Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay.

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.

Human parainfluenza virus circulation, United States, 2011-2019.

BACKGROUND: Human parainfluenza viruses (HPIVs) cause upper and lower respiratory tract illnesses, most frequently among infants and young children, but also in the elderly. While seasonal patterns of HPIV types 1-3 have been described, less is known about national patterns of HPIV-4 circulation. OBJECTIVES: To describe patterns of HPIVs circulation in the United States (US). STUDY DESIGN: We used data from the National Respiratory and Enteric Virus Surveillance System (NREVSS), a voluntary passive laboratory-based surveillance system, to characterize the epidemiology and circulation patterns of HPIVs in the US during 2011-2019. We summarized the number of weekly aggregated HPIV detections nationally and by US census region, and used a subset of data submitted to NREVSS from public health laboratories and several clinical laboratories during 2015-2019 to analyze differences in patient demographics. RESULTS: During July 2011 - June 2019, 2,700,135 HPIV tests were reported; 122,852 (5 %) were positive for any HPIV including 22,446 for HPIV-1 (18 %), 17,474 for HPIV-2 (14 %), 67,649 for HPIV-3 (55 %), and 15,283 for HPIV-4 (13 %). HPIV testing increased substantially each year. The majority of detections occurred in children aged ≤ 2 years (36 %) with fluctuations in the distribution of age by type. CONCLUSIONS: HPIVs were detected year-round during 2011-2019, with type-specific year-to-year variations in circulation patterns. Among HPIV detections where age was known, the majority were aged ≤ 2 years. HPIV-4 exhibited an annual fall-winter seasonality, both nationally and regionally. Continued surveillance is needed to better understand national patterns of HPIV circulation.

Respiratory Pathogens in Infants Diagnosed with Acute Lower Respiratory Tract Infection in a Tertiary Care Hospital of Western India Using Multiplex Real Time PCR.

OBJECTIVE: To determine the frequency of respiratory pathogens in infants diagnosed with acute lower respiratory tract infections. METHODS: A prospective cross-sectional observational study was conducted in infants hospitalized with a diagnosis of acute lower respiratory tract infection (ALRTI), in a tertiary care hospital in a metropolitan city of Western India. Nasopharyngeal swabs were analyzed by multiplex real time polymerase chain reaction, for 18 viruses and 3 bacteria (H. influenzae type b, C. pneumoniae and M. pneumoniae). The entire data was entered in Microsoft excel sheet and frequencies were determined. RESULTS: One hundred eligible infants were enrolled. Pathogens were detected in 82 samples, which included Respiratory syncytial viruses (RSV) A / B (35.4%), Human rhinovirus (25.6%), Adenovirus (22%), Human Parainfluenza viruses (11%), Human bocavirus (9.8), Human metapneumovirus A / B (8.5%), Influenza A (H1N1) pdm 09 (6.1%), Parechovirus (3.7%), Human coronaviruses (3.66%), Haemophilus influenzae type b (6.1%), Chlamydia pneumoniae (2.4%) and Mycoplasma pneumoniae (2.4%). Influenza A (other than H1N1), Influenza B, Human Coronavirus 229E and Enterovirus were not detected. The rate of coinfection was 34% and rhinovirus was the most common of the multiple pathogens. CONCLUSIONS: Spectrum of viral etiologies of ALRTI is highlighted. Etiological diagnosis of ALRTI would enable specific antiviral therapy, restrict antibiotic use and help in knowing burden of disease.

Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine.

Porcine parainfluenza virus type 1 (PPIV-1) is a member of the genus Respirovirus in the family Paramyxoviridae. The PPIV-1 was initially detected in 2013 from slaughter pigs in Hong Kong, China although its role in respiratory disease has remained unknown without virus isolates for experimental inoculation in swine. The objective of this study was to determine the relative frequency of PPIV-1 detection in diagnostic samples collected from swine in the United States, describe the cell culture isolation of PPIV-1, and characterize PPIV-1 cell culture isolates in vitro. Among 842 porcine specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 2016-2017, 43.3% were PPIV-1 positive by a real-time, reverse transcriptase PCR suggesting PPIV-1 may be common in swine. Two strains of PPIV-1 were successfully isolated in an LLC-MK2 cell line from a PPIV-1 RT-qPCR positive nasal swab (USA/MN25890NS/2016) and lung (USA/IA84915LG/2017). The PPIV-1 cytopathic effect was demonstrated in tissue culture and enveloped viral particles were observed by electron microscopy. The whole genome, F, and HN gene sequences of both isolates share 98.2%, 98.5%, and 98.2% nucleotide homology, respectively, and phylogenetic analysis indicated they are closely related to other PPIV-1 strains detected in swine from the United States. Whole virus PPIV-1-specific monoclonal antibodies were generated for PPIV-1 detection in infected LLC-MK2 cells by indirect immunofluorescence and immunocytochemistry assays. The virus isolates and monoclonal antibodies obtained in the present study can be used to investigate the pathogenesis of PPIV-1 and develop new diagnostic tests.

