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Nitric oxide (NO) in human tears regulates numerous ocular surface processes, such as tear generation, corneal wound healing, conjunctival vascular tone, and so forth. Any deviation from its normal concentration is linked to various ocular syndromes, including microbial keratitis, conjunctivitis, pterygium, dry eye, retinitis, glaucoma, and so forth. Therefore, precise monitoring of NO in tears can be considered as a potential biomarker for ocular diseases. Here, we report a highly sensitive and selective electrochemical NO sensor using carbon ink-based electrodes. Counter, working (WE), and reference electrodes have been designed and painted on a butter paper by using carbon ink. To improve the sensing performance, the WE has been modified with a gold nanoparticle (Au NP)-deposited carbon cloth (CC). Such a paper-based sensor demonstrated high sensitivity of ∼0.34 μA μM -1 cm -2 , ultralow detection limit of ∼2.35 nM, wide linear range of 10 nM-0.4 mM, and fast response time (0.35 s). The sensor also showed excellent stability and selectivity toward the interfering agents in human body fluids. Such a low-cost, flexible paper-based sensor was employed for the detection of NO in artificial tears.

The breakout of the pandemic COVID-19 has affected numerous countries and territories worldwide. As COVID-19 specific medicines yet to be invented, at present the treatment is case specific, hence identification and evaluation of different prevalent treatment options based on various criteria and attributes are very important not only from the point of view of present pandemic but also for futuristic pandemic preparedness. The present study focuses on identifying, evaluation and ranking of treatment options using Multi Criteria Decision Making (MCDM). In this regard, the existing literature, doctors and scientist were interviewed to know the current treatment options in vogue and the scale of their importance with respect to the criteria. The criteria taken are side effect, regime cost, treatment duration, plasma stability, plasma turnover, time of suppression, ease of application, drug-drug interaction, compliance, fever, pneumonia, intensive care, organ failure, macrophage activation syndrome, hemophagocytic syndrome, pregnancy, kidney problem, age. This study extended Hesitant Fuzzy Set (HFS) to Generalized Hesitant Fuzzy Sets (GHFS). Generalized Hesitant Pentagonal Fuzzy Number (GHPFN) is developed. The properties of GHPFN are demonstrated. Two types of GHPFN has been described. The GHPFN (2nd type) along with MCDM tool Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) has been applied to rank the treatment options. The result of the study ranked 'Hydroxychloroquine' as the first alternative followed by, 'Plasma Exchange', 'Tocilizumab', 'Remdesivir' and 'Favipravir'. To check the robustness and steadiness of the proposed methodology, comparative analysis and sensitivity analysis has been conducted., (© 2023 Elsevier Ltd. All rights reserved.)

Glutathione, a tri-peptide (glutamate-cysteine-glycine) with the thiol group (-SH), is most efficient antioxidative agent in eukaryotic cells. The present study aimed to isolate an efficient probiotic bacterium having the potential to produce glutathione. The isolated strain Bacillus amyloliquefaciens KMH10 showed antioxidative activity (77.7 ± 2.56) and several other essential probiotic attributes. Banana peel, a waste of banana fruit, is chiefly composed of hemicellulose with various minerals and amino acids. A consortium of lignocellulolytic enzyme was used for the saccharifying banana peel to produce 65.71 g/L sugar to support the optimal glutathione production of 181 ± 4.56 mg/L; i.e., 1.6 folds higher than the control. So, the studied probiotic bacteria could be an effective resource for glutathione; therefore, the stain could be used as natural therapeutics for the prevention/treatment of different inflammation-related gastric ailments and as an effective producer of glutathione using valorized banana waste that has excellent industrial relevance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In recent times, COVID-19 pandemic has posed certain challenges to transportation companies due to the restrictions imposed by different countries during the lockdown. These restrictions cause delay and/ or reduction in the number of trips of vehicles, especially, to the regions with higher restrictions. In a pandemic scenario, regions are categorized into different groups based on the levels of restrictions imposed on the movement of vehicles based on the number of active cases (i.e., number of people infected by COVID-19), number of deaths, population, number of COVID-19 hospitals, etc. The aim of this study is to formulate and solve a fixed-charge transportation problem (FCTP) during this pandemic scenario and to obtain transportation scheme with minimum transportation cost in minimum number of trips of vehicles moving between regions with higher levels of restrictions. For this, a penalty is imposed in the objective function based on the category of the region(s) where the origin and destination are situated. However, reduction in the number of trips of vehicles may increase the transportation cost to unrealistic bounds and so, to keep the transportation cost within limits, a constraint is imposed on the proposed model. To solve the problem, the Genetic Algorithm (GA) has been modified accordingly. For this purpose, we have designed a new crossover operator and a new mutation operator to handle multiple trips and capacity constraints of vehicles. For numerical illustration, in this study, we have solved five example problems considering three levels of restrictions, for which the datasets are generated artificially. To show the effectiveness of the constraint imposed for reducing the transportation cost, the same example problems are then solved without the constraint and the results are analyzed. A comparison of results with existing algorithms proves that our algorithm is effective. Finally, some future research directions are discussed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Elsevier B.V. All rights reserved.)

