Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Route of Zika virus infection in Aedes aegypti by transmission electron microscopy.

BACKGROUND: Zika fever has been a global health security threat, especially in the tropical and subtropical regions where most of the cases occur. The disease is caused by Zika virus (ZIKV), which belongs to the family Flaviviridae, genus Flavivirus. The virus is transmitted by Aedes mosquitoes, mostly by Aedes aegypti, during its blood meal. In this study we present a descriptive analysis, by transmission electron microscopy (TEM), of ZIKV infection in A. aegypti elected tissues at the 3rd day of infection. ZIKV vertical transmission experiments by oral infection were conducted to explore an offspring of natural infection. RESULTS: Gut and ovary tissues harbored a higher number of viral particles. The ZIKV genome was also detected, by RT-qPCR technique, in the organism of orally infected female mosquitoes and in their eggs laid. CONCLUSIONS: The data obtained suggest that the ovary is an organ susceptible to be infected with ZIKV and that virus can be transmitted from mother to a fraction of the progeny.

Research Note: Anti-inflammatory effects and antiviral activities of baicalein and chlorogenic acid against infectious bursal disease virus in embryonic eggs.

The purpose of this study was to investigate if baicalein and chlorogenic acid could inhibit the inflammatory responses induced by and protect against infectious bursal disease virus (IBDV) in chicken embryonic eggs. Nine-day-old embryonated chicken eggs were randomly divided into 3 groups of 50 eggs per group: 1) treatment with varying concentrations of baicalein, 2) treatment with varying concentrations of chlorogenic acid, or 3) left untreated as a control. Forty-eight hours after hatching, each group was inoculated with a very virulent IBDV isolate, and the survival of the embryo was monitored daily until the embryonic livers were collected 72 h after inoculation. After IBDV infection, the viral loads in the embryonic livers were evaluated using qRT-PCR, and the hepatic content of inflammatory mediators, such as histamine, interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and nuclear factor-kappa B (NF-κB), were examined. Significant antiviral potential was demonstrated at concentrations of 108 and 215 µg/egg of baicalein and chlorogenic acid, respectively. We observed a concentration-dependent response in the antiviral properties of these chemicals. Treating the embryos with baicalein and chlorogenic acid significantly reduced histamine production. Moreover, pretreatment with baicalein and chlorogenic acid significantly inhibited NF-κB activation, and this inhibited the subsequent production of the proinflammatory cytokines TNF-α and IL-1ß in the context of IBDV infection. These findings suggest that baicalein and chlorogenic acid have anti-IBDV properties, and they may be useful in the prevention of inflammation-related diseases.

Population genomic transformations induced by isolation of wild bird avian influenza viruses (Orthomyxoviridae) in embryonated chicken eggs.

Isolation and cultivation of wild-type viruses in model organism cells or tissues is standard practice in virology. Oftentimes, the virus host species is distantly related to the species from which the culture system was developed. Thus, virus culture in these tissues and cells basically constitutes a host jump, which can lead to genomic changes through genetic drift and/or adaptation to the culture system. We directly sequenced 70 avian influenza virus (Orthomyxoviridae) genomes from oropharyngeal/cloacal swabs collected from wild bird species and paired virus isolates propagated from the same samples following isolation in specific-pathogen-free embryonated chicken eggs. The data were analyzed using population genetic approaches including evaluation of single nucleotide polymorphism (SNP) frequencies and divergence with pooled-sequencing analyses, consensus sequence placement in neighbor-joining trees, and haplotype reconstruction and networks. We found that propagation of virus in eggs leads to skewed SNP mutation spectra with some SNPs going to fixation. Both synonymous and nonsynonmous SNP frequencies shifted. We found multiple consensus sequences that differed between the swabs and the isolates, with some sequences from the same sample falling into divergent genetic clusters. Twenty of 23 coinfections detected had different dominant subtypes following virus isolation, thus sequences from both the swab and isolate were needed to obtain full subtype data. Haplotype networks revealed haplotype frequency shifts and the appearance or loss of low-frequency haplotypes following isolation. The results from this study revealed that isolation of wild bird avian influenza viruses in chicken eggs leads to skewed populations that are different than the input populations. Consensus sequence changes from virus isolation can lead to flawed phylogenetic inferences, and subtype detection is biased. These results suggest that for genomic studies of wild bird influenza viruses the biological field should move away from chicken egg isolation towards directly sequencing the virus from host samples.

