Viral Nucleic Acid Sequences Research Articles

A novel syndrome of silent rhinovirus-associated bronchoalveolitis in children with recurrent wheeze

BackgroundRhinovirus (RV) infections trigger wheeze episodes in children. Thus, understanding of the lung inflammatory response to RV in children with wheeze is important. ObjectiveTo examine the associations of RV on lung lavage (BAL) granulocyte patterns and biomarkers of inflammation with age in children with treatment-refractory, recurrent wheeze (n=616). MethodsChildren underwent BAL to examine viral nucleic acid sequences, bacterial cultures, granulocyte counts, and phlebotomy for both general and type-2 inflammatory markers. ResultsDespite the absence of cold symptoms, RV was the most common pathogen detected (30%), and when present, was accompanied by BAL granulocytosis in 75% of children. Compared to children with no BAL pathogens (n=341), those with RV alone (n=127) had greater (p < 0.05) isolated neutrophilia (43% versus 16%), mixed eosinophils and neutrophils (26% versus 11%), and less pauci-granulocytic (27% versus 61%) BAL. Children with RV alone furthermore had biomarkers of active infection with higher total blood neutrophils and serum c-reactive protein (CRP), but no differences in blood eosinophils or total IgE. With advancing age, the log odds of BAL RV alone were lower, 0.82 [0.76-0.88, p < 0.001], but higher, 1.58 [1.01-2.51, p = 0.04], with high-dose daily corticosteroid treatment. ConclusionsChildren with severe recurrent wheeze often (22%) have a silent syndrome of lung RV infection with granulocytic bronchoalveolitis and elevated systemic markers of inflammation. The syndrome is less prevalent by school age, and not informed by markers of type-2 inflammation. We speculate that dysregulated mucosal innate antiviral immunity is a responsible mechanism.

Advancements in Nanopore Technology for Virus Detection

Background: The spread of infectious diseases caused by viruses is always a global concern to public health. Developing affordable, accurate, fast and effective technologies for virus detection is crucial in reducing virus transmission. A nanopore is a sensor that can identify target molecules at a single molecule level, often used for genome sequencing and early disease detection. Nanopores are classified in two types: biological nanopores, ideal for detecting viral nucleic acid sequences, and solid-state nanopores primarily used to detect viral particles. Methods: In this review, we first provide a brief overview of the properties and fundamental principles of these two types of the nanopore. Then, we focus on the application of nanopores in viral nucleic acid sequencing and the quantitative detection of viral nanoparticles. Additionally, we discuss new strategies combining nanopore sensors with other technologies, which greatly improve the sensing performance. Results: A literature review on the application of nanopores in controlling viral epidemics is provided. The pros and cons of biological nanopores and solid-state nanopores are summarized, respectively, and the opportunities of integrating novel technologies with nanopore sensors to enhance the latter are addressed in this paper. Conclusion: Owing to significant advancements in nanotechnology and integration with other technologies such as machine learning, nanopore sensors are becoming widely applied in virusesrelated analysis. In the long term, nanopore sensors are expected to play an important role in the field of virus detection and analysis.

Distance-Regulated Photoelectrochemical Sensor "Signal-On" and "Signal-Off" Transitions for the Multiplexed Detection of Viruses Exposed in the Aquatic Environment.

Photochemical (PEC) sensors were severely limited for multiplex detection applications due to the cross interference between multiplex signals at the single recognition interface. In this work, a distance-regulated PEC sensor was developed for multiplex detection by using an i-Motif sequence with conformational transformation activity as the signal transduction unit. Through dynamic regulation of the spatial distance between the end site of the functional sequence and the electrode material, the photogenerated electrons on the surface of the sensor were directionally transferred. Thus, a PEC sensor with "signal-on" and "signal-off" dual signal output modes was developed for simultaneous detection of multitarget molecules. Combining isothermal nucleic acid amplification, the PEC sensor constructed in this work was successfully applied to the detection of two virus (Norovirus and Rotavirus) nucleic acid sequences. Under the optimal condition, this bioassay protocol exhibits a linear range of 0.01-100 nM for both viruses with detection limits of 0.72 and 0.53 pM, respectively. In this study, a stimulus-mediated distance regulation strategy successfully addressed the transduction of multiplex detection signals at the single recognition interface of the PEC sensor. It is expected that the technical barriers to multiplex detection of PEC sensors will be overcome and the application of PEC sensing technology will be expanded in the field of environmental analysis.