In the current era, the theory of vagueness and multi-criteria group decision making (MCGDM) techniques are extensively applied by the researchers in disjunctive fields like recruitment policies, financial investment, design of the complex circuit, clinical diagnosis of disease, material management, etc. Recently, trapezoidal neutrosophic number (TNN) draws a major awareness to the researchers as it plays an essential role to grab the vagueness and uncertainty of daily life problems. In this article, we have focused, derived and established new logarithmic operational laws of trapezoidal neutrosophic number (TNN) where the logarithmic base μ is a positive real number. Here, logarithmic trapezoidal neutrosophic weighted arithmetic aggregation ( L a r m ) operator and logarithmic trapezoidal neutrosophic weighted geometric aggregation ( L g e o ) operator have been introduced using the logarithmic operational law. Furthermore, a new MCGDM approach is being demonstrated with the help of logarithmic operational law and aggregation operators, which has been successfully deployed to solve numerical problems. We have shown the stability and reliability of the proposed technique through sensitivity analysis. Finally, a comparative analysis has been presented to legitimize the rationality and efficiency of our proposed technique with the existing methods., (© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021.)

The outburst of the pandemic Coronavirus disease since December 2019, has severely impacted the health and economy worldwide. The epidemic is spreading fast through various means, as the virus is very infectious. Medical science is exploring a vaccine, only symptomatic treatment is possible at the moment. To contain the virus, it is required to categorize the risk factors and rank those in terms of contagion. This study aims to evaluate risk factors involved in the spread of COVID-19 and to rank them. In this work, we applied the methodology namely, Fuzzy Analytic Hierarchy Process (FAHP) to find out the weights and finally Hesitant Fuzzy Sets (HFS) with Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) is applied to identify the major risk factor. The results showed that "long duration of contact with the infected person" the most significant risk factor, followed by "spread through hospitals and clinic" and "verbal spread". We showed the appliance of the Multi Criteria Decision Making (MCDM) tools in evaluation of the most significant risk factor. Moreover, we conducted sensitivity analysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Vertically aligned TiO 2 nanorods (NRs) were synthesized on a fluorine-doped tin oxide-deposited glass substrate by the hydrothermal method. A Schottky junction ultraviolet photodetector was fabricated by spin-coating of the poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) polymer on TiO 2 NRs. For the improvement of device performance, the electrical conductivity of the polymer was increased by adding dimethyl sulfoxide (DMSO) and concentrated H 2 SO 4 acid. The diode parameters, i.e., work function, series resistance, and ideality factor, were studied for all devices. Photoresponse behavior of the TiO 2 nanorod/PEDOT:PSS junction was studied upon illumination of white light of intensity 80 mW/cm 2 . Our acid-treated sample demonstrated the highest photocurrent value, which is 10 times larger than that of DMSO-treated and 39 times larger than that of the untreated sample. Our acid-treated device showed superior external quantum efficiency (∼12 560%), responsivity (∼34.43 A/W), and detectivity (∼1.6 × 10 11 Hz 1/2 /W) at ∼340 nm wavelength under -1 V bias.

