Egg Passage Research Articles

Advances in antiviral vaccine development.

Antiviral vaccines have been the most successful biomedical intervention for preventing epidemic viral disease. Vaccination for smallpox in humans and rinderpest in cattle was the basis for disease eradication, and recent progress in polio eradication is promising. Although early vaccines were developed empirically by passage in live animals or eggs, more recent vaccines have been developed because of the advent of new technologies, particularly cell culture and molecular biology. Recent technological advances in gene delivery and expression, nanoparticles, protein manufacturing, and adjuvants have created the potential for new vaccine platforms that may provide solutions for vaccines against viral pathogens for which no interventions currently exist. In addition, the technological convergence of human monoclonal antibody isolation, structural biology, and high-throughput sequencing is providing new opportunities for atomic-level immunogen design. Selection of human monoclonal antibodies can identify immunodominant antigenic sites associated with neutralization and provide reagents for stabilizing and solving the structure of viral surface proteins. Understanding the structural basis for neutralization can guide selection of vaccine targets. Deep sequencing of the antibody repertoire and defining the ontogeny of the desired antibody responses can reveal the junctional recombination and somatic mutation requirements for B-cell recognition and affinity maturation. Collectively, this information will provide new strategic approaches for selecting vaccine antigens, formulations, and regimens. Moreover, it creates the potential for rational vaccine design and establishing a catalogue of vaccine technology platforms that would be effective against any given family or class of viral pathogens and improve our readiness to address new emerging viral threats.

Avian Influenza: Mixed Infections and Missing Viruses

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.

Th2 responses in schistosomiasis

Schistosomiasis is caused by infection with parasitic flatworms of the genus Schistosoma. It is characterized by the development of strong CD4(+) T cell and B cell responses that, during primary infection, fail to eliminate the parasites, but in collaboration with cells of the innate immune system allow survival in the face of ongoing tissue damage caused by the lodging of parasite eggs in the liver and the passage of eggs across the intestinal epithelium. Mounting a tightly controlled Th2 response is key to this outcome, and while this type of response is a risk factor for the development of fibrosis, it also underpins the development of resistance to further infection; as such, understanding how Th2 responses are induced and regulated in schistosomiasis remains a critical area of research.

Evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the "sweating" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

Fat flu: the obese host in influenza virus evolution

During the 2009 influenza pandemic, obesity was identified as an independent risk factor for increased disease severity and mortality. These epidemiological findings have been confirmed using obese mouse models. Previous studies in mice have demonstrated that nutritional status can affect virulence of RNA viruses including influenza. The global epidemic of obesity combined with increased disease severity in obese subjects prompted us to question whether obesity could influence influenza virus evolution. To determine if the obese lung microenvironment could differentially affect viral adaptation, A/California/04/2009 influenza virus was passaged five times in either lean or diet‐induced obese (DIO), C57Bl/6 male mice. Lean and DIO mice were then infected with parental and passaged viruses and monitored for morbidity and mortality. Interestingly, virus passaged in DIO mice induced greater morbidity, pathology and mortality in both lean and DIO animals compared to lean‐passaged virus. Studies are underway to define the molecular determinants associated with the enhanced pathogenicity; however, the obese virus retained increased pathogenicity following egg passage suggesting genetic changes to the virus itself. These studies suggest that the expanding obese population may not only be more susceptible to disease, but may differentially regulate viral evolution towards more virulent viral strains.Grant Funding Source: NIH NIAID contract number HHSN266200700005C and the American Lebanese Syrian Associated Charities (ALSAC)

Current applications of oogram methodology in experimental schistosomiasis; fecundity of femaleSchistosoma mansoniand egg release in the intestine of AKR/J mice following immunomodulatory treatment with pentoxifylline

