Exploiting the Legacy of the Arbovirus Hunters.

In recent years, it has become evident that a generational gap has developed in the community of arbovirus research. This apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. On the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. This paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. In this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. To this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead.

Electron Microscopy in Discovery of Novel and Emerging Viruses from the Collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA).

Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been an important and widely used technique for discovery, identification and characterization of new viruses. Using TEM, viruses can be differentiated by their ultrastructure: shape, size, intracellular location and for some viruses, by the ultrastructural cytopathic effects and/or specific structures forming in the host cell during their replication. Ultrastructural characteristics are usually sufficient for the identification of a virus to the family level. In this review, we summarize 25 years of experience in identification of novel viruses from the collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA).

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Insect-specific viruses detected in laboratory mosquito colonies and their potential implications for experiments evaluating arbovirus vector competence.

Recently, there has been a dramatic increase in the detection and characterization of insect-specific viruses in field-collected mosquitoes. Evidence suggests that these viruses are ubiquitous in nature and that many are maintained by vertical transmission in mosquito populations. Some studies suggest that the presence of insect-specific viruses may inhibit replication of a super-infecting arbovirus, thus altering vector competence of the mosquito host. Accordingly, we screened our laboratory mosquito colonies for insect-specific viruses. Pools of colony mosquitoes were homogenized and inoculated into cultures of Aedes albopictus (C6/36) cells. The infected cells were examined by electron microscopy and deep sequencing was performed on RNA extracts. Electron micrograph images indicated the presence of three different viruses in three of our laboratory mosquito colonies. Potential implications of these findings for vector competence studies are discussed.

Quaranfil, Johnston Atoll, and Lake Chad viruses are novel members of the family Orthomyxoviridae.

Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae.

Ticks associated with macquarie island penguins carry arboviruses from four genera.

Macquarie Island, a small subantarctic island, is home to rockhopper, royal and king penguins, which are often infested with the globally distributed seabird tick, Ixodes uriae. A flavivirus, an orbivirus, a phlebovirus, and a nairovirus were isolated from these ticks and partial sequences obtained. The flavivirus was nearly identical to Gadgets Gully virus, isolated some 30 year previously, illustrating the remarkable genetic stability of this virus. The nearest relative to the orbivirus (for which we propose the name Sandy Bay virus) was the Scottish Broadhaven virus, and provided only the second available sequences from the Great Island orbivirus serogroup. The phlebovirus (for which we propose the name Catch-me-cave virus) and the previously isolated Precarious Point virus were distinct but related, with both showing homology with the Finnish Uukuniemi virus. These penguin viruses provided the second and third available sequences for the Uukuniemi group of phleboviruses. The nairovirus (for which we propose the name Finch Creek virus) was shown to be related to the North American Tillamook virus, the Asian Hazara virus and Nairobi sheep disease virus. Macquarie Island penguins thus harbour arboviruses from at least four of the seven arbovirus-containing genera, with related viruses often found in the northern hemisphere.

Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini.

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.

[The arboviruses of genus alphavirus family togaviridae were isolated from mosquitoes captured from Yantai].

OBJECTIVE: To isolate arbovirus from mosquitoes caught from Yantai. METHODS: The isolated viruses were tested for their physico- chemical properties, examined by electron microscopy and specific immuno-reactivity. RESULTS: Fifteen strains of virus were isolated from mosquitoes in 1994 from Yantai. Three of them were further assayed. The results showed that the viruses could multiply on C6/36 cell and produce typical cytopathogenic effect. The viruses couldn't cause regular sickness and death of suckling mice by intracerebral inoculation. The viruses were sensitive to ether, but resistant to 5, -Idu. One sample was examined with electron microscope, and spherical virus particles were observed. The diameter of the virus particles is about 55 +/- 2. 3nm. It did not react with the JBE virus and Bunyaviridae group specific immuno-ascitic fluids but cross-reacted with the group A Togaviridae specific immuno-ascitic fluid. CONCLUSIONS: The results showed that the viruses isolated from Yantai belong to alphavirus of togaviridae.

Mayaro virus proteins

Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied

Investigations on the structure and morphogenesis of some arboviruses isolated in the U.S.S.R.

The morphology and some stages of morphogenesis in suckling mouse brains of hitherto serologically unclassified viruses Kaspiy (LEIV-A-63), Zavashan (LEIV-Ap-6158), Artashat (LEIV-A-2366), and Paramushir (LEIV-C-2268), isolated in the U.S.S.R., of GM-710 virus isolated in Scotland, and of Sokuluk (LEIV-K-400) virus belonging to the genus Flavivirus (family Togaviridae) were studied. Virion sizes were determined and changes in infected cells described. Based on their structure and morphogenesis, the viruses Kaspiy, Artashat, Zavashan and Paramushir were referred to the family Bunyaviridae and GM-710 virus was referred to the genus Orbivirus (family Reoviridae).

