Discovery and sequencing of novel and identified mosquito-associated viruses and genetic determinants of flavivirus host specificity

The advent of globalization and the continuing effects of climate change have exposed an even greater percentage of the world to disease agents carried by vectors which were once thought to be restricted to tropical or subtropical areas. Arthropods vectors such as mosquitoes and ticks harbor a number of emerging and re-emerging pathogens of public and veterinary health importance worldwide. These pathogens include viruses from four major families including the Flaviviridae, Bunyaviridae, Togaviridae and Reoviridae families. The Flavivirus genus is one of four genera in the Flaviviridae family, with over 70 species, the great majority of which are maintained in horizontal transmission cycles between hematophagous arthropod vectors and vertebrate hosts. However, within this genus also exists two smaller subsets of viruses which are restricted to either insects (insect-specific flaviviruses) or vertebrates (no known vector flaviviruses). Since it is likely that dual-host flaviviruses may have evolved from single-host precursors, investigative studies into the genetic elements which modulate flavivirus host specificity may elucidate the evolutionary hurdles necessary to make the leap from a single- to a dual-host flavivirus. Such studies could have major implications for efficacious vaccine or antiviral drug development. In this dissertation, I report the creation and characterization of the first bat-associated flavivirus chimeras, designed to investigate host restriction in no known vector (NKV) flaviviruses at the level of attachment and entry. These chimeras were created using two representative dual-host flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV) whose attachment and entry proteins (premembrane and envelope) were substituted with that of a bat-associated NKV flavivirus, Rio Bravo virus (RBV). The result was the generation of two different chimeric viruses, both of which were able to replicate in both insect and vertebrate cell lines, indicating that NKV flavivirus restriction is dictated by a post-attachment/entry event. Additional studies examining restriction at downstream stages in the viral life cycle may prove valuable in determining the genetic elements that modulate host restriction in this group. In the chapters that follow, we also report the discovery and identification of four novel and two previously identified viruses from mosquitoes collected in the Yucatan Peninsula of Mexico using metagenomics. We also report full sequence data for two insect-specific flaviviruses (ISFs) and for the large segment of two bunyaviruses for which full sequence data was not available

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Discovery and sequencing of novel and identified mosquito-associated viruses and genetic determinants of flavivirus host specificity

The advent of globalization and the continuing effects of climate change have exposed an even greater percentage of the world to disease agents carried by vectors which were once thought to be restricted to tropical or subtropical areas. Arthropods vectors such as mosquitoes and ticks harbor a number of emerging and re-emerging pathogens of public and veterinary health importance worldwide. These pathogens include viruses from four major families including the Flaviviridae, Bunyaviridae, Togaviridae and Reoviridae families. The Flavivirus genus is one of four genera in the Flaviviridae family, with over 70 species, the great majority of which are maintained in horizontal transmission cycles between hematophagous arthropod vectors and vertebrate hosts. However, within this genus also exists two smaller subsets of viruses which are restricted to either insects (insect-specific flaviviruses) or vertebrates (no known vector flaviviruses). Since it is likely that dual-host flaviviruses may have evolved from single-host precursors, investigative studies into the genetic elements which modulate flavivirus host specificity may elucidate the evolutionary hurdles necessary to make the leap from a single- to a dual-host flavivirus. Such studies could have major implications for efficacious vaccine or antiviral drug development. In this dissertation, I report the creation and characterization of the first bat-associated flavivirus chimeras, designed to investigate host restriction in no known vector (NKV) flaviviruses at the level of attachment and entry. These chimeras were created using two representative dual-host flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV) whose attachment and entry proteins (premembrane and envelope) were substituted with that of a bat-associated NKV flavivirus, Rio Bravo virus (RBV). The result was the generation of two different chimeric viruses, both of which were able to replicate in both insect and vertebrate cell lines, indicating that NKV flavivirus restriction is dictated by a post-attachment/entry event. Additional studies examining restriction at downstream stages in the viral life cycle may prove valuable in determining the genetic elements that modulate host restriction in this group. In the chapters that follow, we also report the discovery and identification of four novel and two previously identified viruses from mosquitoes collected in the Yucatan Peninsula of Mexico using metagenomics. We also report full sequence data for two insect-specific flaviviruses (ISFs) and for the large segment of two bunyaviruses for which full sequence data was not available.</p

Evidence that Lokern virus (family Peribunyaviridae) is a reassortant that acquired its small and large genome segments from Main Drain virus and its medium genome segment from an undiscovered virus

Abstract Background Lokern virus (LOKV) is a poorly characterized arthropod-borne virus belonging to the genus Orthobunyavirus (family Peribunyaviridae). All viruses in this genus have tripartite, single-stranded, negative-sense RNA genomes, and the three RNA segments are designated as small, (S), medium (M) and large (L). A 559 nt. region of the M RNA segment of LOKV has been sequenced and there are no sequence data available for its S or L RNA segments. The purpose of this study was to sequence the genome of LOKV. Methods The genome of LOKV was fully sequenced by unbiased high-throughput sequencing, 5′ and 3′ rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. Results The S and L RNA segments of LOKV consist of 952 and 6864 nt. respectively and both have 99.0% nucleotide identity with the corresponding regions of Main Drain virus (MDV). In contrast, the 4450-nt. M RNA segment has only 59.0% nucleotide identity with the corresponding region of MDV and no more than 72.7% nucleotide identity with all other M RNA segment sequences in the Genbank database. Phylogenetic data support these findings. Conclusions This study provides evidence that LOKV is a natural reassortant that acquired its S and L RNA segments from MDV and its M RNA segment from an undiscovered, and possibly extinct, virus. The availability of complete genome sequence data facilitates the accurate detection, identification and diagnosis of viruses and viral infections, and this is especially true for viruses with segmented genomes because it can be difficult or even impossible to differentiate between reassortants and their precursors when incomplete sequence data are available

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

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

Discovery of a novel Tymoviridae-like virus in mosquitoes from Mexico.

A novel Tymoviridae-like virus, designated Ek Balam virus, was isolated from male Culex quinquefasciatus mosquitoes collected in Yucatan, Mexico. The genome was fully sequenced and shown to have no more than 69% nt sequence identity to its closest known relative. Mosquito cells were permissive to Ek Balam virus replication, but mammalian and avian cells were refractory, suggesting that vertebrates are not involved in the maintenance of the virus in nature

Detection of novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico using metagenomics and characterization of their in vitro host ranges.

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics

Arbovirus Surveillance near the Mexico–U.S. Border: Isolation and Sequence Analysis of Chikungunya Virus from Patients with Dengue-like Symptoms in Reynosa, Tamaulipas

A total of 1,090 residents of the city of Reynosa, Tamaulipas, on the Mexico–U.S. border presented at hospitals and clinics of the Secretariat of Health, Mexico, in 2015 with symptoms characteristic of dengue. Dengue virus (DENV) antigen was detected by enzyme-linked immunosorbent assay in acute sera from 134 (12.3%) patients. Sera from select patients (N = 34) were also tested for chikungunya virus (CHIKV) RNA by quantitative reverse transcription–polymerase chain reaction. Thirteen (38.2%) patients, including five DENV antigen-positive patients, were positive. Sera from three CHIKV RNA-positive patients were further assayed by virus isolation in cell culture and CHIKV was recovered on each occasion. The genome of one isolate and structural genes of the other two isolates were sequenced. In conclusion, we present evidence of CHIKV and DENV coinfections in patients who live near the Mexico–U.S. border and provide the first genome sequence of a CHIKV isolate from northern Mexico