Kinetic stability modulation of polymeric nanoparticles for enhanced detection of influenza virus via penetration of viral fusion peptides.

Specific interactions between viruses and host cells provide essential insights into material science-based strategies to combat emerging viral diseases. pH-triggered viral fusion is ubiquitous to multiple viral families and is important for understanding the viral infection cycle. Inspired by this process, virus detection has been achieved using nanomaterials with host-mimetic membranes, enabling interactions with amphiphilic hemagglutinin fusion peptides of viruses. Most research has been on designing functional nanoparticles with fusogenic capability for virus detection, and there has been little exploitation of the kinetic stability to alter the ability of nanoparticles to interact with viral membranes and improve their sensing performance. In this study, a homogeneous fluorescent assay using self-assembled polymeric nanoparticles (PNPs) with tunable responsiveness to external stimuli is developed for rapid and straightforward detection of an activated influenza A virus. Dissociation of PNPs induced by virus insertion can be readily controlled by varying the fraction of hydrophilic segments in copolymers constituting PNPs, giving rise to fluorescence signals within 30 min and detection of various influenza viruses, including H9N2, CA04(H1N1), H4N6, and H6N8. Therefore, the designs demonstrated in this study propose underlying approaches for utilizing engineered PNPs through modulation of their kinetic stability for direct and sensitive identification of infectious viruses.

Detección e identificación de Salmonella spp. en huevos para consumo humano, provenientes de diferentes localidades de Bogotá, Colombia, 2015 / Salmonella spp. isolation and identification in eggs for human consumption from different urban areas in Bogotá, Colombia, 2015

Objetivo: Determinar la prevalencia de Salmonella spp. en muestras de huevos para consumo humano en localidades de Bogotá. Materiales y métodos: Se obtuvieron 96 muestras en tiendas y plazas de mercado de 4 localidades de la ciudad. Se procesó de forma separada la cáscara y el contenido interno mediante métodos microbiológicos y moleculares para aislamiento e identificación Salmonella spp. Resultados: Se determinó una prevalencia total de Salmonella spp. de 9,4% (n=9), de ésta el 55% (n = 5) provenían del contenido interno y 44% (n = 4) de cáscara, sin embargo no se logró tipificar el serovar de Salmonella enterica presente. Las localidades con mayor presencia del patógeno fueron Usaquén y Fontibón. Discusión: Estudios realizados en Colombia evidencian bajas prevalencias de Salmonella spp. (0 -1,74%) en muestras de huevos, sin embargo los hallazgos de este estudio evidencian una mayor recuperación, lo que podría asociarse con inadecuadas condiciones de manejo y/o almacenamiento del producto. Conclusión: Se estableció la presencia de Salmonella spp. en las muestras de huevo evaluadas, lo que implica un riesgo potencial para la salud pública, por lo que es necesario ampliar este tipo de estudios para conocer la situación real a nivel nacional frente a este patógeno.

Objective: To determine the prevalence of Salmonella spp presence in eggs for human consumption in urban areas in Bogota. Materials and methods: 96 samples were collected from convenience stores and markets in 4 urban areas in the city. The eggshells were separated from the egg's internal content and both were processed separately using microbiological and molecular techniques to isolate and identify Salmonella spp. strains. Results: A Salmonella spp. prevalence of 9.4% (n=9) was found. Salmonella spp was isolated from the egg's internal content in 55% (n=5) of samples and 44% (n=4) from the eggshells. The Salmonella enterica serovar could not be identified. The pathogen was more frequently isolate in samples from Usaquén and Fontibón urban areas. Discussion: Studies of Salmonella spp. prevalence in eggs done in Colombia have shown it to be low (0-1.74%); However, this study determined a higher prevalence. These results suggest that inadequate handling/storage conditions could have been associated with them. Conclusion: Salmonella spp. was isolated from the egg samples from 4 different urban areas in Bogotá. These findings suggest the existence of a public health risk; therefore, there is a need to perform wider and more complete studies to determine the actual situation of Salmonella ssp. egg contamination in the country.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Analysis by high throughput sequencing of Specific Pathogen Free eggs.

Specific Pathogen Free (SPF) embryonated eggs are used for the production of many veterinary and human vaccines. We have used High Throughput Sequencing to screen allantoic fluids and embryos for the presence of encapsidated viral genomes and viral transcripts, respectively. SPF eggs from two different producers were tested. We evidenced sequences corresponding to known endogenous retroviruses and sequences of Avian Leukosis Virus, but no sequence that might suggest a productive infection of eggs with a virus even distant from known viruses. Our results strongly suggest that SPF eggs such as those used for this study represent a safe substrate for the production of vaccines.