Success rates of inoculation of the various compartments of embryonated chicken eggs at different incubation days.

Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2 ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38 mm needles.

Seasonal risk of low pathogenic avian influenza virus introductions into free-range layer farms in the Netherlands.

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free-range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free-range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free-range layer farms, to determine the high-risk period. Data from the LPAI serological surveillance programme for the period 2013-2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015-2016, longitudinal follow-up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014-2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk-based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

Newcastle disease virus vectored rabies vaccine induces strong humoral and cell mediated immune responses in mice.

Rabies is a devastating disease affecting almost all mammalian animal species including humans. Vaccines are available to combat the disease. Protection against the disease is rendered by assessing the humoral immune response. Recent reports suggest the role of cell mediated immune response (CMI) in assessing vaccine efficacy. In the present study, two live vectored vaccine candidates containing glycoprotein G of rabies virus were generated using the mesogenic Newcastle disease virus (NDV) strain R2B and another with NDV with an altered fusion protein cleavage site as backbones. The efficacy of these vaccine candidates on testing in experimental mouse model indicated generation of robust humoral and CMI responses. The recombinant NDV containing the altered fusion protein cleavage site with glycoprotein G showed the highest CMI response in mice indicating its usage as a potential live vectored vaccine candidate against the disease.

Vertical transmission of zika virus in Aedes albopictus.

BACKGROUND: Zika virus (ZIKV) is an arthropod-borne flavivirus transmitted by Aedes mosquitoes. Aedes albopictus is an important vector of ZIKV worldwide. To date, most experiments have focused on the vertical transmission of ZIKV in Ae. aegypti, while studies on Ae. albopictus are very limited. To explore vertical transmission in Ae. albopictus, a series of laboratory studies were carried out. METHODOLOGY/PRINCIPAL FINDINGS: In this study, Ae. albopictus were blood-fed with ZIKV-infectious blood, and the ovaries and offspring viral infection rates were analyzed by reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). ZIKV was detected in the ovaries and oviposited eggs in two gonotrophic cycles. The minimum filial egg infection rates in two gonotrophic cycles were 2.06% and 0.69%, and the effective population transmission rate was 1.87%. The hatching, pupation, and emergence rates of infected offspring were not significantly different from those of uninfected offspring, indicating that ZIKV did not prevent the offspring from completing the growth and development process. ZIKV was detected in three of thirteen C57BL/6 suckling mice bitten by ZIKV-positive F1 females, and the viremia persisted for at least seven days. CONCLUSIONS/SIGNIFICANCE: ZIKV can be vertically transmitted in Ae. albopictus via transovarial transmission. The vertical transmission rates in F1 eggs and adults were 2.06% and 1.87%, respectively. Even though the vertical transmission rates were low, the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to suckling mice through bloodsucking. This is the first experimental evidence of offspring with vertically transmitted ZIKV initiating new horizontal transmission. The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases.

Evidence of vertical transmission of novel duck orthoreovirus in ducks.

