REPORT – COVID-19: transmission by insect bite

I wrote this article trying to analyze the problem of COVID-19 contagion and its potential evolutions / mutations in relation to a vector common to many diseases – insects.
Specialized research in the field of epidemiological diffusion has for decades highlighted the ability of mosquitoes and related to function as a capillary propagation tool for viruses and bacteria.
Although expressed very technically, I have tried to represent this reality from different angles.
There is also clear evidence of “evolutions” in the viral families of the famous coronavirus, which can herald epidemics of even more devastating scale if not taken seriously.

Let’s start by understanding that pointing the finger at a question “COVID-19 is transmitted by mosquitoes” is very reductive!
We must ask ourselves … can mosquitoes transmit this class of virus? are there evolutions? how can we deal with the problem? Where can we start from?

Let’s start

Coronaviridae, along with Arteriviridae and Roniviridae, belong to the order Nidovirales.

Viruses belonging to these families are large positive strand RNA viruses and are known to infect mammals, birds, fish and arthropods [1].

Entry into a host cell is usually mediated by an interaction between the virus spike glycoprotein and a cellular receptor [2]. After entry, the virus disassembles and a replication/transcription complex forms on double-membraned vesicles ([3] and references within).

New subgenomic RNA is produced by a mechanism known as discontinuous transcription [4].

Coronavirus replication requires the production of negative-strand RNA from which positive-strand RNA is produced. Viral proteins are produced from the positive-strand subgenomic RNAs and from the positive-strand full-length RNA.

The two largest open reading frames, ORF1a and ORF1a/b, are translated from the full-length RNA. These open reading frames (ORFs) encode polyproteins pp1a and pp1ab which are cleaved by self-encoded proteases.

The proteins encoded in ORF1a and ORF1a/b function as the replicase, making subgenomic RNAs and new copies of the genomic RNA [5]. Production of the pp1ab polyprotein requires the translating ribosome to change reading frame at the frameshift signal that bridges ORF1a and ORF1a/b.

Like most viral frameshift signals, frameshifting at the coronavirus signal leads to expression of an RNA-dependent RNA polymerase (RdRP), a protein essential for viral replication (for review, see [6]).

The proteins upstream of the frameshift signal include the predicted proteases and other uncharacterized proteins [5]. We have previously suggested that the ratio of the pp1a and pp1ab proteins might affect the regulation and production of genomic and subgenomic RNA [7].

The SARS coronavirus frameshift signal has a seven nucleotide ‘slippery sequence’ and a stimulatory pseudoknot separated by a spacer region.

During programmed -1 ribosomal frameshifting (-1PRF), the tRNAs positioned on the slippery site uncouple from the mRNA and reconnect in the new reading frame.

The second stem of the stimulatory pseudoknot is formed by the distal 3’ sequence base-pairing with residues in the loop region of the first stem loop.

Unlike other frameshift-stimulating pseudoknots the SARS pseudoknot contains an additional internal stem loop [8,9,10]. The function of this structure, called stem 3, is unknown.

We have shown that alterations to the SARS coronavirus frameshift signal affect frameshifting efficiency [9,11].

Reduction in frameshifting efficiency is expected to result in decreased expression of the frameshift proteins, including the RdRP. Some mutations that reduced frameshifting were associated with a several-fold reduction in the amount of genomic RNA [7].

The order Nidovirales

The order Nidovirales [21] includes positive-sense singlestranded RNA (ssRNA?) viruses of three families: Arteriviridae [22] (12.7–15.7-kb genomes; ‘‘small-sized nidoviruses’’), Coronaviridae [23] and Roniviridae [24] (26.3–31.7 kb; the last two families are jointly referred to as
‘‘large-sized nidoviruses’’) [5].

All other known ssRNA? viruses have genome sizes below 20 kb. Recently, two closely related viruses, Cavally virus (CAVV) and Nam Dinh virus (NDiV), were discovered by two independent groups of researchers in Coˆte d’Ivoire in 2004 and in Vietnam in 2002, respectively [26, 27].

CAVV was isolated from various mosquito species belonging to the genera Culex, Aedes,
Anopheles and Uranotaenia [27].

It was most frequently found in Culex species, especially Culex nebulosus. Except for Culex quinquefasciatus, which circulates worldwide, the other mosquito species are endemic to Africa.

NDiV was isolated from Culex vishnui, which is endemic to Asia, and Culex tritaeniorhynchus, which circulates in Asia and Africa [6], and there are indications that it may infect more mosquito species (Nga, unpublished data).

