The resurgence of Shamonda virus, an African Simbu group virus of the genus Orthobunyavirus, in Japan

Summary. Six virus isolations were made from Culicoides biting midges and blood samples of sentinel cattle in Kagoshima and Miyazaki Prefectures, the southern part of Japan, in 2002. Serological and genetical tests identified these viruses as isolates of Shamonda virus (SHAV), which belongs to the Simbu group of the genus Orthobunyavirus of the family Bunyaviridae. Initially, SHAV was isolated from cattle and Culicoides biting midges in Nigeria in the 1960s, and its presence has not been reported until this study. The present results indicate a wider distribution of SHAV than previously assumed.     

Characterization of Mycobacterium tuberculosis isolated from cancer patients with suspected tuberculosis infection in Egypt: identification,prevalence, risk factors and resistance pattern

Data are sparse on Mycobacterium tuberculosis infection among patients with cancer in Egypt. We sought to detect the presence of tuberculosis (TB) disease among patients with malignant conditions and suspected TB and to study the main risk factors. Also, we compared different diagnostic procedures and detected the antimicrobial susceptibility of M. tuberculosis isolates against rifampin and isoniazid. One hundred patients were included in this study, all of them had malignant conditions and were suspected by the clinicians of having TB. Identification of M. tuberculosis in different specimens was performed by smear microscopy, followed by Lowenstein–Jensen medium and Mycobacterium growth indicator tube (MGIT) cultures and artus^® real-time PCR. In addition, an indirect MGIT anti-TB susceptibility test was carried out against rifampin and isoniazid. A total of 76% of studied cases were found to be TB positive. The frequencies of TB-positive cases in the bronchogenic, haematological and solid tumour malignancy groups were 21%, 25% and 30%, respectively. Significant differences between pulmonary and extrapulmonary TB in different malignancy groups were recorded. Real-time PCR showed the highest overall diagnostic efficiency. Multidrug-resistance of M. tuberculosis to both rifampin and isoniazid was detected in 28.6% of examined isolates. Infection in cancer patients with TB was significantly more often recorded among elderly patients and those suffering from poverty. Pulmonary TB is more common than extrapulmonary TB in patients with malignancy. Real-time PCR is the most accurate and rapid method for TB diagnosis. MGIT-rifampin resistance may be used as a reliable marker for detection of multidrug-resistant TB. Diagnosis and instituting treatment course for active or latent TB infection are crucial before starting anticancer therapy.     

Identification of California serogroup viruses (Bunyaviridae, Bunyavirus) using monoclonal antibodies to Inkoo virus

Five types of monoclonal antibodies to inkoo virus were used to detect antigenic relationships between 12 Inkoo-like strains and the prototype Inkoo virus strain. Eight strains were found antigenically very close or identical to Inkoo virus, and the rest 4 are probably original variants (viruses) of California serogroup.     

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: comparison with the middle RNA of Bunyamwera and California serogroup viruses

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50-52%) and amino acid (30-32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743-748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.     

Antigenic and genetic comparisons of Japanese and Australian Simbu serogroup viruses: evidence for the recovery of natural virus reassortants

The antigenicity and RNA genome structures of five Simbu serogroup bunyaviruses isolated in Japan and Australia were analyzed using monoclonal antibodies (Mabs) raised to Akabane (AKA) virus and oligonucleotide fingerprinting. The virion surface glycoprotein (G1) and the nucleocapsid (N) protein of heterologous viruses showed no reactivity to the Mabs, while the AKA-derived anti-G1 Mab (2F1) reacted with Peaton virus and all three AKA anti-N Mabs reacted with Tinaroo (TIN) virus at almost the same antibody titers as the homologous virus. Oligonucleotide fingerprinting analyses indicated that the three RNA species of all the viruses were unique and distinguishable. However, AKA and TIN viruses exhibited very similar S RNA oligonucleotide fingerprints, while the L and M RNA fingerprints were quite different. The S RNA sequence of TIN virus has been determined and compared with that of AKA and Aino viruses. The results revealed 95.1% S sequence homology between the AKA and TIN viruses. The antigenic and genetic comparisons of AKA and TIN viruses suggest that the two viruses may represent naturally occurring reassortant viruses.     

Salt- and pH-dependent hemagglutination with Kasba virus,a member of the Palyam serogroup of the genus Orbivirus

Kasba virus grown in BHK21-WI2 cells was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 25°C and 37°C. HA was observed at all temperatures with cattle, sheep and goat but not with swine, chicken, and goose erythrocytes. The HA was dependent on not only the NaCl concentration but also the pH of the diluent. The HA titer significantly improved by increasing the NaCl molarity to 0.6 M and standardizing pH to 7.5. The HA titer was 16- or 32-fold higher in 0.4 M or 0.6 M solution than in 0.2 M solution of not only NaCl but also several other salts. The HA reaction was inhibited by specific antibody.     

