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Abstract Background Despite dengue virus (DENV) outbreak in Gabon a decade ago, less is known on the potential circulation of DENV serotypes in the country. Previous studies conducted in some areas of the country, are limited to hospital-based surveys which reported the presence of some cases of serotype 2 and 3 seven years ago and more recently the serotype 1. As further investigation, we extend the survey to the community of Moyen Ogooué region with the aim to assess the presence of the dengue virus serotypes, additionally to characterize chikungunya (CHIKV) infection and describe the symptomatology associated with infections. Method A cross-sectional survey was conducted from April 2020 to March 2021. The study included participants of both sexes and any age one year and above, with fever or history of fever in the past seven days until blood collection. Eligible volunteers were clinically examined, and blood sample was collected for the detection of DENV and CHIKV using RT-qPCR. Positive samples were selected for the target sequencing. Results A total of 579 volunteers were included. Their mean age (SD) was 20 (20) years with 55% of them being female. Four cases of DENV infection were diagnosed giving a prevalence of 0.7% (95%CI: 0.2–1.8) in our cohort while no case of CHIKV was detected. The common symptoms and signs presented by the DENV cases included fatigue, arthralgia myalgia, cough, and loss of appetite. DENV-1was the only virus detected by RT-qPCR. Conclusion Our results confirm the presence of active dengue infection in the region, particularly DENV-1, and could suggest the decline of DENV-2 and DENV-3. Continuous surveillance remains paramount to comprehensively describe the extent of dengue serotypes distribution in the Moyen-Ogooué region of Gabon.

Background: Despite dengue virus (DENV) outbreak in Gabon a decade ago, less is known on the potential circulation of DENV serotypes in the country. Previous studies conducted in some areas of the country, are limited to hospital-based surveys which reported the presence of some cases of serotype 2 and 3 seven years ago and more recently the serotype 1. As further investigation, we extend the survey to the community of Moyen Ogooué region with the aim to assess the presence of the dengue virus serotypes, additionally to characterize chikungunya (CHIKV) infection and describe the symptomatology associated with infections. Method: A cross-sectional survey was conducted from April 2020 to March 2021. The study included participants of both sexes and any age one year and above, with fever or history of fever in the past seven days until blood collection. Eligible volunteers were clinically examined, and blood sample was collected for the detection of DENV and CHIKV using RT-qPCR. Positive samples were selected for the target sequencing. Results: A total of 579 volunteers were included. Their mean age (SD) was 20 (20) years with 55% of them being female. Four cases of DENV infection were diagnosed giving a prevalence of 0.7% (95%CI: 0.2–1.8) in our cohort while no case of CHIKV was detected. The common symptoms and signs presented by the DENV cases included fatigue, arthralgia myalgia, cough, and loss of appetite. DENV-1was the only virus detected by RT-qPCR. Conclusion: Our results confirm the presence of active dengue infection in the region, particularly DENV-1, and could suggest the decline of DENV-2 and DENV-3. Continuous surveillance remains paramount to comprehensively describe the extent of dengue serotypes distribution in the Moyen-Ogooué region of Gabon. [ABSTRACT FROM AUTHOR]

Abstract Background Acute respiratory infections (ARI) are a threat to human health and survival, resulting in many paediatric hospitalisations. However, the epidemiological and clinical severity characteristics of the human parainfluenza virus (PIV), one of the most prevalent respiratory viruses, are not well understood in children. Methods To identify the epidemiological features of PIV infection, in 2019, hospitalised children with ARI were screened using multiplex polymerase chain reaction (PCR) for PIV and 10 other common respiratory pathogens. Subtyping of randomly selected PIV-positive samples was performed using reverse transcription-PCR. Demographics, epidemiology, clinical manifestations, diagnosis, and outcomes were compared between PIV subtypes. Results The annual detection rate for PIV was 14.9%, with a peak from April to September. Children under one year of age had the highest rate of PIV infection (45.5%) compared to other age groups. Of the 121 sequenced samples, 58.7%, 36.4% and 4.9% were positive for PIV-3, PIV-1 and PIV-2, respectively, and no PIV-4 was detected. Severe infections were associated with pre-existing underlying diseases and co-infections, but not with PIV serotype. After excluding cases of co-infection, we found that PIV-2 infection was associated with upper respiratory tract infections, whereas PIV-1 and PIV-3 mainly caused lower respiratory tract infections. Apart from the proportion of patients with fever, there were no significant differences among the three subtypes in terms of clinical symptoms, severity, and outcome. Conclusion Here, PIV was the main pathogen causing ARI in hospitalised children. Appropriate attention should be paid to children with underlying diseases and co-infections to prevent the worsening of severe PIV infection.

Background: Acute respiratory infections (ARI) are a threat to human health and survival, resulting in many paediatric hospitalisations. However, the epidemiological and clinical severity characteristics of the human parainfluenza virus (PIV), one of the most prevalent respiratory viruses, are not well understood in children. Methods: To identify the epidemiological features of PIV infection, in 2019, hospitalised children with ARI were screened using multiplex polymerase chain reaction (PCR) for PIV and 10 other common respiratory pathogens. Subtyping of randomly selected PIV-positive samples was performed using reverse transcription-PCR. Demographics, epidemiology, clinical manifestations, diagnosis, and outcomes were compared between PIV subtypes. Results: The annual detection rate for PIV was 14.9%, with a peak from April to September. Children under one year of age had the highest rate of PIV infection (45.5%) compared to other age groups. Of the 121 sequenced samples, 58.7%, 36.4% and 4.9% were positive for PIV-3, PIV-1 and PIV-2, respectively, and no PIV-4 was detected. Severe infections were associated with pre-existing underlying diseases and co-infections, but not with PIV serotype. After excluding cases of co-infection, we found that PIV-2 infection was associated with upper respiratory tract infections, whereas PIV-1 and PIV-3 mainly caused lower respiratory tract infections. Apart from the proportion of patients with fever, there were no significant differences among the three subtypes in terms of clinical symptoms, severity, and outcome. Conclusion: Here, PIV was the main pathogen causing ARI in hospitalised children. Appropriate attention should be paid to children with underlying diseases and co-infections to prevent the worsening of severe PIV infection. [ABSTRACT FROM AUTHOR]

Background: Dengue is an arboviral disease that has a large effect on public health in subtropical and tropical countries. Rapid and accurate detection of dengue infection is necessary for diagnosis and disease management. We previously developed highly sensitive immunochromatographic devices, the TKK 1st and TKK 2nd kits, based on dengue virus (DENV) nonstructural protein 1 detection. However, these TKK kits were evaluated mainly using DENV type 2 clinical specimens collected in Bangladesh, and further validation using clinical specimens of other serotypes was needed. Methods: In the present study, one of the TKK kits, TKK 2nd, was evaluated using 10 DENV-1, 10 DENV-2, 4 DENV-3, 16 DENV-4, and 10 zika virus-infected clinical specimens collected in Bangkok, Thailand. Results: The TKK 2nd kit successfully detected all four DENV serotypes in patient serum specimens and did not show any cross-reactivities against zika virus serum specimens. The IgM and/or IgG anti-DENV antibodies were detected in seven serum specimens, but did not seem to affect the results of antigen detection in the TKK 2nd kit. Conclusion: The results showed that the TKK 2nd kit successfully detected all four DENV serotypes in clinical specimens and confirmed the potential of the kit for dengue diagnosis in endemic countries. [ABSTRACT FROM AUTHOR]