Analysis of abundance patterns of 39 CAVV isolates in different habitat types along an anthropogenic disturbance gradient has indicated an increase in virus prevalence from natural to modified habitat types [28].

A significantly higher prevalence was found especially in human settlements. Analysis of habitatspecific virus diversity and ancestral state reconstruction demonstrated an origin ofCAVVin a pristine rainforest with subsequent spread into agriculture and human settlements [27].

Notably, it was shown for the first time that virus diversity decreased and prevalence increased during the process of emergence from a pristine rainforest habitat into surrounding areas of less host biodiversity due to anthropogenic modification [27].

Both viruses were propagated in Aedes albopictus cells and characterized using different techniques. A number of common properties place CAVV and NDiV in the order Nidovirales.

These properties include (i) the genome organization with multiple open reading frames (ORFs), (ii) the predicted proteomes (Fig. 1), (iii) the production of enveloped, spherical virions, and (iv) the synthesis of genome-length and subgenome-length viral RNAs in infected cells [6, 7].

Particularly, the two viruses were found to encode key molecular markers characteristic of all nidoviruses: a 3C-like main protease (3CLpro, also known as Mpro) flanked by two transmembrane (tM) domains encoded in replicase ORF1a, as well as an RNA- dependent RNA polymerase (RdRp) and a combination of a Zn-binding module (Zm) fused with a superfamily 1 helicase (HEL1) encoded in ORF1b.

As in other nidovirus genomes, ORFs 1a and 1b were found to overlap by a few nucleotides in both CAVV and NDiV. The ORF1a/1b overlap region includes a putative -1 ribosomal frameshift site (RFS) that is expected to direct the translation of ORF1b by a fraction of the ribosomes that start translation at the ORF1a initiation codon.

Thus, a frameshift just upstream of the ORF1a termination codon mediates the production of a C-terminally extended polyprotein jointly encoded by ORF1a and ORF1b. Combined, these markers form the characteristic nidovirus constellation: tM-3CLpro-tM_RFS_RdRp_Zm- HEL1 (Fig. 1) [21, 25].

Likewise, virion proteins are encoded in ORFs that are located downstream of ORF1b and expressed from a set of subgenomic mRNAs. No similarities were found between the (putative) structural proteins of CAVV and NDiV and those of other nidoviruses [26, 27].

The most distinctive molecular characteristic of CAVV and NDiV, however, is the *20-kb genome size, that is inter- mediate between the size ranges of small-sized and large- sized nidovirus genomes. Consequently, each of the two viruses has been proposed to prototype a new nidovirus family [26, 27].

In this study, we compared the genomes of CAVV (GenBank accession number HM746600) and NDiV (GenBank accession number DQ458789) to assess their relationship and use this insight for taxonomic classification of these viruses.

To date, only very limited biological information is available for CAVV and NDiV (see above), and in general, biological properties may be affected pro- foundly by a few changes in the genome.

In view of these considerations and in line with the accepted taxonomic approach to viruses of the family Coronaviridae [23], comparative sequence analysis was considered the most reliable basis for classification.

The overall similarity between the CAVV and NDiV genomes was found to be strikingly high: nearly identical sizes (20,187 and 20,192 nt, respectively), conservation of ORFs with sequence identities ranging from 87.8 to 96.1% at the amino acid level and from 88.3 to 93.7% at the nucleotide level (Table 1).

Given this high similarity, prior assignments of domains and genetic signals were cross-checked to produce a unified description.

There was complete agreement between the two studies [26, 27] on the mapping of all nidovirus-wide conserved domains in CAVV and NDiV, as well as on the identifi- cation of GGAUUUU as a plausible slippery sequence in RFS (see above).

Additionally, our analysis showed that the NDiV-based assignment [6] of 30-to-50 exoribonuclease (ExoN) and 20-O-methyltransferase (OMT), two replicative domains characteristic for large-sized nidoviruses [25], and N7-methyltransferase (NMT) [29] in ORF1b extends to CAVV. Likewise, CAVV may lack a uridylate-specific endonuclease (NendoU), as has previously been observed for NDiV [26].

The synthesis of subgenomic RNAs from which ORFs 2a to 4 are predicted to be expressed appears to be controlled by transcription-regulating sequences (TRSs) [30–32] identified upstream of ORF2a/2b, ORF3a and ORF4 (collectively designated as body TRSs).

Other putative TRSs were identified downstream of the leader region located at the 50-end of the viral genome [26, 27].