Metagenomic Next-Generation Sequencing Reveals the Profile of Viral Infections in Kidney Transplant Recipients During the COVID-19 Pandemic.

BackgroundTo study the clinical application of metagenomic next-generation sequencing (mNGS) in the detection of viral infections in kidney transplant recipients (KTRs) during the COVID-19 pandemic.MethodsUsing mNGS technology, 50 human fluid samples of KTRs were detected, including 20 bronchoalveolar lavage fluid (BALF) samples, 21 urine samples and 9 blood samples. The detected nucleic acid sequences were compared and analyzed with the existing viral nucleic acid sequences in the database, and the virus infection spectrum of KTRs was drawn.ResultsThe viral nucleic acids of 15 types of viruses were detected in 96.00% (48/50) of the samples, of which 11 types of viruses were in BALF (95.00%, 19/20), and the dominant viruses were torque teno virus (TTV) (65.00%; 13/20), cytomegalovirus (CMV) (45.00%; 9/20) and human alphaherpesvirus 1 (25.00%; 5/20). 12 viruses (95.24%, 20/21) were detected in the urine, and the dominant viruses were TTV (52.38%; 11/21), JC polyomavirus (52.38%; 11/21), BK polyomavirus (42.86%; 9/21), CMV (33.33%; 7/21) and human betaherpesvirus 6B (28.57%; 6/21). 7 viruses were detected in the blood (100.00%, 9/9), and the dominant virus was TTV (100.00%; 9/9). Four rare viruses were detected in BALF and urine, including WU polyomavirus, primate bocaparvovirus 1, simian virus 12, and volepox virus. Further analysis showed that TTV infection with high reads indicated a higher risk of acute rejection (P < 0.05).ConclusionsmNGS detection reveals the rich virus spectrum of infected KTRs, and improves the detection rate of rare viruses. TTV may be a new biomarker for predicting rejection.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome

IntroductionThe diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model.ObjectivesHere we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient’s infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased).MethodsHigh resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created.ResultsThe predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74–0.91) and 0.76 (CI 0.67–0.86).ConclusionPrognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

The Interactions between Nanoparticles and the Innate Immune System from a Nanotechnologist Perspective

The immune system contributes to maintaining the body’s functional integrity through its two main functions: recognizing and destroying foreign external agents (invading microorganisms) and identifying and eliminating senescent cells and damaged or abnormal endogenous entities (such as cellular debris or misfolded/degraded proteins). Accordingly, the immune system can detect molecular and cellular structures with a spatial resolution of a few nm, which allows for detecting molecular patterns expressed in a great variety of pathogens, including viral and bacterial proteins and bacterial nucleic acid sequences. Such patterns are also expressed in abnormal cells. In this context, it is expected that nanostructured materials in the size range of proteins, protein aggregates, and viruses with different molecular coatings can engage in a sophisticated interaction with the immune system. Nanoparticles can be recognized or passed undetected by the immune system. Once detected, they can be tolerated or induce defensive (inflammatory) or anti-inflammatory responses. This paper describes the different modes of interaction between nanoparticles, especially inorganic nanoparticles, and the immune system, especially the innate immune system. This perspective should help to propose a set of selection rules for nanosafety-by-design and medical nanoparticle design.

Transient blindness associated with mild encephalitis/encephalopathy with a reversible splenial lesion (MERS): a case report and review of literature

