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Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed., Author summary Glanders is a highly contagious and life-threatening disease caused by Burkholderia mallei, a Tier 1 select agent, with no available vaccine. The disease is endemic in the Middle East, Asia, Africa and South America with sporadic outbreaks and mainly occurs in horses, donkeys and mules, although it has also been reported in camels, tigers, lions, and even humans. As the bacterium is not easily isolated from clinical specimens and correct identification based on clinical signs is difficult, it is thus important to develop serological tests which can quickly diagnose B. mallei infection. In this study, we generated a monoclonal antibody against B. mallei lipopolysaccharide and used it to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the serodiagnosis of B. mallei infection. The developed cELISA was optimized and evaluated using glanders-free and glanders-positive horses, donkeys and mice from Hong Kong and the Middle East, and was shown to be highly sensitive and specific for the detection of glanders in different animals. A simple and inexpensive test to allow for the early detection and diagnosis of suspected clinical cases as well as the screening of apparently asymptomatic animals will be helpful in controlling the spread and elimination of the disease.

In 2019, the World Health Organization declared 3 billion to be at risk of developing Crimean Congo Hemorrhagic Fever (CCHF). The causative agent of this deadly infection is CCHFV. The data related to the biology and immunology of CCHFV are rather scarce. Due to its indispensable roles in the viral life cycle, NP becomes a logical target for detailed viral immunology studies. In this study, humoral immunity to NP was investigated in CCHF survivors, as well as in immunized mice and rabbits. Abundant antibody response against NP was demonstrated both during natural infection in humans and following experimental immunizations in mice and rabbits. Also, cellular immune responses to recombinant NP (rNP) was detected in multispecies. This study represents the most comprehensive investigation on NP as an inducer of both humoral and cellular immunity in multiple hosts and proves that rNP is an excellent candidate warranting further immunological studies specifically on vaccine investigations., Author summary Crimean Congo Hemorrhagic Fever Virus (CCHFV) is the most lethal human pathogen of medical importance after the dengue virus among arboviruses. The increasing geographic spread of Hyalomma ticks, which are responsible for viral transmission widespread, threatens billions of people. WHO currently declares the field of research on CCHFV as the second most urgently needed areas of investigations on emerging pathogens. About 10 to 40% of those infected with the virus lose their life due to the rapidly developing severe clinical manifestations. Pandemic potential and the lack of any approved treatment or vaccine make raise the studies on CCHFV as critical. The studies on CCHFV are challenging due to the necessities of BSL-4 facilities and the immunological characterization of individual structural proteins will lay the groundwork for the steps to be taken to treat and prevent this emerging disease. As is known from other RNA viruses, nucleoprotein (NP) has crucial roles in the viral life cycle, both in viral replication and transcription and in the formation of the virion structure. So far, detailed and comprehensive immunological characterizations on NP in multiple are not undertaken. Our study was set out to embark such detailed investigation. The strong humoral and cellular immune response to NP demonstrated by this study indicates that NP might be an excellent candidate for future scrutinies on vaccines and diagnostic reagents.

Background Snakebite is a neglected tropical disease that causes high global rates of mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapeutic for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. Methodology/Principal findings In this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Two different immunogen mixtures, consisting of seven and twelve haemotoxic venoms sourced from geographically diverse and/or medically important snakes, were used to raise ovine polyclonal antibodies, prior to characterisation of their immunological binding characteristics and in vitro neutralisation profiles against each of the venoms. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom. These findings suggest that a core set of venom immunogens may be sufficient to stimulate antibodies capable of broadly neutralising a geographically diverse array of haemotoxic snake venoms, and that adding additional venom immunogens may impact negatively on the dose efficacy of the resulting antivenom. Conclusions/Significance Although selection of appropriate immunogens that encapsulate venom toxin diversity without diluting antivenom potency remains challenging and further optimisation is required, the findings from this pilot study suggest that the generation of pathology-specific antivenoms with global utility is likely to feasible, thereby highlighting their promise as future modular treatments for the world’s tropical snakebite victims., Author summary Snakebite is a major public health concern that causes extensive death and disability in the tropical world. Variation in venom composition causes major challenges for snakebite therapy by typically restricting antivenom efficacy to only the few snake species that each product was designed against, meaning that many geographically restricted therapeutics are manufactured for different parts of the world. In this study, we explored the feasibility of generating a global pathology-specific antivenom by using two different sets of haemotoxic venoms as immunogens, one including seven venoms and the other twelve venoms, to generate ovine polyclonal antibodies. Our in vitro immunological cross-reactivity and venom functional-neutralisation findings demonstrated broadly comparable venom recognition and inhibition against the various venom immunogens, irrespective of whether they were included in the immunising mixture. However, assessments of murine in vivo protection against venom-induced lethality revealed that the antivenom generated with fewer immunogens outperformed the comparator, and provided broader preclinical efficacy, even against some venoms not used to raise the antibodies. These findings suggest that careful selection of venoms used as immunogens is crucial for maintaining antivenom potency, and that a small subset of venoms may be capable of generating an antivenom with potential global efficacy against a specific pathology commonly observed after snakebite.

