Sensitivity of infl uenza virus strains isolated from various regions of Kazakhstan in 2018–2019 to antiviral drugs

The purpose of the study was to examine susceptibility of the Kazakhstan strains of infl uenza A/H1N1 and type B viruses, isolated from various regions of Kazakhstan in 2018–2019, to antiviral drugs. Materials and methods. The susceptibility analysis of 20 strains of infl uenza A/H1N1 and B viruses was carried out with chemotherapeutic agents including Remantadine, Tamifl u, Arbidol, and Ingavirin. Viruses were cultured in the allantoic cavity of developing 10-day-old chicken embryos for 48 hours at 36 °C. The hemagglutinating activity was determined according to the conventional method on 96-well plates using 0.75% chicken red blood cell suspension; the infectivity was calculated by the Reed-Muench method. The sensitivity of virus strains to diff erent concentrations of antiviral drugs was evaluated by the level of reproductive suppression of 100 lg EID50/0.2 ml of virus in chicken embryos. Statistical analysis was performed with the use of Microsoft Offi ce Excel 2010 software. Results. A study of sensitivity to chemotherapeutic agents demonstrated heterogeneity of Kazakhstan 2018–2019 infl uenza A and B viruses population on this feature. The sensitivity to Tamifl u was found in all Kazakhstan strains of infl uenza A/H1N1 virus and three type B strains (inhibitory concentration was 0.44–25.38 μg/mL). The reproduction of most viruses was eff ectively inhibited by tamifl u at a concentration of 0.68–3.23 μg/mL. The inhibitory concentration for the three strains of A/H1N1 virus was 7.23–25.38 μg/mL. Remantadin inhibited the reproduction of viruses at higher doses (12.60–25.55 μg/mL). All viruses under study were resistant to Arbidol and Ingavirin. One type B infl uenza virus was found to be weakly sensitive to Ingavirin. Conclusion. The heterogeneity of the infl uenza virus population in their sensitivity to antiviral drugs indicates the need for constant epidemiological surveillance in order to identify drug-resistant variants.

Filamentous versus Spherical Morphology: A Case Study of the Recombinant A/WSN/33 (H1N1) Virus

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin–Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually “nonfilamentous” virus strains.

Isolation and molecular identification of wild Newcastle disease virus isolated from broiler farms of Diyala Province, Iraq

Background and Aim: Newcastle disease virus (NDV) remains a major viral disease of poultry. The morbidity and mortality rates of chickens vaccinated with NDV in broiler farms in Diyala Province were 100% and 80%, respectively, rates due to suspected infection with the highly virulent NDV. The present study aimed to isolate and identify the NDV virus and evaluate its pathogenicity in infected broiler chickens at poultry farms. Materials and Methods: Broiler chickens at two commercial poultry farms were suspected of being infected with virulent NDV due to high mortality rates. Virus isolated from samples of intestinal tissues, lungs, trachea, spleen, kidneys, and air sacs was adapted in the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. The NDV pathotype was determined based on the mean death time (MDT) in eggs as well as the intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index pathogenicity indexes of the isolated samples. Broilers were experimentally infected by inoculation with fluids collected from the allantoic cavities of 60 broilers aged 35 days. Serological and molecular tests were followed by enzyme-linked immunosorbent assay to determine levels of anti-NDV immunoglobulin G, and isolates were identified using a hyperimmune (HI) test and real-time polymerase chain reaction (RT-PCR). Results: Suspected and isolated NDV field samples propagated in the allantoic cavity of 10-day-old fertile SPF chickens were NDV positive in the rapid hemagglutination test within a few seconds. Pathogenicity indices and MDT showed that the isolated NDV was viscerotropic and velogenic. The virus was identified as NDV by the HI test using specific anti-LaSota HI serum and RT-PCR with specific primers and probes. Propagation of the virus in the allantoic cavity of embryonated hen eggs produced a viral titer of 109.5 EID50/0.1 mL. Conclusion: The virus isolated from broiler chicken farms in Diyala Province, Iraq, was viscerotropic and velogenic according to the pathogenicity indices and RT-PCR. The isolated NDV caused 100% morbidity and 90% mortality in NDV-vaccinated and experimentally infected broiler chickens.