Graphene quantum dot (GQD)-sensitized ZnO nanorods/poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) Schottky junction has been fabricated for visible-blind ultraviolet (UV) photodetector applications. Schottky diode parameters such as ideality factor, effective work function, and series resistance have been studied for GQD-modified and pristine ZnO nanorod-based devices. Under illumination of broadband light of intensity 80 mW/cm 2 , GQD-sensitized samples showed 11 times higher photocurrent value compared to pristine ZnO at -0.75 V external bias. GQD-modified detector demonstrated maximum photocurrent at UV region (wavelength ∼340 nm) for all reverse bias voltages. ZnO nanorods/polymer Schottky junction UV detectors revealed high external quantum efficiency (EQE) more than 100%. Interestingly, GQD sensitized nanorod-based device demonstrated high EQE value of 13,161% at -1 V bias (wavelength ∼340 nm), which is eight times higher than pristine ZnO NR-based detector. GQD-modified detectors also showed superior responsivity (36 A/W), detectivity (1.3 × 10 12 Hz 1/2 /W) at -1 V bias under incident of light of wavelength 340 nm. Even at very low intensity of UV light (0.07 mW/cm 2 ), GQD-modified UV detectors showed high photocurrent (∼7.0 mA/cm 2 ).

CdS decorated ZnO nanorods have been grown by a combination of hydrothermal method and successive ion layer absorption and reaction (SILAR) method. Optical absorption and emission properties of ZnO nanorods have been studied after sensitization with CdS nanoparticles. Current-voltage characteristics of ZnO nanorods and CdS sensitized ZnO nanorods have been studied in an electrochemical cell. The spectral dependent photocurrent and photopotential behaviors of ZnO nanorods and CdS sensitized ZnO nanorods have been investigated using monochromatic light of wavelength 300-700 nm. The photopotential recovery time have been estimated for CdS sensitized nanorods and pristine nanorods.

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

Anthrax, foot and mouth disease (FMD), haemorrhagic septicaemia (HS), peste des petits ruminants (PPR) and rabies are considered to be endemic in Bangladesh. This retrospective study was conducted to understand the geographic and seasonal distribution of these major infectious diseases in livestock based on data collected through passive surveillance from 1 January 2010 to 31 December 2012. Data analysis for this period revealed 5,937 cases of anthrax, 300,333 of FMD, 13,436 of HS, 247,783 of PPR and 14,085 cases of dog bite/rabies. While diseases were reported in almost every district of the country, the highest frequency of occurrence corresponded to the susceptible livestock population in the respective districts. There was no significant difference in the disease occurrences between districts bordering India/Myanmar and non-border districts (p>0.05). Significantly higher (p<0.01) numbers of anthrax (84.5%), FMD (88.3%), HS (84.9%) and dog bite/rabies (64.3%) cases were reported in cattle than any other species. PPR cases were reported mostly (94.8%) in goats with only isolated cases (5.2%) in sheep. The diseases occur throughout the year with peak numbers reported during June through September and lowest during December through April, with significant differences (p<0.01) between the months. The annual usages of vaccines for anthrax, FMD, HS and PPR were only 7.31%, 0.61%, 0.84% and 11.59% of the susceptible livestock population, respectively. Prophylactic vaccination against rabies was 21.16% of cases. There were significant differences (p<0.01) in the administration of anthrax, FMD and HS vaccines between border and non-border districts, but not PPR or rabies vaccines. We recommend that surveillance and reporting of these diseases need to be improved throughout the country. Furthermore, all suspected clinical cases should be confirmed by laboratory examination. The findings of this study can be used in the formulation of more effective disease management and control strategies, including appropriate vaccination policies in Bangladesh.

