Serological Cross-reactivity Research Articles

Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry

Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus.

Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue, a flavivirus closely related to ZIKV. Here we investigate the serological crossreaction between the two viruses. Dengue immune plasma substantially crossreacted with ZIKV and could drive antibody-dependent enhancement of ZIKV infection. Using a panel of human anti-dengue monoclonal antibodies we showed that most antibodies reacting to dengue envelope protein also reacted to ZIKV. Antibodies to linear epitopes including the immunodominant fusion loop epitope while able to bind ZIKV could not neutralize the virus but instead promoted ADE. These data indicate that dengue immunity may drive higher ZIKV replication and have clear implications for disease pathogenesis and future ZIKV and dengue vaccine programs.

Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23.

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains.

BackgroundAt least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa.ResultsA U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree.ConclusionsThese data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.

Human Infections by Multiple Spotted Fever Group Rickettsiae in Tennessee.

Rocky Mountain spotted fever is the most common tick-borne disease in Tennessee. However, Rickettsia rickettsii has rarely been isolated from endemic ticks, suggesting rickettsioses may be caused by other species. A total of 56 human serum samples that were serologically positive for exposure to Rickettsia were obtained from commercial laboratories in 2010 and 2011. In addition, 20 paired sera from patients with encephalitis and positive Rickettsia serology were obtained from the Tennessee Unexplained Encephalitis Surveillance (TUES) study. Using an immunofluorescence assay, reactivity of the sera to R. rickettsii, Rickettsia montanensis, Rickettsia parkeri, and Rickettsia amblyommii was tested, and a comparison of endpoint titers was used to determine the probable antigen that stimulated the antibody response. Cross-absorption was conducted for 94.8% (N = 91) of the samples due to serologic cross-reactivity. Of the commercial laboratory samples, 55.4% (N = 31) had specific reactivity to R. amblyommii and 44.6% (N = 25) were indeterminate. Of the paired TUES samples, 20% (N = 4) had specific reactivity to R. amblyommii, 5% (N = 1) to R. montanensis, and 5% (N = 1) to R. parkeri Patients with specific reactivity to R. amblyommii experienced fever (75%), headache (68%) and myalgia (58%). Rash (36%) and thrombocytopenia (40%) were less common. To our knowledge, this is the first time R. amblyommii has been reported as a possible causative agent of rickettsioses in Tennessee.

Alpha proteobacteria of genus Anaplasma (Rickettsiales: Anaplasmataceae): Epidemiology and characteristics of Anaplasma species related to veterinary and public health importance.

The Anaplasma species are important globally distributed tick-transmitted bacteria of veterinary and public health importance. These pathogens, cause anaplasmosis in domestic and wild animal species including humans. Rhipicephalus, Ixodes, Dermacentor and Amblyomma genera of ticks are the important vectors of Anaplasma. Acute anaplasmosis is usually diagnosed upon blood smear examination followed by antibodies and nucleic acid detection. All age groups are susceptible but prevalence increases with age. Serological cross-reactivity is one of the important issues among Anaplasma species. They co-exist and concurrent infections occur in animals and ticks in same geographic area. These are closely related bacteria and share various common attributes which should be considered while developing vaccines and diagnostic assays. Movement of susceptible animals from non-endemic to endemic regions is the major risk factor of bovine/ovine anaplasmosis and tick-borne fever. Tetracyclines are currently available drugs for clearance of infection and treatment in humans and animals. Worldwide vaccine is not yet available. Identification, elimination of reservoirs, vector control (chemical and biological), endemic stability, habitat modification, rearing of tick resistant breeds, chemotherapy and tick vaccination are major control measures of animal anaplasmosis. Identification of reservoirs and minimizing the high-risk tick exposure activities are important control strategies for human granulocytic anaplasmosis.

Genetic Analysis and Detection of fliC H1 and fliC H12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli.

The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliCH1 and fliCH12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliCH1 and fliCH12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliCH1 and fliCH12, respectively. The real-time PCR for common detection of fliCH1 and fliCH12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliCH1 and fliCH12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources.

A comparative hospital-based observational study of mono- and co-infections of malaria, dengue virus and scrub typhus causing acute undifferentiated fever.