Oogram methodology permitted a direct assessment of the fecundity of Schistosoma mansoni and the passage of parasite eggs from mesenteric vessels into the intestinal lumen in AKR/J mice that had been infected via the intraperitoneal route with 60 cercariae of the trematode and later subjected to short-term subcutaneous treatments with pentoxifylline (PTX). The administration of PTX did not change oviposition kinetics, the individual fecundity of female parasites (as determined by the study of the ratio of second-stage S. mansoni eggs per g intestine/number of female parasites recovered from the portal system), nor the number of mature and dead eggs retained in the host tissue, though the drug has known immunomodulatory effects, as shown previously in experimental schistosomiasis. A better appraisal was also carried out, including the study of statistical parameters, concerning the utilization of the ratios of each stage of immature eggs (first to fourth) per g intestine/number of female worms from the portal system. The second-stage eggs had the lowest variability, confirming that the utilization of this stage as an indicator of the individual fecundity of parasite females is indeed viable. In the light of our findings, current uses of oogram methodology are discussed. Moreover, additional consideration is given to data obtained in the present study concerning intraperitoneal infection with S. mansoni cercariae in both untreated and treated mice of the AKR/J strain, such as the recovery of mature worms, eggs and free granulomas from the peritoneal cavity of these rodents.

Modification of the avian coronavirus infectious bronchitis virus for vaccine development

Infectious bronchitis virus (IBV) causes an infectious respiratory disease of domestic fowl that affects poultry of all ages causing economic problems for the poultry industry worldwide. Although IBV is controlled using live attenuated and inactivated vaccines it continues to be a major problem due to the existence of many serotypes, determined by the surface spike protein resulting in poor cross-protection, and loss of immunogenicity associated with vaccine production. Live attenuated IBV vaccines are produced by the repeated passage in embryonated eggs resulting in spontaneous mutations. As a consequence attenuated viruses have only a few mutations responsible for the loss of virulence, which will differ between vaccines affecting virulence and/or immunogenicity and can revert to virulence. A new generation of vaccines is called for and one means of controlling IBV involves the development of new and safer vaccines by precisely modifying the IBV genome using reverse genetics for the production of rationally attenuated IBVs in order to obtain an optimum balance between loss of virulence and capacity to induce immunity.

Downstream passage of fish larvae and eggs through a small-sized reservoir, Mucuri river, Brazil

In South America, one important symptom of the failure of fish passages to sustain fish migratory recruitment is the inability of eggs and larvae to reach the nurseries. This is especially so when the breeding areas are located upstream of a reservoir, and the floodplain is downstream of the dam. Therefore, the transport of fish larvae and eggs across reservoir barriers is a key factor in the development of effective conservation strategies. In this paper, we evaluate the potential for migratory fish larvae and egg transportation across a small size reservoir in eastern Brazil. We sampled fish daily between 15th October 2002 and 15th February 2003 (spawning period) in the Mucuri River, immediately upstream of the reservoir and downstream of the Santa Clara Power Plant dam. Our study was the first to indicate the possibility of successful larval passage through the reservoir of a hydroelectric reservoir and dam in South America, and showed that the passage of migratory fish larvae was associated significantly with residence time of water in the reservoir. The relatively short water residence time and elevated turbidity of the Santa Clara's reservoir waters during the rainy season certainly contributed to the successful passage, and can be considered as key factors for a priori evaluations of the feasibility of a downstream larval passage.

Improving diagnosis of urogenital schistosome infection

Evaluation of: Ibironke OA, Phillips AE, Garba A, Lamine SM, Shiff C. Diagnosis of Schistosoma haematobium by detection of specific DNA fragments from filtered urine samples. Am. J. Trop. Med. Hyg. 84(6), 998–1001 (2011).Schistosomiasis (commonly known as bilharzia or snail fever) is the second (to malaria) most important human parasitic disease in tropical and subtropical in regions. In Africa, Schistosoma haematobium, the causative agent of urogenital schistosomiasis, is the most prevalent species causing human disease and is responsible for most of the schistosome-related disease in the region. Diagnosis of morbidity in field settings mainly relies on the detection of hematuria (blood in the urine) and proteinuria (protein in the urine) which results from the passage of parasite eggs through the bladder wall. Ultrasound scans of the urinary tract are also used to detect morbidity but are less practical in the majority of field settings owing to the requirement of specialized equipment and trained personnel. Current diagnosis of infection relies on detecting excreted eggs and excreted or circulating parasite products. Diagnostic methods include microscopic examination of eggs in urine (currently considered the gold standard), microscopic examination of tissue biopsies, serological and reagent strip diagnosis of circulating parasite proteins detectable in blood and urine and, more recently, detection of parasite DNA in urine or vaginal lavage samples. All currently used diagnostic methods have limitations associated with them. In particular, the gold standard microscopic enumeration of eggs in urine is less sensitive in low infections and does not detect single sex or prepatent infections, which makes it particularly inaccurate in young children harboring light infections and in older individuals with chronic infections who both excrete low levels of eggs. The detection of parasite DNA in urine samples by PCR described in the article by Ibironke et al. improves on this limitation. This article reviews the method described by Ibironke et al., compares it with current methods and discusses its potential use in field settings.