Since 2017, duck spleen necrosis caused by new variant duck orthoreovirus (N-DRV) infection had been observed in several provinces in China. This disease retards the growth and development of ducks, thereby reducing feed return rate. N-DRV infection causes damage to duck spleen and other immune organs, leading to immunosuppression and susceptibility to other pathogens. In this study, we successfully constructed a breeding duck artificial infection model and found that N-DRV infection can cause pathologic changes, such as ovarian hemorrhage, follicle atrophy, and fallopian tube bleeding in breeding ducks, resulting in significantly reduced fertilization rate and egg hatching rate. Viral RNA was present in egg vitelline membrane, duck embryo, and duckling's spleen samples, as determined through quantitative polymerase chain reaction (qPCR). Autopsy revealed obvious pathologic changes in the spleen and other organs, although there were no obvious early clinical symptoms observed in ducklings. Sequence distance and phylogenetic analysis confirmed that N-DRV-SD19 re-isolated from the spleen samples of ducklings was consistent with the strain N-DRV-XT18 used for infecting breeding ducks. The findings in this study confirmed that N-DRV can be vertically transmitted through eggs, which provide an important reference for the disease prevention and control.

Assessing the vertical transmission potential of dengue virus in field-reared Aedes aegypti using patient-derived blood meals in Ho Chi Minh City, Vietnam.

BACKGROUND: Dengue viruses (DENV) can be transmitted from an adult female Aedes aegypti mosquito through the germ line to the progeny; however, there is uncertainty if this occurs at a frequency that is epidemiologically significant. We measured vertical transmission of DENV from field-reared Ae. aegypti to their F1 progeny after feeding upon blood from dengue patients. We also examined the transmission potential of F1 females. METHODS: We examined the frequency of vertical transmission in field-reared mosquitoes, who fed upon blood from acutely viremic dengue patients, and the capacity for vertically infected females to subsequently transmit virus horizontally, in two sets of experiments: (i) compared vertical transmission frequency of field-reared Ae. aegypti and Ae. albopictus, in individual progeny; and (ii) in pooled progeny derived from field- and laboratory-reared Ae. aegypti. RESULTS: Of 41 DENV-infected and isofemaled females who laid eggs, only a single female (2.43%) transmitted virus to one of the F1 progeny, but this F1 female did not have detectable virus in the saliva when 14 days-old. We complemented this initial study by testing for vertical transmission in another 460 field-reared females and > 900 laboratory-reared counterparts but failed to provide any further evidence of vertical virus transmission. CONCLUSIONS: In summary, these results using field-reared mosquitoes and viremic blood from dengue cases suggest that vertical transmission is uncommon. Field-based studies that build on these observations are needed to better define the contribution of vertical DENV transmission to dengue epidemiology.

Simple, Low-Cost and Long-Lasting Film for Virus Inactivation Using Avian Coronavirus Model as Challenge.

The COVID-19 infection, caused by SARS-CoV-2, is inequitably distributed and more lethal among populations with lower socioeconomic status. Direct contact with contaminated surfaces has been among the virus sources, as it remains infective up to days. Several disinfectants have been shown to inactivate SARS-CoV-2, but they rapidly evaporate, are flammable or toxic and may be scarce or inexistent for vulnerable populations. Therefore, we are proposing simple, easy to prepare, low-cost and efficient antiviral films, made with a widely available dishwashing detergent, which can be spread on hands and inanimate surfaces and is expected to maintain virucidal activity for longer periods than the current sanitizers. Avian coronavirus (ACoV) was used as model of the challenge to test the antivirus efficacy of the proposed films. Polystyrene petri dishes were covered with a thin layer of detergent formula. After drying, the films were exposed to different virus doses for 10 min and virus infectivity was determined using embryonated chicken eggs, and RNA virus quantification in allantoic fluids by RT-qPCR. The films inactivated the ACoV (ranging from 103.7 to 106.7 EID50), which is chemically and morphologically similar to SARS-CoV-2, and may constitute an excellent alternative to minimize the spread of COVID-19.

Unlike Zika, Chikungunya virus interferes in the viability of Aedes aegypti eggs, regardless of females' age.