Objectives Trichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure. Methods A competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-like protein (rCLP-cELISA) as well as monoclonal antibodies. The sensitivity and accuracy of the rCLP-cELISA were quantified using swine (n = 1316), mice (n = 189) and human (n = 157) serum samples. T. spiralis-antibody targeting test ability of the rCLP-cELISA in swine (n = 22) and human (n = 36), instead of other parasites or viruses antibodies, was evaluated. Results The rCLP-cELISA showed high agreement with commercial ELISA kits in field swine sera assessed by Cohen’s kappa value (κ = 0.7963). And it showed 100% specificity in human trichinellosis detection with sensitivity of 96.49%, no cross-reaction with other parasite or virus infections, and high positive detection rate of 87.5% in low-dose infected swine. Besides, the rCLP-cELISA exhibited potential in the detection of T. spiralis, T. nelsoni and Trichinella T8 infections. Conclusions The rCLP-cELISA can be used for T. spiralis-associated antibody test in multihost sera., Author summary Infections with T. spiralis that lives in host muscles for long periods of time are commonly difficult to diagnosis without causing a strong immune response or symptoms. The habit of eating raw/undercooked pork meat accidentally introduces humans into the exposure of T. spiralis that circulates between herds and rodents. There is an urgent need for serological antibody test of T. spiralis to monitor the infection of humans as well as hosts in the food chain, which is restricted by the mixture type of current used antigens and species-specific secondary antibodies for different hosts. We developed a novel recombinant cystatin-like protein-based competition enzyme-linked immunosorbent assay (rCLP-cELISA) employing monoclonal antibody. The proposed method showed considerable sensitivity and specificity in filed swine sera and human serum samples with good versatility in mice. Taking advantage of its controllable quality stability, the rCLP-cELISA had potential applications for screening of T. spiralis infections for multihost sera in one test. With the development of monoclonal antibody modification strategies and the discovery of antigenic proteins from parasitic pathogens, the proposed competition ELISA also provides useful reference for the improvement of serological assay for monitoring the exposure of zoonotic parasites.

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19., Author summary SARS-CoV-2 continues to threaten global health, and development of SARS-CoV-2 vaccine is an urgent priority to halt COVID-19 epidemic. Here, we developed DNA vaccines containing the spike gene from SARS-CoV-2 and delivered via electroporation. We observed that the DNA vaccine induced a long-lasting humoral response against SARS-CoV-2, and the elicited serum antibodies efficiently blocked the binding of SARS-CoV-2 RBD to its entry receptor (ACE2). Importantly, the DNA vaccine induced strong Th1-biased immune responses against SARS-CoV-2, which could provide protective effect with low side effects. Finally, we demonstrated that SARS-CoV-2 DNA vaccine conferred protective efficacy against SARS-CoV-2 infection in Syrian hamsters. Our findings suggested that DNA vaccination could be a useful approach for controlling COVID-19 pandemic in near future.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most widespread zoonotic arthropod-borne viruses in many parts of Africa, Europe and Asia. It belongs to the family of Nairoviridae in the genus of Orthonairovirus. The main reservoir and vector are ticks of the genus Hyalomma. Livestock animals (such as cattle, small ruminants and camels) develop a viremias lasting up to two weeks with absence of clinical symptoms, followed by seroconversion. This study was carried out to assess risk factors that affect seroprevalence rates in different species. In total, 928 livestock animal samples (cattle = 201; sheep = 247; goats = 233; camels = 247) from 11 out of 13 regions in Mauritania were assayed for CCHFV-specific immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assays (ELISA) (including a novel indirect camel-IgG-specific CCHFV ELISA). Inconclusive results were resolved by an immunofluorescence assay (IFA). A generalized linear mixed-effects model (GLMM) was used to draw conclusions about the impact of certain factors (age, species, sex and region) which might have influenced the CCHFV antibody status of surveyed animals. In goats and sheep, about 15% of the animals were seropositive, whereas in cattle (69%) and camels (81%), the prevalence rate was significantly higher. On average, cattle and camels were up to twice to four times older than small ruminants. Interestingly, the seroprevalence in all species was directly linked to the age of the animals, i.e. older animals had significantly higher seroprevalence rates than younger animals. The highest CCHFV seroprevalence in Mauritania was found in camels and cattle, followed by small ruminants. The large proportion of positive animals in cattle and camels might be explained by the high ages of the animals. Future CCHFV prevalence studies should at least consider the age of surveyed animals in order to avoid misinterpretations., Author summary Crimean-Congo hemorrhagic fever virus (CCHFV) is a silent threat that repeatedly causes severe hemorrhagic disease in humans who have been in close contact with livestock of endemic countries. The detection of CCHFV IgG antibodies in livestock can be a first indication whether the virus circulates in a given region and is thus a valuable diagnostic tool for determining the endemic status. Interestingly, earlier data from Mauritania showed a noticeable difference in IgG prevalence between sheep (18%) and cattle (67%). In contrast to sheep and cattle, current monitoring data on CCHFV IgG antibody presence in camels and goats in Mauritania is very limited. This study was conducted to provide a comprehensive up-to-date overview of CCHFV seroprevalences in the four most important Mauritanian livestock species (cattle, sheep, goats and camels). It attempts to highlight the role of potential risk factors responsible for deviating prevalences. In addition, we developed a camel-specific IgG ELISA, which can be used in future CCHFV seroprevalence studies. Furthermore, findings of this study contribute to a better understanding of the current epidemiological CCHFV situation in sub-Saharan Africa and which role different livestock species play regarding the viral circulation in endemic regions.

