Genomic characterization of 99 viruses from the bunyavirus families Nairoviridae, Peribunyaviridae, and Phenuiviridae, including 35 previously unsequenced viruses.

Bunyaviruses (Negarnaviricota: Bunyavirales) are a large and diverse group of viruses that include important human, veterinary, and plant pathogens. The rapid characterization of known and new emerging pathogens depends on the availability of comprehensive reference sequence databases that can be used to match unknowns, infer evolutionary relationships and pathogenic potential, and make response decisions in an evidence-based manner. In this study, we determined the coding-complete genome sequences of 99 bunyaviruses in the Centers for Disease Control and Prevention's Arbovirus Reference Collection, focusing on orthonairoviruses (family Nairoviridae), orthobunyaviruses (Peribunyaviridae), and phleboviruses (Phenuiviridae) that either completely or partially lacked genome sequences. These viruses had been collected over 66 years from 27 countries from vertebrates and arthropods representing 37 genera. Many of the viruses had been characterized serologically and through experimental infection of animals but were isolated in the pre-sequencing era. We took advantage of our unusually large sample size to systematically evaluate genomic characteristics of these viruses, including reassortment, and co-infection. We corroborated our findings using several independent molecular and virologic approaches, including Sanger sequencing of 197 genome segments, and plaque isolation of viruses from putative co-infected virus stocks. This study contributes to the described genetic diversity of bunyaviruses and will enhance the capacity to characterize emerging human pathogenic bunyaviruses.

Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections.

Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.

Laboratory Diagnosis of Chikungunya Virus Infections and Commercial Sources for Diagnostic Assays.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >5 days of illness. Commercially available CHIKV IgM-detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Measuring Haitian children's exposure to chikungunya, dengue and malaria.

OBJECTIVE: To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti. METHODS: We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinant Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014. FINDINGS: Of the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean. CONCLUSION: Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015.

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Laboratory Validation of a Real-Time RT-PCR Assay for the Detection of Jamestown Canyon Virus.

The neuroinvasive disease caused by Jamestown Canyon virus (JCV) infection is rare. However, increasing incidence and widespread occurrence of the infection make JCV a growing public health concern. Presently, clinical diagnosis is achieved through serological testing, and mosquito pool surveillance requires virus isolation and identification. A rapid molecular detection test, such as real-time RT-PCR, for diagnosis and surveillance of JCV has not been widely utilized. To enhance testing and surveillance, here, we describe the development and validation of a real-time RT-PCR test for the detection of JCV RNA. Three primer and probe sets were evaluated for analytical sensitivity and specificity. One probe set, JCV132FAM, was found to be the most sensitive test detecting 7.2 genomic equivalents/µL. While less sensitive, a second probe set JCV231cFAM was the most specific test with limited detection of Keystone virus at high RNA loads. Taken together, these data indicate both probe sets can be utilized for a primary sensitive screening assay and a secondary specific confirmatory assay. While both primer and probe sets detected high viral loads of Keystone virus, these assays did not detect any virus in the California encephalitis virus clade, including negative detection of the medically important La Crosse virus (LACV) and snowshoe hare virus (SSHV). The real-time RT-PCR assay described herein could be utilized in diagnosis and surveillance in regions with co-circulation of JCV and LACV or SSHV to inform public health action.

Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics.

Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.

Correction: Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics.

[This corrects the article DOI: 10.1371/journal.pntd.0010487.].

Genomic Evaluation of the Genus Coltivirus Indicates Genetic Diversity among Colorado Tick Fever Virus Strains and Demarcation of a New Species.

The type species of the genus Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.

Genetic Characterization of Frijoles and Chilibre Species Complex Viruses (Genus Phlebovirus; Family Phenuiviridae) and Three Unclassified New World Phleboviruses.

The genus Phlebovirus is a diverse group of globally occurring viruses, including tick-, mosquito-, and sand fly-borne pathogens. Phleboviruses have historically been classified by serological methods. However, molecular methods alone have been used to identify emergent novel and related strains in recent years. This makes reconciling the classification of historically and newly characterized viruses challenging. To address this in part, we describe the characterization of the genomes of the Frijoles and Chilibre species complex phleboviruses, and three unclassified phleboviruses isolated in the Americas: Caimito, Itaporanga, and Rio Grande viruses that had previously only been described at the serological level. With the exception of Itaporanga virus, the phleboviruses sequenced in this study are phylogenetically related to the current species Frijoles phlebovirus, Bujaru phlebovirus, or the Chagres antigenic complex. Unexpectedly, molecular and phylogenetic analysis suggests Chilibre and Caimito viruses are taxonomically related to the family Peribunyaviridae. These viruses have a genomic architecture similar to peribunyaviruses and form monophyletic groups within the genus Pacuvirus. Our data highlight the importance of reconciling serological and molecular taxonomic classification. In addition, we suggest the taxonomy of Chilibre and Caimito viruses should be revised.

Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management.

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Microcarrier culture of COS-1 cells producing Japanese encephalitis and dengue virus serotype 4 recombinant virus-like particles.

Two stably transfected COS-1 cell lines that secrete recombinant Japanese encephalitis and dengue virus serotype 4 virus-like particles (VLPs) have been adapted to grow on Cytodex 3 microcarriers in an orbital shaker flask platform. The VLPs are used as antigens in diagnostic enzyme-linked immunosorbent assays to detect anti-arboviral IgM and IgG antibodies in human serum samples. Converting from a stationary flask batch system to a microcarrier fed-batch system has led to increases in antigen concentration while decreasing costs by reducing the amount of cell culture medium, disposables, and labor. The cell culture longevity was increased by 48 days in this optimized system, which may be due to a continual supply of nutrients resulting in prolonged survival of the cells on the microcarrier surface. An initial trial using a serum-free medium with this cell line was promising and may lead to reductions in cost, while reducing the variability between batches introduced by fetal bovine serum.

Detection of Colorado Tick Fever viral RNA in acute human serum samples by a quantitative real-time RT-PCR assay.

A quantitative real-time RT-PCR assay for the detection of Colorado Tick Fever (CTF) viral RNA in human clinical samples is presented. The sensitivity of this assay has been shown to be greater than that of the isolation of virus in Vero cells by standard plaque assay in a direct comparison. The specificity of the CTF quantitative real-time RT-PCR assay was determined by the exclusive detection of CTF viral RNAs when applied to a diverse panel of CTF viral isolates and reference strain agents known to circulate in areas of CTF virus transmission. Lastly, the quantitative real-time RT-PCR assay demonstrated exceptional sensitivity for the detection of CTF viral RNA in acute human serum. The quantitative real-time RT-PCR assay is efficient, sensitive and specific and as such is useful for the detection of CTF viral RNA in the diagnostic or research laboratory.

A comparison of concentration methods applied to non-infectious flavivirus recombinant antigens for use in diagnostic serological assays.

Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times.

First Complete Genome Sequences of Anopheles A Virus of the Genus Orthobunyavirus.

Here, we report the first complete genome sequence of Anopheles A virus (ANAV) that was isolated from Colombia in 1940, and we include the first description of the medium and large segments. The ANAV medium and large segments share the highest identity with serogroup member Lukuni virus, which causes human infection.

Molecular characterization of human pathogenic bunyaviruses of the Nyando and Bwamba/Pongola virus groups leads to the genetic identification of Mojuí dos Campos and Kaeng Khoi virus.

BACKGROUND: Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types. CONCLUSIONS/SIGNIFICANCE: On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.

The genome sequence of Lone Star virus, a highly divergent bunyavirus found in the Amblyomma americanum tick.

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.

Evaluation of IgM antibody capture enzyme-linked immunosorbent assay kits for detection of IgM against Japanese encephalitis virus in cerebrospinal fluid samples.

Infection with Japanese encephalitis virus (JEV) is a major public health problem in Asia. Detection of JEV-specific IgM in serum and cerebrospinal fluid (CSF) by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is currently the most widely used diagnostic method to detect JEV infection. Because of the possible presence of IgM cross-reactivity with other flaviviruses in serum and the high ratio of inapparent-to-apparent JEV infections, a positive result in serum only suggests a recent infection and not necessarily an encephalitic illness caused by JEV. Consequently, detection of JEV-specific IgM in CSF assumes great diagnostic relevance. We evaluated two commercial JEV MAC-ELISA kits using 60 CSF samples obtained from patients with acute encephalitis syndrome. The Panbio and XCyton kits had sensitivities of 65-80% and 95% and specificities of 90% and 97.5%, respectively. Performance information on these commercial JEV MAC-ELISA kits for CSF should assist in laboratory-based JE surveillance programs.

Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay.

The dengue (DEN) viruses are positive-strand RNA viruses in the genus Flavivirus. Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by infection with one of four closely related but serologically distinct DEN viruses, designated DEN-1, DEN-2, DEN-3, and DEN-4 viruses. All four DEN serotypes are currently co-circulating throughout the subtropics and tropics, and genotypic variation occurs among isolates within a serotype. A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction (0.4 to 1.2 x 10(6) PFU/ml). Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.