New clinical and seasonal evidence of infections by Human Parainfluenzavirus.

Human Parainfluenzaviruses (PIVs) account for a significant proportion of viral acute respiratory infections (ARIs) in children, and are also associated with morbidity and mortality in adults, including nosocomial infections. This work aims to describe PIV genotypes and their clinical and epidemiological distribution. Between December 2016 and December 2017, 6121 samples were collected, and submitted to viral culture and genomic quantification, specifically Parainfluenza 1-4 (PIV1-4), Influenza A and B, Respiratory Syncytial Virus (RSV) A and B, Adenovirus, Metapneumovirus, Coronavirus, Rhinovirus, and Enterovirus. Normalized viral load, as (log10) copies/103 cells, was calculated as virus Ct, determined by multiple qRT-PCR, as a function of the Ct of ß-globin. PIV was confirmed in 268 cases (4.37%), and linked to both upper and lower respiratory tract disease, being more frequent in children than in adults (5.23 and 2.43%, respectively). PIV1 and PIV3 were most common (31 and 32.5%, of total PIV positive samples, respectively), with distribution being similar in children and adults, as was viral load. PIV type was correlated with seasonality: PIV3 being more frequent in winter and spring, PIV1 in summer, and PIV 4 in fall. No correlation between vial load and clinical severity was found. Novel findings were that PIV viral load was higher in fall than in other seasons, and PIV4, classically linked to mild respiratory symptoms, was circulating, in children and adults, at all levels of symptoms throughout the year.

Rapid detection of three rabbit pathogens by use of the Luminex x-TAG assay.

BACKGROUND: Domestic rabbits especially New Zealand white rabbits play an important role in biological research. The disease surveillance and quality control are essential to guarantee the results of animal experiments performed on rabbits. Rabbit hemorrhagic disease virus, rabbit rotavirus and Sendai virus are the important pathogens that needed to be eliminated. Rapid and sensitive method focus on these three viruses should be established for routine monitoring. The Luminex x-TAG assay based on multiplex PCR and fluorescent microsphere is a fast developing technology applied in high throughput detection. Specific primers modified with oligonucleotide sequence/biotin were used to amplify target fragments. The conjugation between oligonucleotide sequence of the PCR products and the MagPlex-TAG microspheres was specific without any cross-reaction, and the hybridization products could be analyzed using the Luminex 200 analyzer instrument. Recombinant plasmids were constructed to estimate the detection limit of the viruses. Furthermore, 40 clinical samples were used to evaluate the efficiency of this multiplex PCR based Luminex x-TAG assay. RESULTS: According to the results, this new method showed high specificity and good stability. Assessed by the recombinant plasmids, the detection limit of three viruses was 100copies/µl. Among 40 clinical specimens, 18 specimens were found positive, which was completely concordant with the conventional PCR method. CONCLUSIONS: The new developed Luminex x-TAG assay is an accurate, high throughput method for rapid detection of three important viruses of rabbits.

Dynamics of Sendai Virus Spread, Clearance, and Immunotherapeutic Efficacy after Hematopoietic Cell Transplant Imaged Noninvasively in Mice.

