Maguari Virus Research Articles

Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

Complete nucleotide sequences of the large RNA genome segments of Main Drain and Northway viruses (family Peribunyaviridae).

The large RNA genome segments of Main Drain virus (MDV) and Northway virus (NORV) were fully sequenced and shown to consist of 6860 and 6875 nucleotides, respectively. Sequence alignments revealed that the large RNA segment of MDV is most closely related to the corresponding region of NORV, with 76.8% nucleotide sequence identity, and the large RNA segment of NORV is most closely related to the corresponding region of Maguari virus, with 79.1% identity.

Neutralizing antibodies for orthobunyaviruses in Pantanal, Brazil.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

Maguari Virus Associated with Human Disease.

Despite the lack of evidence for symptomatic human infection with Maguari virus (MAGV), its close relation to Cache Valley virus (CVV), which does infect humans, remains a concern. We sequenced the complete genome of a MAGV-like isolate (OBS6657) obtained from a febrile patient in Pucallpa, Ucayali, Peru, in 1998. To facilitate its classification, we generated additional full-length sequences for the MAGV prototype strain, 3 additional MAGV-like isolates, and the closely related CVV (7 strains), Tlacotalpan (1 strain), Playas (3 strains), and Fort Sherman (1 strain) viruses. The OBS6657 isolate is similar to the MAGV prototype, whereas 2 of the other MAGV-like isolates are located on a distinct branch and most likely warrant classification as a separate virus species and 1 is, in fact, a misclassified CVV strain. Our findings provide clear evidence that MAGV can cause human disease.

PREVALÊNCIA DE ANTICORPOS CONTRA ARBOVÍRUS DA FAMÍLIA <italic>Bunyaviridae</italic> EM BÚFALOS DE ÁGUA

<title>Resumo</title><p>O Estado do Pará corresponde a 26% da Amazônica brasileira, onde uma grande quantidade de Arbovírus tem sido descrito. O presente trabalho teve como objetivo determinar a prevalência de anticorpos detectados pela técnica de inibição de hemaglutinação contra nove tipos diferentes de arbovírus da família <italic>Bunyaviridae,</italic> sendo oito do gênero <italic>Orthobunyavirus: vírus Guaroa, vírus Maguari, vírus Tacaiuma, vírus Utinga, vírus Belem, vírus Caraparu, vírus Oropouche</italic> e <italic>vírus Catu</italic>e um do gênero <italic>Phlebovirus: vírus Icoaraci,</italic> em soros de búfalos de água no Estado do Pará, Brasil. Para todos os Arbovírus investigados houve presença de anticorpos, com exceção do <italic>vírus Belém.</italic>Anticorpos para o <italic>vírus Maguari</italic> foram mais prevalentes (7,33%). O rebanho bubalino do presente estudo mostrou variáveis níveis de anticorpos em reações heterotípicas e monotípicas podendo indicar que há circulação da maioria dos bunyavírus estudados em búfalos domésticos no estado do Pará, e que o vírus Maguari é o de maior circulação. Por isso, são necessários outros estudos para investigar o papel dos búfalos de água na manutenção e dispersão de arbovírus, assim como se esses vírus podem causar enfermidades na referida espécie, principalmente, em casos de defeitos congênitos e abortamentos.</p>

Orthobunyaviruses, a Common Cause of Infection of Livestock in the Yucatan Peninsula of Mexico