BackgroundMild encephalitis/encephalopathy with a reversible splenial lesion (MERS) is a clinical-radiological syndrome that can be related to infectious and non-infectious conditions. The most prominent neurological symptoms are disturbance of consciousness, abnormal speech, delirious behavior, seizures, muscle weakness, ophthalmoplegia, facial nerve paralysis and headache. Here we report the case of a child with MERS presenting with the unusual symptom of bilateral transient blindness.Case presentationA 4-year-old female patient, with a history of fever, abdominal pain, loss of appetite and cough lasted for a few days, experienced 3 episodes of transient bilateral loss of vision with difficulty in walking. Her physical examination showed absence of focal neurological and meningeal irritation signs, although responsiveness was slightly impaired. The ophthalmologic evaluation, including a fundus oculi examination, was negative.The electroencephalogram showed slow activity in the temporo-occipital regions, more evident in the right hemisphere. A lumbar puncture was performed and cerebrospinal fluid analysis revealed normal glycorrhachia, cell counts, protein levels and IgG index.Magnetic resonance imaging of the brain showed a signal alteration in the splenium of the corpus callosum, without contrast enhancement. This finding was suggestive of a reversible cytotoxic lesion. Empiric antiviral treatment with acyclovir and intravenous dexamethasone was initiated. Polymerase chain reaction search for neurotropic viral nucleic acid sequences in the cerebrospinal fluid was negative, while a low number of HHV-6 DNA copies was detected in the blood.Electroencephalograms were repeated in the following days, showing a progressive normalization of the pattern. The child was discharged without symptoms after 10 days of treatment with oral corticosteroids. After 40 days, brain magnetic resonance imaging showed a complete normalization of the signal alteration in the splenium of the corpus callosum.ConclusionTransient blindness was reported as an initial symptom of MERS in a few children. To date, there is no evidence of effective treatment methods. Nonetheless, MERS diagnosis provides pediatricians with valuable prognostic information in order to reassure patients and their families about the good outcome of this disease.

Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR

The new beta-coronavirus, which caused Severe Acute Respiratory Coronavirus-2 Syndrome (SARS-CoV-2), a major respiratory outbreak in Wuhan, China in December 2019, is now prevalent in many countries around the world. Identifying PCR-based viruses is a well-known and relatively stable protocol. Unfortunately, the high mutation rates may lead to widespread changes in viral nucleic acid sequences, and so using specific primers for PCR can be recommended. In this study, we evaluated the power of a conventional RT-PCR to detect SARS-CoV-2 RNA among the five set primer sets. The five genomic regions of the Coronavirus SARS-2 virus including Nucleocapsids (N), Envelope (E), RNA depended RNA Polymerase (RdRp), ORF1ab and Spike (S) were selected for primer designing. A conventional RT-PCR was performed to compare sensitivity, specificity and other analytical characteristics of primers designed against two Real Time PCR commercial kits. The result of the comparative analysis showed that the ORF1ab, N and RdRp primers had a sensitivity, specificity and positive predictive value higher than other primers. A significant difference in the analytical sensitivity between the studied primer sets in RT-PCR kits was observed. In this study, the ORF1ab, Nucleocapsid and RdRp regions have the best primers for identifying the SARS-CoV-2 RNA between different genes that have been suggested.

Imported B3 genotype measles virus isolated in Fujian province

Objective: We isolated and identified the genotypes and molecular characteristics of the imported B3 measles virus (MeV) in Fujian province in 2018. Methods: Throat swab specimens were collected from clinically diagnosed measles patients and tested for viral RNA, using the real-time reverse transcription-polymerase chain reaction after the RNA extraction. Reverse transcription-polymerase chain reaction method was undertaken to amplify the 634 nucleotide acids of 3-terminal of the nucleoprotein gene. A phylogenetic tree was constructed and similarities in homology assessed. Results: We successfully isolated and obtained two measles virus strains and eighteen viral nucleic acid sequences. The Fujian strains were clustered within the same genotype group of WHO genotype B3 reference strains. Compared to the major circulating measles strain genotype B3 in the world, two Fujian strains MV18-41 and MV18-42 showed 100.0% nucleic acid homology to HongKong.CHN/35.18 strain which was isolated from Hong Kong in 2018. The remaining 16 Fujian strains showed the highest homology (99.9%) with the Mvs/Osaka.JPN/38.18/B3 strain isolated from Japan in 2018. Compared with other 23 WHO genotype reference strains, homology on both nucleotide and amino acid of the Fujian strain and the B1 genotype reference strain were the smallest, as 95.1%-95.4% and 95.3%, respectively. The differences of homology between the Fujian strain and H1 genotype reference strain were the largest, as 88.7%-89.0% and 87.3%, respectively. In addition, there were 13 mutation sites between the Fujian strain and the vaccine strain (Shanghai-191) at the 150 amino acid position of carboxy terminus on N protein, However, these sites did not cause functional changes in the protein region. Conclusions: In Fujian province, two strains of B3 genotype measles virus were obtained successfully, which were considered to be new genotype measles virus found in 2018. These findings showed it is necessary to strengthening the monitoring program on imported cases for better control and eliminate the measles virus.