Reduction of high pathogenicity avian influenza virus in eggs from chickens once or twice vaccinated with an oil-emulsified inactivated H5 avian influenza vaccine.

The negative impact of high pathogenicity avian influenza virus (HPAIV) infection on egg production and deposition of virus in eggs, as well as any protective effect of vaccination, is unknown. Individually housed non-vaccinated, sham-vaccinated and inactivated H5N9 vaccinated Once or Twice adult White leghorn hens were challenged intranasally/intratracheally 3-weeks post-vaccination with H5N2 HPAIV. The non-/sham-vaccinated layers experienced 100% mortality (0% survivability) within 3-4 days post-challenge (DPC), and major changes to reproductive parameters including precipitous drops in egg production (79-0% in <5 days), production of soft and thin-shelled eggs, and deposition of virus in albumin and yolk, and on the egg shell surface of 53% of eggs. By comparison, the three H5-vaccinated groups had 83%, 100% and 100% survivability after challenge; the two H5-vaccinated Once hens that died had low pre-challenge HI titers (GMT=16). H5-vaccinated Once or Twice groups maintained egg production after challenge (63%), but there was a mild and significant reduction in egg production as compared to pre-challenge egg production (79%). H5-vaccinated groups had reduced number of virus contaminated eggs (28%), and in most groups, reduced quantity of virus in contaminated eggs compared to non-/sham-vaccinated groups. No HPAIV-positive eggs were laid on or after 5 DPC. In conclusion, HPAIV infection had major negative impact on egg production and other reproductive parameters. H5-vaccination Once or Twice prevented declines in egg production after HPAIV challenge, reduced number of virus-infected eggs, and typically reduced the titer of virus in internal contents and on eggshell surface.

Establecimiento de una línea celular primaria a partir de huevos con embrión de Toxocara canis / Establishing a primary cell line from Toxocara canis embryonated eggs

Introducción. Toxocara canis es el segundo nematelminto más prevalente en perros a nivel regional y entre los tres más frecuentes en algunos países de la región. Debido a que la fuente de contaminación es el perro, éste se convierte en un nematodo con gran potencial zoonótico. Por esta razón, consideramos importante disponer de una línea celular de este helminto para el estudio de los aspectos básicos, así como para el desarrollo de técnicas diagnósticas. Objetivo. Obtener una línea celular primaria a partir de huevos con embrión de T. canis. Métodos. Los parásitos se extrajeron del intestino delgado de perros menores de un año. Las células embrionarias se obtuvieron mediante la embriogénesis de los huevos de los nematodos adultos, en cuatro diferentes medios; dos ricos en sustancias nutritivas, el tercero con formol al 1 % y el cuarto con agua destilada. Las células se obtuvieron mediante disociación mecánica de los huevos con embrión mediante la utilización de jeringas 30G. Resultados. El tiempo estimado de obtención de la línea celular fue de 15 días, en los que siete eran utilizados en la embriogénesis de los huevos. Las células respondieron positivamente a los métodos de crioconservación luego de dos días, e inclusive dos meses después, permitiendo fases de replicación de cuatro pases. Conclusiones. Se logró obtener una línea celular de T. canis a partir de huevos con embrión de este helminto. Esta línea celular ayudará al entendimiento de las relaciones patógenas, posibles blancos terapéuticos y para el desarrollo de métodos diagnósticos.

Introduction: Toxocara canis is the second most prevalent nemathelminthes in dogs at regional level and among the three most frequent in some countries in the region. Due to the fact that the dog is the contamination source, it becomes a nematode with a high zoonotic potential, so we consider it important to be able to use the cell line of this helminth to study the basic aspects, as well as the development of diagnostic techniques. Objective: To obtain a primary cell line from embryonated eggs of T.canis. Methods: The parasites were extracted from the small intestines of dogs under one year old. Embryonic cells were obtained by embryogenesis of the eggs secreted by adult worms in four different media; two were rich in nutrients, one was 1% formaldehyde, and the other was distilled water. The cells were obtained by mechanical dissociation of embryonated eggs using 30G needles. Results: The estimated time for obtaining the cell line was fifteen days, from which seven were used for egg embryogenesis. The cells responded positively to the cryopreservation methods after two days or even two months, allowing a replication phase with four passes. Conclusions: We managed to obtain a cell line from T. canis embryonated eggs. This cell line will help the understanding of pathogenic relationships, potential therapeutic targets and for developing diagnostic methods.

