Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

Growth and Maintenance of Wolbachia in Insect Cell Lines

The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate hosts in the clade Ecdysozoa. Originally described in Culex pipiens mosquitoes, Wolbachia is currently represented by 17 supergroups and is believed to occur in half of all insect species. In mosquitoes, Wolbachia acts as a gene drive agent, with the potential to modify vector populations; in filarial worms, Wolbachia functions as a symbiont, and is a target for drug therapy. A small number of Wolbachia strains from supergroups A, B, and F have been maintained in insect cell lines, which are thought to provide a more permissive environment than the natural host. When transferred back to an insect host, Wolbachia produced in cultured cells are infectious and retain reproductive phenotypes. Here, I review applications of insect cell lines in Wolbachia research and describe conditions that facilitate Wolbachia infection and replication in naive host cells. Progress in manipulation of Wolbachia in vitro will enable genetic and biochemical advances that will facilitate eventual genetic engineering of this important biological control agent.

A recombinant baculovirus TnGV in Trichoplusia ni larvae using the PIG bombardment method and the CRISPR/Cas9 system

Baculoviruses have been used for the expression of heterologous proteins of biotechnological interest. However, most of these proteins are obtained by homologous co-transfection recombination in cell lines, limiting their use. Recently, the CRISPR/Cas9 system has excelled in its high efficiency in editing specific sequences without the need for insect cell lines. In this work, the CRISPR/Cas9 system was used to edit the genome of Trichopusia ni granulovirus (TnGV) and transformation of insects by the PIG bombardment method. A homologous repair vector (pTnGV101) was designed with regions orf5 and orf7, as well as sgRNA flanking TnGV P10 of this virus. The bombardment transformation was carried out at 175 psi with 40% of infected T. ni larvae, of which 38% expressed the reporter protein EGFP. These results demonstrate that the CRISPR/Cas9 system and PIG bombardment can be used for genetic modification of baculovirus in vivo.

Susceptibility of Midge and Mosquito Vectors to SARS-CoV-2

SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2-infected bloodmeal. Species tested included Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae) biting midges, as well as Culex tarsalis (Coquillett) and Culex quinquefasciatus (Say) mosquitoes (Diptera: Culicidae), all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2-spiked blood and at a time point postinfection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Aedes aegypti (Linnaeus) (Diptera: Culicidae) (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines evaluated support SARS-CoV-2 replication, suggesting that these species are unable to be biological vectors of SARS-CoV-2.

Susceptibility of midge and mosquito vectors to SARS-CoV-2 by natural route of infection

SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2infected blood meal. Species tested included Culicoides sonorensis biting midges, as well as Culex tarsalis and Culex quinquefasciatus mosquitoes, all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2 spiked blood and at various time points post infection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Ae. aegypti (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines support SARS-CoV-2 replication. We conclude, that biting insect do not pose a risk for transmission of SARS-CoV-2 to humans or animals following a SARS-CoV-2 infected blood meal.

Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.