Characterization of some ungrouped viruses.

Bangui, Bobaya, Kowanyama, Upolu and Zinga are enveloped spherical RNA viruses sensitive to ether and acid pH; morphologically resembling Bunyaviridae, their diameters range from 90 to 100 nm and their bouyant density is 1.17 to 1.18 g/ml in sucrose. St. Floris, a bunya-like virus, serologically related to Rift Valley Fever, has a diameter ranging from 90 to 110 nm. Triniti, Zingilamo, IPYM 120 and virus strain AnB4268 are enveloped spherical RNA viruses sensitive to ether and acid pH, have diameters of 65, 55, 55 and 60 nm and bouyant densities of 1.18, 1.24, 1.20 and 1.18 g/ml in sucrose, respectively; morphologically they resemble Togaviridae. Salanga is a poxvirus measuring 190 X 225 nm. Virus strain AnY1444 is a spherical, non-enveloped RNA virus measuring 85 nm, morphologically resembling Reoviridiae; it is restraint to both ether and acid pH and shows two-peak densities of 1.32 and 1.36 g/ml in caesium chloride. Bangoran, Keuraliba and Yata resemble Rhabdoviridae measuring 60 X 175, 65 X 195 and 60 X 185 nm, respectively.

[Properties of the RNA of the new Okhotsk orbivirus]. / Izuchenie nekotorykh svoistv RNK novogo orbivirusa Okhotskii.

Electron microscopic examination of Okhotsky virus RNA showed it to have double-stranded fragmented structure. Investigation of the kinetics of virus-specific RNA synthesis in chick embryo fibroblast cell cultures inoculated at a high multiplicity of infection demonstrated that the synthesis of virus-specific RNAs of Okhotsky virus reached maximum by 7 hours postinfection. A decrease in the multiplicity of infection slowed down the kinetics of virus-specific RNAs synthesis. The sedimentation analysis in sucrose density gradient showed the virus-specific RNAs of Okhotsky virus to sediment with a peak in 14.5 S band.

[Arboviruses - structure and classification (author's transl)]. / Arbovirus - structure et classification.

The larger, biologically defined, set of arboviruses contains sub-sets representative of a number of different taxons. Arboviruses classified on serological grounds into Groups A and B are now placed respectively into the genera alphavirus and flavivirus, which form part of the family Togaviridae. The recently defined family Bunyaviridae contains almost 200 different arboviruses, serologically divisible into at least 24 different serogroups. The family Reoviridae includes the genus orbivirus which covers at least 17 serological groups, and the family Rhabdoviridae is another major family which contains a significant number of arboviruses. Outside these four families individual arboviruses are placed are placed in the families Iridoviridae, Poxviridae, Picornaviridae and Coronaviridae, but a least 50 arbovirueses remain unclassified.

Neuropathology of S. Paulo south coast epidemic encephalitis (Rocio flavivurus).

The neuropathology of 8 cases of S. Paulo south coast epidemic encephalitis (Rocio flavivirus), a new arbo B virus encephalitis, is described. The topographic pattern of the lesions appears to be almost specific. The gray matter is predominantly affected. Interstitial mononuclear infiltration, microglial proliferation and perivascular lymphocytic cuffing were seen. Neuronophagia was seldom seen except during the acute phases of the disease. Throughout the neuraxis, the gray matter was affected to a greater degree than white matter. The more damaged structures, in descending order, were as follows: thalamus, dentate nucleus, substantia inominata, brain stem, spinal cord and basal nuclei. Most of the cases exhibited thalamic inflammatory necrosis. Electron microscopy disclosed in one case virus-like particles, resembling those described in other arbo B viruses in the cytoplasm of thalamic neurons. In this case, virus was isolated from the brain and an immunofluorescence test also showed antigenic material in the thalamic neurons.

Electron microscopy of Lednice virus in chick embryo cells.

Replication of Lednice virus in chick embryo cells was studied for 72 hr after inoculation by infectivity titration and the indirect immunofluorescence technique. At 24 and 48 hr after inoculation, electron microscopy revealed spherical virions of uniform morphology, 80-105 nm in diameter, which were localized mostly extracellularly.

Separation of alphavirus nucleocapsids from envelope fragments.

Methods for the preparation of alphavirus nucleocapsids and envelope fragments for biochemical, electron microscopical and serological investigations are described. Difficulties which have been encountered previously in attempts to isolate viral nucleocapsids for morphological studies, namely disruption of the fragile particles, have been overcome by treatment with formaldehyde.