Chikungunya and Zika are arboviruses transmitted by the mosquito Aedes aegypti. Mosquito fecundity and egg viability are important parameters of vectorial capacity. Here we aim to understand, comparatively, the effects of Chikungunya virus (CHIKV) and Zika virus (ZIKV) infections on the fecundity and fertility of young and old Aedes aegypti females. Using artificial infection blood feeding experiments we observed that both CHIKV and ZIKV do not alter the number of eggs laid when compared to uninfected females, although the egg fertility significantly decreases in both young and old CHIKV-infected females. There is an upward trend of null females (infertile females) from 2.1% in young to 6.8% in old ZIKV-infected females. Together, our data revealed that CHIKV and ZIKV affects differently Ae. aegypti physiology, that may be related to different viral spread in nature.

Effects of an H7 Highly Pathogenic and Related Low Pathogenic Avian Influenza Virus on Chicken Egg Production, Viability, and Virus Contamination of Egg Contents and Surfaces.

Both highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) can cause decreases or even cessation of egg production in chickens and turkeys. Production of abnormal eggs (deformed, thin-shelled, soft-shelled) can also be caused by AIV infection. Additionally, egg surfaces and contents may also be contaminated with virus. Because data quantifying these effects are lacking, white Plymouth Rock hens were inoculated with HP or LP AIV while in production. No decreases in egg production or abnormal eggs were observed with LPAIV-infected hens. No lesions or viral antigen staining in ovary and oviduct were observed in LPAIV-infected hens 3 days postchallenge. LPAIV RNA was detected on eggs collected from 12 hr to 11 days postinoculation (PI) and was on or in 6.4% (15/234) of the eggs. Titer equivalents of LPAIV ranged from 1.3-2.5 log10 50% egg infectious doses (EID50). No virus was detected in embryo tissue from eggs laid by LPAIV-infected hens. In contrast, egg production by HPAIV-inoculated hens decreased at 72 hr PI and 18.4% (16/87) of the eggs were abnormal. However, viability was similar to that of the sham inoculates. HPAIV RNA was detected in or on 11.1% (9/81) of the eggs from 36 hr through 96 hr PI, when the hens were euthanatized. HPAIV RNA was detected on 6.2% of eggshells, in 4.2% of albumin/yolk samples, and in 8.3% of embryo tissue. Forty percent of the abnormal eggs were positive for HPAIV RNA. Titer equivalents on or in HPAIV-contaminated eggs ranges from 1.0-4.0 log10 EID50. Lesions and viral antigen staining were present in the ovary and all sections of the oviduct of infected hens 3 days postchallenge. These data will inform models using production-based triggers for LPAIV monitoring and for risk assessments to determine the disposition of eggs from flocks infected with LPAIV or HPAIV.