Generation of a recombinant rabies Flury LEP virus carrying an additional G gene creates an improved seed virus for inactivated vaccine production.

The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75–43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-011-0668-7) contains supplementary material, which is available to authorized users.

Parasitological and morphological study of Schistosoma mansoni and diabetes mellitus in mice

Schistosomes are blood-dwelling flukes which are highly dependent on the host metabolism. The aim of this study was to investigate possible relationship between streptozotocin-induced diabetes and the outcome of acute murine schistosomiasis mansoni. Male and female SW mice were treated by a single intraperitoneally injected dose of streptozotocin (180mg/kg). Seven days after induction, both control and diabetic animals were infected with 70 Schistosoma mansoni cercariae (BH strain). Diabetics and their controls were weighed 45days after birth and for the last time prior to killing. Susceptibility to infection was evaluated twice a week by quantifying fecal egg excretion 7–9weeks post-infection by the Kato–Katz’ thick smear method. Mice were euthanized the day after the last fecal examination was performed. Adult worms were recovered from the portal system and mesenteric veins, whereas liver and intestine were removed for enumeration of egg load. No differences in worm length or in measurements of the reproductive organs, tegument, and suckers were detected. Also oviposition was unaffected as the total number of eggs per female worm from the liver, the small and the large intestine was the same in both groups. An oogram evaluation revealed a lower percentage of mature (23.0% vs. 40.7%) and a higher percentage of immature (69.1% vs. 51.7%) eggs in the small intestine of the diabetic mice. We suggest that principally a hampered egg passage through the intestine tissue caused this reduction and that probably both the eggs and the impaired host response play a role.

Bilateral Ovulation Fossa Inclusion Cysts in Miranda Jennets

Multiple cysts ranging from 2-111 mm were noted bilaterally in the ovulation fossa of 11 mature Miranda jennets. These ovulation fossa inclusion cysts (OFICs) were lined by a simple low to columnar epithelium that included many ciliated cells. Although most cases were incidental findings, two of the jennets were presented with reduced fertility. Extensive cyst formation could have been responsible for the reproductive problems because they replaced most of the ovarian parenchyma. Due to their close proximity to the ovulation fossa, the OFICs may have mechanically interfered with passage of eggs into the oviduct. OFICs are histologically common in equids, but are reported uncommonly as gross lesions in either mares or jennets. Ovarian inclusion cysts are associated with neoplasia in women; however, these OFICs showed no evidence of epithelial hyperplasia or cellular atypia and no evidence of independent growth, therefore they were considered to be non-neoplastic. The bilateral occurrence and high incidence of OFICs in Miranda jennets, a breed with limited genetic variability, suggests that the lesion has a genetic causation.

Passage of fish larvae and eggs through the Funil, Itutinga and Camargos Reservoirs on the upper Rio Grande (Minas Gerais, Brazil)

The objective of this study was to evaluate the passage of fish eggs and larvae through the Funil, Itutinga and Camargos Reservoirs, located in the upper Rio Grande basin, Minas Gerais, Brazil. Samples were taken downstream and upstream of the dams using a conical ichthyoplankton net and were collected every two weeks, twice per sampling day, between November 2008 and March 2009. Although eggs and larvae were abundant immediately upstream of the reservoirs, no ichthyoplankton were captured immediately downstream of the dams, possibly indicating that eggs and larvae do not pass through the reservoirs. The arrival of ichthyoplankton in the reservoirs without its effective passage downstream makes the survival of these eggs and larvae unlikely. Furthermore, this lack of downstream movement may compromise the recruitment of species to downstream stretches, especially in the case of the Funil Reservoir (because of the presence of a fish pass in this dam). We emphasize that the fish lift operation at the Funil Dam must be carefully assessed, considering not only its efficiency but also its short- and long-term effects on the recruitment of migratory fish species from the river.