Circulating exosomes from human lung transplant recipients with respiratory viral infections contain nucleic acids potential to activate innate immune signaling (cGAS/STING and RIG-1 Pathways)

Abstract Exosomes are 50–200nm size vesicles released by cells into the body fluids. We reported the presence of circulating exosomes with lung self-antigens (SAgs) (Collagen-V,K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Respiratory viral infections (RVI) is a known risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant. We postulated immune mechanism by which RVI increases risk for CLAD is by induction of exosomes with SAgs containing viral DNA/RNA capable of activating innate immune signaling via DNA/RNA sensing. Exosomes were isolated using ultracentrifugation. Exosomal DNA and RNA were used to generate libraries using Kapa Biosystem’s kit. Illumina 2×150bp pair-end reads were checked on FastQC, aligned to human/viral genome from CHIP seeker Database. Role of exosomes inducing cell signaling in human airway epithelial cells (KCC266) and Hep2 were analyzed by incubating exosomes from LTxRs with RVI or stable. Viral nucleic acid sequences were higher in exosomes from LTxRs with RVI while comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in exosomes from RVI. We observed upregulation of proteins associated with cGAS/STING and RIG1 in KCC266 and Hep2 cells (STING; 1.5, cGAS; 1.9, pIRF3; 2.5, pTBK; 3.1, MAVS; 1.7 and IFNβ; 3.8 fold respectively) incubated with exosomes from LTxRs with RVI. We conclude that LTxRs with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling. This can lead to activation of innate immune signaling resulting in adaptive immune responses to viral and donor antigens resulting in CLAD.

Circulating Exosomes from Human Lung Transplant Recipients Having Respiratory Viral Infections Contain Nucleic Acids and Activate Signaling Pathways CGAS/STING and RIG-1

PurposeExosomes are membrane bound vesicles are released by cells into body fluids. Our laboratory demonstrated the presence of circulating exosomes with lung self-antigens (Collagen-V and K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Since respiratory viral infections (RVI) is a risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant, we postulated that RVI can lead to induction of exosomes with self-antigens containing viral DNA/RNA capable of activating innate immune signaling via cGAS/STING and RIG1 pathways, a mechanism leading to immune activation resulting in CLAD.MethodsExosomes were isolated using ultracentrifugation. Size (50-200nm) was determined using nanosight. DNA and RNA were isolated using kits and quantified on the Nanodrop. Libraries were generated using Kapa Biosystem's library kit. The raw Illumina 2x150bp pair-end reads were checked on FastQC and were aligned to the human and viral genome build from CHIPseeker Database. Validation was done using antibodies and primers for respiratory syncytial virus, coronavirus, and rhinovirus. To determine the role of exosomes to induce cell signaling and endoplasmic stress (ER), airway epithelial cells (KCC266), and Hep2 cells were incubated with exosomes from LTxRs with RVI or stable.ResultsViral nucleic acid sequences were noted in higher levels in exosomes from LTxRs with RVI in comparison to stable. Comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in RVI LTxRs. Further, we identified upregulation of proteins associated with cGAS/STING and RIG1 (MAVS, MDA5, IFNβ) and ER stress (PERK, ATF4 and BiP) in KCC266 and Hep2 cells incubated with exosomes from LTxRs with RVI, but not stable. In contrast, exosomes from stable LTxRs had 91 sequences for MAP kinase and cell death signaling pathways.ConclusionWe conclude that LTxRs diagnosed with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling and ER stress. This can lead to activate innate immune signaling via cGAS/STING and RIG1 pathways resulting in immune responses to viral and donor antigens resulting in CLAD.

Viral and subviral derived small RNAs as pathogenic determinants in plants and insects.

The phenotypic manifestations of disease induced by viruses and subviral infectious entities are the result of complex molecular interactions between host and viral factors. The viral determinants of the diseased phenotype have traditionally been sought at the level of structural or non-structural proteins. However, the discovery of RNA silencing mechanisms has led to speculations that determinants of the diseased phenotype are caused by viral nucleic acid sequences in addition to proteins. RNA silencing is a gene regulation mechanism conserved within eukaryotic kingdoms (with the exception of some yeast species), and in plants and insects it also functions as an antiviral mechanism. Non-coding RNAs of viral origin, ranging in size from 21 to 24 nucleotides (viral small interfering RNAs, vsiRNAs) accumulate in virus-infected tissues and organs, in some cases to comparable levels as the entire complement of host-encoded small interfering RNAs. Upon incorporation into RNA-induced silencing complexes, vsiRNAs can mediate cleavage or induce translational inhibition of nucleic acid targets in a sequence-specific manner. This review focuses on recent findings that suggest an increased complexity of small RNA-based interactions between virus and host. We mainly address plant viruses, but where applicable discuss insect viruses as well. Prominence is given to studies that have indisputably demonstrated that vsiRNAs determine diseased phenotype by either carrying sequence determinants or, indirectly, by altering host-gene regulatory pathways. Results from these studies suggest biotechnological applications, which are also discussed.

