Virus Isolation Methods Research Articles

Development of a novel reverse transcription PCR and its application to field sample testing for feline calicivirus prevalence in healthy stray cats in Korea.

BackgroundFeline calicivirus (FCV) is a major and highly infectious pathogen in cats worldwide. However, there have been limited studies about the status of FCV infections in Korea.ObjectivesTo investigate the current status of FCV infections in stray cats in Korea.MethodsA novel reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved nucleotide sequences of reported FCV strains. Field swab samples were collected from 122 cats (2 hospital admitted cats and 120 stray cats) in 2016 and 2017. All the samples were tested by virus isolation and 2 different RT-PCRs, including the novel RT-PCR, for the detection of FCV.ResultsThe novel RT-PCR assay showed no cross-reactivity to the nucleic acids of the other feline pathogens tested, and the limit of detection was calculated as 100 TCID50/mL based on an in vitro assessment. The novel RT-PCR assay detected 5 positive samples from the 122 field samples, which showed perfect agreement with the results of the virus isolation method. In contrast, another RT-PCR assay used in a previous study in Korea detected no positive samples. The prevalence of FCV infection in stray cats was 2.5% (3/120) based on the results of virus isolation and the novel RT-PCR assays.ConclusionsThe current study is the first report of the detection and prevalence of FCV in stray cats in Korea. The novel RT-PCR assay developed in this study showed high sensitivity and specificity, which indicates a useful diagnostic assay to identify FCV infection in cats.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses

BackgroundIn 2017–2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans.MethodsIn this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV.ResultsThe results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry.ConclusionThe duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

Acetylcholinesterase is not a generic marker of extracellular vesicles

ABSTRACTAcetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.

Improved detection of influenza A virus from blue-winged teals by sequencing directly from swab material.

The greatest diversity of influenza A virus (IAV) is found in wild aquatic birds of the orders Anseriformes and Charadriiformes. In these birds, IAV replication occurs mostly in the intestinal tract. Fecal, cloacal, and/or tracheal swabs are typically collected and tested by real‐time RT‐PCR (rRT‐PCR) and/or by virus isolation in embryonated chicken eggs in order to determine the presence of IAV. Virus isolation may impose bottlenecks that select variant populations that are different from those circulating in nature, and such bottlenecks may result in artifactual representation of subtype diversity and/or underrepresented mixed infections. The advent of next‐generation sequencing (NGS) technologies provides an opportunity to explore to what extent IAV subtype diversity is affected by virus isolation in eggs. In the present work, we evaluated the advantage of sequencing by NGS directly from swab material of IAV rRT‐PCR‐positive swabs collected during the 2013–14 surveillance season in Guatemala and compared to results from NGS after virus isolation. The results highlight the benefit of sequencing IAV genomes directly from swabs to better understand subtype diversity and detection of alternative amino acid motifs that could otherwise escape detection using traditional methods of virus isolation. In addition, NGS sequencing data from swabs revealed reduced presence of defective interfering particles compared to virus isolates. We propose an alternative workflow in which original swab samples positive for IAV by rRT‐PCR are first subjected to NGS before attempting viral isolation. This approach should speed the processing of samples and better capture natural IAV diversity.OPEN RESEARCH BADGES This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.3h2n106.

Replacement of neuraminidase inhibitor-susceptible influenza A(H1N1) with resistant phenotype in 2008 and circulation of susceptible influenza A and B viruses during 2009-2013, South Africa.

BackgroundData on the susceptibility of influenza viruses from South Africa to neuraminidase inhibitors (NAIs) are scarce, and no extensive analysis was done.ObjectivesWe aimed to determine oseltamivir and zanamivir susceptibility of influenza A and B virus neuraminidases (NAs), 2007‐2013, South Africa.Patients/MethodsWe enrolled participants through national influenza‐like illness surveillance, 2007‐2013. Influenza diagnosis was by virus isolation and quantitative polymerase chain reaction (qPCR). Drug susceptibility was determined by chemiluminescence‐based NA‐STAR/NA‐XTD assay. Sanger sequencing was used to determine molecular markers of NAI resistance.ResultsForty percent (6341/15 985) of participants were positive for influenza viruses using virus isolation (2007‐2009) and qPCR (2009‐2013) methods. A total of 1236/6341 (19.5%) virus isolates were generated of which 307/1236 (25%) were tested for drug susceptibility. During 2007‐2008, the median 50% inhibitory concentration (IC50) of oseltamivir for seasonal influenza A(H1N1) increased from of 0.08 nmol/L (range 0.01‐3.60) in 2007 to 73 nmol/L (range 1.56‐305 nmol/L) in 2008. Influenza A isolates from 2009 to 2013 were susceptible to oseltamivir [A(H3N2) median IC50 = 0.05 nmol/L (range 0.01‐0.08); A(H1N1)pdm09 = 0.11 nmol/L (range 0.01‐0.78)] and zanamivir [A(H3N2) median IC50 = 0.56 nmol/L (range 0.47‐0.66); A(H1N1)pdm09 = 0.35 nmol/L (range 0.27‐0.533)]. Influenza B viruses were susceptible to both NAIs. NAI resistance‐associated substitutions H275Y, E119V, and R150K (N1 numbering) were not detected in influenza A viruses that circulated in 2009‐2013.ConclusionsWe confirm replacement of NAI susceptible by resistant phenotype influenza A(H1N1) in 2008. Influenza A and B viruses (2009‐2013) remained susceptible to NAIs; therefore, these drugs are useful for treating influenza‐infected patients.