Characterization of a variant virus isolated from neural cell culture after infection of mouse coronavirus JHMV

Our previous experiments showed that a variant virus with a larger envelope glycoprotein encoded by a larger mRNA3 (cl-2) multiplied predominantly in the brain of rats after wild type (wt) JHMV infection (F. Taguchi, S. Siddell, H. Wege, and V. ter Meulen, 1985, J. Virol. 54, 429-435). We could isolate similar but not identical variant virus after infection of cultured neural cells from rat brain with wt JHMV (designated CNS virus), which also had a larger mRNA3 and produced larger envelope E2 glycoprotein in infected cells. CNS virus multiplied to a higher degree in cultured astrocytes from rat than wt JHMV and cl-2. During infection with these variant viruses in neural cells, virus populations generated did not change, in contrast to consistent selection of viruses with larger mRNA3 after wt JHMV infections. CNS virus produced abundant mRNA2a as well as 65K glycoprotein while the productions of both were trace in cl-2 infected cells. The present experiments, together with our previous observation, suggest that the larger E2 glycoprotein may be of importance for the replication in rat brain cells.     

Sequence analysis of the medium RNA segment of three Simbu serogroup viruses,Akabane, Aino,and Peaton viruses

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.     

Transmission of a newly recognized virus (Bunyaviridae, Bunyavirus) isolated from Aedes albopictus (Diptera: Culicidae) in Potosi, Missouri

Aedes albopictus (Skuse) mosquitoes collected in Potosi, Mo., were tested for their ability to transmit a newly recognized Bunyamwera sero group virus isolated from the same mosquito population. Mosquitoes were fed artificial blood meals containing 4.5-6.2 log10 TCID50 of virus per ml. After 7-29 d at 25 degrees C, 79-99% of the mosquitoes had disseminated infections and 0-26% transmitted virus to fluid-filled capillary tubes. Transmission was first observed after 7 d of extrinsic incubation. Tests failed to detect transovarial transmission in 5,145 progeny from ovarian cycles 2-4. Following parenteral inoculation with 5.3-6.0 log10 TCID50 of virus, four of nine adult hamsters developed viremia. Ten of 16 suckling mice died following intracerebral inoculation of 5.0 log10 TCID50 of virus (fifth Vero cell passage); the average survival time was 8.8 d (SD, 3.5). No mortality occurred in 10 suckling mice inoculated with 3.6 log10 TCID50 of virus (second Vero cell passage).     

Characterization of Rabies virus isolated from canids and identification of the main wild canid host in Northeastern Brazil

The rabies cases in dogs and wild canids in Northeastern Brazil are a public and animal health problem. This paper describes the identities of the coding region of the N-gene of Rabies virus (RABV) isolated in canids from Northeastern Brazil. The genetic tree generated using the sequence data described here divided the cluster BRAZILAN CANIDS into two subclusters (DOG-RELATED STRAINS and WILD CANID-RELATED STRAINS) with identities greater than those already described. The two subclusters are sub-divided into geographic groups related to the origin of the isolates, suggesting a long-standing ecological coexistence of the sequence types characteristic of the groups. This article also analyzes the 513-nucleotide stretch of the mitochondrial DNA control region of rabies-positive canids from Northeastern Brazil with a view to identifying the main RABV host among them. Among the four species of wild canids found in the region, two (Cerdocyon thous and Pseudalopex vetulus) are frequently associated with rabies. Phylogenetic analysis of sequence data generated from mtDNA suggests that C. thous is the main wild canid host in the region. The results obtained in this study are in concordance with the zoology and ecology of wild canids, and thus, help improve epidemiologic vigilance of rabies and allow a more targeted control of the disease.     

Teratogenicity of Australian Simbu serogroup and some other Bunyaviridae viruses: the embryonated chicken egg as a model.

The use of embryonated chicken eggs as a model for assessing the teratogenic potential of animal viruses was investigated with 12 members of the Bunyaviridae family. Infection of 4-day-old embryonated chicken eggs via the yolk sac with 10 of the viruses resulted in deaths or congenital deformities that were similar to those observed in Akabane virus infections of fetal ruminants and included arthrogryposis, scoliosis, mandible defects, and retarded development. Statistical analysis showed that the viruses fell into three main groupings, namely, those that caused both death and deformities (Akabane, Aino, Tinaroo, and Belmont viruses), those that mainly caused death (Peaton, Thimiri, and Facey's Paddock viruses), and those that required very high doses to cause either death or deformities (Douglas and CSIR0296 viruses). In addition, two viruses (Kowanyama and Mapputta viruses) caused neither death nor deformities. A difference in the pathogenic potential between two Akabane isolates (B8935 and CSIR016) in the embryonated chicken egg model was found to correlate with differences previously observed in experimentally infected sheep; Akabane CSIR016 was the more pathogenic. It is concluded that the embryonated chicken egg model should also be of value in assessing the teratogenic potential of other Bunyaviridae and attenuated vaccine viruses, although it does not assess the ability of the virus to cross the placenta.     