Palmitoylation of CM2 is dispensable to influenza C virus replication.

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.

Detection of avian leukosis virus subgroups in albumen of commercial and native fowl eggs using RT-PCR in Iran.

Avian leukosis viruses (ALVs) belong to Alpharetrovirus genus of the family Retroviridae that are widespread in nature. Different subgroups of ALV commonly infect egg-laying hens. They are responsible for economic losses due to both mortality and depressed performance in chickens. To investigate the presence of these viruses in chickens in Iran, 560 egg albumens were selected from different farms of Fars province, Iran. These eggs were obtained from flocks of two research centers of native fowl production (60 eggs), a broiler grandparent farm (100 eggs), three broiler breeder farms (300 eggs), and a commercial layer flock (100 eggs). Firstly, for primary screening a degenerative primer set (PU1 and PU2) were used in reverse transcriptase-polymerase chain reaction (RT-PCR). Positive cases were detected in 47 of 300 (15.7%) samples from three broiler breeders, 40 of 100 (40%) samples from commercial layer, 53 of 60 (88.3%) samples from flocks of two research centers of native fowl production, and none from the samples of broiler grandparent. Then RT-PCR was undertaken with primers PA1 and PA2 on the positive samples. RT-PCR analysis detected ALVs in two of 47 (4.3%) samples from three broiler breeders, 13 of 40 (32.5%) samples from commercial layer, and 19 of 53 (35.8%) samples from flocks of two research centers of native fowl production. The sequencing results showed that subgroup E of ALV was the most detected virus among chicken eggs and subgroup B was more prevalent in the eggs of native fowls. This is the first report of the ALV subgroup B and E in egg albumen in Iran.

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells.

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.

A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo.

The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not well-characterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400-2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9Delta4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9.

Survey of endogenous virus and TVB* receptor status of commercial chicken stocks supplying specific-pathogen-free eggs.

Endogenous avian leukosis virus (ALVE) and the ALVE receptor (TVB*S1) status of six commercial chicken lines supplying specific-pathogen-free eggs were analyzed. All commercial chicken lines are certified free of the avian leukosis virus (ALV) by screening for expression of the p27 protein using the standard enzyme-linked immunosorbent assay. The commercial chicken lines A, E, and F contained replication competent ALVE inserts. Line A was fixed for ALVE21, and lines E and F were segregating for ALVE10. In addition, ALVE1 was detected in all the chicken lines. Chicken lines B, D, and F were essentially fixed for the TVB*S1 allele that confers susceptibility to ALVE, whereas lines A, C, B, and E were resistant, containing either the TVB*S3 or TVB*R alleles. The results show that lines selected to be ALV p27 negative give rise to two different genotypes. One genotype lacks the TVB*S1 receptor for ALVE. Chicken lines with the TVB*S1 negative genotype can retain replication competent endogenous virus inserts such as ALVE2, 10, or 21 and still display the p27 negative phenotype. These replication competent ALVE viruses are phenotypically p27 negative in the absence of the TVB*S1 receptor because their chromosomal integration sites restrict transcription and subsequent production of the p27 protein and virus particles to levels below the detection limit. If the TVB*S1 receptor is present, the limited production of ALVE virus particles reinfects and integrates into more productive chromosomal locations in the cell. Increased production of infective virus particles and detectable levels of p27 follow this reinfection and integration into more active regions of the cells genome. The other genotype observed in the commercial lines retains the ALVE receptor (TVB*S1) but either lacks replication competent inserts or expresses the envelope encoded protein from defective inserts such as ALVE3 or ALVE6. In this phenotype, the env-coded glycoprotein encoded by the defective inserts binds to the TVB*S1 receptor and blocks the reinfection of the replication competent ALVE virus. This receptor interference stops reinfection and subsequent production of detectable virus particles and the p27 protein. Mixtures of different p27 negative phenotypes can result in the p27 positive phenotype and ALVE virus production. For example, mixtures of ALVE receptor positive (TVB*S1) but ALVE negative (p27 negative and envelope negative) chick embryo fibroblasts (CEFs) with fibroblasts that are receptor negative but ALVE positive could generate cells expressing high levels of p27 and ALVE virus. In this situation, the undetectable levels of ALVE virus from the receptor negative CEFs would infect and integrate into the receptor positive CEFs and produce detectable levels of ALVE virus. The implications of these findings for vaccine manufacturers and regulatory agencies are discussed.

