic H5N1 influenza virus using reverse genetics.
Results and Conclusions Chickens immunized with the vaccine were protected from lethal infections with homologous and heterologous H5N1 or H9N2 influenza viruses. Specific antibody against H5N1 virus was detected up to 11 weeks after vaccination (the endpoint of this study). In vaccinated chickens, IgA and IgG antibody subtypes were induced in lung and intestinal tissue, and CD4+ and CD8+ T lymphocytes expressing interferon-gamma were induced in the splenocytes. These data suggest that a live attenuated H5N1 vaccine with cold-adapted H9N2 internal genes can protect chickens from infection with H5N1 and H9N2 influenza viruses by eliciting humoral and cellular immunity.
Although the use of vaccines against avian influenza viruses in birds has been discouraged over the years, the unprecedented occurrence of outbreaks caused by avian influenza (AI) viruses in recent times has required review of this policy. A variety of products are now available on the market, ranging from inactivated conventional to live recombinant products. The general consensus on the use of vaccination is that if complying to GMP standards and properly administered, birds will be more resistant to field challenge and will exhibit reduced shedding levels in case of infection. However, viral circulation may still occur in a clinically healthy vaccinated population. This may result in an endemic situation and in the emergence of antigenic variants. In order to limit these risks, monitoring programmes enabling the detection of field exposure in vaccinated populations are recommended by international organisations and are essential to allow the continuation of international trade. Adequate management of a vaccination campaign, including monitoring, improved biosecurity and restriction is essential for the success of any control program for AI.
Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.
Zoonotic viruses, such as highly pathogenic avian influenza (HPAI), present a significant threat to both the poultry industry and public health. The present method of controlling avian influenza (AI) relies on good farming practice with limited use of vaccination in some countries. However, new ways to control disease outbreaks might be possible with additional knowledge of the natural host response to virus. Moreover, manipulation of the innate immune system in mammals improves the outcomes following viral infection. A similar approach might be applied to the chicken, nevertheless, a greater knowledge of the chicken innate immune system is required. This review outlines important mammalian antiviral mechanisms that have been modulated to strengthen viral immunity and highlights the potential application of these strategies in the chicken, especially in regards, to AI.
ABSTRACT: H9N2 influenza virus is endemic in many Asian countries and is regarded as a candidate for the next human pandemic. Knowledge of the induction of inflammatory responses and toll-like receptors (TLRs) in chickens infected with H9N2 is limited. Here, we show that H9N2 induces pro-inflammatory cytokines such as transforming growth factor-beta 3; tumor necrosis factor-alpha; interferon-alpha, -beta, and gamma; and TLR 1, 2, 3, 4, 5, 7, and 15 in trachea, lung, and intestine of infected chickens. In the lung, TLR-15 was dominantly induced. Taken together, it seems that H9N2 infections efficiently induce inflammatory cytokines and TLRs in trachea, lung and intestine of chickens.
The global need for a pandemic influenza vaccine is large. High-income countries have stated their intent to provide universal access for pandemic influenza vaccine to their populations. Assuming that a two-dose schedule would be needed, providing universal coverage globally would represent approximately 6.5 billion two-dose courses or 13 billion doses. In the best case scenario, should an outbreak of pandemic influenza occur in the near term, using H5N1 as a proxy for the pandemic virus, the total available doses for the global population within six months of an out break would be only 1.2 billion courses or 2.4 billion doses. In addition, current stockpiles of pandemic influenza vaccine are limited. However, promising developments are occurring with respect to global capacity, technological innovation, and global conviction that offer potential solutions to the problem of pandemic influenza vaccine supply for the world's population.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Baculoviruses were engineered to express hemagglutinin (HA) genes of recent avian influenza (AI) isolates of the H5 and H7 subtypes. The proteins were expressed as either intact (H7) or slightly truncated versions (H5). In both cases purified HA proteins from insect cell cultures retained hemagglutination activity and formed rosettes in solution, indicating proper folding. Although immunogenic in this form, these proteins were more effective when administered subcutaneously in a water-in-oil emulsion. One or two-day-old specific pathogen free (SPF) White Rock chickens, free of maternal AI antibodies, responded with variable serum HI titers, but in some cases the titers were comparable to those achieved using whole virus preparations. Vaccination of three-week-old chickens with 1.0 μg of protein per bird generated a more consistent serum antibody response with an average geometric mean titer (GMT) of 121 (H5) and 293 (H7) at 21 days postvaccination. When challenged with highly pathogenic strains of the corresponding AI subtypes, the vaccinated birds were completely protected against lethal infection and in some cases exhibited reduced or no cloacal shedding at 3 days postinfection. Vaccine protocols employing these recombinant HA proteins will not elicit an immune response against internal AI proteins and thus will not interfere with epidemiological surveys of natural influenza infections in the field.