To determine the seroprevalence of selected orthobunyaviruses in livestock in the Yucatan Peninsula of Mexico, a serologic investigation was performed using serum samples from 256 domestic animals (182 horses, 31 sheep, 1 dog, 37 chickens, and 5 turkeys). All serum samples were examined by plaque reduction neutralization test using Cache Valley virus (CVV), Cholul virus (CHLV), South River virus (SOURV), Kairi virus, Maguari virus, and Wyeomyia virus. Of the 182 horses, 60 (33.0%) were seropositive for CHLV, 48 (26.4%) were seropositive for CVV, 1 (0.5%) was seropositive for SOURV, 60 (33.0%) had antibodies to an undetermined orthobunyavirus, and 13 (7.1%) were negative for orthobunyavirus-specific antibody. Of the 31 sheep, 6 (19.3%) were seropositive for CHLV, 3 (9.7%) were seropositive for CVV, 4 (12.9%) were seropositive for SOURV, 16 (51.6%) had antibodies to an undetermined orthobunyavirus, and 2 (6.5%) were negative for orthobunyavirus-specific antibody. The single dog was seropositive for SOURV. Four (11%) chickens had antibodies to an undetermined orthobunyavirus, and 1 (20%) turkey was seropositive for CHLV. These data indicate that orthobunyaviruses commonly infect livestock in the Yucatan Peninsula.

Characterization of Maguari orthobunyavirus mutants suggests the nonstructural protein NSm is not essential for growth in tissue culture

Maguari virus (MAGV; genus Orthobunyavirus, family Bunyaviridae) contains a tripartite negative-sense RNA genome. Like all orthobunyaviruses, the medium (M) genome segment encodes a precursor polyprotein (NH 2-Gn-NSm-Gc-COOH) for the two virion glycoproteins Gn and Gc and a nonstructural protein NSm. The nucleotide sequences of the M segment of wild-type (wt) MAGV, of a temperature-sensitive (ts) mutant, and of two non-ts revertants, R1 and R2, that show electrophoretic mobility differences in their Gc proteins were determined. Twelve amino acid differences (2 in Gn, 10 in Gc) were observed between wt and ts MAGV, of which 9 were maintained in R1 and R2. The M RNA segments of R1 and R2 contained internal deletions, resulting in the removal of the N-terminal 239 residues of Gc (R1) or the C-terminal two thirds of NSm and the N-terminal 431 amino acids of Gc (R2). The sequence data were consistent with analyses of the virion RNAs and virion glycoproteins. These results suggest that neither the N-terminal domain of Gc nor an intact NSm protein is required for the replication of MAGV in tissue culture.

Identification of Simbu, California and Bunyamwera serogroup bunyaviruses by nested RT-PCR

We describe a reverse transcription-polymerase chain reaction (RT-PCR) with primers that anneal to the 5′ and 3′ ends and amplify the Bunyavirus S RNA segments. The RT-PCR was done on the fluids of C6/36 cells infected with each of 21 bunyaviruses. The bunyaviruses studied, with the exception of Catu virus, produced amplicons having 700 to 1300 base pairs and probably contained the whole S RNA segment sequence. A nested PCR performed with these amplicons distinguished California and most Bunyamwera serogroup viruses from other bunyaviruses by use of BBC specific internal primers for the S RNA segment, and distinguished Simbu serogroup viruses from others by use of BS specific internal primers. The nested-PCR amplicons of Guaroa, Maguari, California encephalitis, Bunyamwera, and Oropouche viruses were sequenced. The sequences were aligned with previously known sequences of the S RNA segment of the same viruses, showing a high degree of homology and thus confirming the specific origin of these amplicons. The nested RT-PCR is suitable as a specific screening for most California and Bunyamwera serogroup and Simbu serogroup viruses depending on the use of BBC or BS internal primers. Oropouche virus is an important public health problem in Brazil and the nested PCR with BS primers could be used for the detection of this virus in tissue culture and mouse brain isolates as well as in clinical samples.

Circulation of eastern equine encephalitis, western equine encephalitis, Ilhéus, Maguari and Tacaiuma viruses in equines of the Brazilian Pantanal, South America.