Localized conserved regions of the S RNA gene products of bunyaviruses are revealed by sequence analyses of the Simbu serogroup Aino virus

The complete nucleotide sequence has been determined for the S RNA of Aino virus, a member of the Simbu serogroup (Bunyavirus genus, family Bunyaviridae). The S RNA is 850 nucleotides long (2.76 × 10⁵ dallons) and in the viral complementary sequence has a short 5' non-coding region of 34 nucleotides and a more extensive 3' non-coding region of 117 nucleotides. The 3'-5' complementarity of the Aino S RNA is about 25 residues long, depending on the arrangement. The Aino sequence predicts that, like snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses (Bishop D.H.L., et al. (1982) Nucleic Acids Res., 10, 3703–3713; Akashi H. and Bishop D.H.L. (1983) J. Virol. 45, 1155–1158), there are two S coded gene products, a nucleoprotein N, and a non-structural protein, NS_S, that are read from overlapping reading frames in the viral complementary sequence. The Aino N primary gene product is composed of 233 amino acids (26.2 × 10³ daltons) and is 45% homologous in sequence with that of LAC virus. The NS_S protein of Aino virus is composed of 91 amino acids (10.5 × 10³ daltons) and is 35% homologous in sequence with the LAC NS_S protein. Unlike those viruses there are no uridylate tracts longer than 4 residues in the 5' non-coding region of the S viral RNA that could function as a template for polyadenylation of Aino S mRNA species.     

Molecular characterization of Nipah virus, a newly emergent paramyxovirus

Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.     

The emergence of Nipah virus, a highly pathogenic paramyxovirus

Nipah virus first emerged in Malaysia and Singapore between 1998 and 1999, causing severe febrile encephalitis in humans with a mortality rate of close to 40%. In addition, a significant portion of those recovering from acute infection had relapse encephalitis and long-term neurological defects. Since its initial outbreak, there have been numerous outbreaks in Bangladesh and India, in which the mortality rate rose to approximately 70%. These subsequent outbreaks were distinct from the initial outbreak, both in their epidemiology and in their clinical presentations. Recent developments in diagnostics may expedite disease diagnosis and outbreak containment, while progress in understanding the molecular biology of Nipah virus could lead to novel therapeutics and vaccines for this deadly pathogen.     

Characterization of hydrangea chlorotic mottle virus, a new member of the genus Carlavirus

An isolate of the tentative carlavirus species Hydrangea chlorotic mottle virus (HdCMV) was found in New Zealand (NZ) in 2007. The host range, serological properties and complete genome sequence of this isolate were determined in this study. While the NZ isolate shared 98% nucleotide sequence identity with the US isolate of HdCMV, differences in titre and host range were found. The HdCMV-NZ genome sequence of 8,433 nt possessed a typical carlavirus organisation with six open reading frames. HdCMV is most closely related (60.4% nt identity) to blueberry scorch virus, a relationship also suggested by serology. These data suggest that HdCMV is a new carlavirus species.     

Latent and lytic infection of isolated guinea pig enteric ganglia by varicella zoster virus

Varicella zoster virus (VZV) has been demonstrated to infect guinea pig enteric neurons in vitro. Latent infection of isolated enteric neurons is established when the cultures predominantly consist of neurons and they are exposed to cell-free VZV. Neurons harboring latent infection survive for weeks in vitro and express mRNA encoding ORFs 4, 21, 29, 40, 62, and 63, but not 14(gC) or 68 (gE) (although DNA encoding the glycoproteins is present). The expressed proteins are the same as those that are also expressed in human sensory neurons harboring latent VZV. In addition to mRNA, the immunoreactivities of ORFs 4, 21, 29, 62, and 63 can be detected. ORF 62 and 29 proteins are cytoplasmic and not intranuclear. VZV does not preferentially infect and/or become latent in intrinsic enteric primary afferent neurons indicating that the virus is latent in these neurons. Lytic infection occurs when mixed cultures of neurons and non-neuronal cells of the bowel wall are exposed to cell-free VZV or when isolated enteric neurons are exposed to cell-associated VZV. When lytic infection occurs, enteric neurons die within 48 hr. Prior to their death, neurons express VZV glycoproteins, including gE and gB, and ORF 62 and 29 proteins are intranuclear. This new animal model should facilitate studies of VZV latency and the efficacy of therapies designed to prevent VZV infection, latency, and reactivation.     

Nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis

In 1998, an outbreak of acute encephalitis with high mortality rates among pig handlers in Malaysia led to the discovery of a novel paramyxovirus named Nipah virus. A multidisciplinary investigation that included epidemiology, microbiology, molecular biology, and pathology was pivotal in the discovery of this new human infection. Clinical and autopsy findings were derived from a series of 32 fatal human cases of Nipah virus infection. Diagnosis was established in all cases by a combination of immunohistochemistry (IHC) and serology. Routine histological stains, IHC, and electron microscopy were used to examine autopsy tissues. The main histopathological findings included a systemic vasculitis with extensive thrombosis and parenchymal necrosis, particularly in the central nervous system. Endothelial cell damage, necrosis, and syncytial giant cell formation were seen in affected vessels. Characteristic viral inclusions were seen by light and electron microscopy. IHC analysis showed widespread presence of Nipah virus antigens in endothelial and smooth muscle cells of blood vessels. Abundant viral antigens were also seen in various parenchymal cells, particularly in neurons. Infection of endothelial cells and neurons as well as vasculitis and thrombosis seem to be critical to the pathogenesis of this new human disease.