Unique among nidoviruses, NDiV and CAVV may use different leader TRSs during the synthesis of different subgenomic RNAs, although further analysis is required to clarify the basis for some discrepancies between the TRS assignment in NDiV and CAVV.

Also, it remains to be shown why the high sequence conservation of virion pro- teins of the two viruses (Table 1) was not manifested in the morphology observed upon EM analysis of virus particles [26, 27]. In this respect, it may be relevant that Zirkel et al. [27] noticed two types of particles in CAVV-infected cells, one of which carried club-shaped surface projections compatible with viral glycoproteins .

This latter type of particles was also observed in infected cell culture supernatant. Ultimately, the origin of the particles of both types, and their relationship to the particles isolated from the medium of NDiV-infected C6/36 cells by Nga et al. [26] should be revealed by future research efforts.

Furthermore, we evaluated the phylogenetic position of CAVV and NDiV in relation to other nidoviruses. We con- ducted a phylogenetic analysis as described in ref. [26]. The study indicates that CAVV and NDiV consistently, albeit very distantly, cluster with viruses of the family Roniviridae, the only other known nidoviruses infecting invertebrates (Fig. 2).

Quantitatively, this Bayesian posterior probability phylogeny illustrates that CAVV and NDiV form a deeply rooted lineage in the nidovirus tree with an evolutionary divergence from other nidoviruses comparable to that sep- arating viruses of the families Coronaviridae and Roniviri- dae (Fig. 2).

Together, these characteristics of CAVV and NDiV (insect host, intermediate genome size, deeply rooted phylogenetic lineage) provide a compelling basis for the creation of a new nidovirus family.

We propose to name this new family Mesoniviridae, where meso is derived from the Greek word ‘‘mesos’’ (in English ‘‘middle’’ or ‘‘in the mid- dle’’) and refers to a key distinctive characteristic of these viruses, namely their intermediate-sized genomes.

The second component of the acronym, ni, refers to nidoviruses, as has been done previously for roniviruses [33] and bafini- viruses [34].

Next, we sought to establish species demarcation criteria to decide whether CAVV and NDiV prototype separate species or belong to a single species. Commonly, this question cannot be answered (reliably) on the basis of only two full genome sequences and otherwise very limited biological data.

To solve this dilemma, we exploited information available for other nidoviruses in our analysis. In order to evaluate the genetic similarity between CAVV and NDiV in the context of sequence divergence of lineages representing previously established nidovirus species, we applied a state-of-the-art framework for a genetics- based classification [35].

This recently introduced classification approach has been shown to recover and refine the taxonomy of picornaviruses [36], and it was also used to revise the taxonomy of coronaviruses extensively (Lauber & Gorbalenya, in preparation) [23].

In addition to CAVV and NDiV, a representative set of 152 large-sized nidoviruses was included in the analysis. Two sets of proteins were used: the first included proteins conserved in all nidoviruses (3CLpro, RdRp, HEL1) (dataset D1), while the second set additionally included ExoN and OMT, which are conserved in large-sized nidoviruses and CAVV/NDiV (dataset D2).

For both datasets a concatenated, multiple amino acid alignment was produced, which formed the basis for compiling pairwise evolutionary distances (PEDs) between all pairs of viruses (Fig. 3ab; for details see ref. [35]).

It was found that the PED separating CAVV and NDiV is within the range of intra-species virus divergence in the families Coronaviridae and Roniviridae for both datasets (Fig. 3cd). Specifically, CAVV and NDiV show a distance (0.016 and 0.029 for D1 and D2, respectively) that is below the genetic divergence of members of several established nidovirus species (maximum of 0.032 and 0.37 for D1 and D2, respectively).

For both datasets, these viruses include gill-associated virus and yellow head virus (species Gill-associated virus, family Roniviridae) [24] and the coronaviruses feline coronavirus, transmissible gastroenteritis virus, and porcine respiratory coronavirus (species Alphacoronavirus 1), IBV (species Avian coronavirus), murine hepatitis virus (species Murine coronavirus), and

Rousettus bat coronavirus HKU9 (species Rousettus bat coronavirus HKU9) [23]. For the dataset comprising the three nidovirus-wide conserved proteins (Fig. 3ac), Mini- opterus bat coronavirus 1 also showed a maximum genetic divergence exceeding that of the CAVV-NDiV pair.

Together, these observations show that CAVV and NDiV belong to the same species, representing a single genus in the family. We propose to name this genus Alphamesoni- virus and the species Alphamesonivirus 1, thereby fol- lowing a naming convention recently applied to the subfamily Coronavirinae [23], which is expected to facili- tate the accommodation of future expansions of the family.