Background: As frequent viral outbreaks continue to pose threat to public health, the unavailability of antiviral drugs and challenges associated with vaccine development underscore the need for antiviral drugs discovery in emergent moments (endemic or pandemic). Plants in response to microbial and pest attacks are able to produce defence molecules such as antimicrobial peptides as components of their innate immunity, which can be explored for viral therapeutics. Methods: In this study, partially purified peptide-rich fraction (P-PPf) were obtained from aqueous extracts of seven plants by reverse-phase solid-phase extraction and cysteine-rich peptides detected by a modified TLC method. The peptide-enriched fractions and the aqueous (crude polar) were screened for antiviral effect against three non-polio enterovirus species C members using cytopathic effect reduction assay. Results: In this study, peptide fraction obtained from Euphorbia hirta leaf showed most potent antiviral effect against Coxsackievirus A13, Coxsackievirus A20, and Enterovirus C99 (EV-C99) with IC50 < 2.0 µg/mL and selective index ≥ 81. EV-C99 was susceptible to all partially purified peptide fractions except Allamanda blanchetii leaf. Conclusion: These findings establish the antiviral potentials of plants antimicrobial peptides and provides evidence for the anti-infective use of E. hirta in ethnomedicine. This study provides basis for further scientific investigation geared towards the isolation, characterization and mechanistic pharmacological study of the detected cysteine-rich peptides. [ABSTRACT FROM AUTHOR]

Background: The foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses. Methods: Specific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group). Results: High virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4–5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge. Conclusions: This study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Performance of the nonstructural 1 Antigen Rapid Test for detecting all four DENV serotypes in clinical specimens from Bangkok, Thailand." The authors of the original article have requested an update to the Ethics approval and consent to participate section. The incorrect information stated that the study was reviewed and approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (TMEC 19–051), and that patient consent was exempt since only leftover specimens were used after anonymization. The correct information is that the study was reviewed and approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (MUTM 2020-024-01), and patient consent was exempt for the same reasons. The correction notice is provided by multiple authors. [Extracted from the article]

Background: HIV infection in Cameroon is characterized by a great viral diversity with all HIV-1 groups (M, N, O, and P) and HIV-2 in circulation. HIV group determination is very important if tailored viral load analysis and treatments are to be applied. In our laboratory, HIV viral load is carried out using two platforms; Biocentric and Abbott depending on the HIV group identified. Biocentric which quantifies HIV-1 group M is a cheap and open system useful in resource limited settings. The objective of this study was to compare the viral load analyses of serologically group-indeterminate HIV samples using the two platforms with the view of reducing cost. Methods: Consecutive samples received between March and May 2014, and between August and September 2014 in our laboratory for HIV viral load analysis were included. All these samples were analyzed for their HIV groups using an in-house ELISA serotyping test. All HIV-1 group M samples were quantified using the Biocentric test while all other known atypical samples (HIV-1 groups N, O and P) were analyzed using the Abbott technique. HIV group-indeterminate samples (by serotyping) were quantified with both techniques. Results: Among the 6355 plasma samples received, HIV-1 group M was identified in 6026 (94.82%) cases; HIV-1 group O, in 20 (0.31%); HIV-1 group M+ O, in 3 (0.05%) and HIV-2, in 3 (0.05%) case. HIV-group indeterminate samples represented about 4.76% (303/6355) and only 231 of them were available for analysis by Abbott Real-Time HIV-1 and Generic HIV Viral Load techniques. Results showed that 188 (81.39%) samples had undetectable viral load in both techniques. All the detectable samples showed high viral load, with a mean of 4.5 log copies/ml (range 2.1-6.5) for Abbott Real-Time and 4.5 log copies/ml (range 2-6.4) for Generic HIV Viral Load. The mean viral load difference between the two techniques was 0.03 log10 copies/ml and a good correlation was obtained (r2 = 0.89; P < 0.001). Conclusion: Our results suggest that cheaper and open techniques such as Biocentric could be useful alternatives for HIV viral load follow-up quantification in resource limited settings like Cameroon; even with its high viral diversity. [ABSTRACT FROM AUTHOR]

Background: Conditionally replicative oncolytic adenoviruses (CRAds) display significant anti-tumor effects. However, the traditional adenovirus of serotype 5 (Ad5) entering cancer cells via coxsackie virus and adenovirus receptor (CAR) can't be utilized for bladder cancer with low expression of CAR, which limits the application of Ad5. Methods: We utilized Ad5/F11p containing the chimeric fiber gene encoding the Ad5 fiber tail domain and Ad11p fiber shaft and knob domains to construct bladder cancer-specific chimeric type viruses Ad5/F11p-PSCAE-UPII-E1A, which can infect bladder cancer cells mediated by CD46 molecule. We carried out series of experiments in vitro to research anti-tumor effect of Ad5/F11p-PSCAE-UPII-E1A and the interaction in combination with cisplatin. Results: The results demonstrated Ad5/F11p-PSCAE-UPII-E1A could infect bladder cancer cells (T24, EJ and 5637) in a CAR-independent way, and exert anti-tumor effect by blocking the cancer cells in G1 phase and inducing apoptosis. Ad5/F11p-PSCAE-UPII-E1A plus cisplatin enhanced the anti-proliferative effect and increased the number of apoptotic cells compared with viruses or cisplatin alone. Ad5/F11p-PSCAE-UPII-E1A plus cisplatin could upregulate the proteins expression of p53, Bax, and cleaved caspase-3, and downregulated Bcl-2 protein expression in T24, EJ and 5637 cells. Conclusion: We constructed a bladder cancer-specific oncolytic adenovirus and provided new combination treatment strategies for bladder cancer. [ABSTRACT FROM AUTHOR]

Background: Transmission of dengue virus (DENV) through blood transfusion has been documented and hence screening for DENV during blood donation has been recently recommended by the American Association of Blood Banks and Centres of Disease Control and Prevention. DENV is endemic in the Western province of the Kingdom of Saudi Arabia (KSA) and serotypes 1, 2 and 3, but not 4, have been detected. However, little is known regarding the rates of DENV during blood donation in the kingdom. The aim of this study was therefore to measure the prevalence of dengue virus and its serotypes in eligible Saudi blood donors in the endemic Western region of KSA. Methods: This was a cross-sectional study and serum samples were collected from 910 eligible Saudi male blood donors. DENV IgM and IgG antibodies were measured serologically by ELISA while viral serotypes were detected by a single step IVD CE certified multiplex RT-PCR kit. Results: The overall prevalence was 39 and 5.5% for IgG+ and IgM+, respectively. There were 12 (1.3%) with exclusively IgM+, 317 (34.8%) exclusively IgG+ and 38 (4.2%) with dual IgM+/IgG+ donors. The overall prevalence was 3.2% (n = 29) and 2.3% (n = 21) for primary and secondary infections. PCR was positive in 5.5% (n = 50) and, DENV-2 (n = 24; 48%) was the most frequent serotype and was significantly higher than DENV-1 (20%; P= 0.02) and DENV-3 (2%; P= 0.1×10-5) but not DENV-4 (30%; P = 0.2). There was no significant difference between both DENV-4 and DENV-1 (P = 0.4). The combination of the PCR and serology findings showed that 22 (2.4%) and 28 (3.1%) donors had primary and secondary viremic infections, respectively. Conclusions: The detected rates of DENV by PCR suggest a potential high risk of viral transmission by blood transfusion. To the best of our knowledge, this study is the first to report the detection of DENV-4 serotype in Saudi Arabia. More studies are required to measure the precise prevalence of DENV serotypes and their potential transmission rate during blood donation in the kingdom. [ABSTRACT FROM AUTHOR]

