First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

A Duplex Quantitative Real-Time Reverse Transcription-PCR for Simultaneous Detection and Differentiation of Flaviviruses of the Japanese Encephalitis and Ntaya Serocomplexes in Birds.

High impact, mosquito-borne flaviviruses such as West Nile virus (WNV), Usutu virus (USUV), Japanese encephalitis virus (JEV), Tembusu virus (TMUV), and Bagaza/Israel turkey meningoencephalomyelitis virus (BAGV/ITV) are emerging in different areas of the world. These viruses belong to the Japanese encephalitis (JE) serocomplex (JEV, WNV, and USUV) and the Ntaya serocomplex (TMUV and BAGV/ITV). Notably, they share transmission route (mosquito bite) and reservoir host type (wild birds), and some of them co-circulate in the same areas, infecting overlapping mosquito and avian population. This may simplify epidemiological surveillance, since it allows the detection of different infections targeting the same population, but also represents a challenge, as the diagnostic tools applied need to detect the whole range of flaviviruses surveyed, and correctly differentiate between these closely related pathogens. To this aim, a duplex real-time RT-PCR (dRRT-PCR) method has been developed for the simultaneous and differential detection of JE and Ntaya flavivirus serocomplexes. The method has been standardized and evaluated by analyzing a panel of 49 flaviviral and non-flaviviral isolates, and clinical samples of different bird species obtained from experimental infections or from the field, proving its value for virus detection in apparently healthy or suspicious animals. This new dRRT-PCR technique is a reliable, specific and highly sensitive tool for rapid detection and differentiation of JE and Ntaya flavivirus groups in either domestic or wild animals. This novel method can be implemented in animal virology diagnostic laboratories as screening tool in routine surveillance and in the event of bird encephalitis emergence.

Seeking Flavivirus Cross-Protective Immunity.

The Flavivirus genus is composed by viral serocomplexes with relevant global epidemiological impact. Many areas of the world present both, vector fauna and geographical conditions compatible with co-circulation, importing, emergence, and epidemics of flaviviruses of different serocomplexes. In this study, we aimed to identify both, immunological determinants and patterns of immune response possibly involved in flavivirus serocomplex cross-protection. We searched B and T cells epitopes which were thoroughly shown to be involved in flavivirus immunological control. Such epitopes were analyzed regarding their conservation, population coverage, and location along flavivirus polyprotein. We found that epitopes capable of eliciting flavivirus cross-protective immunity to a wide range of human populations are concentrated in proteins E, NS3, and NS5. Such identification of both, immunological determinants and patterns of immune response involved in flavivirus cross-protective immunity should be considered in future vaccine development. Moreover, cross-reactive epitopes presented in this work may be involved in dynamics of diseases caused by flaviviruses worldwide.

The distribution of important sero-complexes of flaviviruses in Malaysia.

Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented.

Epizootiology of Tacaribe serocomplex viruses (Arenaviridae) associated with neotomine rodents (Cricetidae, Neotominae) in southern California.

The objective of this study was to advance our knowledge of the epizootiology of Bear Canyon virus and other Tacaribe serocomplex viruses (Arenaviridae) associated with wild rodents in California. Antibody (immunoglobulin G [IgG]) to a Tacaribe serocomplex virus was found in 145 (3.6%) of 3977 neotomine rodents (Cricetidae: Neotominae) captured in six counties in southern California. The majority (122 or 84.1%) of the 145 antibody-positive rodents were big-eared woodrats (Neotoma macrotis) or California mice (Peromyscus californicus). The 23 other antibody-positive rodents included a white-throated woodrat (N. albigula), desert woodrat (N. lepida), Bryant's woodrats (N. bryanti), brush mice (P. boylii), cactus mice (P. eremicus), and deer mice (P. maniculatus). Analyses of viral nucleocapsid protein gene sequence data indicated that Bear Canyon virus is associated with N. macrotis and/or P. californicus in Santa Barbara County, Los Angeles County, Orange County, and western Riverside County. Together, analyses of field data and antibody prevalence data indicated that N. macrotis is the principal host of Bear Canyon virus. Last, the analyses of viral nucleocapsid protein gene sequence data suggested that the Tacaribe serocomplex virus associated with N. albigula and N. lepida in eastern Riverside County represents a novel species (tentatively named "Palo Verde virus") in the genus Arenavirus.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²7ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Diversity among Tacaribe serocomplex viruses (Family Arenaviridae) associated with the southern plains woodrat (Neotoma micropus).

The southern plains woodrat (Neotoma micropus) is the principal host of Catarina virus in southern Texas and a natural host of other North American Tacaribe serocomplex viruses. The objectives of this study were to increase our knowledge of the genetic diversity among Tacaribe serocomplex viruses associated with N. micropus and to define better the natural host relationships of these viruses. Pairwise comparisons of complete glycoprotein precursor gene sequences and complete nucleocapsid protein gene sequences revealed a high level of genetic diversity among Tacaribe serocomplex viruses associated with N. micropus in western Oklahoma, southern New Mexico, and northern and southern Texas. Collectively, the results of Bayesian analyses of nucleotide sequences and pairwise comparisons of amino acid sequences confirmed that the arenaviruses associated with N. micropus in Oklahoma and New Mexico should be included in the Whitewater Arroyo species complex, and indicated that that the arenaviruses associated with N. micropus in northern Texas are strains of a novel arenaviral species--tentatively named "Middle Pease River virus". Together, the results of assays for arenavirus and assays for anti-arenavirus antibody in 54 southern plains woodrats and 325 other rodents captured at 2 localities suggested that the southern plains woodrat is the principal host of Middle Pease River virus in northern Texas.