A taxonomic proposal for family, genus, and species recognition has been available on-line at the ICTV website (http://talk.ictvonline.org/files/proposals/taxonomy_proposals_ invertebrate1/m/default.aspx) since August 2011. It has been approved by the chairs of the ICTV Arteriviridae, Coronaviridae, and Roniviridae Study Groups and the Executive Committee of the ICTV, and will be considered again at the next EC-ICTV meeting, to be held in Leuven, Belgium, in July 2012.

The recognition of CAVV and NDiV as a single virus species can be contrasted with the detection of these viruses in many mosquito host species and their spread to different continents (Africa and Asia, respectively) [26, 27].

The underlying mechanisms of this broad dispersal are unknown but might include the crossing of the host species barrier rather than virus-host cospeciation. Further research, including the characterization of biological properties of CAVV and NDiV and the extension of surveillance studies to other regions of the world, is needed to understand the ecology, host tropism and medical and/or economic relevance of mesoniviruses.

Zoonosis

Zoonosis (zoo-e-no-sis) is an infectious disease that may be transmitted from animals (wild and domestic) to humans or from humans to animals.

The word zoonosis is derived from the Greek, zoon (animal) (pronounced as zoo-on) and nosos (disease). Of the 1415 microbial diseases affecting humans, 61% are zoonotic (Taylor et al., 2001) and among emerging infectious diseases, 75% are zoonotic with wildlife being one of the major sources of infection (Daszak et al., 2001).

A new virus has been emerging almost every year since last two decades (Woolhouse and Sequeria, 2005).

Of 534 zoonotic viruses (belonging to 8 families) identified 120 cause human illnesses with or without the involvement of intermediate host/vectors. In the past 15 years, many zoonotic viral infections are of emerging and re-emerging in nature (Wilke and Hass, 1999) and haemorrhagic fever causing viruses transmitted by insect vectors (arboviruses i.e., yellow fever virus) (Khan et al., 1988), rodents i.e., Hanta viruses (Peters and Khan, 2002) and also by direct contact i.e., Filoviruses (Payling, 1996).

Thus, they pose a great challenge to both veterinary and public health professionals. It is essential to investigate the complex interactions between pathogens, host, vectors and environment to curtail these infections.

This review focuses on description of the important zoonotic viral infections with especially the recently emerging and reemerging diseases and their causes, transmission, clinical manifestations, distribution and preventive measures, to abreast the knowledge on zoonoses.

Transmission

Zoonotic viruses are transmitted to humans either directly or indirectly.

Direct transmission involves contact between the infected and susceptible individual (orf), bite (rabies) and handling of the affected animal’s tissues or materials (Orf).

Indirect transmission involves transmission through the bite of a hematophagous (blood-sucking) arthropod after replicating in the reservoir animal host (Japanese encephalitis, yellow fever).

Most viral zoonoses require blood-sucking arthropods for their transmission to humans. Among them, mosquitoes (Equine encephalitis complex) are the most common followed by ticks (Powassan virus), sand flies (Vesicular stomatitis) and midges (bluetongue).

The arthropod vector becomes infected when it feeds the blood of a viraemic animal.

In most of the cases, virus replicates in the arthropod tissues and reaches their salivary glands. The arthropod then transmits the virus to a new susceptible host when it injects infective salivary fluid while taking a blood meal.

The extrinsic incubation period (time between ingestion and transmission of the virus) is usually 8 to 12 days.

This period depends on the virus, the environment and the vector species involved (Hubalek and Halouzka, 1999). Arthropod-borne viruses generally remain undetected until humans encroach on the natural enzootic focus or until the virus escapes the primary cycle via a secondary vector or vertebrate host.

Wild birds are important to public health as they carry various zoonotic pathogens and they either act as reservoir hosts or help in disseminating the infected arthropod vectors (Reed et al., 2003).

In addition, bird migration provides a mechanism for the establishment of new endemic foci of disease at great distances from where an infection was acquired (avian influenza).

There has been a change in the transmission pattern especially in the occurrence and incidence of diseases due to broadening of host range (Monkey pox and Nipah viruses), high mutation rate (avian influenza, FMD) and anthropogenic environmental changes viz., ecological imbalance and change in agricultural practices (Wilke and Haas, 1999).

Role of Wildlife in Zoonosis

The significance of wild life as animal reservoir for zoonotic viruses has been traced long back with two important ancient diseases such as rabies and West Nile virus and represent as large spectrum of transmission mode (Marr and Calisher, 2003).

