a sucrose gradient and inactivated using binary ethylenimine (BEI). 20 g body weight) were purchased from a commercial supplier (Bomholdtgard). ( ) Relationship between transforming growth factor beta1 and. The combination of these two variables allows the manufacturer to either . significant deviation from Salk's linear relationship was observed (Gard a, b; AE () Rabies virus inactivation by binary ethylenimine: new method for. template, and that the question on production and supply of reference binary ethyleneimine (BEI) This is usually prepared by dissolving, to a . relation to the vaccine strain being tested and the serological method being.
Furthermore, both strains showed significant anti-fibrotic activity in two rat models of liver fibrosis. However, iORFV strain NZ2 appeared more potent compared to strain D in both its antiviral and antifibrotic activity based on dosage calculations using tissue culture infectious dose 50 TCID50 of active virus. Debris was removed by centrifugation.
The supernatant was decanted and centrifuged Beckman, SW 28 for 6 hours at The virus was purified through a sucrose gradient and inactivated using binary ethylenimine BEI. Experimental results have been confirmed in independent experiments.
In order to reduce edge effects, the 36 wells at the outer rim of the well plate were not used, but were filled with assay medium. After three days cells were rinsed with phosphate buffered saline and NS3 protease activity as an accurate readout for HCV replication was determined by using a FRET assay which has been described previously . Animals HBV transgenic mice: The mice used in the experiments produced — HBV genome equivalents per ml plasma as determined by quantitative real time PCR .
For induction of liver fibrosis with either pig serum or Carbon tetrachloride CCl4 in rats, female Sprague Dawley rats at a body weight of — g Moellegaard, Lille Skensved, Denmark were used.
All efforts were made to minimize suffering. Quantitative analysis of hepadnaviral nucleic acid in livers of HBV-transgenic mice was performed using dot-blot analysis and HBV DNA in plasma was analyzed by quantitative PCR essentially as described previously .
However, the full dose response in HBV-transgenic mice did include three instead of four dosages in the repeat although in individual experiments each dose has been repeatedly studied. Pig Serum-induced Liver Fibrosis ORFV prevents both the development of fibrosis and the activation of collagen-producing cells in rat models of liver fibrosis .
Rats were sacrificed after 7 weeks and livers were subjected to collagen determination. Serial sections were stained with Sirius Red fibrous collagen and scored using a three class system normal histology, beginning fibrosis with sprouting, unconnected septae and full-blown fibrosis with a network of interconnected septae.
Hydroxyproline was measured as described recently Picosirius stained scar collagen was quantified as described below for CCl4-induced liver fibrosis. Immunohistology was performed as described . The percentage of a standardized centrilobular area stained was determined for each animal by an automated, blinded procedure. Histology and morphometric analysis was performed essentially as described recently .
Briefly, for morphometry of picosirius stained scar collagen we used a Leica QWin image analysis and processing system with motorized stage. Reproducibility of the results has been confirmed in independent experiments. LSEC were seeded onto collagen type I coated well tissue culture plates at a density of Vaccines rely on the effective stimulation of the immune response against the virus, mostly against the surface glycoprotein hemagglutinin HAthe primary immunogen of influenza viruses.
Influenza vaccines were a high priority for the U. However, it was not until that influenza vaccines became available for the general population www. In this review, we will discuss the use of vaccines to prevent and control influenza in animals and humans. We will highlight challenges faced to establish an effective and standardized vaccination program and discuss new approaches that are being tested to address these issues. Also on the membrane, the matrix protein 2 M2 in IAV and BM2 in IBV acts as a unidirectional proton pump, which is essential early in the infection cycle by allowing the release of vRNPs from the endosome into the cytoplasm and subsequent migration to the nucleus.
The non-structural protein NS1 carries out multiple functions during infection with overall antiviral antagonistic activity Knipe et al. Top Schematic structure and genome organization of influenza A and B viruses. Matrix protein 1 M1 is associated with the membrane. Non-structural nuclear export protein NEP.
On the left panels each monomer is shown with a different color. The receptor binding site RBS is highlighted in red. On the right panels, residue conservation for every position in the protein sequence is shown in a color scale, and visualized using the 3D tool available at the Influenza Research Database www.
The HA ectodomain also consists of a globular head domain, and the stalk or stem domain, which are responsible for receptor binding and membrane fusion, respectively Knipe et al. The NA is a type 2 glycoprotein present as mushroom-shaped homotetramers. IAV are further classified into H H and N N subtypes according to the antigenic characteristics of the HA and NA, most of them detected in wild aquatic birds, considered the natural hosts of influenza.
Only H1N1 and H3N2 subtypes currently circulate in humans. H1 and H3 subtypes combined with either N1 or N2 subtypes are endemic in pigs. In addition, permanent lineages of IAVs of the H3 subtype circulate in horses and dogs. A wider range of IAV subtypes have become established in land-based birds of the order Galliformes e.