Prevalence and molecular characterization of infectious bronchitis virus isolated from chicken in Bangladesh

Aim: The present study was aimed to determine the prevalence of infectious bronchitis virus (IBV) as well as virus isolation, identification, and molecular characterization of various strains circulating in Bangladesh. Materials and Methods: A total of 371 swabs and organ samples were collected from four types of chicken including layer, Sonali (local), broiler, and broiler breeder under eight districts (Rangpur, Bogura, Tangail, Dhaka, Gazipur, Mymensingh, Jamalpur, and Cumilla) during 2014-2016 in Bangladesh. Results: Out of 371 samples, 65 samples were positive in reverse transcriptase polymerase chain reaction (RT-PCR) for molecular identification of IBV. The overall prevalence was 17.52% recorded and among the selected types of chicken, the highest prevalence of IBV was found in layer that was 42.22% followed by 17.24% in Sonali, 14.93% in broiler breeder, and lowest prevalence was 11.94% in broiler chicken, respectively. Moreover, the prevalence of IBV was recorded highest in aged chicken at 41-60 weeks, which was 54.55% in layer, 27.27% in Sonali, and, afterward, 14.68% was found in broiler breeder, respectively. Frequency of IBV more frequently in winter (22.67%) followed by rainy (15.87%) and summer season (11.58%). The highest prevalence of IBV was found Tangail district (41.67%) followed by Mymensingh (24.42%), Gazipur (19.32%), Dhaka (15.38%), Jamalpur (16.67%), Bogura (13.68%), Cumilla (5.88%), and Rangpur (9.26%), respectively. Samples that were found high positive in IBV RT-PCR (Ct value below 30) were subjected to inoculation into chicken egg embryo to observe characteristic changes in chicken embryo. Swabs and organ samples were processed and passaged in 9-day-old embryonated chicken eggs through allantoic cavity route. IBV virus suspected samples inoculated into chicken egg embryos after 3-5 passages showed dwarfing and curling of the embryos which are characteristic lesions of IBV. Allantoic fluid was collected from all inoculated eggs and performed partial sequencing of S1 gene for three isolates. After sequencing, the phylogenetic tree was constructed from the nucleotide sequences of IBV isolates. Two of the isolates are 4/91 IBV and another one matched with QX-like IBV. Conclusion: The results revealed that the three isolates from different places in Bangladesh were identified for the 1st time as which will help for IBV control strategy.

A Case of Newcastle Disease Virus in Red-Headed Lovebird in Sudan

Two diseased red-headed lovebirds were presented for diagnosis to the Department of Avian Diseases and Diagnosis,Veterinary Research Institute, aged 37 days and 4 years. The symptoms were dyspnea, cyanosis of the comb, diarrhea, and fever. Postmortem lesions included pale liver and bloody enteritis. Newcastle disease virus was isolated from lungs, trachea, and intestines following inoculation in the allantoic cavity of 10-day-old fertile eggs; the NDV was identified by the means of HA&HI tests using specific NDV antisera (Lasota strain). The isolate agglutinated equine RBCs but failed to agglutinate sheep and bovine RBCs. The pathogenicity of the NDV isolate was studied, the mean death time was 96 hours, and the intracerebral pathogenicity index (ICPI) value was 0.9, indicating the isolate of lentogenic type.