In this study, three novel luminescent nanofibrous metal-organic gels (MOGs) have been synthesized by the reaction of 1,3,5-tris(3-pyridylmethoxyl)benzene (L) with chloride salts of Cd(II), Hg(II), and Cu(II). The metal-ligand coordination, intermolecular π-π stacking and several other weak interactions found to play an important role in the formation of nanofibrous materials. The gel materials are characterized by rheology, diffuse reflectance spectra and various microscopic techniques such as TEM, FESEM, and AFM. The gels MOG-1 and MOG-2 were found to exhibit significant white photoluminescence, whereas the MOG-3 exhbits green emission upon excitation at 325 nm. Furthermore, the MOG-1 has shown its application as a chemosensor for the remarkable detection of nitroaromatics such as nitrobenzene (NB), 2,4-dinitrophenol (DNP). The significant quenching response for NB and DNP is attributed to the strong charge-transfer interactions between the electron-deficient aromatic ring of NB and the electron rich aromatic group of L in MOG-1. The crystal structure of Cd(II) complex of L reveals the formation one-dimensional network which contains strong π-π interactions within and between the networks and these strong π-π interactions generate the free charge carrier in all these nanofibrous gels.

Highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in Bangladesh since its first isolation in February 2007. Phylogenetic analysis of the haemagglutinin (HA) gene of HPAI H5N1 viruses demonstrated that 25 Bangladeshi isolates including two human isolates from 2007-2011 along with some isolates from neighbouring Asian countries (India, Bhutan, Myanmar, Nepal, China and Vietnam) segregate into two distinct clades (2.2 and 2.3). There was clear evidence of introduction of clade 2.3.2 and 2.3.4 viruses in 2011 in addition to clade 2.2 viruses that had been in circulation in Bangladesh since 2007. The data clearly demonstrated the movement of H5N1 strains between Asian countries included in this study due to migration of wild birds and/or illegal movement of poultry across borders. Interestingly, the two human isolates were closely related to the clade 2.2 Bangladeshi chicken isolates indicating that they have originated from chickens. Furthermore, comparative amino acid sequence analysis revealed several substitutions (including 189R>K and 282I>V) in HA protein of some clade 2.2 Bangladeshi viruses including the human isolates, suggesting there was antigenic drift in clade 2.2.3 viruses that were circulating between 2008 and 2011. Overall, the data imply genetic diversity among circulating viruses and multiple introductions of H5N1 viruses with an increased risk of human infections in Bangladesh, and establishment of H5N1 virus in wild and domestic bird populations, which demands active surveillance., (© 2013 Blackwell Verlag GmbH.)

The growing demand for biomaterials for electrical and optical devices is motivated by the need to make building blocks for the next generation of printable bio-electronic devices. In this study, transparent and flexible resistive memory devices with a very high ON/OFF ratio incorporating gold nanoparticles into the Bombyx mori silk protein fibroin biopolymer are demonstrated. The novel electronic memory effect is based on filamentary switching, which leads to the occurrence of bistable states with an ON=OFF ratio larger than six orders of magnitude. The mechanism of this process is attributed to the formation of conductive filaments through silk fibroin and gold nanoparticles in the nanocomposite. The proposed hybrid bio-inorganic devices show promise for use in future flexible and transparent nanoelectronic systems.

Polymer capped CdSe nanoparticles have been grown using a simple chemical process. Transmission electron microscopy study revealed the formation of isolated CdSe nanoparticles encapsulated in polyvinyl alcohol matrix. The optical band gap of nanoparticle has been found to be blue shifted from the bulk value due to quantum confinement of carriers. The recombination dynamics of polymer encapsulated nanoparticles has been studied in details using time resolved photoluminescence measurements. Room temperature photoluminescence decay has been used to explain the biexponential decay characteristics originated from the delocalized and localized carriers. The spectral photocurrent measurements of CdSe-polymer composite materials have also been studied.