Background: Avian paramyxovirus serotype 1 (APMV-1) viruses are globally distributed, infect wild, peridomestic, and domestic birds, and sometimes lead to outbreaks of disease. Thus, the maintenance, evolution, and spread of APMV-1 viruses are relevant to avian health. Methods: In this study we sequenced the fusion gene from 58 APMV-1 isolates recovered from thirteen species of wild birds sampled throughout the USA during 2007-2014. We analyzed sequence information with previously reported data in order to assess contemporary genetic diversity and inter-taxa/inter-region exchange of APMV-1 in wild birds sampled in North America. Results: Our results suggest that wild birds maintain previously undescribed genetic diversity of APMV-1; however, such diversity is unlikely to be pathogenic to domestic poultry. Phylogenetic analyses revealed that APMV-1 diversity detected in wild birds of North America has been found in birds belonging to numerous taxonomic host orders and within hosts inhabiting multiple geographic regions suggesting some level of viral exchange. However, our results also provide statistical support for associations between phylogenetic tree topology and host taxonomic order/region of sample origin which supports restricted exchange among taxa and geographical regions of North America for some APMV-1 sub-genotypes. Conclusions: We identify previously unrecognized genetic diversity of APMV-1 in wild birds in North America which is likely a function of continued viral evolution in reservoir hosts. We did not, however, find support for the emergence or maintenance of APMV-1 strains predicted to be pathogenic to poultry in wild birds of North America outside of the order Suliformes (i.e., cormorants). Furthermore, genetic evidence suggests that ecological drivers or other mechanisms may restrict viral exchange among taxa and regions of North America. Additional and more systematic sampling for APMV-1 in North America would likely provide further inference on viral dynamics for this infectious agent in wild bird populations. [ABSTRACT FROM AUTHOR]

Background: Foot-and-mouth disease (FMD) is an economically devastating disease that severely limits international trade of animals. Of the seven FMD virus (FMDV) serotypes, serotype A is one of the most widespread cross the world. Currently antibodies to FMDV are detected in animals using the virus neutralization test (VNT) and the enzyme-linked immunosorbent assay (ELISA). The VNT is laborious, time-consuming and reliant on live virus and cell cultures, while ELISA has the advantage of using inactivated antigens and often provides more reproducible results. The aim of this study was to develop a reliable and rapid competitive ELISA (cELISA) for the detection of antibodies to FMDV serotype A (FMDV/A). Results: A panel of FMDV/A specific monoclonal antibodies (mAbs) was generated and their ability to compete with a polyclonal serum from FMDV/A-infected cattle was examined. Two mAbs inhibited the binding of a polyclonal serum to FMDV/A viruses. The binding epitopes of each were determined as conformational and located on the VP2 viral capsid protein. The FMDV/A cELISA was developed using these two mAbs and FMDV/A inactivated virus as antigen. The diagnostic specificity and sensitivity were 99.7 and 99.3% (98.5-100%) respectively, based on a predetermined cut-off of 50% inhibition. When analysing sera from animals experimentally infected with FMDV/A, the cELISA detected antibodies from 5-days post infection (dpi) and remained positive for at least 21-28 days post infection. Comparison based on the Kappa coefficient showed strong agreement (90-94%) between cELISA and VNT. Conclusion: The cELISA results are comparable to the VNT for antibody detection making it a simple and reliable test to detect antibodies against FMDV/A. [ABSTRACT FROM AUTHOR]

In several parts of China, there have been a large number of hydropericardium syndrome (HPS) outbreaks caused by serotype 4 fowl adenovirus (FAdV4) in broiler chickens since 2015. These outbreak-associated FAdV-4 strains were distinct from previous circulating strains which did not lead to severe HPS outbreaks. To better understand the molecular epidemiology of the currently circulating FAdV strains for effective diagnosis and treatment of HPS, we isolated 12 HPS outbreak-associated FAdV-4 strains from different regions in central China and investigated their molecular characteristics by performing phylogenetic analyses based on the hexon genes. Our results indicated the FAdV-4 strains in this study all belonged to serotype FAdV-4, species FAdV-C. And in comparison with ON1, KR5, MX-SHP95, PK-01, PJ-06 strains within the cluster where outbreak-associated FAdV-4 strains were located, the nucleotide sequence divergence were 1.31, 1.10, 1.42, 2.77 and 2.84%, respectively. Phylogenetic analyses revealed the hexon genes of the 12 outbreak-associated strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor and we suggested that these outbreak-associated FAdV-4 strains originate from earlier strains in India. [ABSTRACT FROM AUTHOR]

Background: Dengue fever, a mosquito-borne disease, is associated with illness of varying severity in countries in the tropics and sub tropics. Dengue cases continue to be detected more frequently and its geographic range continues to expand. We report the largest documented laboratory confirmed circulation of dengue virus in parts of Kenya since 1982. Methods: From September 2011 to December 2014, 868 samples from febrile patients were received from hospitals in Nairobi, northern and coastal Kenya. The immunoglobulin M enzyme linked immunosorbent assay (IgM ELISA) was used to test for the presence of IgM antibodies against dengue, yellow fever, West Nile and Zika. Reverse transcription polymerase chain reaction (RT-PCR) utilizing flavivirus family, yellow fever, West Nile, consensus and sero type dengue primers were used to detect acute arbovirus infections and determine the infecting serotypes. Representative samples of PCR positive samples for each of the three dengue serotypes detected were sequenced to confirm circulation of the various dengue serotypes. Results: Forty percent (345/868) of the samples tested positive for dengue by either IgM ELISA (14.6 %) or by RT-PCR (25.1 %). Three dengue serotypes 1-3 (DENV1-3) were detected by serotype specific RT-PCR and sequencing with their numbers varying from year to year and by region. The overall predominant serotype detected from 2011-2014 was DENV1 accounting for 44 % (96/218) of all the serotypes detected, followed by DENV2 accounting for 38.5 % (84/218) and then DENV3 which accounted for 17.4 % (38/218). Yellow fever, West Nile and Zika was not detected in any of the samples tested. Conclusion: From 2011-2014 serotypes 1, 2 and 3 were detected in the Northern and Coastal parts of Kenya. This confirmed the occurrence of cases and active circulation of dengue in parts of Kenya. These results have documented three circulating serotypes and highlight the need for the establishment of active dengue surveillance to continuously detect cases, circulating serotypes, and determine dengue fever disease burden in the country and region. [ABSTRACT FROM AUTHOR]