Background Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators’ interactions, and vector exposure intensity associated with CVL resistance and susceptibility. Methodology/Principal findings A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11–2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. Conclusions/Significance The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way., Author summary Visceral Leishmaniasis (VL) is a disease that can affect humans and dogs, caused by a parasite called Leishmania transmitted through the bite of sandfly insects. During the bite, together with the parasite, the insects also inoculate their saliva into the host. The host immune response produces molecules to the sandfly saliva, such as antibodies and cytokines that can impact VL resistance or susceptibility. The presence of these molecules also indicates if the insects bit the hosts. We followed dogs of a VL endemic area for two years to study Canine Visceral Leishmaniasis (CVL) and immune response to sandfly saliva. Dogs were evaluated at 6-month intervals to determine Leishmania infection, clinical manifestations, parasite load, immune response, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. Dogs living in the endemic area were intensely bitten, as at the beginning of the study, 62% of the dogs present anti-saliva antibodies, reaching 100% after one year. Our findings revealed a biosignature of CVL susceptibility characterized by elevated parasite load, interaction of cytokines, and higher exposure to the sandfly. This data reinforced that CVL is a complex disease affected by several factors related to each other.

Russell’s vipers (RVs) envenoming is an important public health issue in South-East Asia. Disseminated intravascular coagulopathy, systemic bleeding, hemolysis, and acute renal injury are obvious problems that develop in most cases, and neuromuscular junction blocks are an additional problem caused by western RV snakebite. The complex presentations usually are an obstacle to early diagnosis and antivenom administration. Here, we tried to produce highly specific antibodies in goose yolks for use in a paper-based microfluidic diagnostic kit, immunochromatographic test of viper (ICT-Viper), to distinguish RVs from other vipers and even cobra snakebite in Asia. We used indirect ELISA to monitor specific goose IgY production and western blotting to illustrate the interaction of avian or mammal antibody with venom proteins. The ICT-Viper was tested not only in prepared samples but also in stored patient serum to demonstrate its preliminary efficacy. The results revealed that specific anti-Daboia russelii IgY could be raised in goose eggs effectively without inducing adverse effects. When it was collocated with horse anti-Daboia siamensis antibody, which broadly reacted with most of the venom proteins of both types of Russell’s viper, the false cross-reactivity was reduced, and the test showed good performance. The limit of detection was reduced to 10 ng/ml in vitro, and the test showed good detection ability in clinical snake envenoming case samples. The ICT-Viper performed well and could be combined with a cobra venom detection kit (ICT-Cobra) to create a multiple detection strip (ICT-VC), which broadens its applications while maintaining its detection ability for snake envenomation identification. Nonetheless, the use of the ICT-Viper in the South-East Asia region is pending additional laboratory and field investigations and regional collaboration. We believe that the development of this practical diagnostic tool marks the beginning of positive efforts to face the global snakebite issue., Author summary For a long time, many scientists have made efforts to develop more effective antivenoms or to expand the limits of existing antivenoms. Such endeavors are challenging but worth the effort in the long run. However, to use precious antivenom more effectively, a snake species discrimination tool might be helpful for treatment by ensuring accurate antivenom usage and dosage. In this study, we revealed that avian IgY might be an economical alternative for collocation with the equine F(ab’)2 fragment in the development of a diagnostic device with improved performance. Only a few avian eggs (about 10–15) would be necessary to produce 10,000 kits. Immunochromatographic test (ICT) is a simple, easy, fast, and low-cost point-of-care lateral flow assay device, and especially adapted to perform in resource-deficient areas. Such an antibody collocation strategy might be beneficial to overcome the urgent need for snakebite diagnosis as soon as possible. More cooperative investigations across Asia continent might be initiated in the future to face the challenge of snakebites together.

Although Japanese encephalitis virus genotype Ib (JEV GIb) has replaced JEV GIII as the dominant genotype in endemic areas of Asia, no JEV GIb has been isolated from JE cases and natural mosquitoes at the same time in an outbreak of JE. In this study, we conducted virological and molecular biological laboratory tests on JE case samples (serum/cerebrospinal fluid) and locally collected mosquito samples from the 2018 JE outbreak in Ningxia, China. The result of JEV IgM antibody detection showed that 96% (67/70) of the suspected cases were laboratory-confirmed JE cases. Of the mosquitoes collected from local environments, 70% (17400/24900) were Culex tritaeniorhynchus of which 4.6% (16 /348 of the pools tested) were positive for JEV, other mosquitoes were negative. JEVs isolated from both the human cases and C. tritaeniorhynchus specimens belong to JEV GIb and are in the same evolutionary clade according to molecular evolution analyses. JEV GIb was detected simultaneously from specimens of JE cases and mosquito samples collected in nature in this study, suggesting that the JE outbreak that occurred in Ningxia in 2018 was due to infection of JEV GIb., Author summary Japanese encephalitis virus (JEV) is recognized as an important encephalitis pathogen all over the world. Its genotype is divided into GI-V. In recent years, JEV GIb (a temperate genotype) has gradually replaced GIII as the prevalent strain in JE endemic areas. Although JEV GIb originated from tropical Asia along with JEV GIa, it has rapidly spread for its advantages in wintering and infecting vectors. Although there have been epidemics caused by JEV GI and GIII, there have been no reports of a JE outbreak caused by JEV GIb alone in northeastern Asia. However, a JE outbreak occurred in the Ningxia Hui Autonomous Region in northern China in summer 2018 which was the first outbreak in Ningxia in recent decades. This paper presents a series of laboratory and field studies of this outbreak. The strain isolated from JE cases as well as JEV detected in Culex tritaeniorhynchus collected from local areas in nature all belonged to JEV GIb and were in the same evolutionary clade. This is the first report of a JE outbreak caused by JEV GIb infection in northeastern Asia (latitude 35 ° 14’– 39 ° 23’ N, longitude 104 ° 17’– 107 ° 39’ E), which used to be a low endemic area of JEV GIII.