Isolation, identification and molecular characterization of Newcastle disease viruses in vaccinated chickens from commercial farms in the Sultanate of Oman

Newcastle disease (ND) remains an important enzootic disease in chickens in several parts of the world. With the increasing reports of virulence and genetic diversity of the causative agent; Newcastle disease virus (NDV), there is a need to identify the circulating NDV in specific regions. In Oman, to this moment, such information is still lacking. The aim of this study was to isolate and characterize the NDV from ND outbreaks from commercial farms in Oman. Following suspected outbreaks of ND in three commercial farms in 2017, a total of 30 carcasses (10 from each flock) of adult chickens were subjected to necropsy for gross and histopathological examination, virus isolation and molecular methods. Specifically, haemagglutination inhibition (HI) test and reverse transcription-polymerase chain reaction (RT-PCR) assay were used for the virus detection and confirmation, respectively. Lesions were suggestive of viscerotropic velogenic form of ND based on gross and histopathological examinations. Isolation of NDV was present in 4 cases and further confirmed by RT-PCR following the target of the partial fusion protein gene of the viral genome. The sequence of the partial fusion gene was determined and phylogenetic tree was constructed based on the partial length F gene of 4 Omani isolates and 65 previously published NDVs. The findings predicted that the Omani isolates had high homology (99%) with the isolate from Pakistan belonging to genotype VII. Subsequently, the isolated pathotype was identified as the virulent NDV. This study serves as a basic work for further research on the analysis and phenotyping of NDV in the Sultanate of Oman. Improved monitoring and surveillance of the disease is important for proper preventive measures.

Viral, Serological, and Antioxidant Investigations of Equine Rhinitis A Virus in Serum and Nasal Swabs of Commercially Used Horses in Poland.

Background Equine rhinitis A virus (ERAV) is considered to be an important pathogen in horses, but relatively few studies are available. Aims The purpose of this study was to verify ERAV seroprevalence in selected horses in Poland, in addition to correlation between ERAV and age and sex of analysed animals and the antioxidant status. Methods The material collected from clinically healthy horses was tested using the VNT (353 serum samples) and virus isolation method (44 nasal swabs). 27 serum samples with antibody titers between 0 and ≥1 : 2048 were chosen for further analysis. The study was conducted in group 1 (ERAV titer ≤ 64) and group 2 (ERAV titer > 64). Results Seroneutralisation tests showed positive results in 72% of serum samples. No significant correlation between ERAV seropositive results and selected biochemical indicators was observed. Group 2 had statistically higher concentrations of SOD and CuZnSOD than the analysed group 1. Conclusions ERAV was not detected in the nasal swab samples. Antioxidant parameters did not significantly vary between horses of different breed, sex, or age. The ERAV virus had an impact on plasma total SOD and Cu/Zn SOD activity in horses in early stages of convalescence.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I.

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

The first external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016

BackgroundThe isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays. ObjectivesTo conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions. Study designTwenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification. ResultsAll NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories. ConclusionThis first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.

Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

Comparison of ELISA and dual stage real time RT-PCR to differentiate Sabin like and non-Sabin like poliovirus isolates.

Environmental surveillance of polioviruses has been used as an important tool in monitoring circulation of wild polioviruses and/or Vaccine derived polioviruses in sewage samples. It is important to distinguish Sabin like isolates from non-Sabin like; ELISA & dual stage real time RT-PCR have been used for the same. Current study was carried out on sewage isolates to compare ELISA & RT-PCR with sequencing to distinguish Sabin like from non-Sabin like. Out of 468 sewage specimens, 91 (19.44%) were non-polio enteroviruses positive and 377 (80.56%) were polio positive by virus isolation method. A total of 488 polio virus isolates were detected by L20B and RD route which were further subjected to ELISA and RT-PCR. The results were compared with sequencing. On comparison, the specificity of ELISA was only 66.67% in spite of very low sensitivity (3.43%). The sensitivity of RT-PCR was 97.71% which makes it a good primary screening test for detection of non-Sabin like viruses. However, the specificity was only 33.33%. RT-PCR appears to be a sensitive tool for detecting non-Sabin like viruses however; the isolates which are non-Sabin like by RT-PCR may not necessarily be mutated viruses. ELISA cannot be used for differentiation of Sabin likes from non-Sabin likes due to low sensitivity.

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.

Dengue Virus Isolation in Mosquito Aedes albopictus Captured During an Outbreak in Tokyo, 2014, by a Method Relying on Antibody-Dependent Enhancement Mechanism Using FcγR-Expressing BHK Cells.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.