Lyophilized visually readable loop-mediated isothermal reverse transcriptase nucleic acid amplification test for detection Ebola Zaire RNA

Recent viral outbreaks highlight the need for reliable, yet broadly deployable diagnostics for detection of epidemic and emerging pathogens. In this study we designed and optimized methods to visually detect viral nucleic acid by isothermal amplification and SYBR dye intercalation. We designed and tested loop-mediated isothermal amplification (LAMP) primers and lyophilized reactions to optimize the detection of Zaire Ebola Virus (ZEBOV) and further evolved the LAMP platform to allow room-temperature storage for deployment in resource limited settings. Our results demonstrated excellent sensitivity and specificity for viral nucleic acid sequences with lower limits of detection of less than 100 copies. Moreover, lyophilized reaction mixtures retained activity for prolonged periods under dry conditions at room temperature. This approach offers a way for detection of emerging viruses in resource limited settings.

Virus discovery in bats

Comprising approximately 20% of known mammalian species, bats are abundant throughout the world1. In recent years, bats have been shown to be the reservoir host for many highly pathogenic viruses, leading to increased attempts to identify other zoonotic bat-borne viruses. These efforts have led to the discovery of over 200 viruses in bats and many more viral nucleic acid sequences from 27 different viral families2,3 (Table 1). Over half of the world’s recently emerged infectious diseases originated in wildlife15, with the genetic diversity of viruses greater in bats than in any other animal16. As humans continue to encroach on the habitat of bats, the risk of spillover of potentially zoonotic viruses is also continuing to increase. Therefore, the surveillance of bats and discovery of novel pathogens is necessary to prepare for these spillover events17.

Synonymous Codon Changes in Measles (HMV) and Canine Distemper (CDV) Viral Nucleic Acid Sequences Result in Gene‐Specific Changes in Levels of Viral Protein Expression

The purpose of this study was to characterize the effects of human and canine codon optimization on expression of viral proteins from HMV and CDV nucleic acid sequences. All the codon changes made were synonymous in that the amino acid coded for was unchanged. The human and canine CUB optimized HMV and CDV sequences had increased nucleic acid and codon identity as compared to the WT sequences. Protein expression from human and canine CUB optimized and sub‐optimized constructs of the N, F, M, H and P/C proteins was assessed in transfected human (293 HEK) and canine (A‐72) cells using fluorescence, Cellomic and, for proteins for which antibodies were available, western analysis.Results1) codon optimization increased protein expression of HMV and CDV N and F proteins in both cell lines; 2) synonymous codon changes did not affect expression of the HMV and CDV P and C and the HMV M proteins, but both optimization and sub‐optimization to canine CUB increased expression of the CDV M protein in human cells; 3) synonymous codon changes increased expression of the HMV H protein in both cell lines, but did not significantly affect CDV H protein expression. Overall both CUB optimization and sub‐optimization affected protein expression independent of viral infection in ways that varied with the gene and cell type, and differed between HMV and CDV. These results indicate that synonymous codon changes in viral nucleic acid sequences can alter protein expression in a variety of ways, some of which are likely to impact viral pathogenicity.Support or Funding InformationThis project was sponsored by the Department of Defense Threat Reduction Agency. The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred.