Background: Dengue virus is circulating in Pakistan since 1994, which causes major and minor outbreaks in many areas of the country. The incidence of dengue in Pakistan in past years mainly restricted to parts of Sindh and Punjab provinces. As such, a severe dengue outbreak appeared in Pakistan in 2011, particularly in Punjab province with Lahore as the most hit city (290 deaths). In 2013, for the first time in the history of Pakistan, dengue outbreak erupted in Swat District, Khyber Pakhtunkhwa, which claimed more than 57 lives. Hence this study was conducted to document circulating serotypes of dengue virus in Pakistan in 2011 and 2013 dengue outbreaks in two different territories/areas of the country. Methods: In total, 1340 blood samples from people having dengue (ELISA positive) and/or dengue like symptoms from various cities/areas of Punjab and Swat, Khyber Pakhtunkhwa (KP) were collected and analyzed by reverse transcription polymerase chain reaction (RT-PCR) using serotype specific primers. Results: The results indicated that all the four dengue virus serotypes were circulating in Punjab Province with highest frequency of DENV-2 (41.64 %) and DENV-3 (41.05 %). Similarly, DENV-2 (41.66 %) and DENV-3 (35.0 %) were dominant serotypes detected in KP-based people lived in Punjab. On the other hand only DENV-2 (40.0 %) and DENV-3 (60.0 %) were detected in Swat District. Furthermore an important observation noted in this study was mixed infection of DENV-2 and DENV-3 in Punjab in 2011 (3.81 %) and in people from KP infected in Punjab (8. 33 %) which may account for the high mortality and morbidity rates as compared to previous outbreaks. Over all male population was mostly infected as compared to females and people in the age group between 15 to 45 was the highest infected group. Conclusions: The findings of this study indicate that all four serotypes of dengue virus are circulating in Punjab whereas serotypes 2 and 3 introduced for the first time into Swat, KP in 2013; about 600 km away from Lahore, Punjab. Overall dengue virus serotypes 2 and 3 were the major outbreak-causing serotypes in Pakistan in 2011 and 2013. Dengue outbreak in Swat may be the continuation of previous dengue outbreaks in Punjab but it needs further research and investigation. [ABSTRACT FROM AUTHOR]

Background: Hand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing. We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9-33.3) were used for assay evaluation. Results: Our assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94-100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible. Conclusions: In conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples. [ABSTRACT FROM AUTHOR]

Background: We report the isolation and characterization of dengue virus (DENV) serotype 4 from a resident of Santa Fé, state of Paraná, South Brazil, in March 2013. This patient presented with hemorrhagic manifestations, high viral load and, interestingly, a mixed Th1/Th17 cytokine profile. Case presentation: The patient presented with classical dengue symptoms, such as fever, rash, myalgia, arthralgia, and hemorrhagic manifestations including petechiae, gum bleeding and a positive tourniquet test result. A serum sample obtained 1 day after the initial appearance of clinical symptoms was positive for NS1 viral antigen, but this sample was negative for both IgM and IgG against DENV. Dengue virus infection was confirmed by isolation of the virus from C6/36 cells, and dengue virus serotyping was performed via one-step RT-PCR. The infection was confirmed to be caused by a serotype 4 dengue virus. Additionally, based on multiple alignment and phylogeny analyses of its complete genome sequence, the viral strain was classified as genotype II (termed LRV13/422). Moreover, a mixed Th1/Th17 cytokine profile was detected in the patient's serum, and this result demonstrated significant inflammation. Biological characterization of the virus via in vitro assays comparing LRV13/422 with a laboratory-adapted reference strain of dengue virus serotype 4 (TVP/360) showed that LRV13/422 infects both vertebrate and invertebrate cell lines more efficiently than TVP/360. However, LRV13/422 was unable to inhibit type I interferon responses, as suggested by the results obtained for other dengue virus strains. Furthermore, LRV13/422 is the first completely sequenced serotype 4 dengue virus isolated in South Brazil. Conclusion: The high viral load and mixed Th1/Th17 cytokine profile observed in the patient's serum could have implications for the development of the hemorrhagic signs observed, and these potential relationships can now be further studied using suitable animal models and/or in vitro systems. [ABSTRACT FROM AUTHOR]

Background: Infectious bronchitis virus (IBV), a major pathogen of commercial poultry flocks, circulates in the form of several serotypes/genotypes. Only a few amino-acid changes in the S1 subunit of wild-type IBVS proteins may result in mutants unaffected by current vaccines. Methods: Partial S1 gene sequences of 3 IBV isolates of the Moroccan Italy 02 genotype from vaccinated and unvaccinated broiler chicken flocks, located in southern and central regions of Morocco, were amplified by RT-PCR, sequenced, and aligned for phylogenetic and amino-acid similarity analyses. Results: The three isolates were found genetically highly distant from known avian IBV based on partial sequences of their S1 genes: gammaCoV/chicken/Morocco/I01/2011(IBV/Morocco/01), gammaCoV/chicken/Morocco/I30/2010 (IBV/Morocco/30), and gammaCoV/chicken/Morocco/I38/2013 (IBV/Morocco/38), nucleotide sequence identities reached 89.5 % to 90.9 % among the three isolates. The deduced protein sequence identities ranged from 29.7 % (between IBV/Morocco/38 and Egypt SCU-14/2013-1) to 78.2 % (between IBV/Morocco/01 and Spain/05/866). Amino acid sequence comparison and phylogenetic analysis indicated the emergence of a new Moroccan genotype, clustering with regionally related isolates from Spain (Spain/05/866) and belonging to a new sub-genotype. Conclusion: Our sequencing results demonstrate a co-circulation of wild-type infectious bronchitis viruses in broiler chickens. These results justify permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the evolving field situation. [ABSTRACT FROM AUTHOR]

Background: There are over 100 serotypes of enterovirus species A-D, which are the common cause of various symptoms in infants, such as meningitis, encephalitis and hand foot mouth disease (HFMD). This study aims to investigate the epidemiological characteristics of enteroviruses in Hangzhou, Zhejiang province, China, and to provide relevant information to guide public health responses and interventions. Methods: Systematic surveillance was conducted on enterovirus infections. Samples were collected from children admitted to the inpatient wards and outpatient departments between January 2010 and December 2012 in the Children's Hospital, Zhejiang University School of Medicine. Enteroviruses from all specimens were detected by RT-PCR using a commercialized detection kit. Results: From 13026 samples collected and examined, 2673 (21.21%) were found positive for enteroviruses. The annual enterovirus-positive rate decreased from 32.78% in 2010 to 14.23% in 2012. Positivity rate for enteroviruses was highest among children aged less than 5 years. The monthly positivity rate for enterovirus infection ranged from 2.6% to 34.83%, with a peak in June and July. Serotypes causing severe symptoms such as HFMD including EV71 and CA16 were decreasing, while the proportion of unidentified EV serotypes causing herpangina and viral encephalitis were on the rise. Conclusions: EV infection is highly prevalent among young children in Hangzhou, as it is in the most other parts of the world. Further surveillance using methods that can subtype all EVs is warranted to better monitor these infections and their etiology. [ABSTRACT FROM AUTHOR]