Herpes simplex virus type 1 (HSV-1) encodes a dUTPase which has been shown to be dispensable for normal viral replication in cultured cells (S. J. Caradonna and Y. Cheng, J. Biol. Chem. 256:9834-9837, 1981; F. B. Fisher and V. G. Preston, Virology 148:190-197, 1986). However, the importance of this enzyme in vivo has not been determined. In this report, HSV-1 strain 17 syn+ and two isogenic engineered dUTPase-negative mutants were characterized in the mouse model. Both mutants replicated with wild-type kinetics and achieved wild-type titers in cultured cells. The mutants were 10-fold less neurovirulent than 17 syn+ following intracranial inoculation and more than 1,000-fold less virulent following footpad inoculation. The dUTPase- mutants replicated with wild-type kinetics in the footpad and entered and replicated efficiently in the peripheral nervous system of the mouse. However, their replication in the central nervous system was significantly reduced. The dUTPase- strains established latent infections but displayed a greatly reduced reactivation frequency in vivo. Neurovirulence, neuroinvasiveness, and reactivation frequency were all restored by recombination with wild-type dUTPase sequences. These results have important implications with regard to anti-herpesvirus therapeutic strategies.
To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico.
The genome of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) comprises ca. 155 kbp of which ca. one-third have been sequenced so far. To gain additional sequence information we analyzed two stretches of 15.5 and 1.9 kbp of the ILTV unique long (U(L)) genome region. The larger fragment contains homologs of the herpes simplex virus (HSV) UL23 (thymidine kinase) and UL22 (glycoprotein H) genes followed by five open reading frames (ORF) encoding putative proteins of 334 to 410 amino acids which exhibit no homology to any known herpesvirus protein. RNA analyses showed that these unique ILTV genes are indeed expressed. An origin of replication separates this cluster of unique genes from a conserved gene cluster consisting of the UL45, UL46, UL48, UL49, UL49.5, and UL50 homologs. The absence of UL47 from this position coincides with the localization of a UL47-homologous ORF within the unique short (U(S)) region of the ILTV genome (M. Wild, S. Cook, and M. Cochran, Virus Genes 12:107-116, 1996). Within the second analyzed region the ILTV UL21 homolog was found adjacent to the UL44 gene. We thus identified five novel herpesvirus genes in ILTV and present evidence for a large internal inversion in the ILTV U(L) region, in contrast to the collinear genomes of other alphaherpesviruses. Interestingly, a similar inversion is also present in the porcine alphaherpesvirus pseudorabies virus.
The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.
We report the clear observation and characterization of room-temperature resonant tunneling in thin-barrier (35-Å) GaAs/AlGaAs multiple-quantum-well (MQW) structures (containing as many as 100 quantum wells) and discuss the effects on electroabsorption modulators. The characteristics of similar MQW devices with both thick (60-Å) and thin (35-Å) quantum-well barriers are studied and compared experimentally and found to be similar, except near the thin-barrier resonant-tunneling field. We present data suggesting a novel exploitation of resonant tunneling for high-power modulators by aligning the resonant-tunneling field with the appropriate wavelength for high absorption. Data from an asymmetric Fabry–Perot electroabsorption modulator are also presented. We also discuss the implications of resonant tunneling on optically bistable self-electro-optic effect devices.
Between the month of October 1997 and January 1998, eight outbreaks of highly pathogenic avian influenza were diagnosed in the Veneto and Friuli-Venezia Giulia regions in north-eastern Italy. For each of the eight outbreaks, influenza A virus of subtype H5N2 was isolated and the inoculation of susceptible chickens confirmed these viruses to be extremely virulent with intravenous pathogenicity indices in 6-week-old chickens of 2.98 to 3.00. Although it was not possible to trace the origin of infection, the epidemiological investigation revealed connections between several outbreaks and emphasized the well-known risk factors for avian influenza such as bird movement, rearing of mixed populations and contact with migratory waterfowl. Control measures listed in European Union directive 92/40/EEC were implemented promptly and spread of the infection to intensively-reared domestic poultry was avoided.