Efectos de un virus de la influenza aviar altamente patógeno y un virus de baja patogenicidad H7 relacionados en la producción de huevos de gallina, la viabilidad y con la contaminación viral del contenido y las superficies del huevo. Tanto el virus de la influenza aviar (AIV) altamente patógeno (HP) como el de baja patogenicidad (LP) pueden causar una disminución o incluso el cese de la producción de huevos en pollos y pavos. La producción de huevos anormales (con cascaron deforme o delgado, o con cascarón blando) también puede ser causada por la infección por el virus de la influenza aviar. Además, las superficies y el contenido del huevo también pueden estar contaminados con virus. Debido a la falta de datos que cuantifiquen estos efectos, gallinas Plymouth Rock blancas fueron inoculadas con virus de alta o baja patogenicidad mientras estaban en producción. No se observaron disminuciones en la producción de huevos o huevos anormales en las gallinas infectadas con el virus de influenza aviar de baja patogenicidad. No se observaron lesiones ni la presencia del antígeno viral en el ovario y oviducto de gallinas infectadas con el virus de baja patogenicidad a los tres días después del desafío. El ARN del virus de baja patogenicidad se detectó en los huevos recolectados de 12 horas a 11 días después de la inoculación y estaba sobre o dentro del 6.4% (15/234) de los huevos. Los títulos equivalentes del virus de baja patogenicidad variaron de 1.3 a 2.5 log10 dosis infecciosas 50% para embrión de huevo (EID50). No se detectó virus en el tejido embrionario de los huevos puestos por gallinas infectadas con el virus de alta patogenicidad. En contraste, la producción de huevos por gallinas inoculadas con el virus de alta disminuyó a las 72 horas después de la inoculación y el 18.4% (16/87) de los huevos fueron anormales. Sin embargo, la viabilidad fue similar a la de los controles negativos inoculados. Se detectó ARN del virus de alta patogenicidad en el 11.1% (9/ 81) de los huevos desde las 36 horas hasta 96 horas después de la inoculación, cuando las gallinas se sacrificaron. Se detectó ARN del virus de alta patogenicidad en el 6.2% de los cascarones de huevo, en el 4.2% de las muestras de albúmina/yema y en el 8.3% del tejido embrionario. Cuarenta por ciento de los huevos anormales fueron positivos para ARN del virus de alta patogenicidad. Los títulos equivalentes sobre o por dentro de los huevos contaminados con influenza aviar de alta patogenicidad variaron de 1.0­4.0 log10 EID50. Las lesiones y la presencia del antígeno viral estaban presentes en el ovario y en todas las secciones del oviducto de las gallinas infectadas tres días después del desafío. Estos datos servirán como fuente de información para los modelos que utilizan disparadores basados en la producción para el monitoreo del virus de influenza aviar de baja patogenicidad y para las evaluaciones de riesgos para determinar la eliminación de los huevos de las parvadas infectadas con virus de influenza aviar de baja o de alta patogenicidad.

Development and Evaluation of Real-Time Reverse Transcription Polymerase Chain Reaction Test for Quantitative and Qualitative Recognition of H5 Subtype of Avian Influenza Viruses.

Avian influenza viruses (AIV) affect a wide range of birds and mammals, cause severe economic damage to the poultry industry, and pose a serious threat to humans. Highly pathogenic avian influenza viruses (HPAI) H5N1 were first identified in Southeast Asia in 1996 and spread to four continents over the following years. The viruses have caused high mortality in chickens and various bird species and deadly infections in humans. Multiple conventional methods have been so far introduced for the detection and identification of avian influenza viruses. Traditional virus isolation methods are gold standard protocol in AI detection; nonetheless, virus isolation in embryonating chicken eggs (ECE) is not a rapid method for the detection of influenza viruses since it is time-consuming and labor-intensive. Furthermore, the isolation of highly pathogenic viruses, such as H5, needs BSL3 laboratories. Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR) is a sensitive and specific method for the detection of influenza viruses. The application of these nucleic acid-based techniques has increased our ability to identify and perform influenza virus care programs, especially in surveillance programs. The current study aimed to detect H5 subtype of avian influenza (AI) virus using fast, specific, and sensitive TaqMan RRT-PCR. Notably, single step RRT-PCR was used to prevent possible laboratory contamination. The specificity of this test was evaluated using nucleic acid extracted from several poultry pathogenic microorganisms and negative clinical specimens from AI-uninfected birds. The sensitivity analysis of the RRT-PCR assay was performed using in vitro-transcribed RNA copy and 10-fold serial dilution of standard AI virus with specific titer. The results indicated the high sensitivity of this method and the lowest detectable dilution of this method based on RNA copies and 1:10 serial dilutions of the standard virus was 10 1.9 EID50 /100.

New PA/1220/98-like variant of infectious bronchitis virus in Poland.