Background Leptospirosis is a zoonotic disease that can be transmitted by contact with the urine of infected mammals. Rodents play a mayor role in the transmission of leptospires to humans. The province of Santa Fe reports the greatest number of cases in Argentina. Yet, in this region, there are still knowledge gaps regarding the diversity of rodent species that may be hosts of pathogenic leptospires. The aims of this study were to evaluate the presence of leptospiral antibodies in rodents from three riverside communities of Santa Fe, and to identify factors associated with leptospiral infection. Methodology/Principal findings Each community was divided into three environmental settings based on the level of human disturbance, and sampled during two springs (Sep-Oct 2014 and 2015) and one autumn (Mar-Apr 2015). Serum samples of captured sigmodontine and murine rodents were tested for leptospiral antibodies by enzyme-linked immunosorbent assay (ELISA), and microagglutination test (MAT) was used to assess the infecting serovar in seropositive individuals. Factors influencing seropositivity were analyzed using logistic regression models. We caught 119 rodents, of which 101 serums were suitable for analysis. Most frequently trapped species were Scapteromys aquaticus, Akodon azarae and Oligoryzomys spp., with seroprevalences of 41.3%, 42.9% and 55% respectively. Seropositivity was higher in individuals with an average body condition score and in those that were sexually mature, but in the latter the differences were marginally significant. Conclusions/Significance Our results suggest that native rodents may be playing a role in the environmental circulation of pathogenic leptospires and provide relevant information for public health policies in the area., Author summary Rodents are considered as the main reservoirs of pathogenic leptospires, which can cause leptospirosis, a disease that can be severe for both humans and domestic animals. Multiple socio-environmental factors have been found to influence the risk of leptospirosis, and many mammal species can play a role in the transmission of the bacteria. The purpose of this study was to investigate the local conditions and rodent fauna that may influence the risk of leptospirosis in communities located in the floodplains of the Parana river, near Santa Fe, Argentina. We conducted a survey of antibodies against leptospires in rodents from the center, border, and outside of three riverside settlements. We observed a relatively high seroprevalence among captured individuals (42%). Animals with an intermediate body condition were more likely to be seropositive. In most sites, native rodents were dominant over introduced rats and mice and lived close to human dwellings. Given the socio-environmental conditions observed in these settlements and the high frequency of floods that affect them, the presence of potentially infected rodents provide ample opportunities for leptospirosis to affect these communities.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 – κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p, Author summary Detection of the snake species responsible for the envenomation in a victim is crucial for clinical and forensic management of poisoning cases. Polyvalent antivenom therapy can seldom lead to immunological complications manifested in the form of symptoms ranging from serum sickness to myalgia. Use of monovalent antivenom therapy is being recommended for targeted venom neutralization in envenomed individuals with minimum side effects. Moreover, the development of field applicable venom detection devices is the need of the hour for enabling orderly detection of poisoning cases at the crime scene, besides testing for illegal trade of snake parts, including venom, protected under the Wildlife Act. The monovalent antivenom therapy and the field level detection of venom conducive to adequate crime scene management is limited by the tools available for species or family-specific identification of the venom under question. For differential detection of the Elapids of the big four snakes from the Viperidae, we have developed a monoclonal antibody-based lateral flow assay kit using recombinant Cytotoxin– 7 protein. The limit of quantitation for the detection of venoms of N. naja and B. caeruleus was ascertained to be 170 pg/μL and 2.1 ng/ μL in spiked buffer samples and 28.7 ng/ μL and 110 ng/ μL in spiked serum samples, respectively. Thus, the kit can effectively detect the venoms of elapids of the big four snakes in both simple and complex matrices of the samples and can be adapted for its use in differential diagnosis.

