The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of the beta coronavirus, an enveloped single-stranded RNA virus with a large genome ~ 30 kb in length. It is known to infect both human and animal species. A study recently published by researchers from Kansas State University and the US Department of Agriculture, which was published on the preprint server bioRxiv * in September 2020, examined whether biting insects pose a risk for the transmission of SARS-CoV-2 to humans or depict animals after a blood meal infected with SARS-CoV-2.
Routes of the SARS-CoV-2 transmission
The SARS-CoV-2 virus is mainly transmitted by droplets and aerosols from infected people directly or via contaminated surfaces. However, it is known that insects transmit numerous diseases to humans by physically transmitting the pathogens and acting as hosts for part of their life cycle.
Do insect vectors transmit the virus?
Both previous viruses, SARS-CoV and MERS-CoV, have not previously been reported to be transmitted by insects. However, scientists are wondering if they can spread the current virus.
To do this, the pathogen must be ingested in a host’s blood meal and cause an infection in the insect’s midgut. Then it must be able to get to the salivary glands and infect them so that the next time a host is fed, the pathogen can be transmitted to the new host.
Previous research shows that the virus cannot multiply in multiple mosquito species. namely, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus when inserted into the thorax. It was not found in either Culex or Anopheles mosquitoes.
Possible transmission in mosquitoes and mosquitoes
The current study is the first to report the susceptibility of three major insect vector species to SARS-CoV-2 infection from a blood meal containing the virus – the natural route of infection. These vectors include biting mosquitoes (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 sensitivity.
In vitro studies give negative results
The researchers first exposed the insect cell lines to the virus in two separate in vitro experiments. The supernatant from the insect cell cultures after 2, 4 and 8 days after infection was added to Vero cells. There was no evidence of cytopathic effect (CPE) in any of the cells.
In vivo studies show viral RNA
Then they allowed the insects to feed on infected blood. They found that out of 200 mosquitoes, 140 survived for up to 10 days. When examined, they observed the presence of SARS-CoV-2 RNA in 85% of the mosquitoes with a mean Ct value of ~ 34. Control mosquitoes on day 10 unanimously showed no signs of viral RNA.
Among the 100 Cx. Tarsalis mosquitoes fed infected blood were only 48 alive on day 10 and 17% were positive for viral RNA with a mean Ct of ~ 31. None of the controls were positive.
From 100 Cx. Quinquefasciatus mosquitoes fed infected blood survived approximately the same number (47) through day 10, and half of them had viral RNA with a mean Ct of ~ 34.
No infectious virus restored
In the next step, they homogenized the insect tissues and incubated them serially on Vero cells. No CPE was observed in mosquitoes even after three consecutive passages. The absence of virus was confirmed by immunofluorescence assay (IFA) testing of the first and third passages. Control cells tested positive by IFA. Control mosquitoes 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 infected blood by VI and IFA.
It is important to realize that the insect will only become infected if the host has virus particles in the blood at the time of feeding. It is known that some infected individuals have viraemia, unlike most animal models tested to date. The exception is hamsters, which typically develop viraemia. This indicates the need for controlled arthropod transmission studies to understand the level of risk and develop reliable epidemiological models for the disease. This is important for the development of public health strategies.
Meaning and implications
The researchers note that their results are consistent with previous studies that rule out a role for Aedes mosquitoes in virus transmission because this cell line did not allow the virus to replicate. They also report that several other cell lines derived from several species of Culex and one species of Culicoides also do not support this infection. Importantly, they used the natural route of inoculation (a virus-spiked blood meal) to determine susceptibility, which increased the relevance of the study.
In addition, the use of this route takes into account the quasi-species characteristic of this virus. Viruses must overcome several genetic and natural selection barriers in order to reproduce and be successfully transmitted by insects. First entering via the blood meal, they then have to detect an infection in the midgut cells, replicate, and then enter the hemocele where they have to survive to reach the salivary glands and eventually replicate. This is the final step before they are released into 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 in order to form a specific population with increased suitability for this host type. These changes could lead to the formation of biotypes of the virus which are more infectious for the salivary gland cells and which are injected into the saliva of the infected insect in greater numbers.
The researchers also report that viral RNA remained detectable for up to 10 days after the blood meal, but that infectious viral particles were not obtained with three serial passages on an appropriate cell culture.
They conclude: “The insect vector species used in this study, which are known to transmit animal and human pathogens, 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. “
* Important NOTE
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.