Background: Dengue has emerged as the most significant of arboviral diseases in the 21st century. It is endemic to >100 tropical and sub-tropical countries around the world placing an estimated 3.6 billion people at risk. It is caused by four genetically similar but antigenically distinct, serotypes of dengue viruses. There is neither a vaccine to prevent nor a drug to treat dengue infections, at the present time. The major objective of this work was to explore the possibility of identifying a small molecule inhibitor of the dengue virus protease and assessing its ability to suppress viral replication in cultured cells. Methods: We cloned, expressed and purified recombinant dengue virus type 2 protease. Using an optimized and validated fluorogenic peptide substrate cleavage assay to monitor the activity of this cloned dengue protease we randomly screened ~1000 small molecules from an 'in-house' library to identify potential dengue protease inhibitors. Results: A benzimidazole derivative, named MB21, was found to be the most potent in inhibiting the cloned protease (IC50 = 5.95 µM). In silico docking analysis indicated that MB21 binds to the protease in the vicinity of the active site. Analysis of kinetic parameters of the enzyme reaction suggested that MB21 presumably functions as a mixed type inhibitor. Significantly, this molecule identified as an inhibitor of dengue type 2 protease was also effective in inhibiting each one of the four serotypes of dengue viruses in infected cells in culture, based on analysis of viral antigen synthesis and infectious virus production. Interestingly, MB21 did not manifest any discernible cytotoxicity. Conclusions: This work strengthens the notion that a single drug molecule can be effective against all four dengue virus serotypes. The molecule MB21 could be a potential candidate for 'hit-to-lead' optimization, and may pave the way towards developing a pan-dengue virus antiviral drug. [ABSTRACT FROM AUTHOR]

Background Dengue viruses (DENVs) are mosquito-borne viruses which can cause disease ranging from mild fever to severe dengue infection. These viruses are endemic in several tropical and subtropical regions. Multiple outbreaks of DENV serotypes 1, 2 and 3 (DENV-1, DENV-2 and DENV-3) have been reported from the western region in Saudi Arabia since 1994. Strains from at least two genotypes of DENV-1 (Asia and America/Africa genotypes) have been circulating in western Saudi Arabia until 2006. However, all previous studies reported from Saudi Arabia were based on partial sequencing data of the envelope (E) gene without any reports of full genome sequences for any DENV serotypes circulating in Saudi Arabia. Findings Here, we report the isolation and the first complete genome sequence of a DENV-1 strain (DENV-1-Jeddah-1-2011) isolated from a patient from Jeddah, Saudi Arabia in 2011. Whole genome sequence alignment and phylogenetic analysis showed high similarity between DENV-1-Jeddah-1-2011strain and D1/H/IMTSSA/98/606 isolate (Asian genotype) reported from Djibouti in 1998. Further analysis of the full envelope gene revealed a close relationship between DENV-1-Jeddah-1-2011strain and isolates reported between 2004-2006 from Jeddah as well as recent isolates from Somalia, suggesting the widespread of the Asian genotype in this region. Conclusions These data suggest that strains belonging to the Asian genotype might have been introduced into Saudi Arabia long before 2004 most probably by African pilgrims and continued to circulate in western Saudi Arabia at least until 2011. Most importantly, these results indicate that pilgrims from dengue endemic regions can play an important role in the spread of new DENVs in Saudi Arabia and the rest of the world. Therefore, availability of complete genome sequences would serve as a reference for future epidemiological studies of DENV-1 viruses. [ABSTRACT FROM AUTHOR]

Background: The plaque reduction neutralization test (PRNT) is currently the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies to dengue virus, and in assessing the immunogenicity of a dengue vaccine. However, the correlation between presence of dengue-neutralizing antibody and protection from infection is not absolute. Findings: In a cohort study in Ratchaburi Province, Thailand, 48 subjects with serologically confirmed symptomatic dengue infection were tested for pre-existing dengue neutralizing antibody using PRNT. Nine subjects had quite high pre-existing PRNT50 titers (titer >90) to subsequent infecting dengue serotypes, but still had symptomatic infections. Conclusion: This report provides evidence that PRNT may not be a good test for predicting protection against subsequent dengue infection. [ABSTRACT FROM AUTHOR]

Background Leukotriene B4, a 5-lipoxygenase product of arachidonic acid with potent chemotactic effects on neutrophils, has not been assessed in dengue patients. In this study, plasma leukotriene B4 and serum high-sensitivity C-reactive protein levels were determined in adult patients during the febrile, convalescent and defervescent stages of dengue serotype-2 (DENV-2) infection, and compared with those of age--matched healthy and non-dengue febrile subjects. In vitro studies were performed to examine the effects of live and heat-inactivated DENV-2 on the activities and expression of 5-lipoxygenase in human neutrophils. Results Plasma leukotriene B4 were elevated during the febrile stages of dengue infection compared to those during convalescence and in study controls. Plasma leukotriene B4 also correlated with serum high-sensitivity C-reactive protein in dengue patients (febrile, r = 0.91, p < 0.001; defervescence, r = 0.87, p < 0.001; convalescence, r = 0.87, p < 0.001). Exposure of human neutrophils to DENV-2 resulted in a significant rise in leukotriene B4; the extent of increase, however, did not differ between exposure to live and heat-inactivated DENV-2. Preincubation of either live or heat-inactivated DENV-2 resulted in reduced release of leukotriene B4 release by neutrophils, indicating that contact with dengue antigens (and not replication) triggers the neutrophil response. Production of leukotriene B4 was associated with an increase in 5-lipoxygenase expression in human neutrophils; addition of MK886 (a 5- lipoxygenase activating protein inhibitor) attenuated further increase in leukotriene B4 production. Conclusion These findings provide important clinical and mechanistic data on the involvement of 5- lipoxygenase and its metabolites in dengue infection. Further studies are needed to elucidate the therapeutic implications of these findings. [ABSTRACT FROM AUTHOR]

Background: Human adenoviruses are promising candidates for addressing health risks associated with enteric viruses in environmental waters. Relatively harmless but common, these DNA viruses persist within the population and are generally considered extremely stable, remaining infectious in water for long periods of time. Group-specific or single species detection of human adenoviruses in environmental samples is usually based on polymerase chain reaction assays. Simultaneous identification of specific species or serotypes needs additional processing. Here we present a simple molecular approach for the monitoring of serotypic diversity in the human adenovirus populations in contaminated water sites. Methods: Diversity patterns of human adenoviruses in environmental samples, collected in an outdoor artificial stream and pond simulation system, were analyzed using a closed tube polymerase chain reaction method with subsequent melting point analysis. Results: Human adenovirus serotype 41 was the most prominent adenovirus serotype detected in environmental water samples, but melting point analyses indicated the presence of additional adenovirus serotypes. Conclusions: Based on investigations with spiked and environmental samples, a combination of qPCR and melting point analysis was shown to identify adenovirus serotypes in sewage contaminated water. [ABSTRACT FROM AUTHOR]