Neutralizing antibodies to EEE (6.7%), WEE (1.2%), ILH (26.6%), MAG (28.2%) and TCM (15.7%) viruses were found in sera of 432 equines of the Brazilian Pantanal, area where undiagnosed horse deaths are frequently observed. A 4-fold rise in CF titer to EEE virus was detected in acute and convalescent sera of an encephalitis horse sacrificed in 1992. Antibodies to EEE, ILH, MAG and TCM viruses were detected in horses less than 2 years old indicating recent circulation of these viruses in the Pantanal. The evidence of recent equine encephalitis associated with rising CF titer to EEE warrants a more intensive study with attempts to isolate virus from horses with clinical manifestations of encephalitis.

Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae

The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3′ and 5′ ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51–53 to a UAG stop codon at bases 6765–6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5′ end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.

Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus

The small (S) RNA segment of the Maguari bunyavirus genome has been cloned as cDNA and its nucleotide sequence determined. The nucleocapsid protein, N, ( M r26K) and a nonstructural protein, NSs, ( M r 11K), are encoded in overlapping reading frames, similar to other bunyavirus S RNA segments. In addition, a third AUG-initiated open reading frame encoding a 9.3K protein was observed. All three polypeptides were translated in cell free systems programmed with RNA transcribed in vitro from the cDNA subcloned downstream of a bacteriophage T7 promoter. The effects on expression of subcloning parts of the cDNA and by site-specific mutagenesis are discussed in relation to the scanning model of initiation of translation. A recombinant baculovirus has been constructed to express the Maguari virus S segment gene products. The N protein was efficiently expressed in infected cells, and a significant amount was in a soluble form. We could not detect the synthesis of NSs nor the 9.3K protein, and the reasons for this are discussed. The 9.3K protein has not been found in Maguari virus-infected cells and so the question of its functional significance remains open.

Cross-neutralization tests among Cache Valley virus isolates revealing the existence of multiple subtypes.

Maguari virus has been classified as a subtype of Cache Valley virus. Seven Bunyamwera serogroup viruses (including prototypes Cache Valley and Maguari viruses), 4 viruses shown in previous tests as close antigenic relatives of Maguari or Cache Valley viruses, and Xingu virus were cross-tested by serum dilution-plaque reduction neutralization. All viruses were distinguishable from prototypes Cache Valley and Maguari viruses. The close antigenic relationships of the Cache Valley-like viruses demonstrate that multiple subtypes of Cache Valley virus exist and suggest that such antigenic variation is a phenotypic expression of considerable genetic diversity.

Human infections with Tensaw virus in south Florida: evidence that Tensaw virus subtypes stimulate the production of antibodies reactive with closely related Bunyamwera serogroup viruses.

Maguari virus, a member of the Bunyamwera serogroup (family Bunyaviridae, genus Bunyavirus) has not been isolated north of Trinidad. Anecdotal information from other investigators has indicated the presence of antibody to Maguari virus in human residents of south Florida. We attributed such antibody to either cross-reactivity with Tensaw virus, the only Bunyamwera serogroup virus known in south Florida, or to cross-reactivity to an antigenic subtype or variant of Tensaw virus. Five strains, identified as Tensaw virus when they were isolated from mosquitoes collected in south Florida more than 20 years ago, were retrieved from storage. They were compared by serum dilution-plaque reduction neutralization tests with Bunyamwera serogroup prototypes Tensaw, Maguari, Cache Valley, and Tlacotalpan viruses. The south Florida isolates were shown to be most closely related to prototype Tensaw virus and most distantly related to prototype Maguari virus. One isolate could not be distinguished from prototype Tensaw virus, and the other 4 appeared to be subtypes of prototype Tensaw virus. More than 300 serum samples from humans in south Florida were tested for neutralizing antibody to prototypes Tensaw and Maguari viruses and to 3 of the field isolates. Thirteen had antibody to prototype Tensaw virus only, 19 to prototype Maguari virus only, and 39 to both. Antibody to all but 6 of these 71 was attributed to infection with Tensaw virus, to a subtype of Tensaw virus, or to travel or birth outside the United States. It is likely that those with antibody to Maguari virus only had been infected with yet another subtype of Tensaw virus, although another, undiscovered, Bunyamwera serogroup virus may exist in south Florida.