There are no approved vaccines or virus-specific treatments for human parainfluenza viruses (HPIVs), which have recently been reclassified into the species Human respirovirus 1, Human respirovirus 3, Human rubulavirus 2, and Human rubulavirus 4 These viruses cause morbidity and mortality in immunocompromised patients, including those undergoing hematopoietic cell transplant (HCT). No small-animal models for noninvasive imaging of respiratory virus infection in the HCT host exist, despite the utility that such a system would offer to monitor prolonged infection, its clearance, and treatment options. We used a luciferase-expressing reporter virus to noninvasively image in mice the infection of murine respirovirus (strain Sendai virus [SeV]), the murine counterpart of HPIV1. Independent of disease severity, the clearance of infection began approximately 21 days after HCT, largely due to the recovery of CD8+ T cells. Immunotherapy with granulocyte colony-stimulating factor (G-CSF) and adoptive transfer of natural killer (NK) cells provided a limited therapeutic benefit. Treatment with a fusion (F) protein-specific monoclonal antibody arrested the spread of lung infection and reduced the disease severity even when treatment was delayed to up to 10 days postinfection but had little observable effect on upper respiratory tract infection. Adoptive transfer of virus-specific T cells at 10 days postinfection accelerated the clearance by 5 days, reduced the extent of infection throughout the respiratory tract, and reduced the disease severity. Overall, the results support investigation of the clinical treatment of respiratory virus infection in the HCT host with monoclonal antibodies and adoptive T-cell transfer; the imaging system should be extendable to other respiratory viruses, such as respiratory syncytial virus and influenza virus.IMPORTANCE Parainfluenza viruses are a major cause of disease and death due to respiratory virus infection in the immunocompromised host, including those undergoing bone marrow transplantation. There are currently no effective treatment measures. We noninvasively imaged mice that were undergoing a bone marrow transplant and infected with Sendai virus, a murine parainfluenza virus (respirovirus). For the first time, we show the therapeutic windows of adoptive T-cell therapy and treatment with a monoclonal antibody to the fusion (F) protein in clearing Sendai virus from the respiratory tract and reducing disease severity. Mice tolerated these treatments without any detectable toxicity. These findings pave the way for studies assessing the safety of T-cell therapy against parainfluenza virus in humans. Adoptive T-cell therapy against other blood-borne viruses in humans has been shown to be safe and effective. Our model of noninvasive imaging in mice that had undergone a bone marrow transplant may be well suited to track other respiratory virus infections and develop novel preventive and therapeutic strategies.

Development of a blocking ELISA for Caprine parainfluenza virus type 3.

Caprine parainfluenza virus type 3 (CPIV3) is a novel pathogen mainly causing respiratory diseases in goats. At present, there are no high throughput and rapid testing methods available for epidemiological investigation. In this study, we designed a modified method for selection of hybridomas that secrete monoclonal antibodies (mAb) specific for CPIV3. The monoclonal antibodies were obtained by combination of indirect enzyme-linked immunosorbent assay (iELISA) and blocking ELISA (bELISA). The technique was efficient to determine each mAb with specificity and sensitivity. One bELISA was validated for the serological diagnosis of CPIV3. After optimization conditions were established, a total of 205 reference goat sera were tested in parallel by bELISA and by virus neutralization (VN) for their relative performances. The cut-off point was ultimately defined as 33.6% by ROC curve analysis. The bELISA specificity and sensitivity were 99.2% and 98.7%, respectively, and agreement with the VN test was >99.0%. Furthermore, testing another 2919 goat sera by bELISA demonstrated 39.3% prevalence in the goat population, more sensitive than HI detection. This new bELISA would offer higher throughput, sensitivity, and specific detection for CPIV3, and will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs in the future.

[Clinical analysis of children with pertussis and significance of respiratory virus detection in the combined diagnosis].

Objective: To analyze the clinical data of children with pertussis and explore the necessity of respiratory virus detection in the combined diagnosis so as to improve the clinician's understanding and standardize the diagnosis and treatment of pertussis in children. Method: Clinical data and laboratory examination of 195 suspected pertussis children between Jan. 2015 and Dec. 2016 in Children's Hospital Affiliated to Capital Institute of Pediatric were analyzed retrospectively. Result: The nasopharyngeal secretions were collected from 195 suspected pertussis children, PCR was employed to detect the nucleic acid of Bordetella pertussis. Meanwhile, 172 of 195 cases were screened for antigens of 7 common respiratory viruses by direct immunofluorescence (DIF) (respiratory syncytial virus(RSV), adenovirus(ADV), influenza virus A and B, parainfluenza viruses type Ⅰ-Ⅲ). (1) Eighty cases were positive in pertussis nucleic acid detection (positive rate was 41.0%), 47 males and 33 females, age ranged from one month to ten years, all of them had paroxysmal cough (100.0%), 50 cases with spasmodic cough (62.5%), 9 cases with vomiting after cough(11.2%), 22 cases with cyanosis after cough(27.5%), 13 cases with roaring after cough(16.2%), 4 cases with dyspnea(5.0%), 18 cases were diagnosed as pneumonia by chest radiography(22.5%) and 1 case was diagnosed as pertussis encephalopathy(1.2%); (2) 172 cases of respiratory virus DIF detection were completed and 69 of them were positive(positive rate was 40.1%), including 32 cases positive for RSV(18.6%), 29 cases for PIVⅢ(16.8%); (3) In 80 confirmed pertussis children, 66 cases of respiratory virus DIF detection were completed and 9 were positive(9/66, 13.6%), including 7 cases positive for PIVⅢ. The clinical manifestations were cyanosis after cough(6 cases), dyspnea(2 cases) and pneumonia were diagnosed by chest radiography in 3 cases, the clinical symptoms of these children were more prominent than children with general pertussis; (4) Patients were divided into three groups according to the pathogens: 57 cases in single pertussis group, 32 cases in RSV infection group, 22 cases in single PIVⅢ infection group.The cases of spasmodic cough in Pertussis group was 35 (61.4%), RSV infection group was 7(21.9%), single PIVⅢ infection group was 8(36.4%), compared with the other two groups, the incidence of spasmodic cough were higher in Pertussis group (χ(2) =12.850, 4.013, P<0.05). The cases of roaring in Pertussis group was 11 (19.3%), RSV infection group was 1(3.1%), single PIVⅢ infection group was 0, and the incidence were higher in Pertussis group (χ(2)=4.596, 4.932, P<0.05). The cases of dyspnea in Pertussis group was 2 (3.5%), RSV infection group was 11(34.4%), single PIVⅢ infection group was 0, and the incidence was higher in RSV infection group (χ(2)=15.654, 9.487, P<0.01). Conclusion: Pertussis is common in children, especially in unvaccinated or incompletely vaccinated infants. The typical clinical manifestation is paroxysmal spasmodic cough; complicated with PIVⅢ infection is a risk factor for sever pertussis. Early detecting of Bordetella by PCR is helpful for the diagnosis of pertussis, RSV and PIVⅢ are the main pathogen for Pertussis-like syndrome. The detection of respiratory virus is helpful for differential diagnosis and medication guidance.

Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model.

Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD.

Air in All the Wrong Places.

Extraluminal air can occur through a wide variety of mechanisms. Often, the free air resides in isolated regions including the thorax, the peritoneum, or the mediastinum. We present a pediatric case where there was extensive extraluminal air simultaneously within several regions, one of which has never been reported in the literature.

Parainfluenza virus type 3 outbreak in a neonatal intensive care unit.

Parainfluenza virus (PIV) is a respiratory pathogen in young children and is second only to the respiratory syncytial virus (RSV) as a cause of lower respiratory tract infection. PIV type 3 (PIV3) is the most severe. Herein we describe an outbreak of PIV3 in three infants in a neonatal intensive care unit. They were diagnosed on virus culture from pharyngeal swabs. We prevented the spread of the virus using standard infection control procedures and isolation of the symptomatic infants. One infant had severe chronic lung disease and was complicated with recurrent wheezing for a long time. Because RSV and PIV have many structural, pathogenic, epidemiologic, and clinical similarities, we speculate that PIV infection causes recurrent wheezing, as observed with RSV infection. Therefore, physicians must consider recurrent wheezing at the time of treatment of PIV infection early in life.

Establishment of reverse transcription polymerase chain reaction assay for simultaneous detection of human parainfluenza virus types 1 to 4 in children with influenza like illnesses.

OBJECTIVES: The aim of this study was to develop an in-house multiplex reverse transcription polymerase chain reaction (mRT-PCR), which can recognize HPIV1-4 in clinical samples. BACKGROUND: Human parainfluenza virus (HPIV) is one of the major causes of viral respiratory infections and can affect people at any age, especially infants and young children. METHODS: Four sets of specific primers targeting conserved areas of hemagglutinin-neuraminidase (HN) genes of HPIV1-4, were designed and tested with type-related plasmid controls. Specificity and sensitivity of mPCR were tested. One-step mRT-PCR was set up using a viral panel containing 10 respiratory viruses, including HPIVs. One hundred nasopharyngeal samples of respiratory infection patients were tested using the set One-step mRT-PCR. RESULTS: The specificity of set mPCR for HPIV1-4 using plasmid positive controls was proved and reaction sensitivity was measured. The specificity of set mRT-PCR was confirmed and 4 and 5 out of 100 clinical samples were HPIV1 and HPIV2 positive, respectively. CONCLUSION: The developed one-step mRT-PCR in this study is an effective and specific assay for clinical diagnosis of HPIV1 to 4 (Tab. 1, Fig. 6, Ref. 28).

A parainfluenza virus type 3 outbreak at a residential aged care facility: The role of microbiologic testing in early identification and antimicrobial stewardship.

We report an outbreak of parainfluenza 3, which had an attack rate of 30%, in a residential care facility in Melbourne, Australia. One-fifth of affected residents required hospitalization, but there were no deaths. The outbreak demonstrated the value of active surveillance and early microbiologic testing and the urgent need for antimicrobial stewardship programs in the aged care setting.