Background: Outbreaks of Foot-and-mouth disease (FMD) have resulted in tremendous economic losses. Thus, the development of a rapid and easily performed test for FMD detection is important for controlling a FMD outbreak and containing its spread. The purpose of this project is to develop a lateral flow immunochromatographic (LFI) strip test for rapid detection of FMD virus serotypes O, A and Asia 1. Methods: Specific monoclonal antibodies (mAbs) against each serotype were produced and used as the capture mAbs. A serotype independent mAb was selected and used as the detection mAb with the aim of subsequently developing a multi-serotype strip test. A new generation of the generic RapidAssay Device (gRAD) was used for the test. Result: Each strip test can specifically detect the FMDV O, A or Asia 1 viruses, but not other vesicular disease viruses. The LFI strip tests for serotypes A and Asia 1 were able to identify all tested serotype A (n= 39) and Asia 1 field isolates (n=17). Whereas the test for serotype O detected 45 out of 46 field isolates. The sensitivity of this strip test was comparable with the double antibody sandwich ELISA for viral antigen detection. All vesicular fluid and epithelium samples collected from experimentally infected animals with serotype O, A and Asia 1 were identified as positive by the LFI strip test. Swab samples (n=11) collected over the lesion area from experimentally inoculated animals (serotype A) were examined. All of them demonstrated positive results using the LFI serotype A strip test and double antibody sandwich (DAS) ELISA. Conclusions: The ability of strip tests to produce rapid results and high specificity makes it a valuable tool for early detection of FMDV O, A and Asia 1 in the field. [ABSTRACT FROM AUTHOR]

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun). The two resulting chimeric proteins, DIII-DENV1-RIgE and DIII-WNVKun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55GagHIV in insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens. [ABSTRACT FROM AUTHOR]

Background: Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods: Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results: Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions: This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed by staff with a basic level of training and storage at low temperatures (e.g., -80°C) is not necessary. These simple, inexpensive (US$ 0.05/sample), robust, sensitive and relatively rapid assays, using improved MAbs such as MAb 2C4.6, should be ideal for the diagnosis of all DENV serotypes in DENV endemic regions. [ABSTRACT FROM AUTHOR]

Background: Dengue epidemics have been reported in Brazil since 1981. In Manaus, a large city in the Amazon region, dengue is endemic with all four-virus serotypes (DENV-1, -2, -3, and -4) simultaneously causing human disease. In 2008, during a surveillance of dengue virus in mosquitoes in the district of Tancredo Neves in Manaus, 260 mosquitoes of Aedes genus were captured, identified and grouped into pools of 10 mosquitoes. Findings: RNA extracts of mosquito pools were tested by a RT-Hemi-Nested-PCR for detection of flaviviruses. One amplicon of 222 bp, compatible with dengue virus serotype 4, was obtained from a pool of Aedes aegypti. The nucleotide sequence of the amplicon indicated that the mosquitoes were infected with DENV-4 of genotype I. This virus of Asian origin has been described in Manaus in 2008 infecting acute febrile illness patients. Conclusion: This is the first report of dengue virus serotype 4 genotype I infecting Aedes aegypti in the Americas. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the leading cause of cancer-related deaths in the US. Recombinant vectors based on adeno-associated virus (AAV) and lentivirus are promising delivery tools for gene therapy due to low toxicity and long term expression. The efficiency of the gene delivery system is one of the most important factors directly related to the success of gene therapy. Methods: We infected SCLC cell lines, SHP-77, DMS 53, NCI-H82, NCI-H69, NCI-H727, NCI-H1155, and NSCLC cell lines, NCI-H23, NCI-H661, and NCI-H460 with VSV-G pseudo-typed lentivirus or 5 AAV serotypes, AAV2/1, AAV2/2, AAV2/4, AAV2/5, and AAV2/8 expressing the CMV promoter mCherry or green fluorescent protein transgene (EGFP). The transduction efficiency was analyzed by fluorescent microscopy and flow cytometry. Results: Of all the serotypes of AAV examined, AAV2/1 was the optimal serotype in most of the lung cancer cell lines except for NCI-H69 and NCI-H82. The highest transduction rate achieved with AAV2/1 was between 30-50% at MOI 100. Compared to all AAV serotypes, lentivirus had the highest transduction efficiency of over 50% at MOI 1. Even in NCI-H69 cells resistant to all AAV serotypes, lentivirus had a 10-40% transduction rate. To date, AAV2 is the most widely-used serotype to deliver a transgene. Our results showed the transduction efficiency of AAVs tested was AAV2/1 > AA2/5 = AAV2/2> > AAV2/4 and AAV2/8. Conclusions: This study demonstrated that VSV-G pseudotyped lentivirus and AAV2/1 can mediate expression of a transgene for lung cancer gene therapy. Overall, our results showed that lentivirus is the best candidate to deliver a transgene into lung cancer cells for treatment. [ABSTRACT FROM AUTHOR]

Background: The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adenoassociated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Methods: Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . Results: We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. Conclusions: In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect. [ABSTRACT FROM AUTHOR]

Background: Local epidemiology of Dengue is defined by the genetic diversity of the circulating Dengue virus (DENV) strains. This important information is not available for the virus strains from most parts of the Indian subcontinent. The present study focused on the genetic diversity of the serotype 3 DENV strains (DENV-3) from India. Results: A total of 22 DENV-3 strains identified by reverse-transcription PCR analysis of serum samples from 709 patients were studied. These samples were collected over a period of 4 years (2008-2011) from dengue fever suspected patients from Kerala, a dengue endemic state in South India. Comparison of a 1740bp nucleotide sequence of the viral Capsid-Pre-membrane-Envelope coding region of our strains and previously reported DENV-3 strains from India, South Asia and South America revealed non-synonymous substitutions that were genotype III-specific as well as sporadic. Evidence of positive selection was detected in the I81 amino acid residue of the envelope protein. Out of the 22 samples, three had I81A and 18 had I81V substitutions. In the phylogenetic analysis by maximum likelihood method the strains from Kerala clustered in two different lineages (lineage III and IV) within genotype III clade of DENV-3 strains. The ten strains that belonged to lineage IV had a signature amino acid substitution T219A in the envelope protein. Interestingly, all these strains were found to be closely related to a Singapore strain GU370053 isolated in 2007. Conclusions: Our study identifies for the first time the presence of lineage IV strains in the Indian subcontinent. Results indicate the possibility of a recent exotic introduction and also a shift from the existing lineage III strains to lineage IV. Lineage shifts in DENV-3 strains have been attributed to dramatic increase in disease severity in many parts of the world. Hence the present observation could be significant in terms of the clinical severity of future dengue cases in the region. [ABSTRACT FROM AUTHOR]