Of the total emerging diseases, 75% are considered zoonotic with wild life as a major source of reservoir. Recent emerging viral diseases which moved into new species such as AIDS, SARS and avian influenza have a strong evidence of wild life origin due to human encroachment and changed international trade and travel patterns.

Commonly the pattern of moving of viral agents from wild animal species to human occurs either as actual transmission being rare (HIV, Influenza A, Ebola and SARS) but will be maintained and has potential of man to man transmission or direct/indirect manner through animal bite and arthropod vectors (rabies, Nipah, West Nile virus and hantavirus) (Bengis et al., 2004).

Many zoonoses with a wildlife origin are spread through insect vectors (Rift Valley fever, equine encephalitis and Japanese encephalitis), whereas, rabies by animal bite and hantaviruses by contact with rodent excreta is common.

The outcome in the form of clinical manifestation in humans depends on the transmission pattern of the agent causing the disease. Direct contact and vector bite lead to the formation of rashes and ulcers, whereas, intake of contaminated meat/water lead to digestive tract problems and diseases transmitted by inhalation of infected foci of dust cause pneumonia like illness (Kruse et al., 2004).

Wild life are basically involved in epidemiology of the disease which is influenced by other factors such as change in agro-climatic conditions, host abundance, movement of pathogens/vector/animal host including migratory birds and anthropogenic factors.

For example, increase in transmission and subsequent spread of Sin Nombre Hantavirus causing Hantavirus Pulmonary Syndrome (HPS) to humans is due to increase in heavy rainfall and host abundance in USA.

Increase in the emergence of some wild life diseases result in high potential of emergence of human pathogens as in the case of West Nile virus spread in USA. A potential threat to human health, animal welfare and species conservation from domesticated and wild life is presented equally by emergence of human and wild life pathogens.

Emerging and Reemerging Zoonoses

The complex interaction between environment/ecology, social, health care, human demographics and behavior influences the emergence and re-emergence of zoonotic viral diseases. Periodic discovery of new zoonoses suggest that the known viruses are only a fraction of the total number that exist in nature.

The RNA viruses are capable of adapting to changing environmental conditions rapidly and are among the most prominent emerging pathogens (Ludwig et al., 2003).

Mutations are more common in RNA viruses (Influenza) than DNA viruses (Pox).

The common mutations are point (insertion/deletion), drift (minor) and shift (major). In addition to these, movement of population, birds, vectors, pathogens and trade contribute to the global spread of emerging infectious diseases (influenza, severe acute respiratory syndrome – SARS).

Other factors viz., human migration, change in land use pattern, mining (disturbance of ecosystem), coastal land degradation, wetland modification, construction of buildings, habitat fragmentation, deforestation, expansion of agents host range, human intervention in wild life resources like hiking, camping and hunting also influence on acquiring zoonotic infections from wildlife (Daszak et al., 2001; Bengis et al., 2004; Patz et al., 2004).

Cessation of vaccination against smallpox since 1980s, emergence of some genetically related orthopoxviruses has been reported throughout the world i.e., monkey pox (Nalca et al., 2005), buffalo pox (Singh et al., 2007) and Bovine Vaccinia (BV) infections (Fernandes et al., 2009).

Despite successful eradication of some viral diseases (small pox and almost polio in humans and rinderpest in cattle) due to intensive research and dedicated coordinated efforts, modern medicine has failed to control many infectious diseases resulting from emerging and reemerging viruses (Table 4).

Some infectious agents already known to be pathogenic have gained increasing importance in recent decades due to change in disease patterns. Several previously unknown infectious agents with a high pathogenic potential have also been identified (Manojkumar and Mrudula, 2006). Several infectious viral agents (DNA and RNA viral families) have been emerged as zoonotic agents (Table 4).

They are associated with flu-like signs (Alkhumra virus infection, influenza A) to respiratory (SARS), pox lesions mostly localized distributed over hairless parts of body namely udder, teats, ears and tail (in buffaloes) and fingers and hands (in humans) due to buffalopox and Orf virus infections in affected goats , hepatitis (hepatitis E virus), haemorrhagic fevers (Ebola, Marburg and hanta virus infections) and encephalitis (Henipa virus complex).

Treatment/prophylaxis is not available to many of these infections.

But some of antiviral compounds, which are under trial, are found to be effective.

Table 3:Viral zoonotic infections causing rashes and arthralgia

Table 4: Emerging and re-emerging zoonotic infections

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