Surveillance, epidemiological, and virological detection of highly pathogenic H5N1 avian influenza viruses in duck and poultry from Bangladesh

Avian influenza viruses (AIVs) continue to pose a global threat. Waterfowl are the main reservoir and are responsible for the spillover of AIVs to other hosts. This study was conducted as part of routine surveillance activities in Bangladesh and it reports on the serological and molecular detection of H5N1 AIV subtype. A total of 2169 cloacal and 2191 oropharyngeal swabs as well as 1725 sera samples were collected from live birds including duck and chicken in different locations in Bangladesh between the years of 2013 and 2014. Samples were tested using virus isolation, serological tests and molecular methods of RT-PCR. Influenza A viruses were detected using reverse transcription PCR targeting the virus matrix (M) gene in 41/4360 (0.94%) samples including both cloacal and oropharyngeal swab samples, 31 of which were subtyped as H5N1 using subtype-specific primers. Twenty-one live H5N1 virus isolates were recovered from those 31 samples. Screening of 1,868 blood samples collected from the same birds using H5-specific ELISA identified 545/1603 (34%) positive samples. Disconcertingly, an analysis of 221 serum samples collected from vaccinated layer chicken in four districts revealed that only 18 samples (8.1%) were seropositive for anti H5 antibodies, compared to unvaccinated birds (n=105), where 8 samples (7.6%) were seropositive. Our result indicates that the vaccination program as currently implemented should be reviewed and updated. In addition, surveillance programs are crucial for monitoring the efficacy of the current poultry vaccinations programs, and to monitor the circulating AIV strains and emergence of AIV subtypes in Bangladesh.

Evaluation of FTA® card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA® cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA® card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA® card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA® card impregnated with clinical samples that had been stored at varying temperature (4–37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA® card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA® card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus (swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like (EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription (RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets (outer and inner) were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit (PFU) mL−1 which was over 104 PFU mL−1 for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Etiology study on severe cases caused by hand-foot-mouth disease in children from Henan province, 2014

To investigate the etiology of severe hand-foot-mouth disease (HFMD) in children in Henan province. A total of 244 HFMD cases admitted to a hospital in Zhengzhou from April to June of 2014 were recruited for research sampling, Real-time RT-PCR, virus isolation, VP1 sequencing and alignment methods were used to test the enterovirus-related etiology. SPSS 17.0 was used in performing statistical analysis. There were 109 severe and 135 mild cases among all the 244 HFMD cases. The number of enterovirus positive stool samples was 229, with positive rate as 93.85%. EV71, Cox A16 and Cox A10 made up 83.84%, 5.68% and 8.30% of the enterovirus etiologicy, strains, respectively. EV71 infection caused 8 HFMD cases with heart-lung failure and 2 death, Cox A10 infection led to 1 HFMD case with heart-lung failure and death. There were statistically differences seen regarding the enterovirus infection rates between severe and the mild HFMD cases (χ(2)=5.312,P=0.021). Statistically significant difference was seen in the constituent ratio of EV71, Cox A16 and the others by Fisher' s exact test (P=0.048). There was statistically significant difference seen between the cardiorespiratory failure rate and the fatality rate by EV71 and Cox A10 infection (χ(2)=0.051,P=0.821; χ(2)=2.198,P=0.138). Cox A10 strains idenfied in Henan in 2014 belonged to genotype 6. The rates on homology of nucleotide and amino acid among the Cox A10 strains in Henan in 2014 were 94.3%-99.7% and 96.3%-100.0% respectively. EV71 still remained the most common pathogen that causing severe HFMD in children, with the increasing Cox A10 percentage in the pathogens spectrum of HFMD infection. Cox A10 strains in Henan in 2014 belonged to genotype 6. Genotype 6 Cox A10 had appeared and widely distributed in Henan for long time, but not yet variated or reconstructed. Cox A10 infection could lead to cardio-respiratory failure thus called for the monitoring program on non-EV71 and non-Cox A16 enterovirus, especially Cox A10 to be strenthened.

Repeated Detection of an Invertebrate Iridovirus in Amphibians

Abstract Invertebrate iridoviruses (IIVs) (family: Iridoviridae) are known pathogens for invertebrates, causing high mortality and reduced fertility in affected insects. Over the past two decades, IIVs have also been increasingly found in lizards in association with nonspecific clinical signs. It has been hypothesized that IIVs from insects can also infect reptiles. From 2010–11, IIVs were repeatedly detected via polymerase chain reaction testing and virus isolation methods in routine diagnostic samples from different amphibians: three blue poison dart frogs (Dendrobates tinctorius azureus), four edible frogs (Pelophylax kl. esculentus), a giant ditch frog (Leptodactylus fallax), an Amazon milk frog (Trachycephalus resinifictrix), mixed organs from agile frogs (Rana dalmatina), a black-spined toad (Bufo melanostictus), and one Lake Urmia newt (Neurergus crocatus). IIVs were found in skin swabs from apparently healthy animals, as well as in multiple organs of frogs that died of unknown causes. Prey insects...