Background: Certain adenovirus serotypes cause severe infections, especially in children. It is important to monitor temporal changes in serotypes causing clinical disease. The objective of this study was to document circulating respiratory adenovirus serotypes by sequencing adenovirus culture isolates from the Greater Toronto Area, Ontario, during December 2008 to April 2010. Methods: Nucleic acid extraction was performed on 90 respiratory tract adenovirus culture isolates. PCR amplification was conducted with primers targeting the adenovirus hexon gene hypervariable region 7. Sanger sequencing and phylogenetic analyses were performed to determine serotype identities. Results: Among 90 clinical respiratory isolates sequenced, eight different serotypes were identified. Serotype 3 (34, 38%), serotype 2 (30, 30%), and serotype 1 (14, 16%) isolates were most common; serotypes 5, 6, 11, 17 and 21 were also observed. Seventeen (50%) of the 34 HAdV-3 isolates were identified between December 2008 and February 2009, while none were identified from December 2009 to February 2010. Between December 2008 and April 2009, the two most common serotypes were HAdV-3 and HAdV-2, detected in 18 (53%) and 8 (24%) of the 34 cultures isolates, respectively. However, from December 2009 to April 2010, there was an increase in HAdV-2, which became the most prevalent serotype, detected in 10 (50%) of the 20 isolates identified (p = 0.05). Conclusions: There was a gradual shift in prevailing adenovirus serotypes during the 17 month study period, from predominantly HAdV-3 to HAdV-2. If an adenovirus vaccine were to be broadly implemented, multiple serotypes should be included. [ABSTRACT FROM AUTHOR]

Hand-foot-and-mouth disease (HFMD) and herpangina are commonly prevalent illness in young children. They are similarly characterized by lesions on the skin and oral mucosa. Both diseases are associated with various enterovirus serotypes. In this study, enteroviruses from patients with these diseases in Korea in 2009 were isolated and analyzed. Demographic data for patients with HFMD and herpangina were compared and all enterovirus isolates were amplified in the VP1 region by reverse transcription-polymerase chain reaction and sequenced. Among the enterovirus isolates, prevalent agents were coxsackievirus A16 in HFMD and coxsackievirus A5 in herpangina. More prevalent months for HFMD were June (69.2%) and May (11.5%), and June (40.0%) and July (24.0%) for herpangina. Age prevalence of HFMD patients with enterovirus infection was 1 year (23.1%), 4 years (19.2%), and over 5 years (19.2%). However, the dominant age group of herpangina patients with enterovirus infection was 1 year (48.0%) followed by 2 years (28.0%). Comparison of pairwise VP1 nucleotide sequence alignment of all isolates within the same serotypes revealed high intra-type variation of CVA2 isolates (84.6-99.3% nucleotide identity). HFMD and herpangina showed differences in demographic data and serotypes of isolated enteroviruses, but there was no notable difference in amino acid sequences by clinical syndromes in multiple comparison of the partial VP1 gene sequence. [ABSTRACT FROM AUTHOR]

Background: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. Methods: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. Results: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/ CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. Conclusions: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine. [ABSTRACT FROM AUTHOR]

The article focuses on the study presented by KwiSung Park and colleagues on identifying the characterization and antiviral activity of common drugs against echovirus 18 isolated in Korea. It mentions that human enteroviruses (HEVs), RNA viruses related to Picornaviridae consists of several immunologically- distinct serotypes that cause human infection. Information on the outbreak of aseptic meningitis caused by the ECV 18 isolate is presented.

The article focuses on the research presented by Hao-tai Chen and colleagues on the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for determining foot-and-mouth disease virus (FMDV) RNA. It mentions that FMDV is a member of the genus Aphthovirus from the family of Picornaviridae which is divided into several serotypes.

The article offers information on the study conducted by the authors related to the analysis of the genomic homologous recombination in Theilovirus based on complete genomes. It states that the Encephalomyocarditis virus (EMCV) and Theilovirus are two distinct species in the Cardiovirus genus of the family Picornaviridae. The EMCVs comprise a single serotype and have a wide host range, while the Theilovirus species, probably includes four serotypes.

The article offers information on the study conducted by the authors related to the development of a serotype colloidal gold strip (CGS) using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease (FMD). It states that the authors developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians.

Background: Cardioviruses are positive-strand RNA viruses in the Picornaviridae family that can cause enteric infection in rodents and also been detected at lower frequencies in other mammals such as pigs and human beings. The Cardiovirus genus consists two distinct species: Encephalomyocarditis virus (EMCV) and Theilovirus (ThV). There are a lot differences between the two species. In this study, the differences of codon usage in EMCV and ThV were compared. Results: The mean ENC values of EMCV and ThV are 54.86 and 51.08 respectively, higher than 40.And there are correlations between (C+G)12% and (C+G)3% for both EMCV and ThV (r = -0.736;r = 0.986, P < 0.01, repectively). For ThV the (C+G)12%, (C+G)3%, axis f'1 and axis f'2 had a significant correlations respectively but not for EMCV. According to the RSCU values, the EMCV species seemed to prefer U, G and C ending codon, while the ThV spice seemed to like using U and A ending codon. However, in both genus AGA for Arg, AUU for Ile, UCU for Ser, and GGA for Gly were chosen preferentially. Correspondence analysis detected one major trend in the first axis (f'1) which accounted for 22.89% of the total variation, and another major trend in the second axis (f'2) which accounted for 17.64% of the total variation. And the plots of the same serotype seemed at the same region at the coordinate. Conclusion: The overall extents of codon usage bias in both EMCV and ThV are low. The mutational pressure is the main factor that determines the codon usage bias, but the (C+G) content plays a more important role in codon usage bias for ThV than for EMCV. The synonymous codon usage pattern in both EMCV and ThV genes is gene function and geography specific, but not host specific. Maybe the serotype is one factor effected the codon bias for ThV, and location has no significant effect on the variations of synonymous codon usage in these virus genes. [ABSTRACT FROM AUTHOR]

To investigate molecular epidemiology of dengue viruses (DENV) in southern China, a total of 14 dengue isolates were collected in southern China during each epidemic year between 1978 and 2006 and their full-length genome sequences were obtained by using RT-PCR method. The E gene sequences from additional 6 dengue fever patients in Guangzhou in 2006 were also obtained by using RT-PCR method. Combined with DENVs sequences published in GenBank, phylogenetic analysis and recombination analysis were performed. One hundred and twenty-five E gene sequences and 60 complete genome sequences published in the GenBank were also involved. Phylogenetic analysis showed that there was a wide genetic diversity of DENVs isolated in southern China. DENV-1 strains exist in almost all of the clades of genotype I and IV except the Asia 1 clade of genotype I; DENV-2 stains are grouped into four of the five genotypes except American genotype. DENV-4 strains are grouped into 2 genotypes (I and II). Phylogenetic analysis also showed that all DENV-4 isolates and two DENV-2 isolates were closely related to the prior isolates from neighboring Southeast Asia countries. The DENV-1 strain isolated during the 2006 epidemic is highly homologous to the strains isolated during the 2001 epidemic. Recombination analysis showed no inter-serotype recombination, but 22 intra-serotype recombination events were found across the 32 complete genomes of all Chinese isolates. The study suggested that dengue fever epidemic in Southern China over the past 30 years presented two important modes, 1) imported-cases-induced endemic prevalence; 2) endogenous epidemic outbreak with natural epidemic focus. Recombination may play an important role in dengue virus evolution and adaptation. [ABSTRACT FROM AUTHOR]