The cross-species transfer of a H5N1 influenza virus from birds to humans, and the systemic spread of this virus in mice, has accelerated the efforts to devise protective strategies against lethal influenza viruses. DNA vaccination with the highly conserved nucleoprotein gene appears to provide cross protection against influenza A viruses in murine models. Whether such vaccines would protect human hosts against different influenza A viruses, including strains with pandemic potential, is unclear. Our aim in this study is to evaluate the ability of a combination DNA vaccine consisting of two plasmids encoding the HA genes from two different subtypes and a DNA vaccine encoding the viral nucleoprotein gene from a H5 virus to induce protection against highly lethal infection caused by H5 and H7 influenza viruses in chickens. Chickens given a single dose of plasmids expressing H5 and H7 hemagglutinins protected the birds from infection by either subtype. However, birds immunized with nucleoprotein DNA and challenged with either A/Ck/Vic/1/85(H7N7) or A/Ty/Ir/1/83 (H5N8) showed definite signs of infection, suggesting inadequate immunity against viral infection. Fifty percent of the nucleoprotein DNA immunized birds survived infection by influenza A/Ty/Ir/1/83 (H5N8) virus (virus of same subtype) while 42% survived infection by influenza A/Ck/Vic/1/85/(H7N7) virus (virus of a different subtype). These studies demonstrate that immunization with DNA encoding a type-specific gene may not be effective against either homologous or heterologous strains of virus, particularly if the challenge virus causes a highly lethal infection. However, the combination of HA subtype vaccines are effective against lethal infection caused by viruses expressing any of the HA subtypes used in the combination preparation.
A recombinant fowlpox vaccine with an H5 hemagglutinin gene insert protected chickens against clinical signs and death following challenge by nine different highly pathogenic H5 avian influenza viruses. The challenge viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant vaccine, and represented diversely geographic and spatial backgrounds; i.e. isolated from four different continents over a 38 year period. The recombinant vaccine reduced detectable infection rates and shedding titers by some challenge viruses. There was a significant positive correlation in hemagglutinin sequence similarity between challenge viruses and vaccine, and the ability to reduce titers of challenge virus isolated from the oropharynx (rs=0.783, P=0.009), but there was no similar correlation for reducing cloacal virus titers (rs=−0.100, P=0.78). This recombinant fowlpox-H5 avian influenza hemagglutinin vaccine can provide protection against a variety of different highly pathogenic H5 avian influenza viruses and frequent optimizing of the hemagglutinin insert to overcome genetic drift in the vaccine may not be necessary to provide adequate field protection.
Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.
Fowlpox virus (FPV) recombinants expressing influenza virus H5 haemagglutinin (HA), nucleoprotein (NP) or co-expressing both of these antigens were tested for vaccine efficacy in chickens. Immunization with the recombinant FPV-HA was highly efficacious but provided no cross protection between subtypes. Bursectomy established that immunity against the H5 subtype was antibody-mediated despite the presence of very low levels of antibody in the vaccinated birds. Immunization with the recombinant FPV expressing the cross-reactive NP antigen did not provide protective immunity despite hyperimmunization and provided no benefit above HA expressed alone. The results suggest that the kinetics of viral replication outpaces immunity induced by NP.
Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.