A newly recognized vesiculovirus, Calchaqui virus, and subtypes of Melao and Maguari viruses from Argentina, with serologic evidence for infections of humans and horses.

In 1983, 17 virus strains were isolated from mosquitoes collected during an outbreak of western equine encephalitis in Santa Fe Province, Argentina. Strains of western equine encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, and Antequera viruses were isolated, as were several bunyaviruses of the California and Bunyamwera serogroups and a new vesiculovirus. Complement fixation and neutralization tests were used to identify the California serogroup virus as a subtype of Melao virus, the Bunyamwera serogroup virus as a subtype of both Maguari and Playas viruses, and the vesiculovirus as a newly recognized agent for which the name Calchaqui virus is proposed. A limited serosurvey of horses and humans in Santa Fe Province and horses from the adjacent Santiago del Estero Province was performed to determine the prevalence of neutralizing antibody to the subtypes of Melao and Maguari viruses and to Calchaqui virus. The high prevalence of antibodies to these three agents indicates the need for further studies of their disease potential in horses, because they are closely related to several other viruses that are known equine pathogens.

Arbovirus Investigations in Argentina, 1977–1980

Serologic surveys of wild and domestic birds, wild mammals, and horses were conducted during arbovirus field studies in Argentina from 1977 through 1980, a non-epizootic interval. The prevalence of neutralizing antibodies to eastern equine encephalitis (EEE) was consistently higher than to western equine encephalitis (WEE) virus in all species and all areas. The presence of antibodies in short-lived avian species and in young unvaccinated horses and the demonstration of seroconversions in horses during the period, indicated that these viruses are either enzootic in, or annually reintroduced into, Argentina. Antibodies to AG80-646, a new subtype of WEE virus isolated in the subtropical north (Chaco Province) from Culex (Melanoconion) mosquitoes, were found in horses and rodents in that region. Antibodies to the TC-83 strain of Venezuelan equine encephalitis (VEE) virus were found in all areas studied. The presence of antibodies in some horses was probably related to vaccination, but the demonstration of seroconversions in sentinel horses and of antibodies in birds and wild mammals indicates active transmission of VEE virus. In 1980 a new enzootic subtype of VEE virus (AG80-663) was isolated from mosquitoes in Chaco; neutralizing antibodies to this virus were prevalent in horses and rodents in this area. Infections with Aura and Una viruses were most common in the subtropical northern provinces. Infection with St. Louis encephalitis was prevalent and widespread, and birds, principally passerine and columbiform species, appear to be the principal hosts. An interesting and unexplained finding was the absence of arbovirus antibodies, in particular SLE antibodies in house sparrows (Passer domesticus). Antibody prevalences in horses exceeded 50% in all areas, and 12% of horses surveyed in Santa Fe Province developed antibody in a 17-month period. Antibodies to other flaviviruses were rare. A high prevalence of immunity to Maguari virus was found in horses; this agent is considered to be a potential equine pathogen. Antibodies to 2 new viruses, Barranqueras and Resistencia, which had been isolated from Cx. (Melanoconion) in Chaco Province, were found in rodents there. Immunity to Gamboa group viruses was prevalent, and birds were implicated as principal hosts.

Identification of nonstructural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae).

The proteins synthesized in BHK cells infected with nine members of the Bunyamwera serogroup (family Bunyaviridae, Bunyavirus genus) were analyzed by polyacrylamide gel electrophoresis. In addition to the virus structural proteins, a number of virus-coded nonstructural proteins were detected. One protein, designated NS1, was shown to be related to the nucleocapsid protein by one-dimensional peptide mapping. A second protein, NS2, was mapped to the M RNA segment by gel electrophoretic analysis of the proteins synthesized in cells infected with reassortants of Batai, Bunyamwera, and Maguari viruses of known genotype. A third protein, NS3, was mapped to the S RNA segment by its pattern of labeling with [35S]cysteine in cells infected with reassortant viruses: the NS3 protein was only labeled when the S RNA segment of Bunyamwera virus was present. The mapping of NS3 was confirmed by in vitro translation of mRNAs which hybridized to recombinant plasmids containing S gene-specific sequences.