A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVYNTN) in China and sequenced the complete genome of the variant PVYNTN-HN2. In this study, the complete genome of isolate PVYNTN-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVYNTN-HN1, like other typical European-PVYNTN isolates such as PVYNTN-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVYNTN-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVYO, PVYN, PVYN:O, PVYNTN-HN1 and PVYNTN-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVYN and PVYNTN-HN1 induced mild symptoms (mainly mild mottling) whereas PVYO, PVYN:O and PVYNTN-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana. [ABSTRACT FROM AUTHOR]

Backgroud: Foot-and-mouth disease virus (FMDV) serotype Asia1 generally infects cattle and sheep, while its infection of pigs is rarely reported. In 2005-2007, FMD outbreaks caused by Asia1 type occurred in many regions of China, as well as some parts of East Asia countries. During the outbreaks, there was not any report that pigs were found to be clinically infected. Results: In this study, a strain of FMDV that isolated from pigs was identified as serotype Asia1, and designated as "Asia1/WHN/CHA/06". To investigate the genomic feature of the strain, complete genome of Asia1/WHN/CHA/06 was sequenced and compared with sequences of other FMDVs by phylogenetic and recombination analysis. The complete genome of Asia1/WHN/CHA/06 was 8161 nucleotides (nt) in length, and was closer to JS/CHA/05 than to all other strains. Potential recombination events associated with Asia1/WHN/CHA/06 were found between JS/ CHA/05 and HNK/CHA/05 strains with partial 3B and 3C fragments. Conclusion: This is the first report of the isolation and identification of a strain of FMDV type Asia1 from naturally infected pigs. The Asia1/WHN/CHA/06 strain may evolve from the recombination of JS/CHA/05 and HNK/CHA/05 strains. [ABSTRACT FROM AUTHOR]

Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3) from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV. [ABSTRACT FROM AUTHOR]

Background: Pneumonia caused by adenovirus infection is usually severe especially with adenovirus serotype 7 commonly associated with lower respiratory tract disease outbreaks. We reported an outbreak of 70 cases of severe pneumonia with one death of infants in Shaanxi Province, China. Sampling showed adenovirus 7 (Ad7) as the primary pathogen with some co-infections. Results: Two strains of adenovirus and two strains of enterovirus were isolated, the 21 pharynx swabs showed 14 positive amplifications for adenovirus; three co-infections with respiratory syncytial virus, two positive for rhinovirus, one positive for parainfluenza 3, and four negative. Adenovirus typing showed nine of the nine adenovirus positive samples were HAdV-7, three were HAdV-3 and two were too weak to perform sequencing. The entire hexon gene of adenovirus was sequenced and analyzed for the two adenovirus serotype 7 isolates, showing the nucleic acid homology was 99.8% between the two strains and 99.5% compared to the reference strain HAdV-7 (GenBank accession number AY769946). For the 21 acute phase serum samples from the 21 patients, six samples had positives results for ELISA detection of HAdV IgA, and the neutralization titers of the convalescent-phase samples were four times higher than those of the acute-phase samples in nine pairs. Conclusions: We concluded adenovirus was the viral pathogen, primarily HAdV-7, with some co-infections responsible for the outbreak. This is the first report of an infant pneumonia outbreak caused by adenovirus serotype 7 in Shaanxi Province, China. [ABSTRACT FROM AUTHOR]

Background: Dengue virus (DENV) is a member of the genus Flavivirus of the family Flaviviridae. DENV are comprised of four distinct serotypes (DENV-1 through DENV-4) and each serotype can be divided in different genotypes. Currently, there is a dramatic emergence of DENV-3 genotype III in Latin America. Nevertheless, we still have an incomplete understanding of the evolutionary forces underlying the evolution of this genotype in this region of the world. In order to gain insight into the degree of genetic variability, rates and patterns of evolution of this genotype in Venezuela and the South American region, phylogenetic analysis, based on a large number (n = 119) of envelope gene sequences from DENV-3 genotype III strains isolated in Venezuela from 2001 to 2008, were performed. Results: Phylogenetic analysis revealed an in situ evolution of DENV-3 genotype III following its introduction in the Latin American region, where three different genetic clusters (A to C) can be observed among the DENV-3 genotype III strains circulating in this region. Bayesian coalescent inference analyses revealed an evolutionary rate of 8.48 × 10-4 substitutions/site/year (s/s/y) for strains of cluster A, composed entirely of strains isolated in Venezuela. Amino acid substitution at position 329 of domain III of the E protein (A→V) was found in almost all E proteins from Cluster A strains. Conclusions: A significant evolutionary change between DENV-3 genotype III strains that circulated in the initial years of the introduction in the continent and strains isolated in the Latin American region in recent years was observed. The presence of DENV-3 genotype III strains belonging to different clusters was observed in Venezuela, revealing several introduction events into this country. The evolutionary rate found for Cluster A strains circulating in Venezuela is similar to the others previously established for this genotype in other regions of the world. This suggests a lack of correlation among DENV genotype III substitution rate and ecological pattern of virus spread. [ABSTRACT FROM AUTHOR]

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery. Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors. Conclusions: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes. [ABSTRACT FROM AUTHOR]

Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in the Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus Orbivirus for which reverse genetics has not been developed yet. [ABSTRACT FROM AUTHOR]

Background: Foot-and-mouth disease (FMD) is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced. Results: Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L), the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/ 1964 and A23/KEN/1965) and serotype O viral isolate (K/117/1999) revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O. Conclusions: Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2), a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A and O has been obtained. In addition to characterization using the VP1 coding region, analyses involving the nonstructural protein coding regions should be performed in order to identify evolutionary processes shaping FMD viral populations. [ABSTRACT FROM AUTHOR]

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) and Streptococcus suis are common pathogens in pigs. In samples collected during the porcine high fever syndrome (PHFS) outbreak in many parts of China, PRRSV and S. suis serotype 7 (SS7) have always been isolated together. To determine whether PRRSV-SS7 coinfection was the cause of the PHFS outbreak, we evaluated the pathogenicity of PRRSV and/or SS7 in a pig model of single and mixed infection. Results: Respiratory disease, diarrhea, and anorexia were observed in all infected pigs. Signs of central nervous system (CNS) disease were observed in the highly pathogenic PRRSV (HP-PRRSV)-infected pigs (4/12) and the coinfected pigs (8/10); however, the symptoms of the coinfected pigs were clearly more severe than those of the HP-PRRSV-infected pigs. The mortality rate was significantly higher in the coinfected pigs (8/10) than in the HPPRRSV- (2/12) and SS7-infected pigs (0/10). The deceased pigs of the coinfected group had symptoms typical of PHFS, such as high fever, anorexia, and red coloration of the ears and the body. The isolation rates of HP-PRRSV and SS7 were higher and the lesion severity was greater in the coinfected pigs than in monoinfected pigs. Conclusion: HP-PRRSV infection increased susceptibility to SS7 infection, and coinfection of HP-PRRSV with SS7 significantly increased the pathogenicity of SS7 to pigs. [ABSTRACT FROM AUTHOR]