Leptospira borgpetersenii Leucine-Rich Repeat Proteins and Derived Peptides in an Indirect ELISA Development for the Diagnosis of Canine Leptospiral Infections.

Domestic and stray dogs can be frequently infected by Leptospira, and thus may represent a source for transmission of this zoonotic disease in Thailand. Here, we have used peptides derived from a recombinant leucine-rich repeat (LRR) protein of Leptospira, rKU_Sej_LRR_2012M, for the development of an indirect enzyme-linked immunosorbent assay (ELISA) aimed at detecting antibodies against Leptospira interrogans, L. borgpetersenii, and L. biflexa, the three major seroprevalences in Thai dogs. The rKU_Sej_LRR_2012M protein is recognized by hyperimmune sera against several leptospiral serovars. The epitope peptides of the rKU_Sej_LRR_2012M showed binding affinities with lower IC50 values than peptides of known antigenic protein LipL32. Four peptides, 2012-3T, 2012-4B, 2012-5B and pool 2012-B, were specifically recognized by rabbit hyperimmune sera against nine serovars from three Leptospira spp. The indirect peptide-based ELISAs with these four peptides were evaluated with the LipL32 ELISA by using a receiver-operator curve (ROC) analysis. All peptides had an area under the curve of ROC (AUC) greater than 0.8, and the sum of sensitivity and specificity for each peptide was greater than 1.5. The degree of agreement of 2012-3T and pool 2012-B and 2012-4B and 2012-5B peptides were in moderate-to-good levels with kappa values of 0.41-0.60 and 0.61-0.80, when compared with LipL32, respectively. This finding would suggest an excellent capability of the 2012-4B and 2012-5B peptide-based ELISAs assay for the diagnosis of canine leptospiral infections.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

Diagnostic imaging features, cytological examination, and treatment of lymphocytic tenosynovitis of the common digital extensor tendon sheath in an eventing horse.

In horses, the structures at the dorsal aspect of the carpus, including the digital extensor tendons, their related tendon sheaths, and bones, are vulnerable to injury because of their superficial location. Injuries to these structures may result in lameness of the affected limb(s) and reduce a horse's athletic performance. A 13-year-old eventing horse that routinely underwent regular exercise exhibited dorsolateral distension of the right carpus. An effusion insensitive to compression was observed in the affected area. No lameness was detected, and the horse exhibited a negative response to the carpal flexion test. Although radiography revealed no abnormal findings in the carpal bones, ultrasonography depicted anechoic fluid and synovial cell proliferation within the common digital extensor tendon sheath. Cytological analysis of the fluid revealed numerous lymphocytes and increased proteinaceous background, suggesting lymphocytic tenosynovitis. The effusion resolved following administration of two intrathecal injections: one injection of corticosteroid combined with hyaluronic acid (HA), and one injection of HA alone. Two weeks after administration of the second injection, daily under-saddle exercise was initiated, consisting of walking and light trotting with a gradual increase in intensity. The horse returned to its habitual intensive training program six weeks following the final injection. In conclusion, the horse was diagnosed with lymphocytic tenosynovitis of the common digital extensor tendon; successful treatment was achieved with administration of corticosteroid and HA. Diagnostic imaging and cytological examination facilitated clinical interpretation and the selection of an appropriate treatment regimen.

Evidence of potential vertical transmission of tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2-day-old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV-free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication.

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques.

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a "mosaic" hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.

Mosaic H5 Hemagglutinin Provides Broad Humoral and Cellular Immune Responses against Influenza Viruses.

UNLABELLED: The most effective way to prevent influenza virus infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza virus (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immunodepletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). Compared to a MVA construct expressing hemagglutinin (HA) from influenza virus A/VN/1203/04 (MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge, whereas MVA-HA-vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8(+) T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine. IMPORTANCE: Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000 to 500,000 deaths annually. Vaccines are still the main intervention to prevent infection, but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza virus A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

UNLABELLED: Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE: Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses.

BACKGROUND: Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses. METHODOLOGY/PRINCIPAL FINDINGS: Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized. CONCLUSIONS/SIGNIFICANCE: Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.

Mucosal administration of raccoonpox virus expressing highly pathogenic avian H5N1 influenza neuraminidase is highly protective against H5N1 and seasonal influenza virus challenge.

We previously generated recombinant poxviruses expressing influenza antigens and studied their efficacy as potential highly pathogenic avian influenza (HPAI) vaccines in mice. While both modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) expressing hemagglutinin (HA) provided strong protection when administered by parenteral routes, only RCN-neuraminidase (NA) showed promise as a mucosal vaccine. In the present study we evaluated the efficacy of RCN-NA constructs by both intradermal (ID) and intranasal (IN) routes. Surprisingly, while RCN-NA completely protected mice when administered by the IN route, it failed to protect mice when administered by the ID route. After challenge, significantly less virus induced pathology was observed in the lungs of mice vaccinated with RCN-NA by the IN route as compared to the ID route. Furthermore, IN administration of RCN-NA elicited neutralizing antibodies detected in bronchoalveolar lavage (BAL) samples. We also determined the role of cellular immune responses in protection elicited by RCN-NA by depleting CD4 and CD8 T cells prior to challenge. Finally, we demonstrated for the first time that antibodies against NA can block viral entry in addition to viral spread in vitro. These studies demonstrate the importance of mucosal administration of RCN viral vectors for eliciting protective immune responses against the NA antigen.

Broad protection against avian influenza virus by using a modified vaccinia Ankara virus expressing a mosaic hemagglutinin gene.

UNLABELLED: A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4(+) and CD8(+) T cells were still detected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity. IMPORTANCE: Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader immunity than a wild-type hemagglutinin.

Display of organophosphorus hydrolase on the cyanobacterial cell surface using synechococcus outer membrane protein a as an anchoring motif.

The display of proteins to cyanobacterial cell surface is made complex by combination of Gram-positive and Gram-negative features of cyanobacterial cell wall. Here, we showed that Synechococcus outer membrane protein A (SomA) can be used as an anchoring motif for the display of organophosphorus hydrolase (OPH) on cyanobacterial cell surface. The OPH, capable of degrading a wide range of organophosphate pesticides, was fused in frame to the carboxyl-terminus of different cell-surface exposed loops of SomA. Proteinase K accessibility assay and immunostaining visualized under confocal laser scanning microscopy demonstrated that a minor fraction of OPH with 12 histidines fused in frame with the third cell-surface exposed loop of SomA (SomAL3-OPH12H) was displayed onto the outermost cell surface with a substantial fraction buried in the cell wall, whereas OPH fused in frame with the fifth cell-surface exposed loop of SomA (SomAL5-OPH) was successfully translocated across the membrane and completely displayed onto the outermost surface of Synechococcus. The successful display of the functional heterologous protein on cell surface provides a useful model for variety of applications in cyanobacteria including screening of polypeptide libraries and whole-cell biocatalysts by immobilizing enzymes.