IBVs are usually restricted to humans, and two antigenically distinct lineages circulate, the Victoria-like and the Yamagata-like lineages.
Influenza A and B viruses accumulate mutations due to the P-complex's lack of proof-reading activity. When these mutations occur in the HA and NA, they lead to antigenic drift, which over time results in escape from earlier immune responses. Antigenic shift is possible through reassortment resulting from the exchange of gene segments between two or more strains.
Reassortment plays a significant role in the evolution of IAVs in the natural reservoir and during the emergence of pandemic strains. The Animal-Human Interface of Influenza Ecology IAVs that typically infect a particular host species can sometimes cross the species barrier and infect a new host. Animal species such as poultry and swine have significant interactions at the animal-human interface, providing ideal environments for zoonotic transmission events of IAV.
These zoonotic transmissions pose a significant threat to public health, not only because of the disease severity and mortality seen in some cases, but also the risk of initiating a pandemic if these viruses become adapted to spread among humans. All human pandemics that happened in the last years originated from viruses with gene segments from animal reservoirs Taubenberger and Morens, Humans usually become infected with animal viruses as a result of close contact with a particular animal species.
Some of the most prominent poultry-adapted IAVs have been responsible for zoonotic outbreaks and are, therefore, of great public health concern. Since the first detection of H5N1 HPAIV in and re-emergence as an epidemic in poultry inhuman infections have been reported, of which were lethal. Most these cases are the result of direct contact with infected poultry World Health Organization, These H7N9 viruses usually result in severe acute respiratory disease, and until September had resulted in laboratory-confirmed human infections and deaths Food Agriculture Organization of the United Nations, Despite the usual mild clinical signs, swine-origin IAVs also represent a real threat to public health due to limited population immunity and potential to become widespread.
The H1N1 pandemic was a result of a swine-origin IAV with a unique combination of gene segments that had not been detected in pigs before, and quickly became globally widespread Smith et al. After the H1N1 pandemic virus H1N1pdm09 established itself in the human population, it transmitted back to pigs, where it continued to reassort with other IAVs of swine origin.
Human infections with vH3N2 IAVs are linked with close contact with infected pigs at agricultural fairs Epperson et al. IAVs infect a range of hosts with the potential to jump between multiple species.
IAVs of swine-origins are often detected in commercial turkeys in the U. IAV transmission events from humans to swine are frequent and have influenced the diversity of viruses circulating in pigs: The influenza vaccine can protect against influenza viruses that are antigenically the same or related to the viruses in the vaccine. Due to the mutating nature of influenza viruses, annual vaccination is recommended.
However, two antigenically distinct lineages of influenza B viruses have circulated globally since the mid 's with limited cross-protection between them Ambrose and Levin, Recommendations for influenza vaccination vary between countries.
Most countries recommend vaccination in children, the elderly, chronically ill, and health care professionals. However, vaccine efficacy can vary widely between seasons, depending particularly on how well matched are vaccine and circulating strains, and on characteristics of the person being vaccinated. Vaccination of pigs is routinely used in several countries, but not all endemic countries vaccinate their herds. In swine, vaccination is typically done in gestating sows to transfer maternally derived antibodies MDA through colostrum to their litters, in two to three doses before farrowing Rajao et al.
Typically, vaccination of sows will protect the litter from clinical signs of disease, and MDAs persist until about 14 weeks of age in piglets Markowska-Daniel et al.
Aziridine | Revolvy
Occasionally and in herds with high virus circulation, vaccination in growing pigs can protect them against infection once MDA have weaned. Vaccination is common in some countries where avian influenza viruses have become endemic to prevent and control at-risk populations, most commonly against H5, H7, and H9 viruses, particularly in Egypt, Vietnam, China, Indonesia, and Mexico Domenech et al.
Vaccination has also been successfully used as a control tool to aid in HPAIV eradication programs in poultry and wild birds in some countries, in situations where the regular stamping-out protocols are not enough to control spread and poses a threat to food supply reviewed in Spackman and Pantin-Jackwood Vaccination of poultry against influenza viruses is still seen with reluctance by many, due to the understanding that vaccines can protect against clinical signs but not infection and, therefore, mask outbreaks and may favor the spread of HPAIV.
The Immune Response Elicited by Vaccination The local innate immune response is critical for limiting viral replication at the initial phase of infection. Particularly, alveolar macrophages Kim et al. Activation of PRRs stimulates production of pro-inflammatory cytokines and type I interferons reviewed in Kreijtz et al.
Inactivated vaccines are poor inducers of innate immunity to give immediate protection. However, a study has demonstrated that binding of UV-inactivated influenza virus to sialic acids SAs can trigger intracellular signals for activation of IFN-inducible genes and cytokine production in primary human dendritic cells Ramos et al. Immunization with traditionally inactivated influenza vaccines primarily induces virus-specific adaptive antibody responses.