A thymidine kinase (TK)-negative (TK-) deletion mutant of the Bucharest (BUK) strain of pseudorabies virus (PRV) was isolated. The mutant, designated as PRV (BUK d13), did not revert to TK-positive (TK+), even when propagated in medium that selected for TK+ viruses. The mutant also replicated equally well at 39.1 C and 34.5 C, and was easily distinguished from other PRV strains by molecular hybridization experiments, restriction nuclease fingerprints, and plaque autoradiography or other assays for the TK phenotype. The PRV (BUK d13) had greatly reduced virulence for mice and rabbits, compared with parental TK+ strains, PRV (BUK-5) and PRV (BUK-5A-R1), and provided mice with solid protection against the TK+ BUK and Aujeszky strains of PRV. Experiments were done in 5- to 6-week-old pigs to assess the safety and efficacy of PRV (BUK d13) in the natural host. In one experiment, pigs were vaccinated IM with 7.5 X 10(8) plaque-forming units of TK- PRV (BUK d13), and were then challenge exposed intranasally (IN) with 4.3 X 10(8) TCID50 of virulent PRV [Indiana-Funkhauser (IND-F)]. Vaccinated pigs did not have clinical signs of illness after vaccination or after challenge exposure. One nonvaccinated control pig died on postchallenge day 4; a 2nd nonvaccinated control pig became moribund, but eventually recovered. Pigs developed virus-neutralizing antibodies after vaccination, and had a secondary immunologic response after challenge exposure; however, PRV was not isolated from the tonsils or trigeminal ganglia of vaccinated pigs at postchallenge exposure day 11.(ABSTRACT TRUNCATED AT 250 WORDS)
To study the immune response of the chicken to specific influenza proteins, we have constructed a recombinant vaccinia virus containing the hemagglutinin gene of influenza A/Turkey/Ireland/1378/83 (H5N8). In mammalian cell culture the hemagglutinin expressed by this recombinant virus was full-length, cleaved into HA1 and HA2 in the absence of trypsin, and transported to the cell surface, confirming that other virus products are not required for cleavage activation. Chickens inoculated through the wing web with the live recombinant virus produced extremely low levels of hemagglutination-inhibiting or infectivity-neutralizing antibody. However, they were protected from lethal H5 influenza virus challenge. Protection extended to the antigenically distinct virulent H5 viruses, Chicken/Pennsylvania/1370/83 and Chicken/Scotland/59. Chemically bursectomized vaccinated chickens were not protected, whereas normal chickens with very low antibody levels (less than 10) obtained by passive transfer were protected in a dose-dependent fashion. This indicates that despite the low antibody titers induced by vaccination, protection was mediated by antibody.
A hepatocellular carcinoma cell line, LMH, has been established from a hepatocellular carcinoma induced in a male leghorn chicken by diethylnitrosamine. The cell line is characterized by well-differentiated morphological and biochemical features including the expression of glucose-6-phosphatase and canalicular ATPase activities and triploid karyotype with six marker chromosomes. The cells have been continuously propagated in culture for 5 yr and are now at about the 120th passage. Morphological change occurred in culture associated with gradual increase in growth rate at about the 40th passage. However, the biochemical and chromosomal features remained constant. This is the first established domestic fowl epithelial cell line and will allow comparative investigation of a number of parameters relevant to chicken hepatocarcinogenesis.
SUMMARY Previous work on the serological relationships of avian influenza A viruses provided an opportunity to assess the protective effects of anti-neuraminidase anti- body in a single species system. Three viruses naturally lethal for the chicken (A/DUTCH, A/TURKEY/ENGLAND/63 and A/TERN/SOUTH AFRICA/6I) were selected so that two (A/DUTCH and A/TURKEY/ENGLAND/63) had an antigenically similar haemagglutinin, two (A/TURKEY/ENGLAND/63 and A/TERN/SOUTHAFRICA/6I) had a similar neuraminidase and two (A/DUTCH and A/TERN/SOUTH AFRICA/6I) were unrelated by surface antigens. Each virus was used to immunize a group of 7-week-old chickens using one dose of killed virus followed by one dose of live virus. Each group was then divided into three subgroups and each subgroup challenged with one of the viruses. The results of cross-challenge with live virus were assessed objectively on mortality. (I) Immunization with any of the viruses pro- tected the birds completely against the homologous virus. (2) Immunization pro- tected the birds completely against a heterologous virus possessing a similar haemagglutinin. (3) Immunization gave partial protection against a heterologous virus possessing a similar neuraminidase and this protection was significantly greater (P : o-oi) than unimmunized controls when birds immunized with A/TURKEY/ENGLAND/63 virus were challenged with A/TERN/SOUTH AFRICA/6I. In birds immunized with A/TERN/SOUTH AFRICA/61 and challenged with A/TURKEY/ ENGLAND/63 the protection was approaching significance (P < o.I > o'o5). The results of testing the sera from the immunized birds confirmed the seriological relationships of the viruses.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.
The susceptibility of three avian cell lines (IQ1A, LMH, and QT-35) to infection by three strains of infectious laryngotracheitis virus (ILTV) was assessed both visually and by hybridization using an ILTV glycoprotein B gene probe. In the chicken liver tumor cell line (LMH), cytopathogenicity was observed at the second passage, and plaque formation was observed at the third passage. The identity of the infectious agent was verified to be ILTV by restriction endonuclease analysis of the virus genome and subsequent Southern hybridization. In contrast to LMH cells, which were a suitable host for ILTV, the quail cell line (IQ1A) was refractory to infection by this virus. Moreover, although LMH cell-adapted ILTV could initially replicate to a limited extent in the other quail cell line (QT-35), this ability was not sustained upon continual passaging.