Background Daboia siamensis (Eastern Russell’s viper) is a medically important snake species found widely distributed across Southeast Asia. Envenomings by this species can result in systemic coagulopathy, local tissue injury and/or renal failure. While administration of specific antivenom is an effective treatment for Russell’s viper envenomings, the availability of, and access to, geographically-appropriate antivenom remains problematic in many rural areas. In this study, we determined the binding and neutralizing capability of antivenoms manufactured by the Thai Red Cross in Thailand against D. siamensis venoms from four geographical locales: Myanmar, Taiwan, China and Thailand. Methodology/Principle findings The D. siamensis monovalent antivenom displayed extensive recognition and binding to proteins found in D. siamensis venom, irrespective of the geographical origin of those venoms. Similar immunological characteristics were observed with the Hemato Polyvalent antivenom, which also uses D. siamensis venom as an immunogen, but binding levels were dramatically reduced when using comparator monovalent antivenoms manufactured against different snake species. A similar pattern was observed when investigating neutralization of coagulopathy, with the procoagulant action of all four geographical venom variants neutralized by both the D. siamensis monovalent and the Hemato Polyvalent antivenoms, while the comparator monovalent antivenoms were ineffective. These in vitro findings translated into therapeutic efficacy in vivo, as the D. siamensis monovalent antivenom was found to effectively protect against the lethal effects of all four geographical venom variants preclinically. Assessments of in vivo nephrotoxicity revealed that D. siamensis venom (700 μg/kg) significantly increased plasma creatinine and blood urea nitrogen levels in anaesthetised rats. The intravenous administration of D. siamensis monovalent antivenom at three times higher than the recommended scaled therapeutic dose, prior to and 1 h after the injection of venom, resulted in reduced levels of markers of nephrotoxicity and prevented renal morphological changes, although lower doses had no therapeutic effect. Conclusions/Significance This study highlights the potential broad geographical utility of the Thai D. siamensis monovalent antivenom for treating envenomings by the Eastern Russell’s viper. However, only the early delivery of high antivenom doses appears to be capable of preventing venom-induced nephrotoxicity., Author summary Snakebite is a major public health concern in rural regions of the tropics. The Eastern Russell’s viper (Daboia siamensis) is a medically important venomous snake species that is widely distributed in Southeast Asia and Southern China, including Taiwan. Envenoming by D. siamensis causes several systemic pathologies, most notably acute kidney failure and coagulopathy. The administration of antivenom is the mainstay therapeutic for treating snakebite, but in remote areas of Southern China access to antivenom is limited, and can result in the use of inappropriate, non-specific, antivenoms and treatment failure. Therefore, maximizing the utility of available efficacious antivenom is highly desirable. In this study, we investigated the utility of the widely available Thai Red Cross antivenoms for binding to and neutralizing D. siamensis venoms sourced from four distinct locales in Asia. Since the effectiveness and antivenom dose required to prevent D. siamensis venom-induced nephrotoxicity has been controversial, we also examined the preclinical efficacy of D. siamensis antivenom at preventing this pathology in experimentally envenomed anaesthetised animals. Our findings suggest that monovalent antivenom from Thailand, which is clinically effective in this country, has highly comparable levels of immunological binding and in vivo venom neutralization to D. siamensis venoms from China, Taiwan and Myanmar. We also show that the early administration of high therapeutic doses of antivenom are likely required to neutralize nephrotoxins and thus prevent acute renal failure following envenoming. Our findings suggest that certain Thai Red Cross antivenoms likely have wide geographical utility against D. siamensis venom and therefore may be useful tools for managing snakebite envenomings by this species in the absence of locally manufactured therapeutics.

The 2013–2016 Ebola virus outbreak in West Africa was the largest and deadliest outbreak to date. Here we conducted a serological study to examine the antibody levels in survivors and the seroconversion in close contacts who took care of Ebola-infected individuals, but did not develop symptoms of Ebola virus disease. In March 2017, we collected blood samples from 481 individuals in Makeni, Sierra Leone: 214 survivors and 267 close contacts. Using commercial, quantitative ELISAs, we tested the plasma for IgG-specific antibodies against three major viral antigens: GP, the only viral glycoprotein expressed on the virus surface; NP, the most abundant viral protein; and VP40, a major structural protein of Zaire ebolavirus. We also determined neutralizing antibody titers. In the cohort of Ebola survivors, 97.7% of samples (209/214) had measurable antibody levels against GP, NP, and/or VP40. Of these positive samples, all but one had measurable neutralizing antibody titers against Ebola virus. For the close contacts, up to 12.7% (34/267) may have experienced a subclinical virus infection as indicated by detectable antibodies against GP. Further investigation is warranted to determine whether these close contacts truly experienced subclinical infections and whether these asymptomatic infections played a role in the dynamics of transmission., Author summary As the causative agent of an often lethal hemorrhagic fever disease in humans and nonhuman primates, Zaire ebolavirus typically causes high fever, severe diarrhea, and vomiting which results in case fatality rates as high as 90%. The 2013–2016 outbreak in West Africa was the largest and most devastating Ebola outbreak to date resulting in over 28,600 identified human cases and 11,300 deaths. Though our knowledge of virus transmission is incomplete, we do know that transmission occurs through direct contact with virus-contaminated body fluids (blood, secretions, or other body fluids), materials such as bedding contaminated with these fluids, and through the handling and preparation of contaminated food. Asymptomatic Ebola virus infections that result in seroconversion in the absence of disease symptoms have been observed both in humans and experimentally in animal models. In the present serology study, we determined a majority of Ebola survivors in our cohort had measurable antibody levels against at least one viral antigen, as expected. In our cohort of close contacts, relatives and health care workers who took care of Ebola-infected individuals during the outbreak, we observed a rate of seroprevalence of 12.7% as indicated by detectable GP antibody levels. Given that Ebola virus is typically associated with a highly lethal disease in humans, it is of great interest to determine the host-virus interactions and transmission dynamics associated with asymptomatic cases.

Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7–20) and IgM (0–12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis., Author summary B. pseudomallei infection, the causative agent of melioidosis, results in severe disseminated or localized infections. A systemic study of the humoral immune response to B. pseudomallei infection using the B. pseudomallei aerosol caprine model would help understand the detectable antigenic proteins as the infection progresses. To study the immune response, IgG and IgM antibody responses to whole cell lysate proteins were identified and analyzed. Antigenic carbohydrates were also studied. From the results, this study suggests that the caprine humoral immune response to aerosolized B. pseudomallei has similarities to human melioidosis and may facilitate the analysis of the temporal antibody responses. In addition, commonly detected immunogenic proteins may be used as biomarkers for the future point of care (POC) diagnostics.