Cadmium sulfide (CdS)-decorated zinc oxide (ZnO) nanorod heterostructures have been grown by a combination of hydrothermal and pulsed laser deposition techniques. Hybrid photovoltaic devices have been fabricated with CdS modified and unmodified ZnO nanorods blended separately with regioregular poly(3-hexylthiophene) (P3HT) polymer as the active layer. The solar cell performance has been studied as a function of ZnO concentration and the casting solvent (chlorobenzene, chloroform, and toluene) in the unmodified ZnO:P3HT devices. The power conversion efficiency is found to be enhanced with the increase of ZnO concentration up to a certain limit, and decreases at a very high concentration. The surface modification of ZnO nanorods with CdS leads to an increase in the open circuit voltage and short-circuit current, with enhanced efficiency by 300% over the unmodified ZnO:P3HT device, because of the cascaded band structure favoring charge transfer to the external circuit.

Infectious bronchitis virus (IBV) is a highly contagious respiratory coronavirus of domestic chickens. Although mortality is low, infection with IBV results in substantial losses for the egg and meat chicken industries. Despite the economic importance of IBV and decades of research into the pathogenesis of infection, significant gaps in our knowledge exist. The aim of this study was to compare the early progression of air sac lesions in birds receiving a vaccine strain of the virus or a more virulent field strain. The air sacs are lined by different types of epithelia and are relatively isolated from the environment, so they represent a unique tissue in which to study virus-induced lesions. Both the pathogenic and vaccine strains of the virus produced significant lesions; however, the lesions progressed more rapidly in the birds receiving the pathogenic strain. Immunohistochemistry demonstrated that in birds infected with the pathogenic strain of virus, IBV spike protein is detected first in the ciliated cells lining the air sac. These preliminary data provide important clues regarding potential mechanisms for IBV tissue tropism and spread and show that the nature of the virus isolate influences the early progression of IBV infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Breath analysis techniques offer a potential revolution in health care diagnostics, especially if these techniques can be brought into standard use in the clinic and at home. The advent of microsensors combined with smart sensor system technology enables a new generation of sensor systems with significantly enhanced capabilities and minimal size, weight and power consumption. This paper discusses the microsensor/smart sensor system approach and provides a summary of efforts to migrate this technology into human health breath monitoring applications. First, the basic capability of this approach to measure exhaled breath associated with exercise physiology is demonstrated. Building from this foundation, the development of a system for a portable asthma home health care system is described. A solid-state nitric oxide (NO) sensor for asthma monitoring has been identified, and efforts are underway to miniaturize this NO sensor technology and integrate it into a smart sensor system. It is concluded that base platform microsensor technology combined with smart sensor systems can address the needs of a range of breath monitoring applications and enable new capabilities for healthcare.

The QT35 cell line, established from 20-methylcholanthrene (MCA)-induced tumours in Japanese quail, is positive for Marek's disease virus (MDV), and therefore we examined whether MDV is important for the development of MCA-induced tumours. Japanese quail were inoculated with the JM16 strain of MDV at 1 or 3 days of age or left uninoculated. At 3 weeks of age, quail were injected in the breast muscle with 4 mg MCA in corn oil or corn oil alone. Quail were observed for tumours three times/week and at post mortem at 11 to 12 weeks of age. MDV DNA was detected by polymerase chain reaction (PCR) in spleens of 14/20 birds inoculated with JM16+corn oil and of 53/71 birds inoculated with JM16+MCA. Interestingly, 1/74 quail was positive in the MCA group alone for MDV DNA. Tumours were collected for histopathology, cell line development, and PCR and reverse transcriptase-PCR for the presence of MDV. Tumours developed in 38/83 MCA-treated and 32/85 JM16+MCA-treated quail. Fibrosarcomas without metastasis were the only tumours observed in the MCA-treated quail, while quail treated with JM16 and MCA developed undifferentiated tumours, fibrosarcomas, lymphosarcomas or combinations with or without metastasis. One out of 20 quail receiving JM16 alone developed a lymphosarcoma. Cell line development was not influenced by JM16. Tumours from MCA-treated quail were negative for MDV, while 19/29 were positive in the JM16+MCA group. MDV transcripts were present in 13/18 tumours examined in the JM16+MCA group. In conclusion, MDV did not affect tumour development but did influence tumour aggression and histological type.