Recently, we demonstrated that direct inoculation of a hemagglutinin 7 (H7)-expressing DNA could vaccinate chickens against a lethal H7 influenza virus challenge. These experiments used a defective-retroviral-based vector to express H7 (p188) (Robinson et al., 1993). Here, we report protective immunizations using a non-retroviral-based vector for H7 expression (pCMV/H7). Unlike the previously used retroviral-based vector, this vector cannot be transmitted as an infectious agent (as a consequence of phenotypic mixing with exogenous or endogenous virus proteins). Vaccination was accomplished by inoculating young, immunocompetent chickens by each of three routes (intravenous, intraperitoneal, and intramuscular) with 100 micrograms of cesium chloride-purified pCMV/H7 DNA in saline. After two immunizations, birds were challenged via the nares with a lethal dose of a highly virulent chicken influenza virus of the H7 subtype. The results of five independent vaccine trials demonstrated protective immunizations in approximately 60% of the pCMV/H7 DNA-inoculated chickens. By contrast, only 3% of the chickens inoculated with control DNA survived the lethal challenge.
We have cloned and sequenced the KpnI L and M restriction fragments of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1) DNA, which are localized adjacently at the right end of the unique long region of the genome. Within the 6930 bp DNA sequence six complete open reading frames (ORFs) were identified. The predicted amino acid sequences of four of them exhibit significant homologies to the UL5 (helicase), UL4, UL3 and UL2 (uracil-DNA glycosylase) genes, which are conserved in similar arrangement in all alphaherpesvirus genomes characterized up to now. A short ORF of 72 codons between UL3 and UL4 of ILTV is positionally homologous to the UL3.5 gene present in the genomes of different members of the Varicellovirus genus of alphaherpesviruses, but not in herpes simplex virus. The predicted ILTV protein encoded upstream of UL2 possesses limited sequence homology to the UL1 gene product of herpesviruses, the structural glycoprotein L. The presence of a N-terminal signal sequence, a conserved N-glycosylation site and two conserved cysteine residues indicates a similar function of the putative ILTV protein. Upstream of the UL1 gene of ILTV the C-terminal part of an additional ORF designated as ULO was identified, which exhibits no significant homology to known herpesvirus genes. RNA analyses indicate the expression of all detected ILTV genes including ULO.
We present a genomic map of infectious laryngotracheitis virus (ILT) and an 18,912 bp sequence containing the entire unique short region and a portion of the flanking short repeats. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains nine potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2, protein kinase, and glycoproteins G, D, I, and E (ORF 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (ORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine-rich, degenerate repeat sequence. This gene appears to be unique to ILT among sequenced herpesviruses. Two ORFs were identified within the short repeat (SR) region. SRORF 1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF 2 has homology to HSV US10.
Pseudorabies virus (PrV) is the causative agent of Aujeszky's disease which results in significant losses in pig husbandry. Recently we identified the gene encoding the deoxyuridine-triphosphatase (dUTPase) of PrV as the homolog of the UL50 gene of herpes simplex virus type 1. The PrV UL50 gene product was characterized and a UL50 negative PrV mutant (PrV UL50-) was generated by insertion of a lacZ expression cassette into the UL50 open reading frame (Jöns and Mettenleiter, J. Virol. 70, 1242-1245). Here we show that replication of PrV UL50- in cell culture was only slightly impaired as compared to wild-type PrV strain Ka. After intranasal infection of young pigs PrV UL50- proved to be substantially attenuated, whereas severe clinical signs and death occurred after infection with wild-type PrV. Challenge infection with the highly virulent NIA-3 strain of PrV showed that prior infection with PrV UL50- conferred protection against Aujeszky's disease. Innocuity and efficacy make UL50-negative PrV an attractive candidate for a live PrV vaccine.
The 24 kbp KpnI restriction fragment A from the unique long genome region of infectious laryngotracheitis virus (ILTV, gallid herpesvirus-1) has been sequenced. The analysed region contains 14 open reading frames sharing homology with conserved alphaherpesvirus genes. Arrangement of the UL6 to UL20 homologues of ILTV is almost identical to that found in the herpes simplex virus type 1 genome. As in other herpesviruses the UL15 gene consists of two exons and is expressed from a spliced mRNA. However, the UL16 gene, which is usually localized within the intron sequence of UL15, is not conserved at this position of the ILTV genome. Another unique feature is the absence of any putative N-glycosylation motifs within the deduced ILTV UL10 gene product, which is the homologue of the conserved herpesvirus glycoprotein M. After preparation of a monospecific antiserum, two distinct UL10 proteins with apparent molecular masses of 36 and 31 kDa were identified in ILTV-infected cells as well as in purified virions. None of these UL10 gene products is modified by N- or O-linked glycosylation. Isolation of a green fluorescent protein-expressing UL10 deletion mutant of ILTV revealed that this gene is not required for virus replication in cell culture.