Rapid detection of novel caprine parainfluenza virus type 3 (CPIV3) using a TaqMan-based RT-qPCR.

Parainfluenza virus type 3 (PIV3) is one of the most important respiratory pathogens for humans and many animals. A novel caprine PIV3 (CPIV3) was recently identified and isolated from Chinese goat flocks with respiratory disease. In order to develop rapid and sensitive methods for CPIV3 detection in infected goats, a TaqMan RT-qPCR was established in this study based on the primers and probe designed to amplify a 150 nucleotide-long region located within the M gene of the virus. The method was able to detect about 1.0×10(1) DNA copies/µL with an efficiency of 99.6% and a R(2) value of 0.997. There were no cross-reaction observed using this technique against peste des petits ruminants virus (PPRV), border disease virus (BDV), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). One hundred and fourteen samples, including nasal swabs, feces swabs, sera, hearts, livers, spleens, lungs, kidneys, tracheas and hilar lymph nodes (HLNs) from six challenged goats, were evaluated by this technique. Using TaqMan RT-qPCR, CPIV3 was positively detected in 51 of 114 samples (44.74%), which was higher than RT-PCR (27.19%, 31/114) and virus isolation (14.9%, 17/114), respectively. The method also gave higher positive detection rate (35%, 42/120) than RT-PCR (28.33%, 34/120) from clinical samples. These data indicated that this method could be used for faster and more accurate monitoring of viral load, disease progression and vaccination efficacy of CPIV3 in goat flocks.

Parainfluenza Virus Types 1, 2, and 3 in Pediatric Patients with Acute Respiratory Infections in Beijing During 2004 to 2012.

BACKGROUND: Although human parainfluenza virus (HPIV) has been determined as an important viral cause of acute respiratory infections (ARIs) in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China. METHODS: Nasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay. RESULTS: Out of these specimens, 1675 (6.50%, 1675/25,773) showed HPIV positive, including 261 (1.01%, 261/25,773) for HPIV1, 28 (0.11%, 28/25,773) for HPIV2, and 1388 (5.39%, 1388/25,773) for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old (352/4077, 8.63%). HPIV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection (12.49%, 157/1257), followed by bronchitis (11.13%, 176/2479), asthma (9.31%, 43/462), bronchiolitis (5.91%, 150/2536), pneumonia (6.06%, 1034/17,068), and those with underlying diseases (1.0%, 15/1506). HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group. CONCLUSIONS: HPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses.

A surface plasmon resonance (SPR)-based biosensor was developed for specific detection of nine common respiratory virus, including influenza A and influenza B, H1N1, respiratory syncytial virus (RSV), parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and severe acute respiratory syndrome coronavirus (SARS). The SPR biosensor was developed by immobilizing nine respiratory virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin was used to label the PCR primer and further amplify the signal by introducing streptavidin after hybridization. Throat swab specimens representing nine common respiratory viruses were tested by the innovative SPR-based biosensor to evaluate the sensitivity, specificity and reproducibility of this method. Results suggest that this biosensor has the potential to simultaneously identify common respiratory viruses.

Development and evaluation of two truncated recombinant NP antigen-based indirect ELISAs for detection of bovine parainfluenza virus type 3 antibodies in cattle.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens in both young and adult cattle. Nucleocapsid protein (NP) is the most abundant viral protein and the main regulator of virus replication and transcription. In this study, amino acid sequence data of BPIV3 NP was used to identify potential linear epitopic regions, which were subsequently used to design truncated recombinant NP antigens. The amino-terminal region (aa 9-157, NP-N) and the carboxy-terminal region (aa 391-500, NP-C) were selected, and these two truncated recombinant BPIV3 NP proteins were expressed in Escherichia coli based on the results of prediction studies. Furthermore, Enzyme-Linked Immunosorbent Assays (ELISAs) were established using the truncated recombinant BPIV3-N proteins as antigens, and 154 clinical samples were used to evaluate the newly established ELISA systems in comparison with a virus neutralisation test (VNT) as a reference. The results showed that a high coincidence rate was observed for the data that were obtained by the two methods. The sensitivity of NP-N ELISA and NP-C ELISA were 98.4% and 94.6%, respectively, and the specificity of both ELISAs was 100% with reference to the VNTs. Our data indicated that both ends of NP have high immunogenicity during BPIV3 infection and that they were good targets for serodiagnosis. The ELISAs based on the two truncated proteins were especially suitable for use in large-scale epidemiological investigations.