Background Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. Methodology/principal findings We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, Author summary Dengue is mosquito-borne viral disease which is currently a global public health problem. It is caused by four different types of dengue viruses. Nearly a 100 million people a year suffer from overt sickness, which may range from mild fever to potentially fatal disease. A virus-based dengue vaccine was launched for the first time in late 2015. Unexpectedly, this vaccine mimics the dengue viruses in that it appears to elicit disease-enhancing antibodies. To reduce such risk, safer vaccines that eliminate viral proteins responsible for undesirable antibodies are needed. We focused our attention on a small domain of the dengue virus surface protein known as envelope domain III (EDIII). Humans make only a small amount of antibodies against EDIII, but these antibodies are effective in blocking dengue virus from entering cells. We used a yeast expression system to display EDIIIs of all four types of dengue viruses on the surface of non-infectious virus-like particles (VLPs). These VLPs elicited antibodies, in mice and monkeys, which blocked all four dengue virus types and their variants from entering cells in culture. Importantly, these antibodies did not enhance dengue infection in a mouse model.

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization., Author summary East Coast fever, caused by the tick-borne protozoan parasite Theileria parva, is a disease that results in significant bovine morbidity, mortality, and production losses in regions of sub-Saharan Africa. Susceptible cattle develop clinical signs within a 7–14 days of exposure, which often progress to severe pulmonary edema and death. Control of East Coast fever in affected regions of Africa is largely prohibited by the lack of an affordable and efficacious vaccine. Furthermore, pastoralist farmers in affected regions of Africa often lack resources to prevent losses due to East Coast fever, so these production losses play a significant role in food security and protein availability. Experimental immunization of cattle with a recombinant T. parva-derived antigen, p67, has shown promise in preventing East Coast fever, but this antigen is extremely difficult to produce in full-length in sufficient quantities, and results of immunization studies using truncated recombinant p67 products are highly inconsistent. In this study, p67 antigen production was optimized and produced for use in future immunization studies. Optimization of p67-based immunization strategies is an important step forward in the development of a sustainable, next-generation vaccine against T. parva, which is urgently needed to minimize losses associated with East Coast fever.

Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by Monkeypox virus (MPXV), the most virulent Orthopoxvirus affecting humans since the eradication of Variola virus (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (Funisciurus spp.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [1,2]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via in vivo bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions., Author summary Monkeypox virus (MPXV) is a virus closely related to Variola virus, the cause of smallpox, and it causes a similar, though less severe, disease in humans in Central and West Africa. This disease is particularly dangerous for children and people with compromised immune systems and the mortality rate is estimated to be about 10% in the more pathogenic Central African strains. Unlike Variola virus, MPXV is primarily transmitted to humans from animals, but it is not known which species commonly carry and spread this virus. Rope squirrels in the genus Funisciurus have previously been linked to the virus in Central Africa. In this study, rope squirrels were experimentally infected with MPXV and subsequently monitored for signs of disease, as well as the amount of virus they shed in their bodily fluids. The results of this study showed that rope squirrels became moderately ill but not all died. They shed large amounts of virus in oral, nasal, rectal, and ocular secretions. This information helps public health doctors and epidemiologists understand the potential risks of contacting rope squirrels for local populations in Africa and will help disease ecologists understand how MPXV is maintained and transmitted between animals and to humans.

Bacillus cereus biovar anthracis (Bcbva) is a member of the B. cereus group which carries both B. anthracis virulence plasmids, causes anthrax-like disease in various wildlife species and was described in several sub-Saharan African rainforests. Long-term monitoring of carcasses in Taï National Park, Côte d’Ivoire, revealed continuous wildlife mortality due to Bcbva in a broad range of mammalian species. While non-lethal anthrax infections in wildlife have been described for B. anthracis, nothing is known about the odds of survival following an anthrax infection caused by Bcbva. To address this gap, we present the results of a serological study of anthrax in five wildlife species known to succumb to Bcbva in this ecosystem. Specific antibodies were only detected in two out of 15 wild red colobus monkeys (Procolobus badius) and one out of 10 black-and-white colobus monkeys (Colobus polykomos), but in none of 16 sooty mangabeys (Cercocebus atys), 9 chimpanzees (Pan troglodytes verus) and 9 Maxwell’s duikers (Cephalophus maxwellii). The combination of high mortality and low antibody detection rates indicates high virulence of this disease across these different mammalian species., Author summary Anthrax is a deadly zoonosis, predominantly known to affect wild and domestic herbivores. It has long been assumed that the disease is exclusively caused by B. anthracis, but recently another member of the B. cereus group, Bacillus cereus biovar anthracis (Bcbva), was found to carry both B. anthracis virulence plasmids. Bcbva causes anthrax-like disease in wildlife throughout sub-Saharan Africa and was shown to be an important cause of wildlife mortality in Taï National Park, Cote d’Ivoire, affecting a broad range of mammalian species. While mortality data has routinely been collected in the area for decades, it remains unknown whether non-lethal Bcbva exposure occurs. We therefore conducted a serological study in four primate and one duiker species in which Bcbva-related fatalities were previously documented. Frequent non-lethal exposure should result in a high antibody seroprevalence within wildlife populations, while high lethality would result in low antibody seroprevalence. We found that antibody detection rates were low, suggesting that Bcbva infections in these species are likely often lethal.