Generation of Replication-Competent Recombinant Influenza A Viruses Carrying a Reporter Gene Harbored in the Neuraminidase Segment

Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An "autocleave" 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

Rapid heat-treatment attenuation of infectious bronchitis virus

In the present study we describe the rapid development of an attenuated live vaccine for GA08, a new serotype of infectious bronchitis virus, using a heat-treatment method. Incubation of the GA08 strain of IBV at 56°C and passage in embryonated eggs was used as a method to fast track the attenuation process. The virus was incubated in a 56°C water bath and aliquots were removed every 5 min for up to 1 h, and then each aliquot was inoculated into 10-day-old embryonated eggs. Virus with the longest incubation time that produced lesions in the embryos was harvested, again incubated at 56°C as described and passaged in embryonated eggs. Attenuation of the virus, designated GA08/GA08HSp16/08, was verified in 1-day-old specific pathogen free chicks. A 10x dose of the vaccine was found to be safe for 2-week-old broiler chicks of commercial origin. The efficacy of the heat-treated attenuated virus was determined by vaccinating broiler chicks of commercial origin at 1 and 14 days of age intraocularly/intranasally. Vaccinated birds that were challenged with 104.5 median embryo infectious doses of pathogenic GA08 virus/bird at 28 days of age were protected from the disease, and challenge virus was only detected in the trachea of one of 21 birds by real-time reverse transcriptase-polymerase chain reaction at 5 days post challenge. The attenuation process took 10 weeks to complete, which is a substantially shorter time than required to attenuate infectious bronchitis virus by serial passage in embryonated eggs without heat treatment (38 weeks or more).

Evaluation of a New Technique for Determining Sex of Adult White-Winged Doves (Zenaida asiatica)

Abstract Previous studies have shown that determining sex of adult white-winged doves (Zenaida asiatica) by cloacal characteristics is highly reliable (90%), while using plumage characteristics and body weights are less so. The drawback of the cloacal technique is that the procedure requires use of a modified nasal speculum to probe the cloacal area, which could possibly cause injury to the bird. We hypothesized that females have a wider pelvis than males for the passage of eggs, which should be quantifiable, thereby providing a means of separating adult males and females. We measured pelvic spread (distance from tip to tip of pelvis) of adults (n = 249). Additionally, body masses were taken for a subset (n = 215) of the doves. Sex was positively determined by either cloacal examination of live birds or gonad examination of dead birds. Mean pelvic spread of males was significantly less than that of females. Whole body mass was not correlated with pelvic spread for either sex. Accuracy of the pelvic sp...

The impact of key amino acid substitutions in the hemagglutinin of influenza A (H3N2) viruses on vaccine production and antibody response

Influenza H3N2 viruses have recently drifted from A/Wisconsin/67/05-like to A/Brisbane/10/07-like viruses. The effect of key amino acid substitutions in the hemagglutinin (HA) protein on the replication, antigenicity and immunogenicity of cold-adapted, live attenuated vaccine strains was examined. A/Brisbane/10/07 egg isolate contained a unique combination of G186 and P194 which were required for efficient virus growth in Madin–Darby Canine Kidney (MDCK) cells and embryonated chicken eggs, but the virus induced low level of serum antibody response in ferrets. Substitution of the G186 and P194 in the HA of A/Brisbane/10/07 by V186 and L194 that were present in the HA of A/Wisconsin/67/05-like viruses significantly impaired virus replication but greatly improved the immunogenicity of the vaccine virus to a level comparable to that elicited by the A/Wisconsin/67/05 vaccine. The replication of the variants with impaired growth could be improved by amino acid substitutions at the 195 or 226 residues. The viruses with the G186 and P194 residues were antigenically distinct from the viruses with V186 and L194. Sequence analysis of the HA sequences of the H3N2 viruses from the database and sequencing of the HA gene of a cell-derived A/Brisbane/10/07-like virus before and after egg passage indicated that the P194 residue was most likely derived from egg adaptation. Our results demonstrated the importance of careful evaluation of vaccine strains to ensure that the selected vaccines not only replicate well in eggs, but also retain their antigenicity and are immunogenic in the host.

PB2 and PA genes control the expression of the temperature-sensitive phenotype of cold-adapted B/USSR/60/69 influenza master donor virus

The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 degrees C and 25 degrees C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.

Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.