Positive serology for dengue and/or scrub typhus infection with/without positive malarial smear (designated as mixed or co-infection) is being increasingly observed during epidemics of acute undifferentiated febrile illnesses (AUFIs). We planned to study the clinical and biochemical spectrum of co-infections with Plasmodium sp., dengue virus and scrub typhus and compare these with mono-infection by the same organisms. During the period from December 2012 to December 2013, all cases presenting with AUFIs to a single medical unit of a referral centre in Garhwal region of the north Indian state of Uttarakhand were retrospectively selected and categorised aetiologically as co-infections, malaria, dengue or scrub typhus. The groups thus created were compared in terms of demographic, clinical, biochemical and outcome parameters. The co-infection group (n = 49) was associated with milder clinical manifestations, fewer, milder and non-progressive organ dysfunction, and lesser need for intensive care, mechanical ventilation and dialysis as compared to mono-infections. When co-infections were sub-grouped and compared with the relevant mono-infections, there were differences in certain haematological and biochemical parameters; however, this difference did not translate into differential outcomes. Scrub typhus mono-infection was associated with severe disease in terms of both morbidity and mortality. Malaria, dengue and scrub typhus should be routinely tested in all patients with AUFIs. Co-infections, whether true or due to serological cross-reactivity, appear to be a separate entity so far as presentation and morbidity is concerned. Further insight is needed into the mechanism and identification of the protective infection.

The mysterious Zika virus: Adding to the tropical flavivirus mayhem.

Until now, known as the demure cousin of dengue virus (DENV) inhabiting Africa, Zika virus (ZIKV) has reinvented itself to cause explosive epidemics captivating the Western hemisphere. The outbreak causing potential for ZIKV was realized when it made its way from Africa to Yap Island Micronesia in 2007, and in French Polynesia in 2013. From there, it moved on to Brazil in 2015. Now ZIKV has infected people in more than 33 countries in Central and South America and the Caribbean. Moreover the epidemiological and subsequent virological association with microcephaly cases in Brazil has prompted the World Health Organization to declare a public health emergency of International Concern. ZIKV shares not only its vector Aedes aegypti with dengue and chikungunya but also the geographic distribution and clinical features, which makes the laboratory confirmation mandatory for definitive diagnosis. The serological cross-reactivity with other Flavivirus, particularly with DENV makes laboratory confirmation challenging and will place additional burden on health systems to establish molecular diagnostic facilities. The evidence of additional nonvector modes of transmission, such as perinatal, sexual as well as transfusion has made preventative strategies more difficult. As ZIKV disease continues to mystify us with several unanswered questions, it calls for coordinated effort of global scientific community to address the ever growing arboviral threat to mankind.

Scrub Typhus and Leptospirosis: The fallacy of Diagnosing with IgM Enzyme Linked Immunosorbant Assay

Introduction: Leptospirosis and scrub typhus are important causes of acute febrile illness in India. IgM Enzyme Linked Immunosorbant Assay (ELISA) is the most common diagnostic modality used for their diagnosis. The common epidemiology of both the diseases creates an opportunity of dual infections with these diseases. Therefore, the objective of the study was to detect and evaluate the cases of serological and molecular dual infections. Methodology: A cross sectional diagnostic study from October 2013 to October 2015 was conducted on 258 patients with acute febrile illness. All the samples were subjected to IgM ELISA for scrub typhus and leptospirosis. The samples that were positive for both scrub typhus and leptospirosis were subjected to serological tests for other infections. They were also subjected to PCR assays to find out the cases of molecular dual infections. Results: A total of twenty serum samples were positive by IgM ELISA for leptospirosis while thirty five serum samples were positive by IgM ELISA for scrub typhus. Among these, ten samples were positive for both the serological tests. These dual positives were additionally positive by serology for some other infections [Dengue (n = 2), Mycoplasma pneumoniae (n = 1), Malaria (n = 1), Chlamydia pneumoniae (n = 6), Typhoid (n = 2) and Legionella pneumophila (n = 1)]. Only one case of molecular dual infection was confirmed. Conclusion: The possibility of serological co-infections should be investigated in endemic areas. In a case of serological dual infection, since there are high chances of serological cross reactivity, molecular confirmation should be sought for. The choice of therapy in inconclusive cases should include those drugs that cover for both the infections.

Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity

Neuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8–64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol.

Genetic and Phylogenetic Characterization of Tataguine and Witwatersrand Viruses and Other Orthobunyaviruses of the Anopheles A, Capim, Guamá, Koongol, Mapputta, Tete, and Turlock Serogroups.

The family Bunyaviridae has more than 530 members that are distributed among five genera or remain to be classified. The genus Orthobunyavirus is the most diverse bunyaviral genus with more than 220 viruses that have been assigned to more than 18 serogroups based on serological cross-reactions and limited molecular-biological characterization. Sequence information for all three orthobunyaviral genome segments is only available for viruses belonging to the Bunyamwera, Bwamba/Pongola, California encephalitis, Gamboa, Group C, Mapputta, Nyando, and Simbu serogroups. Here we present coding-complete sequences for all three genome segments of 15 orthobunyaviruses belonging to the Anopheles A, Capim, Guamá, Kongool, Tete, and Turlock serogroups, and of two unclassified bunyaviruses previously not known to be orthobunyaviruses (Tataguine and Witwatersrand viruses). Using those sequence data, we established the most comprehensive phylogeny of the Orthobunyavirus genus to date, now covering 15 serogroups. Our results emphasize the high genetic diversity of orthobunyaviruses and reveal that the presence of the small nonstructural protein (NSs)-encoding open reading frame is not as common in orthobunyavirus genomes as previously thought.