An avian poxvirus from the beak scab of an American flamingo (Phoeniconais ruber rubber) was isolated by inoculation on the chorioallantoic membrane (CAM) of specific-pathogen-free (SPF) chicken embryos. The virus produced multifocal areas of epithelial hyperplasia along with foci of inflammation in the CAM, and rare cells contained small eosinophilic intracytoplasmic bodies. Chickens inoculated with the isolated virus in the feather follicle of the leg did not develop significant lesions. Nucleotide sequence comparison of a PCR-amplified 4.5 kb HindIII fragment of the genome of flamingo poxvirus (FlPV) revealed very high homology (99.7%) with condor poxvirus (CPV), followed by approximately 92% similarity with canary poxvirus (CNPV) and Hawaiian goose poxvirus (HGPV), but less similarity (approximately 69%) to fowl poxvirus (FPV), the type species of the genus Avipoxvirus of family Poxviridae. As in the cases with CPV, CNPV, and HGPV, genetic analysis of FlPV revealed an absence of three corresponding FPV open reading frames (ORF199, 200, and 202) and an absence of any reticuloendotheliosis virus (REV) sequences in this region. There are only nine nucleotide substitutions observed between FlPV and CPV in the 4.5 kb fragment; those were clustered in the ORF201 region, which in FPV genome is a site for integration of REV sequences. Phylogenetic analysis of the predicted amino acid sequences of the ORF201-coded hypothetical protein demonstrated FlPV to be more closely related to CPV, as well as to CNPV and HGPV, than to FPV.

The operation of a nonvolatile memory device is demonstrated using junction-like CdS nanocomposites embedded in a polymer matrix. The capacitance-voltage characteristics of Al/conducting polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene-vinylene]/CdS nanocomposites in a polyvinyl alcohol matrix/indium tin oxide device exhibit hysteresis, which is attributed to the trapping, storage, and emission of holes in the quantized valence band energy levels of isolated CdS nanoneedles. The characteristics at different operating frequencies show that the hysteresis is due to trapping of charge carriers in CdS nanocomposites rather than in the interfacial states. The memory behavior in the inorganic/organic heterostructure is explained on the basis of a simple energy band diagram.

The California 99 (Cal99) variant of infectious bronchitis virus (IBV) was first recovered in 1999 from vaccinated broiler chicken flocks in Central California. The S1 hypervariable region of Cal99 genome was most closely related to Arkansas (Ark) serotype viruses. In this study, the complete genome of Cal99 was sequenced, and the structural protein genes were compared with those of commonly used IBV vaccines as well as those of isolates from naturally occurring outbreaks in different parts of the world, to elucidate potential sources of genetic material. Based on sequence comparison, the prototype Cal99 virus is similar to the apathogenic ArkDPI virus, except in the S1 gene and stretches of sequence in the S2 and M structural protein genes, which are more related to Connecticut (Conn) and Massachusetts (Mass) strain viruses, respectively. We speculate that these two fragments came from a Conn and a Mass virus, respectively, and were incorporated into a virus largely derived from ArkDPI. Since Ark, Conn and Mass strains have been simultaneously used as live vaccines in California, both point mutations and recombination among vaccine strains may have contributed to the emergence of the Cal99 variant virus. Analysis of the structural protein genes of six Cal99 isolates demonstrated that viruses of this serotype may differ substantially in the non-S1 structural genes. Finally, we performed a challenge study with Cal99 and demonstrated that the virus causes late-onset respiratory disease, with a severity comparable to that of the M41 IBV challenge strain.

Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5' end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PL(pro)), main protease (M(pro)), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase gene are important for virus replication. We have sequenced four regions of the replicase genes corresponding to the 5'-terminal sequence, PL(pro), M(pro), and RdRp domains from 20 heterologous IBV strains, and compared them with previously published coronavirus sequences. All the coronavirus 5'-termini and PL(pro) domains were divergent, unlike the M(pro) and the RdRp domains that were highly conserved with 28% and 48% conserved residues, respectively. Among IBV strains, the 5' untranslated region including the leader sequence was highly conserved (>94% identical); whereas, the N-terminal coding region and the PL(pro) domains were highly variable ranging from 84.6% to 100%, and 77.6% to 100% identity, respectively. The IBV M(pro) and RdRp domains were highly conserved with 82.7% and 92.7% conserved residues, respectively. The BJ strain was the most different from other IBVs in all four regions of the replicase. Phylogeny-based clustering based on replicase genes was identical to the antigen-based classification of coronaviruses into three groups. However, the IBV strain classification based on replicase gene domains did not correlate with that of the type-specific antigenic groups. The replicase gene sequences of many IBVs recovered from infected chickens were identical to those of vaccine viruses irrespective of serotype, suggesting that either there has been an exchange of genetic material among vaccine and field isolates or that there is a convergent evolution to a specific replicase genotype. There was no correlation between the genotype of any region of the replicase gene and pathotype, suggesting that the replicase is not the sole determinant of IBV pathogenicity.

We used slot blot hybridization of the hypervariable regions of the S1 subunit of spike peplomer gene to identify and characterize infectious bronchitis virus (IBV) strains. Template DNA was created from six reference strain IBVs of different serotypes and immobilized on a nitrocellulose membrane. We synthesized digoxigenin-labeled probes from reference and unknown field viruses and hybridized them to template DNA. All reference strains could be distinguished and isolates identified by serotype if they were at least 95% identical to a reference strain. This slot blot hybridization procedure was specific and reproducible, and strain typing was consistent with the S1 sequencing of the IBV genome. This study thus provides a simple and rapid method for typing of IBV.

In order to verify a commonly held assumption that only Massachusetts (Mass) serotype of infectious bronchitis virus (IBV) was prevalent in the United States between the 1930s (when IBV was first isolated) and the 1950s (when the use of commercial IBV vaccines began), we examined 40 IBV field isolates from the 1940s. Thirty-eight of those isolates were recognized as Mass serotype viruses based on their reactivity to Mass-specific monoclonal antibody (Mab) and neutralization by Mass-specific chicken serum. The remaining two isolates, N-M24 and N-M39, that did not react with Mass-specific Mab, resisted neutralization by Mass-specific chicken serum, and were neutralized only by homologous chicken antibody were identified as non-Mass IBV. When the first 900 nucleotides (nt) from the 5'-end of the spike (S1) glycoprotein gene and their deduced amino acid (aa) sequences were compared, the two non-Mass isolates differed from each other by 24% and 28%, respectively. In a similar comparison, the non-Mass viruses N-M24 and N-M39 differed from M28, a Mass-type isolate from the 1940s, by 21% and 22% (nt) and 28% and 27% (aa), respectively. These data indicate that antigenic and genetic diversity among IBV isolates existed even in the 1940s. Interestingly, when the N-terminal region of the S1 of M28 was compared to that of M41, a prototype Mass virus that has undergone countless number of in vivo and in vitro host passages, the two viruses differed by only 2% (nt) and 4% (aa). This finding suggests that frequent genetic changes are not inherent in all IBV genomes.

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.

Chicks hatched with high levels of maternal antibody had excellent protection (>95%) against infectious bronchitis virus (IBV) challenge at 1 day of age, but not at 7 days (<30%). This protection significantly (P<0.05) correlated with levels of local respiratory antibody and not with serum antibody.A high percentage of both maternal antibody-positive (Mab+) and maternal antibody-negative (Mab-) chicks failed to produce IBV antibody when vaccinated at 1 day of age by the intraocular route. In addition, Mab+ chickens had a weaker virus-neutralizing antibody response to a second IBV vaccination compared to Mab- birds (P<0.05). Mab+ chicks experienced a more rapid decline (P<0.01) in maternal antibody after 1-day-of-age vaccination compared to their unvaccinated counterparts.A monoclonal antibody-based blocking ELISA that measured antibody levels specific to S1 glycoprotein of IBV correlated well with virus-neutralizing antibody titers.