Antibodies against the HA protein, predominantly the globular head domain, are the major protective response against influenza since they usually correlate with surrogate in vitro assays such as the hemagglutination inhibition HI or virus neutralizing VN assays.
A major hurdle to overcome is that the HA globular head is highly variable and harbors mutations that can lead to antigenic variation. Most HA-head-specific antibodies are only protective against closely antigenically related viruses.
In contrast, the stalk domain is more conserved. Influenza infection can result in low levels of antibodies against the stalk region, but these antibodies can have broadly neutralizing activity Ekiert et al. In pigs, however, vaccine-induced antibodies to a linear epitope located in the stalk domain increased virus fusion and enhanced respiratory disease, possibly involved with a negative effect Khurana et al.
However, in mice passive transfer of post-vaccination polyclonal sera resulted in reduced lung virus replication and weight loss compared to animals receiving pre-vaccination sera Nachbagauer et al. Antibodies against the other surface proteins, NA and M2, are not considered neutralizing by traditional assays but studies showed that they limit virus replication and spread in vivo.
These CTLs are mainly directed against the relatively conserved internal proteins NP, M1, and the P-complex subunits, which confer a high degree of cross-reactive immune response to various influenza strains in humans and swine Heinen et al.
Not only these CTL aid in virus clearance, but also with protection against disease severity. Current traditional inactivated vaccines do not stimulate cellular immune responses and, therefore, are usually less effective against heterologous influenza infections, but novel technologies to stimulate T cell immunity are being tested Soema et al. Antigenic drift requires frequent vaccine updates to match circulating viruses and meet efficient coverage.
Meetings are held twice a year to select vaccine strains for the following influenza season. The meetings take place in February for the Northern Hemisphere and in September for the Southern Hemisphere, at least 6 months before their respective influenza seasons. Based on antigenic data from ferret antisera, paired serologic analysis of human samples, and virologic surveillance data, the GISRS recommends that vaccine strains are either maintained or updated to offer more effective protection.
Usually following WHO recommendations, each country and its regulatory agencies decide about the influenza vaccines licensed in their territory. In general, each vaccine strain in the vaccine requires an update every 2—3 years, with at least one strain updated each year.
Vaccine Strains Selection and Production for Animal Use Except for vaccine strain selection against equine influenza, for systematic vaccine strain selection or updates is lacking for other animal production systems.
A panel of specialists coordinated by the World Organization for Animal Health OIE meets annually to analyze surveillance and epidemiological data related to worldwide circulation of IAVs in horses. Genetic sequences and antigenic data are used for recommendations of suitable vaccine strains for inclusion in commercial equine influenza vaccines Paillot, Both of these efforts have generated consistent flow of genetic data that is useful for vaccine strain choice.
Still lacking are collaborative panels to continuously analyze this surveillance data compared to antigenic characteristics of main circulating strains.
Unlike IAVs in humans, IAVs in pigs do not follow common evolutionary trends and tend to adopt unique evolutionary pathways depending on the country, region, and even at the farm level. The high diversity and heterogeneous distribution of different viral lineages circulating in pigs represents a major obstacle for effective vaccine choices against swine influenza Anderson et al. Therefore, swine influenza vaccine manufacturers make independent decisions about the strains to include in their products, and those formulations are not often updated.
Likewise, despite intense routine worldwide IAV surveillance in intensive poultry producing areas, the lack of structures to better assess the effects of the use of vaccines in the perpetuation and evolution of IAV in poultry often means that the available commercial vaccines available are not antigenically matched, resulting in less-than-optimal protection Spackman and Pantin-Jackwood, This network aims to help recognized emerging strains, provide training and advice, to increase collaboration and data sharing within the scientific community, and to help promote animal influenza research and development.
Conventional Vaccines against Influenza Most influenza vaccines on the market for humans and animals are inactivated influenza vaccines IIV. Regardless of the vaccine strain or manufacturing platform in which the vaccine is made, influenza vaccines are typically produced by growing the target viruses in chicken eggs, which is reliant on continual egg supply.
Unfortunately, most recent influenza viruses do not grow well in eggs which affects the efficiency of the traditional reassortment method.
More importantly, forcing these vaccine viruses to grow in eggs often results in egg-adapted changes associated with antigenic mismatches. In the case of HPAIVs, their inherent high lethality for chicken embryos makes them unsuitable as vaccine strains.
Some of these limitations are overcome by reverse genetics techniques that allow generation of whole recombinant influenza viruses entirely from cloned DNA Neumann et al. Thus, reverse genetics allowed to increase the yield of some of the components of LAIV or to mutate the cleavage site of H5N1 HPAIVs and make them suitable for preparation of vaccine seed stocks for pandemic preparedness or as poultry vaccines.
Whole wild-type viruses are predominantly used for traditional animal vaccines.