Diagnosis of a new variant of soybean yellow mottle mosaic virus with extended host-range in India.

Soybean yellow mottle mosaic virus (SYMMV, genus Carmovirus) was previously known to occur in South Korea and USA causing bright yellow mosaic in soybean. In this study, SYMMV (Car-Mb14 isolate) was isolated from mungbean (Vigna radiata) exhibiting mild mottling and puckering symptoms in the experimental field at Indian Agricultural Research Institute, New Delhi during 2012. The virus isolate, Car-Mb14 induced veinal mottling, mild mottling, chlorotic blotching, local and systemic necrosis in soybean, mungbean, blackgram, French bean and guar bean, respectively. The symptomatology of the present isolate of SYMMV was different from the previously reported South Korean isolate, as the later did not induce symptoms in any of the above legumes other than soybean. The present isolate was phylogenetically distinct and shared 90-93% sequence identity in coat protein (CP) of 52 SYMMV isolates reported from Korea and USA. In order to know the serological relationships, the CP gene of the present isolate was over expressed as a 39kDa protein in E. coli and an antiserum of 1:16,000titer against the recombinant CP was produced. Serological cross reactivity analysis revealed that SYMMV was serologically related to blackgram mottle virus but not to cowpea mottle virus, the other legume infecting carmoviruses. The antiserum was used to detect prevalence of SYMMV in legume crops by ELISA. Out of 145 field samples of legumes (mungbean, blackgram, French bean and soybean) collected from different places in India, SYMMV was detected only in 16 samples of mungbean and one sample of blackgram. The natural infection of SYMMV in mungbean and blackgram was further confirmed based on CP gene sequence. This study provides evidence of occurrence of a new variant of SYMMV with distinct symptom phenotype and extended host-range in India.

Serological cross-reactivity between Strongyloides venezuelensis and Syphacia muris in Wistar rats (Rattus norvegicus)

One of the problems frequently faced in laboratory facilities is the possibility of the natural parasitic infection of lab animals, which can interfere with biomedical research results. The present study aimed to evaluate cross-reactivity among serum samples from Wistar rats (Rattus norvegicus) naturally infected with Syphacia muris and experimentally infected with Strongyloides venezuelensis. Forty rats were divided into four groups of ten animals each. Parasite load was evaluated by quantifying the adult worms from both helminthes species recovered from the intestines and the S. venezuelensis eggs eliminated in feces. Serological cross-reactivity by parasite-specific IgG detection was tested via enzyme linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT) and immunoblotting. The results demonstrated that the quantity of S. venezuelensis eliminated eggs and parthenogenetic females decreased significantly in cases of co-infection with S. muris. ELISA revealed 100% cross-reactivity of serum samples from both species against the opposing antigen. IgG cross-reactivity was confirmed by IFAT using tissue sections of S. venezuelensis larvae and adult S. muris. Immunoblotting showed that IgG antibodies from the sera of animals infected with S. muris recognized eight antigenic bands from S. venezuelensis saline extract and that IgG antibodies from the sera of animals infected with S. venezuelensis recognized seven bands from S. muris saline extract. These results demonstrate the serological cross-reactivity between S. muris and S. venezuelensis in infected rats.

Molecular Allergen-Specific IgE Assays as a Complement to Allergen Extract–Based Sensitization Assessment

Molecular allergen-based component-resolved diagnostic IgE antibody tests have emerged in the form of singleplex assays and multiplex arrays. They use both native and recombinant allergen molecules, sometimes in combination with each other, to supplement allergen extract-based IgE antibody analyses. The total number of available allergenic molecules has reached a diagnostically useful level; however, more molecules are needed to cover all the clinically important allergen specificities. Thus, for the foreseeable future, molecular allergen-specific IgE analyses will remain a supplement for initial allergen extract-based IgE antibody analyses in the diagnostic workup of the allergic patient. As a spin-off, it will enable manufacturers to improve the quality of extracts for in vitro testing. The 2 most exciting diagnostic developments linked to component-resolved diagnostic tests are the possibility to increase diagnostic sensitivity by the inclusion of allergens that are underrepresented in the current extracts and in vitro assays and to increase the diagnostic specificity by taking the information on allergen cross-reactivity into account. Particularly the latter application is still under development. This requires additional studies on the clinical relevance of serological cross-reactivity.