Microfluidic cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection of viral DNA

Portable and simple analytical devices based on microfluidics with chemiluminescence detection are particularly attractive for point-of-care applications, offering high detectability and specificity in a simple and miniaturized analytical format. Particularly relevant for infectious disease diagnosis is the ability to sensitively and specifically detect target nucleic acid sequences in biological fluids. To reach the goal of real-life applications for such devices, however, several technological challenges related to full device integration are still to be solved, one key aspect regarding on-chip integration of the chemiluminescence signal detection device. Nowadays, the most promising approach is on-chip integration of thin-film photosensors.We recently proposed a portable cartridge with microwells aligned with an array of hydrogenated amorphous silicon (a-Si:H) photosensors, reaching attomole level limits of detection for different chemiluminescence model reactions. Herein, we explore its applicability and performance for multiplex and quantitative detection of viral DNA. In particular, the cartridge was modified to accommodate microfluidic channels and, upon immobilization of three oligonucleotide probes in different positions along each channel, each specific for a genotype of Parvovirus B19, viral nucleic acid sequences were captured and detected.With this system, taking advantage of oligoprobes specificity, chemiluminescence detectability, and photosensor sensitivity, accurate quantification of target analytes down to 70pmolL−1 was obtained for each B19 DNA genotype, with high specificity and multiplexing ability. Results confirm the good detection capabilities and assay applicability of the proposed system, prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities.

A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus circulating in Asia and Africa. In 2013, a large outbreak was reported on the archipelago of French Polynesia. In this study, we report the detection and molecular characterization of Zika virus for the first time in Chile from an outbreak among the inhabitants of Easter Island. A total of 89 samples from patients suspected of having ZIKV infection were collected between the period from January to May, 2014. Molecular diagnosis of the virus was performed by RT-PCR followed by the sequencing of the region containing the NS5 gene. A comparison of the viral nucleic acid sequence with those of other strains of ZIKA virus was performed using the MEGA software. Fifty-one samples were found positive for ZIKV by RT-PCR analysis. Further analysis of the NS5 gene revealed that the ZIKV strains identified in Easter Island were most closely related to those found in French Polynesia (99.8 to 99.9% nt and 100% aa sequence identity). These results strongly suggest that the transmission pathway leading to the introduction of Zika virus on Easter Island has its origin in French Polynesia.

Isolation and characterization of a novel Rhabdovirus from a wild boar (Sus scrofa) in Japan

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar (Sus scrofa leucomystax) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan.

Human and Canine Codon Optimization of Measles and Canine Distemper Viral Nucleic Acid Sequences Increases Sequence Identity and Protein Expression Compared to Wild Type and Human Codon Sub‐Optimized Constructs

The role of codon usage bias (CUB) in the host adaptation and pathogenicity of human measles virus (HMV) and canine distemper virus (CDV) is being investigated and compared. Analysis of nucleic acid sequences for HMV and CDV nucleoprotein (N) revealed that human and canine CUB optimized constructs, as compared to wild type (WT) and human CUB sub‐optimized constructs, were characterized by increased nucleic acid and codon identities, and increased protein expression when transfected into human (293 HEK) cells. The purpose of this study was to characterize human and canine codon optimized nucleic acid sequences of the MHV and CDV F, M, H,L and P/C proteins. All human and canine CUB optimized HMV and CDV nucleic acid sequences had increased nucleic acid and codon identity as compared to WT sequences. Protein expression in human cells transfected with human and canine CUB optimized constructs of the F, M, H and P/C proteins was assessed using florescence, Cellomic and where possible western analysis. Higher protein expression was present in cells transfected with the human and canine codon optimized constructs compared to cells transfected with HMV and CDV WT or HMV suboptimized constructs. These results suggest that further adaptation of HMV and CDV to the mammalian CUB could enhance viral protein expression and pathogenesis.

Detection of DNA virus in respiratory samples of children by multiplex PCR combined with RDB

Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples, and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection. Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database, and fixed specific oligonucleotide probes on the nylon membrane. After multiple PCR amplification of virus DNA of human bocavirus (hBOV), karolinska Institutet (KI), adenovirus (AdV), Washington University polyomaviros (WUPyV), and human parvovirus B19 (HPVB19), the denaturalized amplification products were hybridized with various specific probes, followed by visualization and analysis of the results. The sensitivity and specificity were tested. At the same time, 108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection, and compared to the results of culture method. Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen. The sensitivity of RDB hybridization was 1 colony-forming units (CFU). The positive rate of 34.26% (37 cases out of 108 cases) with PCR-RDB method was significantly higher than that 27.78% (30 cases out of 108 cases) with common test method. Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples, which might be a promising tool for clinical application. Key words: DNA viruses/isolation & purification; DNA viruses/genetics; DNA viruses/pathogenicity; Respiratory tract infections/virology; Respiratory tract infections/genetics; Polymerase chain reaction; Gene amplification; Nucleic acid probes; Child