Background Leishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies. Methodology/Principal findings Seventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1–28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67–81 and 122–135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%). Conclusions/Significance Our work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases., Author summary Leishmaniasis is a neglected tropical disease being among the six endemic prioritized diseases in the world. Visceral leishmaniasis (VL) is caused by Leishmania infantum and represents a serious public health problem in Brazil. Dogs are the main source of infection in the urban area and, in Brazil, the main strategies of the Visceral Leishmaniasis Control Program are directed to control the canine reservoir (serological survey and euthanasia of dogs which present reactive serum). In general, diagnosis of canine visceral leishmaniasis (CVL) has been presented as a problem for Brazilian public health services. The issue should be attributed mainly to the following factors: 1- range of similar clinical signs observed in other infectious diseases that affect dogs; 2- large percentage of asymptomatic or oligosymptomatic dogs; 3- nonspecific histopathological changes; 4- nonexistence of a diagnostic test 100% specific and sensitive. In this work, we developed a synthetic bi-epitope peptide as an antigen for immunodiagnostic ELISA to detect CVL. The biepitope used for ELISA assay accurately distinguish (98% sensitivity and 99% specificity) CVL dogs sera from non-infected dogs sera.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus’ low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories’ and methods’ performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories., Author summary The European network of National Reference Laboratories (NRLs) for equine diseases guarantees West Nile virus (WNV) surveillance and warning of the emergence of the disease. The WNV NRL network has gathered together most of the European countries facing WNV outbreaks. In this context, two inter-laboratory proficiency tests (ILPTs) were designed in 2010 and 2013 to evaluate the network’ and methods’ performances in detecting WNV infection through serology. A comparison of these two ILPTs emphasised a substantial improvement in the analytical performance of the WNV antibody detection tools over the years within the European NRLs network. Nevertheless the serological cross-reactions among related flaviviruses, such as the Japanese encephalitis, Usutu or tick-borne encephalitis viruses through IgG detection, associated with the Virus Neutralisation Tests’ (VNT) lower sensitivity, long duration and need for Biosafety Level 3 (BSL-3) facilities are major concerns related to indirect WNV diagnosis. All these remarks plead in favour of the development and implementation of new technologies to provide alternatives to classical methods for serological flavivirus diagnosis.

Background Mammarenaviruses are associated with human hemorrhagic fever diseases in Africa and America. Recently, a rodent mammarenavirus, Wēnzhōu virus (WENV) and related viruses, have been reported in China, Cambodia, and Thailand. Moreover, in Cambodia, these viruses were suspected to be associated with human disease. In China, Yunnan Province is famous for its abundant animal and plant diversity and is adjacent to several South-eastern Asia countries. Therefore, it is necessary to know whether WENV-related viruses, or other mammarenaviruses, are prevalent in this province. Methodology/Principal findings Small mammals were trapped, euthanized, and sampled. Mammarenavirus RNA was detected using a nested reverse transcription polymerase chain reaction (RT-PCR) and quantified by real-time RT-PCR. A total of 1040 small mammals belonging to 13 genera and 26 species were trapped in Yunnan Province. WENV-related mammarenaviruses were detected in 41 rodent liver samples, mainly in brown rats (Rattus norvegicus) and oriental house rats (R. tanezumi).Viral nucleocapsid protein was detected in liver sections by indirect immunofluorescence assay. Full-length-genomes were amplified by RT-PCR and used for phylogenetic analysis with the MEGA package. Recombination analysis was performed using the SimPlot and Recombination Detection Program. Conclusions/Significance WENV related viruses circulated in small mammals in Yunnan Province. Whole genome sequence analysis of five selected viral strains showed that these viruses are closely related to WENVs discovered in Asia and form an independent branch in the phylogenetic tree in the WENV clade. Paying attention to investigate the influence of these viruses to public health is essential in the epidemic regions., Author summary Rodents are natural reservoirs of mammarenavirus. Lymphocytic choriomeningitis virus (LCMV), isolated in Asian countries during the 1990s, has a worldwide distribution and was the first mammarenavirus isolated. In 2014, a second mammarenavirus, Wēnzhōu virus (WENV), was identified in rodents in Zhejiang Province of China and later in Guangdong, Shandong, and Hainan Provinces. Most importantly, WENV or related viruses were reported in Thailand and Cambodia. In Cambodia, the isolated virus was associated with human respiratory diseases. In this study, we detected WENV or related viruses in Yunnan Province and found a high prevalence in rats of two species (Rattus norvegicus and R. tanezumi). Phylogenetic analysis of the complete L and S segments of five strains showed that these viruses form an independent phylogenetic branch in WENV clade most closely related to WENVs found in China and Cambodia. Considering the wide spread distribution of rats and altered distribution patterns due to ecological changes, we propose that these viruses may have a wider prevalence and be found in countries from South-eastern Asia to China. Given that WENV may be associated with human diseases, it is necessary to improve surveillances of these viruses in their natural reservoirs and in humans.