Molecular, serological and in vitro culture-based characterization of Bourbon virus, a newly described human pathogen of the genus Thogotovirus

BackgroundIn June of 2014, a previously healthy man from Kansas with a recent history of tick exposure died from complications related to an illness marked by fever, thrombocytopenia and leukopenia. An isolate was derived from the blood of this patient during the course of diagnostic testing. This isolate was subsequently identified as a novel orthomyxovirus of the genus Thogotovirus by next generation sequencing and was named Bourbon virus after the patient’s county of residence. ObjectivesTo support research and diagnostic aims, we provide a basic description of Bourbon virus at both the molecular and serological levels. Furthermore, to preliminarily identify potential host and vector range associations we have characterized the growth kinetics of Bourbon virus in a variety of vertebrate and invertebrate cell lines. Study designBourbon virus was subjected to next generation-high throughput sequencing, phylogenetic, and basic structural protein analyses as well as 2-way plaque reduction neutralization assays. Also, we inoculated a variety of cell types with Bourbon virus and evaluated the growth kinetics by determining viral titers in the supernatants taken from infected cells over time. ResultsBourbon virus possesses 24–82% identity at the amino acid sequence level and low serological cross-reactivity with other Thogotoviruses. In vitro growth kinetics reveal robust replication of Bourbon virus in mammalian and tick cells. ConclusionsMolecular and serological characterizations identify Bourbon virus as a novel member of the genus Thogotovirus. Results from cell culture analyses suggest an association between Bourbon virus and mammalian and tick hosts.

Bet v 1- and Bet v 2-Associated Plant Food Sensitization in Uganda and Germany: Differences and Similarities

Background: Birch pollen allergy and concomitant plant food sensitization are well documented in Europe. However, there are currently no data available on pollen-associated plant food sensitization or even pollen allergy in tropical Africa. Our study aimed to investigate Bet v 1- and Bet v 2-associated plant food sensitization in atopic patients from Uganda and compare it with sensitization rates in German patients. Methods: Sera from 83 Ugandan and 97 German atopic patients were analysed using UniCAP100™ for allergen-specific IgE against the birch tree pollen allergens Bet v 1 and Bet v 2 as well as the plant foods hazelnut, apple, kiwi, pea, peach, cherry, litchi, peanut, and soy. Results: As expected, sensitization to Bet v 1 and cross-reactive plant food allergens was more common in German atopic patients. In contrast, the prevalence of sensitization against Bet v 2 was remarkably similar in Ugandan and German patients. Interestingly, in Ugandan patients we found IgE-mediated sensitization against plant foods such as hazelnut, pea, peach, cherry, and litchi that are neither cultivated nor consumed in Uganda. Conclusions: For Ugandan atopic patients, sensitization against the Bet v 2 allergen (a plant profilin) may explain cross-reactivity to several plant foods which are not consumed in Uganda. Additionally, it is probable that sensitization of Ugandan atopics to alder pollen (Alnus acuminata, plant family Betulaceae) caused serological cross-reactivity with Betula verrucosa-related allergens.

Further Characterization of Rio Grande Virus and Potential for Serological Cross Reactivity with other Phleboviruses

Members of the genus Phlebovirus (family Bunyaviridae ) are new and emerging disease pathogens of humans and animals. Newly identified viruses include Heartland virus (HRTV), Lone Star virus in the USA, and Severe Fever with Thrombocytopenia Syndrome virus in Asia. Assays to support surveillance, epidemiologic studies, and diagnosis of these viruses may also detect related viruses within the genus, confounding interpretation. Rio Grande virus (RGV) was isolated in 1973 from southern plains woodrats ( Neotoma micropus ) in the United States and has been preliminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora . RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12. The ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays.

New evidence on tick-borne rickettsioses in the Altai region of Russia using primary lesions, serum and blood clots of molecular and serological study

Tick-borne rickettsioses (TBRs) have similar clinical symptoms and can give serological cross-reaction. We firstly found that in the natural foci of North Asian tick typhus (NATT) in the Altai region of Russia, TBRs can be caused by two Rickettsia species: Rickettsia sibirica subsp. sibirica (causative agent of NATT) and Rickettsia heilongjiangensis. Rickettsial DNA was detected in primary lesions, serum samples and blood clots using real-time PCR. Therefore, each case of TBRs should be verified by using molecular typing. TBR caused by R. sibirica subsp. sibirica – NATT dominates on the territory of Altai region.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse’s ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.