The current COVID-19 pandemic is caused by the betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped single-stranded RNA virus with a large genome of ~30 kb length. It is known to infect both humans and animal species. A recent study by researchers at the Kansas State University, and the United States Department of Agriculture and published on the preprint server bioRxiv* in September 2020 looked to see if biting insects pose a risk for transmission of SARS-CoV-2 to humans or animals following a SARS-CoV-2 infected blood meal.
Routes of SARS-CoV-2 Transmission
The SARS-CoV-2 virus is mainly carried by droplet and aerosols from infected individuals, directly or via contaminated surfaces. However, insects are known to carry numerous diseases to humans both by physically transferring the pathogens and by acting as hosts for part of their lifecycle.
Do Insect Vectors Transmit the Virus?
Both earlier viruses, SARS-CoV and MERS-CoV, have not been reported to be transmitted by insects so far. However, scientists are wondering whether they can spread the current virus.
For this to occur, the pathogen must be ingested in a blood meal from a host, and establish infection within the midgut of the insect. Then it must be able to travel to the salivary glands and infect them so that during the next feeding on a host, it will transmit the pathogen to the new host.
Earlier research shows that the virus cannot replicate in several mosquito species; namely, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus, when introduced into the thorax. It was not found in either Culex or Anopheles mosquitoes.
Potential Transmission in Midges and Mosquitoes
The current study is the first to report the susceptibility of three important insect vector species to SARS-CoV-2 infection via a blood meal containing the virus – the natural route of infection. These vectors include biting midges (Culicoides sonorensis), and two important human vectors, Cx. tarsalis and Cx. Quinquefasciatus. They also used four cell lines derived from four other insects: C. sonorensis (W8a), Ae. aegypti (C6/36), Cx. tarsalis (CxTrR2), and Cx. quinquefasciatus (HSU) to determine SARS-CoV-2 susceptibility.
In Vitro Studies Produce Negative Results
The researchers first exposed the insect cell lines with the virus in vitro in two separate experiments. The supernatant from the insect cell cultures after 2, 4 and 8 days from infection was added to Vero cells. There was no sign of a cytopathic effect (CPE) in any of the cells.
In Vivo Studies Show Viral RNA
They then allowed the insects to feed on infected blood. They found that of 200 midges, 140 survived for up to 10 days. On examination, they observed the presence of SARS-CoV-2 RNA in 85% of the midges, with the mean Ct value being ~34. Control midges on day 10 unanimously failed to show any sign of the viral RNA.
Among the 100 Cx. tarsalis mosquitoes fed on infected blood, only 48 were alive at day 10, and 17% were positive for viral RNA at a mean Ct value of ~31. None of the controls were positive.
Of 100 Cx. quinquefasciatus mosquitoes fed on infected blood, roughly the same number (47) survived till day 10, and half of them had viral RNA with a mean Ct of ~34.
No Infectious Virus Recovered
In the next step, they homogenized the insect tissues and incubated them on Vero cells serially. With midges, no CPE was observed even with three successive passages. The absence of the virus was confirmed by testing the first and third passages by immunofluorescence assay (IFA). Control cells tested positive by IFA. Control midges were negative for both IFA and incubation. The same result was obtained with six pooled tissue samples from control Cx. Tarsalis and Cx. quinquefasciatus after feeding them on infected blood by VI and IFA.
It is essential to recognize that only if the host has virus particles in the bloodstream at the time of feeding will the insect be infected. Some infected individuals have been known to display viremia, unlike most animal models tested so far. The exception is hamsters, which typically develop viremia. This indicates the need for controlled arthropod transmission studies to understand the degree of risk and to help develop reliable epidemiological models for the disease. This is important in designing public health strategies.
Importance and Implications
The researchers note that their results agree with earlier studies ruling out a role for Aedes mosquitoes in viral transmission as this cell line failed to allow the virus to replicate. They also report that various other cell lines derived from several Culex species and one Culicoides species also fail to support this infection. Importantly, they used the natural route of inoculation (a virus-spiked blood meal) to determine susceptibility, adding to the relevance of the study.
Moreover, the use of this route takes into account the quasi-species characteristic of this virus. Viruses have to negotiate several genetic and natural selection obstacles to replicate and be transmitted by insects successfully. First entering via the blood meal, they have then to establish infection in the midgut cells, replicate and then enter the hemocele where they must survive, to reach the salivary glands and replicate finally. This is the last step before they are released in saliva during the next blood meal.
RNA viruses are by nature, quasi-species, and natural selection pressures that favor their infection and replication in insects to form a specific population with increased fitness for this host type. These changes could result in the formation of biotypes of the virus that have higher infectivity for the salivary gland cells and which are injected in greater numbers in saliva from the infected insect.
The researchers also report that viral RNA remained detectable for up to 10 days following the blood meal, but infectious viral particles were not recovered with three serial passages on suitable cell culture.
They conclude: “The insect vector species known to transmit animal and human pathogens used in this study are refractory to SARS-CoV-2 infection under experimental conditions and, therefore, most likely do not play a role in the transmission of SARS-CoV-2.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.