Genome subunit reassortment among bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes

A simple and rapid procedure for determining the genotypes of viruses has been applied to analysis of genome subunit reassortment in heterologous crosses of Batai virus, Bunyamwera virus, and Maguari virus, three members of the Bunyamwera serogroup of bunyaviruses. The procedure for determining genotype made use of specific molecular probes to identify the parental origin of the L and M RNA subunits. Complementary DNA copies of the three RNA segments of Bunyamwera virus were prepared by reverse transcription using synthetic oligonucleotide primers for first and second strand synthesis. The cDNA transcripts were inserted into a pBR322 vector and gene-specific probes prepared from nick-translated plasmid DNA. L and M gene-specific probes were identified which could unequivocally discriminate Bunyamwera virus genome subunits in a dot-hybridization test using cytoplasmic RNA extracts immobilised on nitrocellulose filters. None of the S gene-specific probes were sufficiently discriminatory for use in this test. Instead the parental origin of the S RNA subunit was inferred from the electrophoretic mobility of the virion N protein. It was observed that reassortment did not occur at random in heterologous crosses of ts mutants of the three viruses, and only the M RNA subunit appeared to segregate freely. However, unrestricted reassortment was observed when recombinant viruses with nonhomologous subunit combinations were used as the parental viruses. It was concluded, therefore, that restriction was mediated at the gene product level and that nonrandom reassortment was not due to incompatibility of genome subunits.

Relationship between Glycoproteins of the Viral Envelope of Bunyaviruses and Antibody-dependent Plaque Enhancement

Hamster antisera against three parental bunyaviruses, Batai, Bunyamwera and Maguari viruses, and six recombinant viruses which carried the nucleocapsid protein of one parent and the glycoproteins of the other, have been tested for their interaction with each of the nine viruses under study by two assays, plaque reduction neutralization and antibody-dependent plaque enhancement. Neutralization was clearly related to the specificity of the parental glycoproteins rather than the nucleoprotein, but the antibody-dependent plaque enhancement assay showed greater cross-reactivity.

Mutant identifying a third recombination group in a bunyavirus.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.

Genetic interactions among viruses of the Bunyamwera complex.

Seventy-seven temperature-sensitive (ts) mutants belonging to three antigenically distinct and geographically isolated members of the Bunyamwera complex--Batai virus, Bunyamwera virus, and Maguari virus--have been isolated after 5-fluorouracil treatment. High-frequency recombination was observed, and the mutants of each virus were classified into two groups, which were shown to be equivalent by heterologous recombination experiments. In most combinations heterologous recombination was less efficient than homologous recombination, but all crosses of group I and II mutants yielded viable recombinants. Recombination was an early event. Analysis by polyacrylamide gel electrophoresis of the proteins of the wild-type viruses and recombinant clones obtained from the six possible heterologous combinations of group I and II mutants indicated that recombination occurred by reassortment of genome subunits. Group I appeared to correspond to the genome subunit coding for the N protein, and group II corresponded to the G1/G2 determinant. The G1 (or G2 or both) protein was associated with neutralization specificity and plaque diameter, and the N protein was associated with plaque opacity. Complementation was observed between two nonrecombining mutants of Maguari virus belonging to group I, which may indicate that the N genome subunit codes for an additional protein. There appeared to be no genetic barrier to exchange of genetic material between Batai, Bunyamwera, and Maguari viruses in vitro, and it is concluded that the Bunyamwera complex is potentially a single gene pool if geographical and ecological constraints are discounted.