Pacheco’s disease in a Hungarian zoo bird population: A case report

An epizootic of Pacheco’s disease is reported from a zoo bird population. The infection was introduced by wild-captured Patagonian conures ( Cyanoliseus patagonus ) despite 61 days of quarantine. The disease affected several parrot species and, interestingly, three out of seven bearded barbets ( Lybius dubius ). The mortality rate was 30.93%. Autopsy revealed abdominal hyperaemia with liver haemorrhages and, in less rapid cases, yellowish discoloration and fragility of the liver. Death was caused by the collapse of circulation. Histopathology demonstrated liver cell necrosis, disintegration of the lobular structure, and a few intranuclear inclusion bodies. Icosahedral virions were detected by electron microscopy. The virus was isolated in the allantoic cavity of embryonated chicken eggs as well as in chicken embryo fibroblast cell culture. A 281-bp-long fragment of psittacid herpesvirus DNA was detected by PCR in cell culture material and liver samples of the affected birds. To our knowledge this is the first report of Pacheco’s disease in bearded barbets as well as the first occurrence of Pacheco’s disease in Hungary.

The haemagglutinins of influenza A (H1N1) viruses in the ‘O’ or ‘D’ phases exhibit biological and antigenic differences

SummaryInfluenza A (H1N1) viruses when initially isolated in mammalian cell cultures (MDCK cells) had different agglutination reactions with chicken and guinea-pig erythrocytes compared to the same viruses after passage. On first isolation the virus HA resembled the ‘O’ phase viruses described originally by Burnet and Bull and agglutinated mammalian but not avian erythrocytes. After passage, the virus HA resembled a classical ‘D’ phase virus and agglutinated both avian and mammalian erythrocytes. Monoclonal and polyclonal antisera detected antigenic differences between the HAs of the viruses in the ‘O’ and ‘D’ phases. The ‘O’ phase virus HA reacted preferentially with antibodies in post infection human antisera. Viruses in the ‘O’ phase replicated poorly in the allantoic cavity of embryonated hens' eggs whilst ‘D’ phase virus replicated in both MDCK cells and in embryonated hens' eggs. At least three distinguishable subpopulations of influenza A (H1N1) viruses may co-exist in clinical throat swab material, including viruses possessing HAs in the ‘O’ and ‘D’ phases and other ‘D’ phase viruses cultivable in embryonated hens' eggs but antigenically distinguishable from the corresponding ‘D’ phase virus in MDCK cells.

Ovine fetal urachus--physiological and hormonal control of its contractile activity

We have investigated whether the ovine fetal urachus near term has the potential to regulate the passage of urine from the bladder into the allantoic cavity. Experiments were performed primarily on longitudinal urachal muscle. In Krebs bicarbonate buffer (pH 7.4 bubbled with 95% O2-5% CO2 at 37 degrees C) the urachus spontaneously developed a prolonged elevated resting tension (RT, 1.1–1.5 g). Intra-abdominal urachus showed a greater development of tension than extra-abdominal urachus, which was associated with the amount of smooth muscle observed in histological specimens. Isoproterenol (1 microM) produced a significant reversible reduction in RT (1.36 +/- 0.15 to 0.67 +/- 0.08 g, P less than 0.01, mean +/- SE). Replacement of normal-O2 buffer (PO2 420.4 +/- 19.5 mmHg) with a low-O2 buffer (PO2 101.3 +/- 4.7 mmHg), or very low-O2 buffer (PO2 38.2 +/- 2.2 mmHg) while maintaining PCO2 and pH constant, produced a reduction in RT (from 1.35 +/- 0.21 to 0.52 +/- 0.13 g and from 1.20 +/- 0.17 to 0.46 +/- 0.04 g, respectively; both P less than 0.01). This effect was reversed when high PO2 was reinstated. Circular muscle showed a similar response when PO2 was reduced. The urachus showed a significant increase in RT (0.74 +/- 0.08 to 1.71 +/- 0.28 g, P less than 0.01) when prostaglandin F2 alpha (PGF2 alpha; 10 microM) was added to the low-O2 buffer. These results demonstrate that the urachus can develop spontaneous contractile activity and responds to alterations in O2, a beta-agonist, and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)