An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

Current developments in avian influenza vaccines, including safety of vaccinated birds as food.

Until recently, most vaccines against avian influenza were based on oil-emulsified inactivated low- or high-pathogenicity viruses. Now, recombinant fowl pox and avian paramyxovirus type 1 vaccines with avian influenza H5 gene inserts (+ or - N1 gene insert) are available and licensed. New technologies might overcome existing limitations to make available vaccines that can be grown in tissue culture systems for more rapid production; provide optimized protection, as a result of closer genetic relations to field viruses; allow mass administration by aerosol, in drinking-water or in ovo; and allow easier strategies for identifying infected birds within vaccinated populations (DIVA). The technologies include avian influenza viruses with partial gene deletions, avian influenza-Newcastle disease virus chimeras, vectored vaccines such as adenoviruses and Marek's disease virus, and subunit vaccines. These new methods should be licensed only after their purity, safety, efficacy and potency against avian influenza viruses have been demonstrated, and, for live vectored vaccines, restriction of viral transmission to unvaccinated birds. Use of vaccines in countries affected by highly pathogenic avian influenza will not only protect poultry but will provide additional safety for consumers. Experimental studies have shown that birds vaccinated against avian influenza have no virus in meat and minimal amounts in eggs after HPAI virus challenge, and that replication and shedding from their respiratory and alimentary tracts is greatly reduced.

Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction.

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.

Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera.

Serologic screening of avian sera for group-specific antibodies to type A influenza is currently accomplished by using the avian influenza (AI) agar gel immunodiffusion (AGID) test. A competitive enzyme-linked immunosorbent assay (CELISA) was developed using a baculovirus vector, Autographa californica nuclear polyhedrosis virus, expressing the nucleoprotein (NP) gene of A/Ann Arbor/6/60 influenza virus. The recombinant NP was obtained by inoculation of Spodoptera frugiperda (Sf9) insect cells or Trichoplusia ni insect larvae with the recombinant baculovirus. A hybridoma cell line producing monoclonal antibody against influenza virus A nucleoprotein was used to generate mouse ascitic fluid for the CELISA. The nucleoprotein and the monoclonal antibody were used without further purification in a CELISA for detection of avian-origin serum antibodies to type A influenza. The AI AGID and CELISA tests were compared for sensitivity and specificity using 1651 experimental and reference antisera. Samples discrepant in AGID and CELISA test results were further evaluated by the AI indirect fluorescent antibody (IFA), hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) tests. The results demonstrated a high degree of correlation between the AGID and CELISA test results, with the IFA, HI, and NI tests offering additional support of CELISA test specificity. The CELISA is a rapid, economical, sensitive, and specific serodiagnostic method for screening large numbers of avian sera for antibodies to avian influenza virus.

Food animal and poultry retroviruses and human health.

In summary, studies reported to date have largely failed to demonstrate human infection with animal and poultry retroviruses or an association between human diseases and these viruses. A number of studies, most of them serologic, have attempted to demonstrate human infection with these viruses. The lack of antibodies in apparently exposed groups of persons suggests an absence of infection. However, another possible explanation is that humans may be immunologically unresponsive to infection with these viruses. Although attempts to infect normal human cells in vitro with many of these viruses have not been reported, BLV and BIV appear to grow poorly or not at all. On the other hand, ALSV subgroup D infect and transform human cells in vitro. However, the production of infectious virus in vitro has been low or nonexistent. This may explain the absence of antibodies in human populations. Furthermore, many of the methods used to detect infection, either directly or indirectly, have either low sensitivity or problems with specificity. Several epidemiologic studies have tried to show a relationship between human and animal leukemia or lymphoma. In many of these studies the actual exposure to retroviruses is unknown and exposure to animals may merely represent exposure to other risk factors that are more important but were either not considered or are undefined; alternatively, a common exposure may be responsible for malignancy in humans and animals with no interspecies relationship. Based on the reported studies, these viruses appear unlikely to be responsible for any significant occurrence of human disease, particularly lymphoid malignancies. Although a definitive statement of no risk to human health is probably unwarranted, the evidence to date indicates that the risk is low and perhaps nonexistent. Thus, no specific public health recommendations regarding retrovirus-infected animals or poultry are warranted at this time.