The aim of the present study was to report the first detection of a new infectious bronchitis virus (IBV) variant in Polish commercial flocks which is completely different to any previously known in this region. In 2018, samples from Ross 308 breeding hens aged 35 weeks were delivered for IBV diagnosis. IBV presence was detected, but all attempts to amplify the S gene fragment were negative. The field material was analysed using the Illumina MiSeq platform and a 1073-nt fragment of the S1 coding region was obtained. The gCoV/ck/Poland/516/2018 strain shared only 52.7-58.1% nucleotide identity to any known genotype of IBV and shared the highest identity of 81.4% to the unique North American PA/1220/98 variant. Based on the obtained sequence, a specific molecular test was constructed and used for screening of chicken samples from 35 field cases delivered to our laboratory between 2018 and 2019 for IBV diagnosis. Application of this test enabled detection of another three chicken flocks as positive for this new strain. All positives were identified in commercial layers with egg production problems. To date, the virus has not been detected in broiler chickens. Taking into account the proposed criteria for the definition of a new IBV genotype or lineage, it seems that the detected viruses in Poland, together with the unique North American PA/1220/98 variant, may be classified as separate lineages/genotype in the new IBV classification. RESEARCH HIGHLIGHTS The new IBV variant is distantly related to other known GI-GVII IBV genotypes/lineages. It affects long-lived birds causing egg production problems. The detected IBV and the unique North American PA/1220/98 variant are candidates for separate lineages in the new GVIII genotype.

Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs.

Avian influenza virus and some mammalian influenza A viruses can be isolated, propagated, and titrated in embryonated chicken eggs (ECEs). Most sample types can be accommodated in ECE culture with appropriate processing. Isolation may also be accomplished in cell culture, and if a mammalian lineage influenza A is expected to be in an avian sample, for example swine influenza in turkey specimens, mammalian cell may be preferable. Culture in ECEs is highly sensitive but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses can be confirmed by hemagglutination inhibition assay, antigen immunoassay, agar gel immunodiffusion assay, or RT-PCR. Finally, ECEs may be used to propagate and titrate an avian influenza virus.

Isolation of Swine Influenza A Virus in Cell Cultures and Embryonated Chicken Eggs.

Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, vaccine production, and so on. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, influenza A virus in swine (IAV-S) isolation and propagation has been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently, Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for the isolation, propagation, and titration of IAV-S. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when IAV-S isolation in MDCK cells is unsuccessful. Optimal specimens for IAV-S isolation includes nasal swabs, nasopharyngeal swabs, oral fluids, bronchoalveolar lavage, lung tissues, and so on. In this chapter, we describe the procedures of sample processing, IAV-S isolation in MDCK cells and in embryonated chicken eggs, as well as the methods used for confirming the virus isolation results.

Equine Influenza Culture Methods.

Equine influenza viruses are cultured in embryonated chicken eggs or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

Sole recombinant Muscovy duck parvovirus infection in Muscovy ducklings can form characteristic intestinal embolism.

Recombinant Muscovy duck parvovirus (rMDPV) has been recently identified as a novel pathogen circulating in Chinese Muscovy duck flocks in the past two decades. Different from classical MDPV, rMDPV infection can form embolism in the intestinal tract of deceased Muscovy ducklings. However, whether rMDPV acts as the sole causative agent involved in the formation of the characteristic embolism in Muscovy ducklings remains unclear. In this study, an infectious plasmid clone pZW containing the complete genome of strain ZW, a previously characterized rMDPV isolate, was constructed, and a single nucleotide mutation was then introduced in the VP1 gene within pZW as the genetic marker. Transfection of pZW in 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route resulted in the rescue of the infectious virus. The rescued virus exhibited similar biological characteristics to its parental strain ZW, as evaluated by the median embryo lethal dose and the replication kinetics in embryonated Muscovy duck eggs. Muscovy duckling infection tests showed that the rescued virus and parental strain can kill all Muscovy ducklings within 7 days post-infection. Postmortem examination revealed that embolism can be observed in the intestinal tracts of deceased ducklings in the rescued and parental virus infection groups. Collectively, the present study demonstrated that sole rMDPV infection of Muscovy ducklings, without participation of other pathogens, is enough to form characteristic embolism in the intestinal tract.