Background Animal control measures in Latin America have decreased the incidence of urban human rabies transmitted by dogs and cats; currently most cases of human rabies are transmitted by bats. In 2004–2005, rabies outbreaks in populations living in rural Brazil prompted widespread vaccination of exposed and at-risk populations. More than 3,500 inhabitants of Augusto Correa (Pará State) received either post-exposure (PEP) or pre-exposure (PrEP) prophylaxis. This study evaluated the persistence of rabies virus-neutralizing antibodies (RVNA) annually for 4 years post-vaccination. The aim was to evaluate the impact of rabies PrEP and PEP in a population at risk living in a rural setting to help improve management of vampire bat exposure and provide additional data on the need for booster vaccination against rabies. Methodology/Principal Findings This prospective study was conducted in 2007 through 2009 in a population previously vaccinated in 2005; study participants were followed-up annually. An RVNA titer >0.5 International Units (IU)/mL was chosen as the threshold of seroconversion. Participants with titers ≤0.5 IU/mL or Equivalent Units (EU)/mL at enrollment or at subsequent annual visits received booster doses of purified Vero cell rabies vaccine (PVRV). Adherence of the participants from this Amazonian community to the study protocol was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88.0% of the non-boosted participants evaluated at each yearly follow-up visit remaining seroconverted. Similar RVNA persistence profiles were observed in participants originally given PEP or PrEP in 2005, and the GMT of the study population remained >1 IU/mL 4 years after vaccination. At the end of the study, 51 subjects (11.9% of the interviewed population) had received at least one dose of booster since their vaccination in 2005. Conclusions/Significance This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas., Author Summary Animal control measures have decreased the incidence of human rabies in urban regions of Latin America. Currently, most cases of human rabies occur in rural areas and are transmitted by bats. In 2004–2005, rabies outbreaks affecting populations living in remote areas of Brazil prompted the widespread vaccination of exposed and at-risk populations. We evaluated the persistence of the humoral immune response for 4 years after vaccination in a rural population at daily risk of rabies exposure. Our aim was to evaluate the impact of vaccination in a rural setting to help improve management of vampire bat exposure. The participation of this Amazonian community was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88% of the participants evaluated at each yearly follow-up visit remaining seroconverted. Similar neutralizing antibody persistence levels were observed in participants originally given post-exposure or pre-exposure prophylaxis in 2005. This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas.

Background Neurotropic arboviral infections are an important cause of encephalitis. A zoonotic, vector-borne alphavirus, Madariaga virus (MADV; formerly known as South American eastern equine encephalitis virus), caused its first documented human outbreak in 2010 in Darien, Panama, where the genetically similar Venezuelan equine encephalitis virus (VEEV) is endemic. We report the results of a seroprevalence survey of animals and humans, illustrating contrasting features of MADV and VEEV ecology and epidemiology. Methods Small mammals were trapped in 42 sites in Darien, Panama, using Sherman traps, Tomahawk traps, and mist nets for bats. Blood was tested for the presence of neutralizing antibodies to MADV and VEEV. In addition, bird sera collected in 2007 in Chagres, Panama, were tested for MADV and VEEV neutralizing antibodies. Viremia was ascertained by RT-PCR. Human exposure to these two viruses was determined by IgG ELISA, followed by plaque reduction neutralization tests. To identify relevant risk factors for MADV or VEEV exposure, logistic regression analysis was performed, and the most parsimonious model was selected based on the Akaike information criterion. Results The animal survey yielded 32 bats (16 species), 556 rodents (12 species), and 20 opossums (4 species). The short-tailed cane mouse (Zygodontomys brevicauda) found abundantly in pasture and farms, had the highest MADV seroprevalence (8.3%). For VEEV, the shrub and forest-dwelling long-whiskered rice rat (Transandinomys bolivaris) had the highest seroprevalence (19.0%). Viremia was detected in one animal (Z. brevicauda). Of the 159 bird sera (50 species) tested, none were positive for either virus. In humans (n = 770), neutralizing antibodies to MADV and VEEV were present in 4.8% and 31.5%, respectively. MADV seropositivity was positively associated with cattle ranching, farming, and fishing. Having VEEV antibodies and shrubs near the house diminished risk. Age, forest work, farming and fishing were risk factors for VEEV, while having MADV antibodies, glazed windows, waste pick-up and piped water were protective. Conclusion Our findings suggest that the short-tailed cane mouse and the long-whiskered rice rat serve as hosts for MADV and VEEV, respectively. The preferred habitat of these rodent species coincides with areas associated with human infection risk. Our findings also indicate that MADV emerged recently in humans, and that the transmission cycles of these two sympatric alphaviruses differ spatially and in host utilization., Author Summary Arthropod-borne viruses are important causes of encephalitis. In 2010, the first documented human outbreak of the mosquito-borne, zoonotic Madariaga virus (MADV) occurred in the Darien region of Panama. Neither its epidemiology nor its transmission cycle is understood. In this study, the authors searched for possible animal hosts of this virus, and sought to describe its epidemiology. They contrast the findings with those for Venezuelan equine encephalitis virus (VEEV), an endemic, genetically similar virus. Zygodontomys brevicauda, the short-tailed cane mouse, had the highest seroprevalence for MADV. This rodent species is most often found in pasture and farm land. Indeed, the risk factors for human MADV exposure were cattle ranching and farming. The animal with highest seroprevalence for VEEV, the long-whiskered rice rat (Transandinomys bolivaris), commonly occurs in forest, and the epidemiological risk factors included working in the forest. Farming and fishing were risk factors for exposure to both viruses, and having antibodies to one virus diminished the risk of being positive for the other. Increasing prevalence with age was seen for VEEV, confirming that VEEV is endemic in the region. This association was absent for MADV, suggesting that this virus emerged recently to infect humans.