Considerable progress has been made during the last years in understanding the molecular basis of protein function in pseudorabies virus (PrV), the causative agent of Aujeszky's disease (AD). Major topics have been the identification and functional characterisation of viral envelope glycoproteins and cellular virus receptors, elucidation of viral proteins involved in neurovirulence and neuropathogenesis, detection and characterisation of attenuating mutations present in and leading to successful attenuated live vaccines, and the near completion of the genomic sequence of PrV DNA. This review, which follows an article prepared for the 1993 AD symposium in Budapest, Hungary, will briefly summarise those recent developments and update the reader on the current state of the art in PrV research.
Only type A influenza viruses are known to cause natural infections in birds, but viruses of all 15 haemagglutinin and all nine neuraminidase influenza A subtypes in the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of their ability to cause disease. The very virulent viruses cause highly pathogenic avian influenza (HPAI), in which mortality may be as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI. All other viruses cause a much milder, primarily respiratory disease, which may be exacerbated by other infections or environmental conditions. Since 1959, primary outbreaks of HPAI in poultry have been reported 17 times (eight since 1990), five in turkeys and 12 in chickens. HPAI viruses are rarely isolated from wild birds, but extremely high isolation rates of viruses of low virulence for poultry have been recorded in surveillance studies, giving overall figures of about 15% for ducks and geese and around 2% for all other species. Influenza viruses have been shown to affect all types of domestic or captive birds in all areas of the world, but the frequency with which primary infections occur in any type of bird depends on the degree of contact there is with feral birds. Secondary spread is usually associated with human involvement, probably by transferring infective faeces from infected to susceptible birds.
From October 1997 to January 1998, highly pathogenic H5N2 avian influenza viruses caused eight outbreaks of avian influenza in northern Italy. A nonpathogenic H5N9 influenza virus was also isolated during the outbreaks as a result of virological and epidemiological surveillance to control the spread of avian influenza to neighbouring regions. Antigenic analysis showed that the Italian H5N2 isolates were antigenically similar to, although distinguishable from, A/HK/156/97, a human influenza H5N1 virus isolated in Hong Kong in 1997. Phylogenetic analysis of the haemagglutinin (HA) genes showed that the highly pathogenic Italian viruses clustered with the Hong Kong strains, whereas the nonpathogenic H5N9 virus, despite its epidemiological association with the highly pathogenic Italian isolates, was most closely related to the highly pathogenic A/Turkey/England/91 (H5N1) strain. Like the HA phylogenetic tree, the nonstructural (NS) phylogenetic tree showed that the H5N2 Italian virus genes are clearly separate from those of the H5N9 strain. In contrast, results of the phylogenetic analysis of nucleoprotein (NP) genes indicated a closer genetic relationship between the two Italian virus groups, a finding suggesting a common progenitor. Comparison of the HA, NS and NP genes of the Italian H5 strains with those of the H5N1 viruses simultaneously circulating in Hong Kong revealed that the two groups of viruses do not share a recent common ancestor. No virological and serological evidence of bird-to-human transmission of the Italian H5N2 influenza viruses was found.
Sequence of the infectious laryngotracheitis virus (SA-2 strain) unique long region UL28 to UL43, Unpublished
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M.A. Johnson, Sequence of the infectious laryngotracheitis virus (SA-2 strain) unique long region UL28 to UL43, Unpublished; GenBank accession number AF168792.
Complete se-quence of a cDNA clone of the hemagglutinin gene of influenza AH5N1) virus: comparison with contem-porary North American and European strains
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De BK, Brownlee GG, Kendal AP, Shaw MW. Complete se-quence of a cDNA clone of the hemagglutinin gene of influenza A/Chicken/Scotland/59 (H5N1) virus: comparison with contem-porary North American and European strains. Nucleic Acids Res 1988;16:4181 – 2.
Highly pathogenic avian influenza, In: OIE Manual of Standards for Diagnostic Tests and Vaccines
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pp. 155 – 60.